INFORMATION ABOUT PRINCIPAL INVESTIGATORS/PROJECT DIRECTORS(PI/PD) and

co-PRINCIPAL INVESTIGATORS/co-PROJECT DIRECTORS

Submit only ONE copy of this form for each PI/PD and co-PI/PD identified on the proposal. The form(s) should be attached to the original
proposal as specified in GPG Section II.B. Submission of this information is voluntary and is not a precondition of award. This information will
not be disclosed to external peer reviewers. DO NOT INCLUDE THIS FORM WITH ANY OF THE OTHER COPIES OF YOUR PROPOSAL AS
THIS MAY COMPROMISE THE CONFIDENTIALITY OF THE INFORMATION.
PI/PD Name: Stanley A Cohn

Gender: Male Female

Ethnicity: (Choose one response) Hispanic or Latino Not Hispanic or Latino

Race: American Indian or Alaska Native

(Select one or more) Asian
Black or African American
Native Hawaiian or Other Pacific Islander
White

Disability Status: Hearing Impairment

(Select one or more)
Visual Impairment
Mobility/Orthopedic Impairment
Other
None

Citizenship: (Choose one) U.S. Citizen Permanent Resident Other non-U.S. Citizen

Check here if you do not wish to provide any or all of the above information (excluding PI/PD name):

REQUIRED: Check here if you are currently serving (or have previously served) as a PI, co-PI or PD on any federally funded
project
Ethnicity Definition:
Hispanic or Latino. A person of Mexican, Puerto Rican, Cuban, South or Central American, or other Spanish culture or origin, regardless
of race.
Race Definitions:
American Indian or Alaska Native. A person having origins in any of the original peoples of North and South America (including Central
America), and who maintains tribal affiliation or community attachment.
Asian. A person having origins in any of the original peoples of the Far East, Southeast Asia, or the Indian subcontinent including, for
example, Cambodia, China, India, Japan, Korea, Malaysia, Pakistan, the Philippine Islands, Thailand, and Vietnam.
Black or African American. A person having origins in any of the black racial groups of Africa.
Native Hawaiian or Other Pacific Islander. A person having origins in any of the original peoples of Hawaii, Guam, Samoa,
or other Pacific Islands.
White. A person having origins in any of the original peoples of Europe, the Middle East, or North Africa.

WHY THIS INFORMATION IS BEING REQUESTED:

The Federal Government has a continuing commitment to monitor the operation of its review and award processes to identify and address
any inequities based on gender, race, ethnicity, or disability of its proposed PIs/PDs. To gather information needed for this important
task, the proposer should submit a single copy of this form for each identified PI/PD with each proposal. Submission of the requested
information is voluntary and will not affect the organization’s eligibility for an award. However, information not submitted will seriously undermine
the statistical validity, and therefore the usefulness, of information recieved from others. Any individual not wishing to submit some or all the
information should check the box provided for this purpose. (The exceptions are the PI/PD name and the information about prior Federal support, the
last question above.)

Collection of this information is authorized by the NSF Act of 1950, as amended, 42 U.S.C. 1861, et seq. Demographic data allows NSF to
gauge whether our programs and other opportunities in science and technology are fairly reaching and benefiting everyone regardless of
demographic category; to ensure that those in under-represented groups have the same knowledge of and access to programs and other
research and educational oppurtunities; and to assess involvement of international investigators in work supported by NSF. The information
may be disclosed to government contractors, experts, volunteers and researchers to complete assigned work; and to other government
agencies in order to coordinate and assess programs. The information may be added to the Reviewer file and used to select potential
candidates to serve as peer reviewers or advisory committee members. See Systems of Records, NSF-50, "Principal Investigator/Proposal
File and Associated Records", 63 Federal Register 267 (January 5, 1998), and NSF-51, "Reviewer/Proposal File and Associated Records",
63 Federal Register 268 (January 5, 1998).

NSF Form 1225(10/99)

 List of Suggested Reviewers or Reviewers Not To Include (optional)

SUGGESTED REVIEWERS:
Dr. R. Gordon
Dept. of Botany and Radiology
Univ. of Manitoba
Winnepeg R3T 2N2 Canada
204-787-1076 (tel)
e-mail: gordonr@cc.umanitoba.ca

Dr. Michael R. Gretz

Department of Biological Sciences
Michigan Technological University
1400 Townsend Dr.
Houghton, MI 49931-1295
Phone: 906-487-3175
Fax: 906-487-3167
e-mail: mrgretz@mtu.edu

Prof. Dr. Donat-P. H der

Friedrich-Alexander Universitat
Institut fur Botanik und Pharmazeutische Biologie
Staudtstr. 5, D-91058, Erlangen
FRG
09131-858216 (tel)
09131-858215 (fax)
e-mail: dphaeder@biologie.uni-erlangen.de

Dr. Kyle D. Hoagland

University of Nebraska - Lincoln
School of Natural Resources
Dept. of Forestries, Fisheries & Wildlife
Lincoln, Nebraska 68583-0814
Phone: (402) 472-8182
Email: KHOAGLAND1@unl.edu
(khoaglan@unlnotes.unl.edu)

Dr. David G. Mann

Deputy Director - Royal Botanic Garden
20A Inverleith Row
Endinburgh, EH3 5LR, Scotland UK
031-552-7171 (tel)
031-552-0382 (fax)
e-mail: mann@rbge.org.uk

Dr. E.F. Stoermer

E. F. Stoermer
Center for Great Lakes
501 East University
University of Michigan
 List of Suggested Reviewers or Reviewers Not To Include (optional)

Suggested Reviewers contd...

Ann Arbor, MI 48109-1090

Phone 734-764-5238
FAX 734-647-2748
e-mail: stoermer@umich.edu

Dr. C. W. Sullivan
Dept. of Biological Sciences
University of Southern California
Bovard (ADM) - Room 203
Los Angeles, CA 90089
Phone 213-740-6709
FAX 213-740-8919
e-mail: csulliva@usc.edu

Dr. M. J. Sullivan
Dept. of Biology - Box GY
Mississippi State Univ.
Mississippi State, MS 39762
601-325-3120 (tel)
e-mail: mjs2@tut.msstate.edu

REVIEWERS NOT TO INCLUDE:

 COVER SHEET FOR PROPOSAL TO THE NATIONAL SCIENCE FOUNDATION
PROGRAM ANNOUNCEMENT/SOLICITATION NO./CLOSING DATE/if not in response to a program announcement/solicitation enter NSF 99-2 FOR NSF USE ONLY

NSF94-79 07/10/99 NSF PROPOSAL NUMBER

9982897
FOR CONSIDERATION BY NSF ORGANIZATION UNIT(S) (Indicate the most specific unit known, i.e. program, division, etc.)

Program - ECOLOGICAL & EVOLUTIONARY PHYS

DATE RECEIVED NUMBER OF COPIES DIVISION ASSIGNED FUND CODE DUNS# (Data Universal Numbering System) FILE LOCATION

825753379
EMPLOYER IDENTIFICATION NUMBER (EIN) OR SHOW PREVIOUS AWARD NO. IF THIS IS IS THIS PROPOSAL BEING SUBMITTED TO ANOTHER FEDERAL
TAXPAYER IDENTIFICATION NUMBER (TIN) A RENEWAL AGENCY? YES NO IF YES, LIST ACRONYMS(S)
AN ACCOMPLISHMENT-BASED RENEWAL

NAME OF ORGANIZATION TO WHICH AWARD SHOULD BE MADE ADDRESS OF AWARDEE ORGANIZATION, INCLUDING 9 DIGIT ZIP CODE
DePaul University
DePaul University
1 East Jackson Boulevard
AWARDEE ORGANIZATION CODE (IF KNOWN)
Chicago, IL. 606042218
0016717000
NAME OF PERFORMING ORGANIZATION, IF DIFFERENT FROM ABOVE ADDRESS OF PERFORMING ORGANIZATION, IF DIFFERENT, INCLUDING 9 DIGIT ZIP CODE

PERFORMING ORGANIZATION CODE (IF KNOWN)

IS AWARDEE ORGANIZATION (Check All That Apply)

(See GPG II.D.1 For Definitions) FOR-PROFIT ORGANIZATION SMALL BUSINESS MINORITY BUSINESS WOMAN-OWNED BUSINESS
TITLE OF PROPOSED PROJECT Physiological Ecology of Diatom Motility and Adhesion

REQUESTED AMOUNT PROPOSED DURATION (1-60 MONTHS) REQUESTED STARTING DATE SHOW RELATED PREPROPOSAL NO.,
IF APPLICABLE
$ 289,784 36 months 01/01/00
CHECK APPROPRIATE BOX(ES) IF THIS PROPOSAL INCLUDES ANY OF THE ITEMS LISTED BELOW
BEGINNING INVESTIGATOR (GPG 1.A.3) VERTEBRATE ANIMALS (GPG II.D.12) IACUC App. Date
DISCLOSURE OF LOBBYING ACTIVITIES (GPG II.D.1) HUMAN SUBJECTS (GPG II.D.12)
PROPRIETARY & PRIVILEGED INFORMATION (GPG II.D.10) Exemption Subsection or IRB App. Date
NATIONAL ENVIRONMENTAL POLICY ACT (GPG II.D.10) INTERNATIONAL COOPERATIVE ACTIVITIES: COUNTRY/COUNTRIES
HISTORIC PLACES (GPG II.D.10)
SMALL GRANT FOR EXPLOR. RESEARCH (SGER) (GPG II.D.12) FACILITATION FOR SCIENTISTS/ENGINEERS WITH DISABILITIES (GPG V.G.)
GROUP PROPOSAL (GPG II.D.12) RESEARCH OPPORTUNITY AWARD (GPG V.H)
PI/PD DEPARTMENT PI/PD POSTAL ADDRESS
Department of Biological Sciences DePaul University
2325 N. Clifton Avenue
PI/PD FAX NUMBER
Chicago, IL 606143238
773-325-7596 United States
NAMES (TYPED) High Degree Yr of Degree Telephone Number Electronic Mail Address
PI/PD NAME
Stanley A Cohn Ph.D. 1986 773-325-7597 scohn@condor.depaul.edu
CO-PI/PD

CO-PI/PD

CO-PI/PD

CO-PI/PD

NSF Form 1207 (10/98) Page 1 of 2

 CERTIFICATION PAGE

Certification for Principal Investigators and Co-Principal Investigators:

I certify to the best of my knowledge that:

(1) the statements herein (excluding scientific hypotheses and scientific opinions) are true and complete, and
(2) the text and graphics herein as well as any accompanying publications or other documents, unless otherwise indicated, are the original work of the
signatories or individuals working under their supervision. I agree to accept responsibility for the scientific conduct of the project and to provide the
required progress reports if an award is made as a result of this application.

I understand that the willful provision of false information or concealing a material fact in this proposal or any other communication submitted to NSF is a
criminal offense (U.S.Code, Title 18, Section 1001).

Name (Typed) Signature Social Security No.* Date

*ON FASTLANE SUBMISSIONS*

PI/PD

and are not displayed

SSNs are confidential
Stanley A Cohn
Co-PI/PD

Co-PI/PD

Co-PI/PD

Co-PI/PD

Certification for Authorized Organizational Representative or Individual Applicant:

By signing and submitting this proposal, the individual applicant or the authorized official of the applicant institution is: (1) certifying that
statements made herein are true and complete to the best of his/her knowledge; and (2) agreeing to accept the obligation to comply with NSF
award terms and conditions if an award is made as a result of this application. Further, the applicant is hereby providing certifications
regarding Federal debt status, debarment and suspension, drug-free workplace, and lobbying activities (see below), as set forth in Grant
Proposal Guide (GPG), NSF 99-2. Willful provision of false information in this application and its supporting documents or in reports required
under an ensuring award is a criminal offense (U. S. Code, Title 18, Section 1001).

In addition, if the applicant institution employs more than fifty persons, the authorized official of the applicant institution is certifying that the institution has
implemented a written and enforced conflict of interest policy that is consistent with the provisions of Grant Policy Manual Section 510; that to the best
of his/her knowledge, all financial disclosures required by that conflict of interest policy have been made; and that all identified conflicts of interest will have
been satisfactorily managed, reduced or eliminated prior to the institution’s expenditure of any funds under the award, in accordance with the
institution’s conflict of interest policy. Conflict which cannot be satisfactorily managed, reduced or eliminated must be disclosed to NSF.

Debt and Debarment Certifications (If answer "yes" to either, please provide explanation.)
Is the organization delinquent on any Federal debt? Yes No
Is the organization or its principals presently debarred, suspended, proposed for debarment, declared ineligible, or voluntarily excluded
from covered transactions by any Federal department or agency? Yes No

Certification Regarding Lobbying

This certification is required for an award of a Federal contract, grant, or cooperative agreement exceeding $100,000 and for an award of a Federal loan or
a commitment providing for the United States to insure or guarantee a loan exceeding $150,000.

Certification for Contracts, Grants, Loans and Cooperative Agreements

The undersigned certifies, to the best of his or her knowledge and belief, that:
(1) No federal appropriated funds have been paid or will be paid, by or on behalf of the undersigned, to any person for influencing or attempting to influence
an officer or employee of any agency, a Member of Congress, an officer or employee of Congress, or an employee of a Member of Congress in connection
with the awarding of any federal contract, the making of any Federal grant, the making of any Federal loan, the entering into of any cooperative agreement,
and the extension, continuation, renewal, amendment, or modification of any Federal contract, grant, loan, or cooperative agreement.
(2) If any funds other than Federal appropriated funds have been paid or will be paid to any person for influencing or attempting to influence an officer or
employee of any agency, a Member of Congress, an officer or employee of Congress, or an employee of a Member of Congress in connection with this
Federal contract, grant, loan, or cooperative agreement, the undersigned shall complete and submit Standard Form-LLL, ‘‘Disclosure Form to Report
Lobbying,’’ in accordance with its instructions.
(3) The undersigned shall require that the language of this certification be included in the award documents for all subawards at all tiers including
subcontracts, subgrants, and contracts under grants, loans, and cooperative agreements and that all subrecipients shall certify and disclose accordingly.
This certification is a material representation of fact upon which reliance was placed when this transaction was made or entered into. Submission of this
certification is a prerequisite for making or entering into this transaction imposed by section 1352, title 31, U.S. Code. Any person who fails to file the
required certification shall be subject to a civil penalty of not less than $10,000 and not more than $100,000 for each such failure.
AUTHORIZED ORGANIZATIONAL REPRESENTATIVE SIGNATURE DATE
NAME/TITLE (TYPED)
Marjorie P. Piechowski, Ph.D., Director 07/09/99
TELEPHONE NUMBER ELECTRONIC MAIL ADDRESS FAX NUMBER
773-325-2595 mpiechow@wppost.depaul.edu 773-325-7389
*SUBMISSION OF SOCIAL SECURITY NUMBERS IS VOLUNTARY AND WILL NOT AFFECT THE ORGANIZATION’S ELIGIBILITY FOR AN AWARD. HOWEVER, THEY ARE AN
INTEGRAL PART OF THE INFORMATION SYSTEM AND ASSIST IN PROCESSING THE PROPOSAL. SSN SOLICITED UNDER NSF ACT OF 1950, AS AMENDED.

Page 2 of 2
 Directorate for Biological Sciences
Division of Integrative Organismal Biology
Ecological & Evolutionary Physiology
Proposal Classification Form
PI: Cohn, Stanley / Proposal Number: 9982897
CATEGORY I: INVESTIGATOR STATUS (Select ONE)
Beginning Investigator - No previous Federal support as PI or Co-PI, excluding fellowships, dissertations, planning grants,
etc.
Prior Federal support only
Current Federal support only
Current & prior Federal support

CATEGORY II: FIELDS OF SCIENCE OTHER THAN BIOLOGY INVOLVED IN THIS RESEARCH
(Select 1 to 3)
Astronomy Engineering Psychology
Chemistry Mathematics Social Sciences
Computer Science Physics None of the Above
Earth Science

CATEGORY III: SUBSTANTIVE AREA (Select 1 to 4)

BEHAVIORAL STUDIES Keystone Species Agricultural Ecology
BIOENGINEERING COMPARATIVE APPROACHES ENDOCRINE DISRUPTORS/
ENVIRONMENTAL
BIOGEOGRAPHY COMPUTATIONAL BIOLOGY ENDOCRINOLOGY
Island Biogeography CONSERVATION & RESTORATION EPIGENETICS
BIOLOGY
Historical/ Evolutionary Biogeography EXTREMOPHILES
CORAL REEFS
Phylogeography GENOMICS (Genome sequence,
CURATION
Methods/Theory organization, function)
DATABASES Viral
BIOMATERIALS
DEVELOPMENTAL BIOLOGY Microbial
BIOTECHNOLOGY
ECOSYSTEMS LEVEL Fungal
Animal Biotechnology
Physical Structure Plant
Plant Biotechnology Animal
Decomposition
Environmental Biotechnology HUMAN NUTRITION
Biogeochemistry
Marine Biotechnology INFORMATICS
Limnology/Hydrology
Metabolic Engineering MARINE MAMMALS
Climate/Microclimate
CHRONOBIOLOGY MOLECULAR APPROACHES
Whole-System Analysis
COGNITIVE NEUROSCIENCE Molecular Evolution
Productivity/Biomass
COMMUNITY ECOLOGY NANOSCIENCE
System Energetics
Community Analysis ORGANISMAL SYSTEMS
Landscape Dynamics
Community Structure Physiological Approaches
Chemical & Biochemical Control
Community Stability Metabolic Processes
Global Change
Succession Hormonal Regulation/ Integration
Climate Change
Experimental Microcosms/ Mesocosms Stress Responses
Regional Studies Sensory Biology
Disturbance
Global Studies Movement Studies
Deforestation
Forestry PALEONTOLOGY
Patch Dynamics
Resource Management (Wildlife, Floristic
Food Webs/ Trophic Structure Fisheries, Range, Other)

Page 1
 Faunistic Variation Animal Pathology
Paleoecology Microevolution Plant Pathology
Biostratigraphy Speciation Coevolution
Palynology Hybridization Biological Control
Micropaleontology Inbreeding/Outbreeding SPINAL CORD/ NERVE
REGENERATION
Paleoclimatology Gene Flow Measurement
STATISTICS & MODELING
Archeozoic Inheritance/Heritability
Methods/ Instrumentation/ Software
Paleozoic Quantitative Genetics/ QTL Analysis
Modeling (general)
Mesozoic Ecological Genetics
Modeling of Biological or Molecular Systems
Cenozoic Gender Ratios
Computational Modeling
PHOTOSYNTHESIS Apomixis/ Parthenogenesis
Statistics (general)
PLANT BIOLOGY Vegetative Reproduction Multivariate Methods
Arabidopsis-Related Plant Research REPRODUCTIVE ANIMAL BIOLOGY Spatial Statistics & Spatial Modeling
POPULATION DYNAMICS & LIFE SPECIES INTERACTIONS Sampling Design & Analysis
HISTORY Experimental Design & Analysis
Predation
Demography/ Life History STRUCTURAL BIOLOGY
Herbivory
Population Cycles SYSTEMATICS
Omnivory
Distribution/Patchiness/ Marginal Taxonomy/Classification
Interspecific Competition
Populations
Niche Relationships/ Resource Nomenclature
Population Regulation Partititioning Monograph/Revision
Intraspecific Competition Pollination/ Seed Dispersal Phylogenetics
Reproductive Strategies Parasitism Phenetics/Cladistics/ Numerical
Gender Allocation Taxonomy
Mutualism/ Commensalism
Metapopulations Macroevolution
Plant/Fungal/ Microbial Interactions
Extinction NONE OF THE ABOVE
Mimicry
POPULATION GENETICS &
BREEDING SYSTEMS

CATEGORY IV: INFRASTRUCTURE (Select 1 to 3)

COLLECTIONS/STOCK CULTURES
Collection Enhancement
Collection Refurbishment
Living Organism Stock Cultures
Natural History Collections
DATABASES
Database Initiation
Database Enhancement
Database Maintenance & Curation
Database Methods
FACILITIES
Controlled Environment Facilities
Field Stations TOOLS DEVELOPMENT
Field Facility Structure Analytical Algorithm Development
Field Facility Equipment
Other Software Development
LTER Site Informatics Tool Development
GENOME SEQUENCING Technique Development
Arabidopsis Genome Sequencing
TRACKING SYSTEMS
Other Plant Genome Sequencing
Geographic Information Systems
INDUSTRY PARTICIPATION
Remote Sensing
INSTRUMENTATION
TRAINING
Instrument Development
Multi-, Cross-, Interdisciplinary Training
Instrument Acquisition
NONE OF THE ABOVE
Computational Hardware
Development/Acquisition

CATEGORY V: HABITAT (Select 1 to 2)

TERRESTRIAL HABITATS
GENERAL TERRESTRIAL Deciduous Forest Desert
Coniferous Forest SUBTROPICAL
TUNDRA
Rain Forest Rain Forest
BOREAL FOREST
Mixed Forest Seasonal Forest
TEMPERATE Prairie/Grasslands Savanna

Page 2
 Thornwoods Deciduous Forest ISLANDS (except Barrier Islands)
Deciduous Forest Coniferous Forest BEACHES/ DUNES/ SHORES/
Coniferous Forest Desert BARRIER ISLANDS
Desert CHAPPARAL/ SCLEROPHYLL/ CAVES/ ROCK OUTCROPS/ CLIFFS
TROPICAL SHRUBLANDS
CROPLANDS/ FALLOW FIELDS/
Rain Forest ALPINE PASTURES
Seasonal Forest MONTANE URBAN/SUBURBAN
Savanna CLOUD FOREST SUBTERRANEAN/ SOIL/
Thornwoods
RIPARIAN ZONES SEDIMENTS
EXTREME TERRESTRIAL
ENVIRONMENT
AERIAL
AQUATIC HABITATS
GENERAL AQUATIC Open Ocean/Continental Shelf EXTREME AQUATIC ENVIRONMENT
Bathyal
FRESHWATER CAVES/ ROCK OUTCROPS/ CLIFFS
Abyssal
Wetlands/Bogs/Swamps MANGROVES
Estuarine
Lakes/Ponds SUBSURFACE WATERS/ SPRINGS
Intertidal/Tidal/Coastal
Rivers/Streams
Coral Reef EPHEMERAL POOLS & STREAMS
Reservoirs
HYPERSALINE MICROPOOLS (Pitcher Plants, Tree
MARINE Holes, Other)
MAN-MADE ENVIRONMENTS
CELL/TISSUE CULTURE (In Vitro) THEORETICAL SYSTEMS OTHER ARTIFICIAL SYSTEMS
In Silico
NOT APPLICABLE
NOT APPLICABLE

CATEGORY VI: GEOGRAPHIC AREA OF THE RESEARCH (Select 1 to 2)

WORLDWIDE Eastern South America (Guyana, Fr. Guiana, North Africa
Suriname, Brazil)
African South of the Sahara
NORTH AMERICA Northern South America (Colombia,
Venezuela) East Africa
United States
Southern South America (Chile, Argentina, Madagascar
Northeast US (CT, MA, ME, NH, NJ, NY,
PA, RI, VT) Uruguay, Paraguay) South Africa
Northcentral US (IA, IL, IN, MI, MN, ND, Western South America (Ecuador, Peru, West Africa
NE, OH, SD, WI) Bolivia)
AUSTRALASIA
Northwest US (ID, MT, OR, WA, WY) EUROPE
Australia
Southeast US (DC, DE, FL, GA, MD, NC, Eastern Europe
SC, WV, VA) New Zealand
Russia
Southcentral US (AL, AR, KS, KY, LA, MO, Pacific Islands
MS, OK, TN, TX) Scandinavia
Western Europe ANTARCTICA
Southwest US (AZ, CA, CO, NM, NV, UT)
Alaska ASIA ARCTIC
Hawaii Central Asia ATLANTIC OCEAN
Puerto Rico Far East PACIFIC OCEAN
Canada Middle East INDIAN OCEAN
Mexico Siberia OTHER REGIONS (Not defined)
CENTRAL AMERICA (Mainland) South Asia
NOT APPLICABLE
Caribbean Islands Southeast Asia
Bermuda/Bahamas AFRICA
SOUTH AMERICA

CATEGORY VII: CLASSIFICATION OF ORGANISMS (Select 1 to 4)

VIRUSES Plant PROKARYOTES
Bacterial Animal Archaebacteria

Page 3
 Cyanobacteria Fabaceae (Leguminosae) Merostomata (Horseshoe Crabs)
Eubacteria Lamiaceae (Labiatae) Pycnogonida (Sea Spiders)
PROTISTA (PROTOZOA) Rosaceae Scorpionida (Scorpions)
Amoebae Solanaceae Araneae (True Spiders)
Apicomplexa ANIMALS Pseudoscorpionida
(Pseudoscorpions)
Ciliophora INVERTEBRATES
Acarina (Free-living Mites)
Flagellates MESOZOA/PLACOZOA
Parasitiformes (Parasitic Ticks &
Foraminifera PORIFERA (Sponges) Mites)
Microspora CNIDARIA Crustacea
Radiolaria Hydrozoa (Hydra, etc.) Branchiopoda (Fairy Shrimp, Water
Flea)
FUNGI Scyphozoa (Jellyfish)
Ostracoda (Sea Lice)
Ascomycota Anthozoa (Corals, Sea Anemones)
Copepoda
Basidiomycota CTENOPHORA (Comb Jellies)
Cirripedia (Barnacles)
Chytridiomycota PLATYHELMINTHES (Flatworms)
Amphipoda (Skeleton Shrimp,
Mitosporic Fungi Turbellaria (Planarians) Whale Lice, Freshwater Shrimp)
Oomycota Trematoda (Flukes) Isopoda (Wood Lice, Pillbugs)
Yeasts Cestoda (Tapeworms) Decapoda (Lobster, Crayfish,
Zygomycota Monogenea (Flukes) Crabs, Shrimp)
GNATHOSTOMULIDA Hexapoda (Insecta) (Insects)
LICHENS
NEMERTINEA (Rynchocoela) (Ribbon Apterygota (Springtails, Silverfish,
SLIME MOLDS Worms) etc.)
ALGAE ENTOPROCTA (Bryozoa) (Plant-like Odonata (Dragonflies, Damselflies)
Animals)
Bacillariophyta (Diatoms) Ephemeroptera (Mayflies)
ASCHELMINTHES
Charophyta Orthoptera (Grasshoppers, Crickets)
Gastrotricha
Chlorophyta Dictyoptera (Cockroaches, Mantids,
Kinorhyncha Phasmids)
Chrysophyta
Loricifera Isoptera (Termites)
Dinoflagellata
Nematoda (Roundworms) Plecoptera (Stoneflies)
Euglenoids
Nematomorpha (Horsehair Worms) Phthiraptera (Mallophaga &
Phaeophyta Anoplura) (Lice)
Rotifera (Rotatoria)
Rhodophyta Hemiptera (including Heteroptera)
ACANTHOCEPHALA (Spiny-headed (True Bugs)
PLANTS Worms)
Homoptera (Cicadas, Scale Insects,
N0N-VASCULAR PLANTS PRIAPULOIDEA Leafhoppers)
BRYOPHYTA BRYOZOA (Ectoprocta) (Plant-like Thysanoptera (Thrips)
Animals)
Anthocerotae (Hornworts) Neuroptera (Lacewings,
PHORONIDEA (Lophophorates) Dobsonflies, Snakeflies)
Hepaticae (Liverworts)
BRACHIOPODA (Lamp Shells) Trichoptera (Caddisflies)
Musci (Mosses)
MOLLUSCA Lepidoptera (Moths, Butterflies)
VASCULAR PLANTS
Monoplacophora Diptera (Flies, Mosquitoes)
FERNS & FERN ALLIES
Aplacophora (Solenogasters) Siphonaptera (Fleas)
GYMNOSPERMS
Polyplacophora (Chitons) Coleoptera (Beetles)
Coniferales (Conifers)
Scaphopoda (Tooth Shells) Hymenoptera (Ants, Bees, Wasps,
Cycadales (Cycads)
Gastropoda (Snails, Slugs, Limpets) Sawflies)
Ginkgoales (Ginkgo)
Pelecypoda (Bivalvia) (Clams, Chilopoda (Centipedes)
Gnetales (Gnetophytes) Mussels, Oysters, Scallops) Diplopoda (Millipedes)
ANGIOSPERMS Cephalopoda (Squid, Octopus, Pauropoda
Monocots Nautilus)
Symphyta (Symphyla)
Arecaceae (Palmae) ANNELIDA (Segmented Worms)
PENTASTOMIDA (Linguatulida)
Cyperaceae Polychaeta (Parapodial Worms) (Tongue Worms)
Liliaceae Oligochaeta (Earthworms) TARDIGRADA (Tardigrades, Water
Hirudinida (Leeches) Bears)
Orchidaceae
POGONOPHORA (Beard Worms) ONYCHOPHORA (Peripatus)
Poaceae (Graminae)
SIPUNCULOIDEA (Peanut Worms) CHAETOGNATHA (Arrow Worms)
Dicots
ECHIUROIDEA (Spoon Worms) ECHINODERMATA
Apiaceae (Umbelliferae)
ARTHROPODA Crinoidea (Sea Lilies, Feather Stars)
Asteraceae (Compositae)
Cheliceriformes Asteroidea (Starfish, Sea Stars)
Brassicaceae (Cruciferae)

Page 4
 Ophiuroidea (Brittle Stars, Serpent
Stars)
Echinoidea (Sea Urchins, Sand
Dollars)
Holothuroidea (Sea Cucumbers)
HEMICHORDATA (Acorn Worms,
Pterobranchs)
UROCHORDATA (Tunicata) (Tunicates,
Sea Squirts, Salps, Ascideans)
CEPHALOCHORDATA
(Amphioxus/Lancelet)
VERTEBRATES
AGNATHA (Hagfish, Lamprey)
FISHES
Chondrichthyes (Cartilaginous Fishes)
(Sharks, Rays, Ratfish)
Osteichthyes (Bony Fishes)
Sarcopterygia (Lobe-finned Fishes)
(Coelacanth, Lungfish)
Actinopterygia (Ray-finned Fishes)
AMPHIBIA
Anura (Frogs, Toads)
Urodela (Salamanders, Newts)
Gymnophiona (Apoda) (Caecilians)
REPTILIA
Chelonia (Turtles, Tortoises)
Serpentes (Snakes)
Sauria (Lizards)
Crocodylia (Crocodilians)
Rhyncocephalia (Tuatara)
CATEGORY VIII: MODEL ORGANISM (Select ONE)
NO MODEL ORGANISM
MODEL ORGANISM (Choose from
the list or input up to 9 characters)
VIRUS/BACTERIA
Lambda Phage
Rhizobacterium
Escherichia coli
Bacillus subtilis
Cyanobacteria (Selenococcus/Selenobacter)
ANIMAL
PROTISTA
Acetabularia acetabulum
Chlamydomonas reinhardtii
Paramecium
Tetrahymena
FUNGI
Dictyostelium
Neurospora
Saccharomyces cereviseae
Schizosaccharomyces pombe
PLANT
AVES (Birds)
Paleognathae (Ratites)
Sphenisciformes (Penguins)
Procellariiformes (Albatrosses, Petrels,
Fulmars)
Pelecaniformes (Pelicans, Gannets,
Boobies, Tropicbirds)
Ciconiiformes (Herons, Bitterns,
Egrets, Storks, Ibis, Flamingo)
Anseriformes (Ducks, Geese,
Screamers)
Falconiformes (Vultures, Hawks,
Eagles, Condors, Kites, Falcons)
Galliformes (Megapodes, Turkeys,
Quail, Pheasants, Peafowl, etc.)
Gruiformes (Cranes, Rails, Gallinules,
Coots, Bustards, Crakes)
Charadriiformes (Terns, Gulls, Stilts,
Avocets, Plovers, Puffins, etc.)
Columbiformes (Pigeons, Doves)
Psittaciformes (Parrots, Lories,
Cockatoos, Kakapo, Conures, etc.)
Cuculiformes (Cuckoos, Turacos, Anis,
Coucal, Roadrunner, etc.)
Strigiformes (Owls)
Apodiformes (Hummingbirds, Swifts,
Thornbills)
Coraciformes (Kingfishers, Todies,
Bee-Eaters, Rollers, Hornbills, etc.)
Piciformes (Woodpeckers, Toucans,
Jacamars, Barbets, Honeyguides)
Passeriformes (Passerines)
MAMMALIA
Monotremata (Platypus, Echidna)
Marsupalia (Marsupials)
Eutheria (Placentals)
Mouse-Ear Cress (Arabidopsis thaliana)
Ice Plant (Mesembryanthemum spp.)
Barley (Hordeum vulgare)
Corn (Zea mays)
Pea (Pisum sativum)
Tobacco (Nicotiana spp.)
Spinach (Spinacia oleracea)
Alfalfa (Medicago spp.)
Tomato (Lycopersicon spp.)
Nematode (Caenorhabditis elegans)
Sea Slug (Aplysia californica)
Sea Slug (Hermissenda spp.)
Pond Snail (Lymnaea spp.)
Terrestrial Snail (Helix spp.)
Squid/Cuttlefish (Loligo, Sepia, etc.)
Octopus (Octopus spp.)
Leech (Hirudo medicinalis)
Horseshoe Crab (Limulus spp.)
Brine Shrimp (Artemia spp.)
Lobster (Homarus, Panilurus, etc.)
Insectivora (Hedgehogs, Moles,
Shrews, Tenrec, etc.)
Chiroptera (Bats)
Edentata (Anteaters, Sloths,
Armadillos)
Primates
Monkeys
Apes (Gibbons, Orang-utan,
Gorilla, Chimpanzee)
Humans
Rodentia
Laboratory Rodents (Rat, Mouse,
Guinea Pig, Hamster)
Non-Laboratory Rodents
Lagomorphs (Rabbits, Hares, Pikas)
Tubulidenata (Aardvarks)
Carnivora (Bears, Canids, Felids,
Mustelids, Viverrids, Hyena,
Procyonids)
Ungulates
Perissodactyla (Odd-toed
Ungulates) (Horses, Rhinos,
Tapirs, etc.)
Artiodactyla (Even-toed
Ungulates) (Cattle, Sheep, Deer,
Pigs, etc.)
Sirenia (Manatees, Dugongs)
Proboscidea (Elephants)
Marine Mammals (Seals, Walrus,
Whales, Otters, Dolphins, Porpoises)
TRANSGENIC ORGANISMS
FOSSIL OR EXTINCT ORGANISMS
NO ORGANISMS
Crayfish (Procambarus, Astacus, etc.)
Dragonfly (Aeschna, etc.)
Grasshopper/Locust (Schistocerca, etc.)
Cockroach (Periplaneta, Blatta, Blatella, etc.)
Mantis (Mantis, Parasphendale, etc.)
Six-Lined Hawk Moth (Manduca sexta)
Fruitfly (Drosophila melanogaster)
Syrphid Fly (Syrphidae)
Apple Maggot (Rhagoletis spp.)
Mosquito (Culex, Aedes, Anopheles, etc.)
Flour Beetle (Tenebrio spp./Tribolium spp.)
Honeybee (Apis mellifera)
Parasitic Wasp (Braconids, Pteromalids,
etc.)
Sea Urchin (Diadema, Mellita, etc.)
Ascidian (Boltenia, Molgula, etc.)
Lancelet (Amphioxus spp.)
Lamprey (Petromyzon spp.)
Skate (Raja, Myliobatis, etc.)
Croaker (Sciaenid Fishes)
Electric Fish (Eigenmannia, Sternopygus,
etc.)
Page 5
Goldfish (Carassius auratus, etc.)
Perch (Perca spp.)
Zebrafish (Danio (Brachydanio) rerio)
Axolotl (Ambystoma mexicanum)
Mudpuppy (Necturus spp.)
African Clawed Frog (Xenopus laevis)
Bullfrog (Rana catesbeiana)
Grass Frog (Rana pipiens)
Marine Toad (Bufo marinus)
Turtle (Chrysemys, Pseudemys, etc.)
Quail (Coturnix spp.)
Chicken Embryo (Gallus domesticus)
House Sparrow (Passer domesticus)
White-Crowned Sparrow (Zonotrichia
leucophrys)
Zebra Finch (Poephila guttata)
Opossum (Monodelphis, Didelphis, etc.)
Bat (Antrozous, Eptesicus, etc.)
Owl Monkey (Aotus spp.)
Rhesus Monkey (Macaca mulatta)
Tamarin (Sanguinus, Leontopithecus spp.)
Chimpanzee (Pan troglodytes)
Human (Homo sapiens)
Chinchilla (Chinchilla laniger)
Deer Mouse (Peromyscus spp.)
Guinea Pig (Cavia porcellus)
Hamster (Mesocricetus, Phodopus, etc.)
Kangaroo Rat (Dipodomys, etc.)
Mouse, Laboratory
Rat, Laboratory
Vole (Microtus spp.)
Domestic Dog (Canis domestica/familiaris)
Domestic Cat (Felis domestica/cattus)
Ferret (Mustelus spp.)
Farm Animals (Horse, Sheep, Pigs, Cattle,
Goats)
[Enter your own model organism - up to
9 characters]
Page 6
 A. Project Summary

Title: Physiological Ecology of Diatom Motility and Adhesion

The ability of cells and organisms to perform controlled and directed movements within their
environment is critical to their ecological success and survival. This project involves a study
designed to understand cell movement in diatoms, a ubiquitous group of golden algae abundant in
both freshwater and marine communities. Although diatoms are ecologically important as one of
the main primary biomass producers in many aquatic communities, little is known about the
environmental regulators of their motility, and the role that motility plays in their ability to
successfully compete (e.g. with other diatoms and with other algae) for light and nutrients.

Many diatoms display active and directed (e.g. phototactic) gliding movements, despite their
enclosure within hardened glass-like cell walls that prohibit direct contact between the cell
membranes and the sediment over which they move. Recent work from our lab has demonstrated
that different diatom species have significantly different motile behaviors, particularly in response to
environmental light conditions. The experiments outlined in this proposal are designed to help test
two specific hypotheses: 1) that the differences in diatom motile behaviors are generated by
differences in the number, distribution and/or translocation speed of cell/substratum contact sites;
and 2) species-specific differences in cells' motile responses to both environmental conditions and
the cell type composition in the population generate different sets of optimal ecological conditions
for each species and population.

This project is designed to test these hypotheses and further understand the ecological
factors affecting diatom motility by using video microscopy analysis in three sets of experiments:

1) Quantitatively analyzing the changes in the characteristics of motility and adhesion for several
diatom species when different combinations of species are placed together under a variety of
environmental conditions. This will allow us to determine how inter-species competition alters the
types of motility characteristics we have already determined for individual species, and the degree
to which light-stimulated behaviors are altered in mixed populations.

2) Using a laboratory flume set-up to measure attributes of diatom adhesion in flowing water. This
will allow us to place mixed populations of cells under different flow conditions, allowing us to
determine how population makeup affects or alters the characteristic adhesions of single species
previously measured. By altering light and nutrient conditions, as well as water flow, we can begin
to determine which ecological conditions are most favorable for which types of diatom populations.

3) Directly analyzing diatom cell/substratum contact sites using reflection interference microscopy.
By observing these sites while altering the ecological conditions such light and temperature, we can
also directly observe changes in the contact sites for cells under different conditions. We will then
be able to correlate the differences in diatom motile behaviors that we have previously
characterized with differences in the number, distribution or speed of contact sites.

Moreover, while not directly related to the experimental hypotheses, data accumulated from
these studies will also help to determine a number of diatom properties useful in understanding the
conditions for overall diatom viability:
• the environmental cues which can deleteriously affect diatom motile behavior;
• the limits of deleterious environmental conditions (such as nutrient starvation, UV light,
chemical toxins) that can be tolerated by particular diatoms;
• the light, nutrient, and substratum conditions most favorable to the growth and vitality of
various diatom species.
 TABLE OF CONTENTS
For font size and page formatting specifications, see GPG section II.C.

Section Total No. of Page No.*

Pages in Section (Optional)*

Cover Sheet (NSF Form 1207) (Submit Page 2 with original proposal only)

A Project Summary (not to exceed 1 page) 1

B Table of Contents (NSF Form 1359) 1

C Project Description (plus Results from Prior 19

NSF Support) (not to exceed 15 pages) (Exceed only if allowed by a
specific program announcement/solicitation or if approved in
advance by the appropriate NSF Assistant Director or designee)

D References Cited 9

E Biographical Sketches (Not to exceed 2 pages each) 2

F Budget 8
(NSF Form 1030, plus up to 3 pages of budget justification)

G Current and Pending Support (NSF Form 1239) 1

H Facilities, Equipment and Other Resources (NSF Form 1363) 1

I Special Information/Supplementary Documentation 0

J Appendix (List below. )

(Include only if allowed by a specific program announcement/
solicitation or if approved in advance by the appropriate NSF
Assistant Director or designee)

Appendix Items:

*Proposers may select any numbering mechanism for the proposal. The entire proposal however, must be paginated.
Complete both columns only if the proposal is numbered consecutively.
NSF Form 1359 (10/99)
 C. Project Description
Resubmission Response
This proposal is a resubmission of one previously submitted to NSF in 1997 and 1998 and
revised based on reviewers’ comments. Specific modifications made to this submission include:
• Based on reviewers' comments and discussions with the program director, the number of experimental
subsections has been reduced. This includes the elimination of the vertical migration experiments,
which were the most exploratory of the experiments.
• In order to strengthen the rationale and conceptual framework within the proposal, brief explanations
have been added to each experimental section, stating how the described experiments relate to our
previous results and to the goals of the proposal.
• Modifications from earlier versions of the proposal that remain are:
– measurement of growth rates for the four main species of diatoms under our typical culture
conditions ([10] - all species have doubling times over 2 days)
– control experiments for cell growth rate in all experiments lasting more than several hours
–basic staining techniques to analyze the mucilage secretions produced by the diatoms as part of the
flume studies
– additional information on the protocols involved in the flume and adhesion experiments, and on the
distribution of time points used for the observations and measurements used in these experiments
• Earlier reviewers voiced concerns over the level of explanation and activities in the proposal that would
tie the laboratory experiments proposed and relevant field observations, and were divided between
expanding the ecological field experiments in the proposal or eliminating them entirely. In response to
these concerns, the field-based work was cut from the proposal itself, and a section was added at the
end of the proposal which outlines the types of field-based work we plan to undertake both
concurrently and subsequent to accumulating the lab-based data of this proposal. This organization
allows the proposal to more clearly show how we plan to connect the lab results with field
observations.
• Earlier reviewers were also split on the degree to which measurement on additional species was needed,
with some reviewers feeling they diverted time from focusing more extensively on a fewer number of
species. In order to address these concerns, the number of additional new species being tested has been
reduced, with a slightly expanded explanation of why the remaining additional species would be useful.

Introduction and Background

Diatoms are an abundant and ubiquitous group of unicellular algae that are distinctive for
their highly ornamented and often rigid silica-based cell walls, as well as their golden-brown
pigmentation [97]. Diatoms are prevalent in almost all aquatic environments, both freshwater and
marine, and exist in both suspended (planktonic) and sediment-dwelling (benthic) forms. As a
group, diatoms are one of the major sources of primary biomass production. Despite the fact that
these photosynthetic cells are among the most ecologically important members of aquatic
communities, much is unknown about their physiology, behavior, ecology, and evolution
[43,97,98,110]. In this study we plan to contribute to the understanding of diatom motility and
ecology by investigating how changes in the environmental conditions (e.g. light, temperature,
substratum composition) affect the motile characteristics of different diatom species, particularly as
they compete with multiple species in resource-limited environments.

Diatom Structure
Diatom cell walls are composed of two overlapping halves, somewhat like the top and bottom of Petri
plates, which are held together by mucilage. Diatoms secrete large amounts of mucilage, not only for
adhesion of the cell wall components, but for physical protection and adhesion of the cell to the substratum
as well [71,97,98,126,127]. Removal of this mucilage by hydrolytic enzymes renders some cells prone to
rapid osmotic swelling and rupture [9]. The main structural component of each half of the diatom wall, the
valve, is highly ornamented, with species-specific patterns of pores and striations [98,103] that allow the cell
to have access to small molecules, ions and nutrients while still offering the cell great physical protection
from bacteria, small predators, and osmotic shock. The large variety of wall forms is thought to have
derived from the diverse selective pressures on diatoms. Such pressures likely include maximizing
surface/volume ratio for nutrient uptake and adhesive contact, streamlining along the apical axis for reducing
resistance to water flow, regulating colonial attachments and sedimentation rates, and efficiently adhering to
or moving through sediments and other algal colonies [43,49,78,103]. Additional variations in the cell wall
structure, due to physiological responses to changes in environmental conditions or different stages in life
histories, make it difficult to assess the actual number of diatom species [43,103].
The relative inflexibility of the silica cell wall also requires that the plane of cell division occurs
midway between the two halves [90,92,93], such that each of the daughter cells remains associated with one
of the two halves of the parental wall. Soon after cell division, each daughter cell secretes one new half-wall
(valve) from a specialized membranous compartment called the silicalemma, which forms directly beneath
the plasma membrane in the area of the cleavage furrow. The new valves form a protective silica-based
scale over each plasma membrane, after which the two daughter cells enlarge, separating themselves from
one another. In this manner, cells can grow and divide without breaking or rupturing the solid portions of the
cell wall. Since each daughter cell produces and secretes one new valve at each division, each vegetative cell
is surrounded by a cell wall consisting of one new valve and one valve inherited from the parental cell. This
arrangement has been useful in studies investigating cell wall morphogenesis, as valves formed during
experimental treatment can be directly compared to the untreated parental valve present on the same cell
[14,102].
A characteristic slit, the raphe, is usually present along the length of one or both of the valves in motile
benthic diatoms. Depending on the species, benthic diatoms may have a raphe on both valves (biraphid),
only one of the valves (monoraphid), or no raphe slit at all (araphid). The raphe can be along the flattened
surface of the valve (known as the valve face), or along an extended edge of the cell wall (known as the keel).
The exact shape and orientation of the raphe is different in each species [28,30,98,104], but close proximity
between the substratum and the raphe is required for motility [41,54].
Diatom Motility
The structural organization of diatoms, in which the cell is constrained within the confines of a
hardened silica-based cell wall, creates restrictions on cell motility. Major ecological displacements of
diatoms are no doubt driven by waves, tidal forces, and water currents [79, 108], and possibly amplified by
the loss of adhesion under unfavorable conditions [4]. However, many benthic diatoms are actively motile
when in contact with a solid or semi-solid surface [41,97,98]. The speed of diatom movements varies
dramatically with species, with some forms moving as rapidly as 10-20 µm/sec, while others move at rates <
1-2 µm/sec. Motile species can live at or beneath the surface of aquatic sediments, and many types have
well coordinated movements such as diurnal phototactic behavior [54,97] and coordinated alignment of cells
during sexual reproduction [19,91,126]. Motility also allows diatoms to migrate up within the layers of
sediment or surface algae to absorb more sunlight during the day, and settle into areas with higher
concentrations of organic nutrients at night [34].
The hardened cell wall, which completely surrounds the diatom plasma membrane, does not allow for
cytoplasmic extensions or protrusions that could actively propel the cell through the water as cilia or flagella
do. Nor can the cell maintain direct membrane-to-substratum contact that would allow the cell to crawl over
the surface in amoeboid-like movement. The mechanisms responsible for generating and controlling this
motility must therefore overcome these physical restrictions [35,41], and recent evidence supports cellular
proteoglycan secretions as the material generating cell/substratum attachments [68, 69].
Several lines of evidence indicate that mucilage or proteoglycan material secreted through the raphe is
required for motility. For example, asymmetric diatoms tend to move in a direction and curvature that match
that of their raphe [41], strands or filaments extending from the raphe are detected using electron microscopy
when protocols designed to stabilize polysaccharides are used [38,41], and particles adhering to the diatom in
the area of the raphe are observed to be transported bidirectionally along the raphe. In addition, some
diatoms are known to deposit mucilage trails from their raphes as they move [35,41], antibodies directed
against proteoglycan material in the raphe area can inhibit motility and adhesion [68,69], chemicals that
interfere with motility can affect secretion and adhesion [120, 128], and chemicals that inhibit secretion of
extracellular matrix can inhibit motility [119]. Moreover, the motile ability of a diatom is correlated with its
shape and ornamentation (e.g. the shape of its raphe [2,41]). Motility of freshwater diatoms also requires 1-
2 atm osmotic pressure of protoplasts against the cell wall [9] suggesting that the pressure may be required
for proper extrusion of mucilage material through the raphe.
Several models by which secreted mucilage could generate raphe-based motility have been suggested,
including propulsion through rapid mucilage expulsion and/or hydration or directed capillary action
[48,50,106]. Mucilage extrusion alone might be able to explain the slower gliding movement of some algae
such as desmids, photosynthetic bacteria, or some araphid or stalk forming diatoms [52,59,89]. However,
the faster and more responsive motility of many raphid diatoms, as illustrated by their phototactic movements
and lateral pairing of cells prior to sexual reproduction [20,21,22,37,41,75,97,98,126] make such a
mechanism unlikely.
The most extensive model for diatom motility, developed by Edgar and others, is based on several sets
of observations: 1) Two bundles of cytoplasmic filaments, associated with the plasma membrane and running
underneath and parallel to the raphe [39,40], are positively stained with fluorescent phalloidin, a probe
specific for actin [42,116,128]; and 2) protocols that stabilize polysaccharides revealed a number of small
fibrous strands of material extending through the raphe to the exterior of the cell [38]. Edgar speculated that
the fibrous material was composed of adherent mucilage strands secreted from the cell and extruded through
the raphe. Under this model, motile force could be produced by moving membrane-bound mucilage
attachment sites along underlying actin filaments, effectively pulling on the mucilage strands down the length
of the cell. The mucilage attachment to the substratum would act as an anchoring site against which the cell
could generate force, thus allowing the cell to pull itself along [41].
The mechanisms of such movement could be similar to the cytoplasmic streaming of vesicles or
chloroplasts along actin cables in higher plant cells [63,105,129], which is generated by force-producing
"motor" proteins such as the myosins [64,65,66,94]. Bidirectional movement could be generated by the use
of anti-parallel actin filaments, either by having actin filaments of both orientations contained within each
actin cable, or by having each of the two cables uniformly oriented, but in opposite directions.
Microfilaments in diatoms are also involved with other types of intracellular motility, such as cytokinesis and
placement of cell wall material [14,90], so one must be cautious; the actin cables may be associated with
secondary processes, and not directly involved in force generation [48]. Since several forms of microtubule-
based motility in diatoms are well-known [14,15,86,130,131], the role of microtubules in motility also
cannot be ruled out. However, latrunculin, a potent actin inhibitor from sea sponge, causes rapid and
reversible inhibition of diatom motility [128], further supporting the model of actin cables for motility.
Diatom Ecology and Physiology
Diatoms have been objects of experimental investigation ever since the intricately sculptured cell walls
and distinct golden pigmentation caught the attention of the early microscopists, and were among the first
organisms analyzed for mitosis and movement [67,73,106]. These cells are abundant, widespread, and a
major source of primary biomass production in many marine and freshwater aquatic communities [7,97,98].
The significance of diatoms at the start of many food chains can be shown, for example, by the toxic
poisonings of humans and brown pelicans [44,82], both of which were ultimately the result of diatom
toxicity. For the pelicans it was due to high levels of domoic acid in anchovies, a principal pelican food
source, which had fed on a bloom of a toxic diatom, while the human poisonings were the result of domoic
acid accumulation in mussels. Such episodes, while rare, graphically demonstrate the importance of diatoms
in the maintenance and stability of aquatic food chains and why they are crucial to the ecological stability of
many aquatic ecosystems [7,98,110]. Their importance in aquatic ecosystems, along with their sensitivity to
environmental conditions, has made diatoms popular indicators in the study of changes in environmental
water quality and ecological stress [13,95,96,109].
Diatoms are also considered to be ecologically important contributors to the erodibility and stability of
some aquatic sediments. The large amount of mucilage secreted by diatoms (which can vary considerably in
composition [57,132]) allows them to adhere strongly to both natural and man-made surfaces [26,27,55],
and is likely composed of a complex set of secreted materials [127]. The mucilage secretions can affect the
erodibility of some intertidal regions [81], may be responsible for some of the stability of river sediments
[70] and is also thought to affect the successful immigration of diatoms into newly available environments
[84,108,111,112]. Some studies show an apparent correlation between diatom motility and strength of
adhesion to the substratum, as well as substrate-specific differences in adhesion [1,55,77,113]. Adhesion
may also be correlated with the presence or pre-conditioning of the substrates with bacteria or other biofilms
[27,51,85]. Mucilage secretion is also known to be an important factor in inter-species aggregation [31,53].
The ecological regulation of benthic algal communities, and the physiological and competitive
interactions responsible for generating successful diatom communities remain poorly understood [72].
Diatoms can migrate through sediment [80] and during development of algal communities may undergo
stratification, with particular diatoms species being found at characteristic distances beneath the top surface
of the community [60,108,112]. Since light can become limited as the density of the community increases,
benthic diatom survival must often involve behavioral strategies to compensate for diminishing light
availability. Such strategies can include movement upward through the surrounding algae, passive
displacement due to stalk formation or differential adhesion to adjacent material [113], or non-motile
responses such as temporary dormancy or conversion to heterotrophy [115]. The relative contribution of
motility remains undetermined, but is almost certain to be considerable in many circumstances since non-
motile species often remain at the bottom of developing communities [108,112].
Diurnal vertical cell movements may enhance survival of algae such as diatoms by allowing cells to
move upward and capture more light during the day, then resettle downward to the more nutrient rich bottom
of the sediment during the night [110,112]. If this is true, there should be environmental cues (e.g. light,
ions, surface properties), either within the sediment or within the overlying water, which regulate the
movement. However, such regulatory processes are poorly understood in diatoms and other benthic algae
[110]. There have been relatively few studies on chemotactic responses in diatoms, although some responses
to sugars have been reported [24]. Nutrient concentrations are often used by protists for determining the
orientation of motility [52] and diatoms are also known to be sensitive to nutrient limitation, which can affect
their adhesion ability [112,118]. Therefore, the available ionic and nutrient concentrations may be important
ecological regulators of diatom motility.
In particular, calcium may be important in regulating actomyosin-based force generation and/or the
secretion of mucilage through the raphe, since it can regulate actin-based motility in both muscle and non-
muscle cells [6,94,114] as well as fusion of secretory vesicles. Extracellular calcium has been identified as
important for adhesion and motility in marine diatoms [23,25,26]. However, large external concentrations of
calcium are not required for freshwater diatom motility [9], although the treatment of freshwater cells with
inhibitors of calcium channels [32,45,125] inhibits motility in a dose-dependent manner [9]. These
differences may reflect the fact that marine diatoms live in an environment with a large external calcium
concentration, whereas freshwater diatoms do not, so that freshwater diatoms may use internal (rather than
external) stores of calcium for regulation. This may explain why mitochondria (which regulate calcium in
some cells [32]) are often observed in the cytoplasm near the raphe [87,88,90]; they could provide both the
ATP and calcium needed for movement.
Light-stimulated motile responses of diatoms, such as diurnal vertical movements and accumulation of
cells in light spots, have been documented for some time [54,74,76,97,98,99]. Such phototactic movements
of algae usually result from one or more types of behavioral responses to changes in light intensity or
wavelength [52]: alterations of cell speed (photokinesis); reversing direction at light-dark boundaries
(photophobic response); light-stimulated changes in the direction of cell growth (phototropism); or variations
in the frequency of direction changes. For diatoms, light directed movements seem to be due primarily to a
photophobic response to light/dark boundaries [8,10,20,76,121,122,123,124] detected at the tips of the cells
[18]. The presence of actin cables beneath the raphe, and the importance of calcium to motility, suggest that
diatom light responses may be regulated similarly to the light stimulated chloroplast movement in Mugeotia
[56,117] in which calcium regulates chloroplast/actin attachment.
While biraphid pennate diatoms can have considerable differences in motile behaviors and sensitivities
to light [10,20], it is not yet clear how this relates to their ability to exploit different microenvironments. For
example, the intensity and spectral quality of light change throughout the various layers of a developing algal
community [61,62], so that differential light responses may contribute to the stratification of diatom species
within the algal mat [60].
The project described in this proposal outlines a coordinated set of experiments designed to extend our
previous work and further our understanding of the environmental and ecological conditions affecting diatom
motility. Specifically, we plan to: 1) analyze the motility of mixed populations of diatoms to determine
changes in motility and light stimulated effects due to inter-species competition; 2) analyze the changes in
cell adhesion that are similarly affected by changes in environmental conditions or cell populations; and 3)
analyze the actual cell/substratum contact sites in order to correlate the diatom behaviors with the presence,
density, direction and speed of cell contact sites. Such a study should provide significant advances to
understanding the physiological responses of diatoms to their environment.

Results from Prior NSF Support

Grant Number: IBN-9407279
Award Amount/Date: $210,000 - July 1994 to June 1997, extended to 1998
Grant Title: Physiology and Ecology of Diatom Motility
Our previous grant allowed us to focus on some ecologically important behavioral differences
between diatom species, with emphasis on the motility and adhesion of the cells under various
environmental conditions. Most of the work centered on two questions: 1) how do the
characteristics of diatom movement and adhesion differ between species; and 2) what
environmental conditions affect diatom motility and adhesion. Some of our findings are
summarized below.
Characterization of Motile Behavior: We have characterized the motility and adhesion strength of
four species of biraphid pennate diatoms [20], developing quantitative assays for cell speed, path curvature,
and relative adhesion. Qualitatively, the four species can be characterized as: Craticula cuspidata- fast,
weakly adhering, straight path; Pinnularia viridis- slow, moderately adhering, curved path; Stauroneis
phoenicenteron - slow, strongly adhering, straight path; Nitzschia linearis- fast, strongly adhering, straight
path (quantitative values reported in [20]).
Environmental Factors Affecting Motility: High concentrations of calcium, reported to be
required for marine diatom motility, are not required for freshwater diatom motility [9], although some
calcium inhibitors do inhibit motility. Freshwater diatoms require very low external osmolarity for motility,
suggesting that the 1-2 atm osmotic pressure of the cell protoplast against the cell wall is possibly required
for aiding extrusion of mucilage through the narrow raphe.
Correlation of Motility and Adhesion with Temperature: In all four species of diatoms, cell
speed steadily increases with temperature until about 35-37 °C, after which the motility rapidly declines [22].
We are now investigating whether this relationship also holds true for adhesion, and whether temperature-
dependent changes in adhesion are due to changes in the mucilage itself, or due to changes in its secretion.
Investigation of Diatom Phototaxis: Our experiments have indicated that diatoms collect in light
spots of light levels at about 1-50 µmole/s-m 2, using a step-down photophobic response, whereby cells
reverse direction at light/dark boundaries. The diatoms species show differences in both wavelength and
intensity sensitivity [10,21]; initial studies show that irradiation of a single diatom culture with spots of light
with different wavelengths can result in different species distributions in each spot.
In conjunction with the lab of Dr. Jeremy Pickett-Heaps (Univ. of Melbourne) we also investigated the
role of localized high intensity light exposure on diatom direction change [18,22]. We determined that high-
energy light (about 500 µmole/s-m 2 at 500 nm) causes rapid cell reversal in <10 sec when the irradiation
occurs at the leading tip of the cell. In summary, the experiments suggested the following:
• high light level irradiation at the leading tip of a moving diatom causes cell reversal ( > 2 sec exposures
were 100% effective at all wavelengths in our tests)
• irradiation of the trailing end causes no direction change
• high-energy and low-energy light responses have the same spectral profile for Craticula
• after an initial irradiation-induced direction change, the cell is less responsive to a second direction
change generated by a subsequent irradiation at the new leading end.
Adhesion of Diatoms With/Without Water Current: In conjunction with Dr. Tuchman at
Loyola University, Chicago, we designed a flume assay in which diatoms are placed onto a platform over
which water flow (at variable speed) is generated. Initial results [21] suggest that the relative rate of cell loss
due to water current is species specific, and highly dependent on the substrate to which the cells are adhering
[17].
Effect of Toxins and UV Irradiation on Diatom Motility: Since diatoms are such important
contributors to the primary production in many aquatic ecosystems, we developed assays that use diatom
motility as an indicator of deleterious changes in the ecological conditions. We have begun to investigate the
effects of both UV irradiation and toxic sediments [10,13] on diatom motility and growth rates. Initial
results show that using irradiations of 3 mW/cm2, cells rapidly lose motility with exposures > 30 sec.
In summary we have developed several quantitative assays which have demonstrated considerable
differences in behavioral responses among several diatom species, and that measurement of these behaviors
can be used as rapid and accurate assays for the presence of environmental stress.

Publications Resulting from NSF Award (Undergraduate Authors marked with asterisk)
Manuscripts
†
Cohn, S.A. and Disparti, N.C.* (1994). Environmental factors influencing diatom cell motility. J.
Phycol. 30: 818-828.
Cohn, S.A. and Weitzell, R.E. Jr. (1996). Ecological considerations of diatom cell motility: I.
Characterization of motility and adhesion in four diatom species. J. Phycol. 32: 928-939.
‡
Cohn, S.A., Spurck, T.P., and Pickett-Heaps, J.D. (1999). High energy irradiation at the leading tip of
moving diatoms causes a rapid change of cell direction. Diatom Research (in press).
‡
Cohn, S.A. and McGuire J.R.* (1999). Using diatom motility as an indicator for environmental stress:
Effects of toxic sediment elutriates on diatom motility. Diatom Research (submitted, in revision).
§
Cohn, S.A. and Sciortino, S. (1999). Examination of motile diatom loss from different substrates due
to the force of water flow. Diatom Research (to be submitted, August/September 1999).

Abstracts
†
Cohn, S.A. and McGuire, J.R.* (1994). Use of diatom motility assays for toxicity testing. Mol. Biol.
Cell 5(S): 485a.
Cohn, S.A. and Weitzell, R.E. Jr. (1995) Characterization of motility and adhesion in four species of
pennate diatoms. J. Phycol . 31(suppl.): 6.
Cohn, S.A., Weitzell, R.E. Jr., Spurck, T.P., and Pickett-Heaps, J.D. (1995) Characterization of motility
and adhesion in pennate diatoms. Mol. Biol. Cell 6(S): 261a.
Cohn, S.A., Weitzell, R.E. Jr., Norris, A.* and Lazzarotto, M.J.* (1996) Comparative Analysis of
Adhesion and Photo-stimulated Aggregation in Pennate Diatoms. Mol. Biol. Cell 7(S): 232a.
Cohn, S.A., Dunbar, S.A., Skoczylas, C.* and Mucha, J.A.* (1997) Comparative Analysis of Diatom
Motility: Phototactic Behavior and Sensitivity to Ultraviolet Radiation. Mol. Biol. Cell 8(S): 386a.
§
Cohn, S.A. and Sciortino, S. (1998) Examination of motile diatom loss from different substrates due to
the force of water flow. Mol. Biol. Cell 9(S): 34a.
†
The data from this study was predominantly acquired prior to the previous grant, but the grant provided
support during the submission and/or revision period.
‡
The data from this study was predominantly acquired during the period of the previous grant, but the
submission process was carried out after the grant period was over.
§
The data from this study was acquired both during and after the period of the previous grant, using
equipment purchased through the previous grant, but the submission process was carried out after the grant
period was over.

In addition, another manuscript, Cohn, S.A., Weitzell, R.E. Jr. and Wibisono, B. Effect of temperature on
diatom motility and adhesion. is expected to be ready for submission within 12-18 months. The PI has also
submitted the first draft of a chapter on diatom photo-based motility for an upcoming volume on
photomovement edited by Drs. D-P. Häder and M. Lebert; the manuscript needs to be revised and additional
material will be resubmitted shortly.

Experimental Design
The proposed project is designed to expand upon those experiments carried out under the
previous grant. While our previous work has allowed us to quantitatively characterize the general
motility and adhesion in four species of pennate diatoms, we now plan to build upon this work by
asking two basic questions:
1) How are the characteristics for motility and adhesion we have measured changed by the presence of
particular combinations of diatoms? That is, how do different diatom species in mixed populations compete
with one another for limited resources or space. This includes both looking at changes in cell speed and
adhesion as well as changes in cell immigration/emigration onto substrates based on species distributions in
the population.
2) How do the actual cell/substratum contact sites differ between species, and how do their abundance,
speed, or distributions change to give rise to the behavioral effects stimulated by environmental changes? In
other words, what are the differences in cell contact sites between the species that gives rise to their
behavioral differences, and which characteristics of these contact sites change as the cells modify their
behavior based on changes in environmental stimuli.
We plan to address these questions using three types of approaches:
• Analysis of Motility - As with the previous single species measurements, we plan to characterize
the motility and light-stimulated responses (e.g. high light avoidance, low light accumulation) of diatoms
using computer-assisted video microscopy. However, in this case we will analyze changes in the motility
that take place when different mixtures of individual species are put together in multi-species populations.
We also plan to characterize two additional species with significantly different valve shapes and structures
from those previously analyzed, so that we can better assess the role of cell structure in motility and
competitive success. In addition, we will analyze the motile behaviors of cells in multi-species populations
under a variety of ecological conditions (e.g. changes in light, nutrients, temperature, substratum
composition) to better determine which types of conditions are best suited for which diatoms.

• Analysis of Cell Adhesion - Our previous work allowed us to develop assays to measure diatom
adhesion in both a static test (inverted coverslip) as well as in a test designed to measure cell loss due to the
force of flowing water. We also measured the effect of different substrates on the relative adhesion ability of
one cell type (Pinnularia viridis). We plan to expand this study to get a better sense of how different cells
react on different substrates, and how the adhesion of cells may change based on the cell type composition in
the population. Such analysis will allow us to determine the relative immigration and emigration rates of cells
in flowing water as they are removed from different substratum surfaces under conditions where the
temperature, light, and rate of water flow can all be independently altered.

• Analysis of Cell/Substratum Contact Sites - We plan to use reflection interference contrast

optics to directly determine the number, size and speed of cell/substratum contact sites from several different
diatom species, as well as analyze the alteration of these contact sites during changes in the cell's motility
(e.g. direction change) or physical environment (e.g. temperature). We will also analyze changes in the
contact sites that occur as populations with different species distributions are used.
Additional Cell Types
In the experiments described below, we plan to analyze the four diatom species characterized
in our earlier studies (P. viridis, C. cuspidata, N. linearis, S. phoenicenteron) as well as add two
more cell types to many of the experiments, both of which are typical and widespread [30]. The
additional cell types are Surirella sp. and Achnanthes sp. Both are cell types with important
structural/functional differences needed to compare with our previous species. In both cases we
will use the largest cells of the form that we can obtain and culture. The reasons for adding each of
these two types are described below.
• Surirella is a motile form with a peripheral raphe canal that runs circumferentially around an
extended rounded keel. This cell type will allow us to answer some critical questions about both
light stimulation and cell adhesion. Because of the curvature of the raphe canal, analysis of the cell
contact sites in Surirella will allow us to better determine the maximum cell/substratum distances
that still can generate a substratum contact site. These cells will also give us a better understanding
of how cells reorient themselves as cell contact sites move along the length of a raphe. In addition,
Surirella can help us answer several questions about light stimulated motile responses. All of the
four species we have previously tested have had raphe branches that extend outward along the
longitudinal axis, away from the cells’ "central areas" where there is a break between the two raphe
branches. The two distal tips of the raphe are thus separated on opposite ends of the cell, near the
areas where the cells are most sensitive to light. In Surirella, however, the valve and raphe canals
originally form after cell division starting at one tip, with the nascent raphe canals extending along
the periphery of the cell and meeting near the opposite end. Surirella therefore provides a cell type
in which the two “tips” of the raphe branches are adjacent to each other, located at the same end of
the cell. This structural organization thus allows us to ask whether Surirella can in fact respond to
the same light cues as the other cells, and if so if the light receptor sites have been displaced away
from the ends of the raphe.
• Achnanthes is a monoraphid form in which several species have shown active motility
[5,30]. Such a cell type will allow us to determine the relative motility and adhesion of monoraphid
forms (on both the raphe and non-raphe sides) and determine whether there is a difference in cell
contact sites generated on the two sides. If we measure motility in our species, we could determine
if the cells would be able to flip over and reassociate from either side, as is easily accomplished in
biraphid forms [20].
If the work outlined in this proposal proves fruitful, we could address a variety of other cell
and raphe forms in the future (e.g. Nitzschia sigmoidia in which the raphe is sigmoidal,
Gomphonema sp. in which one end of the cell is much larger than the other, or Cymbella sp. in
which the entire cell is curved).

Light Microscopic Analysis of Motility

Our previous work allowed us to develop several assays designed to measure physical and
motile characteristics of cells such as: cell size/shape, average cell speed, path curvature, frequency
of direction change, and attributes of light-stimulated responses. The assay methods are described
below, followed by the competition experiments we propose which will use these assays.
Motile Characterization Assays
Work under the previous grant characterized behavioral differences between four species of
pennate diatoms: Craticula cuspidata, Pinnularia viridis, Stauroneis phoenicenteron, and
Nitzschia linearis [20,21,22 - species determined subsequently]. These species are all large,
relatively linear, biraphid diatoms isolated from the same pond (which was artificially fed from
nearby stream water). The cells were all isolated from the outflow region of the pond, where there
was relatively constant water current, in order to obtain species that were likely to exhibit some
ability to remain adhered in the presence of water flow.

The characterization of cell motility will be accomplished using a computer-assisted video

microscope set-up as used previously for analysis of diatom motility and kinesin-driven microtubule
motility [9,11,12,16,20,58,101]. The set-up consists of a Zeiss Axioskop microscope equipped with
differential interference contrast (DIC) optics and a high resolution Dage newvicon video camera.
The output of the video camera is directly connected to the video output of an Amiga 2000
computer, resulting in a mixed signal in which the computer cursor is superimposed directly over
the video-microscope image. The computer 'mouse' can then be used to move the computer cursor
to desired points on the microscope image as viewed on the video monitor. Using a custom-
written program for the Amiga 2000, the computer cursor can be moved to a desired location, the
mouse button triggered, and the time and relative location of the point marked in the computer. By
following a moving object with the mouse, its velocity can thereby be measured. Initial calibration
between screen distances and actual distances on the microscope stage is obtained using a stage
micrometer.
The computer/video microscope set-up can be used to calculate average velocities, interval
velocities, path lengths, and other motile characteristics, with the measurements made relatively
quickly, in real time. The computer set-up can also take input from a video recorder, allowing the
computer to calculate velocities from previously recorded images; in this case the calibration is
made from recorded images of a stage micrometer. For those conditions (e.g. movement on
opaque surfaces) where transmitted light cannot be used, observations can be made using a high-
resolution dissecting stereomicroscope equipped with a video camera and external fiber optic
illumination.
Photo-response Assays
Previous work has clearly shown that the phototactic response in diatoms is strongly due to
photophobic responses of diatoms at light/dark boundaries [20,74,121]. The low/moderate light
level step-down response causes diatoms to rapidly change direction and move into the light upon
reaching a light/dark boundary, resulting in the accumulation of diatoms in light spots. This
response is sensitive to both light wavelength and intensity, causing the cells to revert to a step-up
(out-of-light) response at high light intensity [18,22]. The response is unique for each species, and
we have been able to quantify the response of individual species to change direction at light/dark
boundaries and accumulate into light spots. We have developed three assays to measure diatom
photo responses: the boundary assay, the accumulation assay, and the avoidance assay.
In the light/dark boundary assay, previously used to determine the wavelength sensitivity of
the photophobic response [20], we measure the frequency at which individual cells change direction
at light/dark boundaries. Cells from culture are mounted on a slide in dimmed room light and then
placed in a light-tight box for 1 hour to reduce any transient effects from room lighting during
mounting. Individual cells are then placed on the microscope and exposed to a small (250-300 µm
diameter) spot created by closing down the field diaphragm. Cells in the spot are allowed to move
to the light/dark boundary, during which their speed can be measured, and then scored for whether
or not the cell changes direction at the boundary; this allows us to determine the frequency of cells
exhibiting direction change. The basal rate of light-independent cell reversal can be determined by
measuring the frequency of cells, illuminated by a fully open diaphragm, reversing direction at a
"mock" spot drawn directly on the video screen. The light intensity can be varied to determine the
minimum intensity required for a photophobic response, and the intensity at which the response
changes from into-light to out-of-light.
In the cell accumulation assay, the lower portion of a small petri plate filled with the desired
population of diatoms is placed into a two piece container which blocks all light except a small (5-7
mm diameter) spot opening in its bottom, and placed on the illuminated base of a dissecting
stereomicroscope. The number of cells within the illuminated spot is counted every 20-30 minutes
by transiently opening the top of the container. A marked spot of the same size (blocked from the
light) is placed inside the container in order to count the cells in a "dark" spot for control. The
intensity of the light is controlled using a variable intensity light source, and the effective
wavelengths are varied using either broad bandpass or interference filters. We can thus determine
the rate of cell accumulation for different species, in both unialgal and mixed populations, as a
function of light intensity, light wavelength, cell density, or species composition.
 A modification of this assay, in which the petri plate is illuminated using focused fiber optic
lighting from above, can be used to illuminate several different areas of a population of cells with
different intensities and/or wavelengths of light. We can then determine if light quality can be used
to differentially alter species distributions within the light spots, and thus whether different diatom
species might use light to segregate themselves into different microenvironments. In all cases, fiber
optic illumination will be used, since this is a cooler form of light and minimizes the changes in
temperature during observation; light intensity will be calibrated with a quantum light meter. Our
initial data [21] indicate that in mixed populations, Craticula cells accumulate much faster in
blue/green light, while Stauroneis accumulate much better in red light.
We are currently developing an epi-irradiation avoidance assay, which is an adaptation of the
irradiation assay previously used to characterize the C. cuspidata step-up (out of light) response at
high light intensities [18,22]. In this assay, we use the epi-illumination filters of a fluorescent
attachment on the Zeiss Axioskop to irradiate the sample slides with 50-100 µm diameter spots of
high intensity light of defined intensity and wavelength. In this way, cells can be observed using
low levels of light from the standard light source from below, and irradiated as desired controlling
the epi-light path from above. Initial tests have shown this method to be able to yield 100%
response in stimulating a direction change in moving Craticula cells using a 2 sec irradiation; more
precise irradiations of < 1 sec will require the shutter system requested in the equipment budget.
This assay can be used to determine the light level, wavelength, and exposure time required to
generate an avoidance (step-up) response in cells.
Competition Experiments
We plan to extend the previous work we have done on single species, concentrating on the
effects of multi-species populations. Large numbers of the desired cultured cells will be rinsed in
diatom medium using serial transfer into spot plate wells using micropipets. Washed cells will then
be placed on a slide containing a small spacer to keep the cells from getting crushed, and allowed to
settle in the dark for 15 min. Initially, we plan to make samples containing relatively equal numbers
of each of the cell types being tested, at a density of about 5-10 cells per mm2. Cells will be placed
on the stage of either a compound microscope (boundary assay, avoidance assay, or motile
characterizations) or a dissecting microscope (accumulation assay) and tested as described above,
with each species in the population being measured independently. We plan to carry out a number
of combinations, comparing sets of dual species, triple species, and quadruple species. After our
initial characterizations of Surirella and Achnanthes, samples containing all six species can also be
carried out. Such tests will allow us to determine the behavioral differences between species in a
mixed population exposed to the same ecological conditions.
By controlling the external environment of the cell populations (e.g. temperature using a
temperature-controlled stage) these experiments will allow us to compare the response of cells in
these mixed populations with the responses measured for individual species. They will also allow
us to determine which types of temperature and light environments are most conducive for the light
accumulation of particular species. We can also modify the chemical environment of the cells (e.g.
altering the pH or ionic composition) to determine the relative activity and viability of the different
cell types under the altered conditions.
These assays will therefore allow us to answer several key questions about diatom populations:
1) Does each diatom species behave differently when part of a mixed population?
2) Do diatoms behave differently when in single species vs. multi-species environments?
3) Do the differential responses of diatom species (e.g. to light intensity or wavelength) allow cells in a
mixed population to segregate into different sub-populations based on light conditions?
Light Microscopic Analysis of Adhesion
In the course of our previous studies, we developed two main assays to functionally measure
diatom adhesion: the inverted coverslip, and the flume assays. These assays allowed us to
determine the relative adhesion of cells by measuring cell loss due to either the force of gravity or
the force of flowing water. We will use these assays to determine if cells in mixed populations
generate different adhesion characteristics than cells in single species populations. Previous
experiments on single species have shown that different species have different adhesion abilities
[17,20,21]. Diatom species are known to secrete different forms of mucilage [57] which may be
modified by other cells or materials [51,119], so it is important to test the relationship between
motility and adhesion, and how differences in the environment and species distributions can alter
the cells’ adhesion ability.
Cell Adhesion on an Inverted Coverslip
In this assay, a small spot of 50-100 cells are placed via micropipet onto a glass coverslip
immersed in diatom medium. The cells are then allowed to settle and adhere for 15 minutes, after
which the coverslip (remaining immersed) is inverted so that all of the cells are attached on the
lower surface. The cells remaining attached to the coverslip are then counted under a stereo-
microscope at 15-30 minute intervals over several hours. Such cells fall off at a rate that decreases
exponentially [20], so that a distinct rate constant can be determined for each species. An
adaptation of this assay places cells on a coverslip in a sealed chamber. Under this method, the
entire chamber is inverted, reducing any loss of cells due to media turbulence.
The idea that adhesion and motility are directly coupled has been supported by work
indicating that inhibition of cell motility results in changes in cell adhesion [69,120,128]. The
inverted coverslip assay allows us to address the issue directly, by measuring cell adhesion under
conditions that alter cell motility. For example, diatoms vary their speed as a function of
temperature [22], and we can determine whether there are similar temperature-dependent changes
of adhesion or contact sites. To determine the effect of temperature on adhesion, we will use a
small double-sided cell chamber attached onto a portable temperature controlled stage. In this
assay, 100-200 cells will be placed onto the coverslip on one side of the chamber, which is then
filled with diatom medium and sealed. The cells are then allowed to adhere onto coverslips at
permissive temperatures (e.g. 25°C), after which the chamber/stage apparatus is inverted. The
initial fall-off rate and motility of the cells can be observed and measured for 30-90 min, after which
the temperature can be shifted, during observation of the cells, to a higher temperature (e.g. 35-
40°C). The fall-off rate and motility of the cells after the temperature shift can then be compared
with the initial rates, and with the rates of untreated cell populations. This will allow us to
determine if higher temperatures inhibit the initial adhesion of cells to substrates, or if it reduces the
adhesion of previously adhered cells.
Similarly, we can use latrunculin, an actin inhibitor from sea sponge that causes rapid and
reversible inhibition of diatom motility [128], to determine the effect of motility inhibition on
adhesion. In these experiments, cells will be allowed to settle and adhere onto a coverslip that had
been sealed onto a drug perfusion chamber such as the type previously used to determine drug
effects on diatoms in vivo [15]. Once the cells have adhered, the chamber will be inverted and the
cells observed in either a stereomicroscope or standard compound microscope to obtain the initial
rate of cell detachment. The chamber will then be flushed with 1 µM latrunculin to stop cell
motility, and the new fall-off rate of inhibited cells determined. The latrunculin-based rate can then
be compared with the pre-treatment fall-off rate, or the rate of untreated cells. This will allow us to
determine the degree to which active cell movement is required for proper cell adhesion.

Cell Adhesion in Water Current

We have previously performed initial experiments in which we determined the rate at which
adhered cells are removed from the substratum by the force of flowing water [21]. In this assay,
(developed with Dr. N. Tuchman of Loyola University, Chicago) cell adhesion is measured in a
flume apparatus in which both the water velocity (measured by a digital flow meter) and the cells'
substratum can be varied. The flume set-up consists of a modified aquarium in which an inner
block of the aquarium is sealed off, resulting in a channel of water surrounding the blocked center.
A variable speed propeller is immersed into one side of the aquarium, propelling the water around
the channel in one direction. A platform is then mounted on the upper surface of the central
compartment (with parallel oriented hollow tubes on either side if needed), so that the water flows
in essentially unidirectional and laminar flow over its surface. Cells are placed onto the platform via
micropipet, allowed to settle and adhere for 15 minutes, then exposed to water flow of various
speeds from 0-0.5 m/sec (calibrated by a water flow meter). The number of cells remaining
adhered to the surface over time is observed and counted using a video stereomicroscope. Initial
experiments suggest that cells detach from the platform at a rate best represented by a power curve
(rather than exponential loss), suggesting that cells become increasingly harder to remove from a
substrate over time. Comparative parameters of the power equation can be determined adequately
by measuring the percentage of cells remaining adhered at 1 min intervals for 5-10 min after the
initiation of water flow, using the average results from at least three replicate trials.
The flume apparatus can also be modified to allow us to change environmental conditions
such as light or temperature. The water in the flume apparatus can be connected to a temperature
controlled circulating water pump, allowing us to vary the water temperature and overhead light
sources can be adjusted and focused onto the platform, in order to determine if the wavelength or
intensity of light alters the ability of cells to adhere to the flume platform.
Cells show differential abilities to adhere to various types of surfaces [17,41,113], and our
initial studies suggest that natural surfaces such as woods are more conducive to diatom adhesion
than synthetic or metal surfaces such as those often contaminated by algal biofouling [26,27]. The
type and texture of the substratum surface is already known to affect algal establishment and
diversity [36], and our flume set-up allows us to directly test which types of surfaces are most
conducive to cell adhesion and colonization. The flume platform can be composed of any of
various materials (e.g. marble, glass, metal, or wood) with the cell loss from each type of surface
measured as described above. We can therefore determine differences in adhesion ability of cells or
mixed populations that are due to surface properties of the substrate. In addition, the surfaces used
can be either untreated, or coated with specific additives to change the surface properties (e.g.
poly-L-lysine to produce a positively charged surface). Since there is evidence that conditioning of
surfaces by bacteria or biofilms may affect the ability of diatoms to adhere and grow [27,51,85,98],
we can also measure the cell adhesion/loss from surfaces in the presence or absence of
conditioning. Acid cleaned glass coverslips can be used as substrates without modification, or be
pre-treated by incubating the coverslips in filtered (non-sterile) pond water in the dark for several
days to produce a biofilm (similar to [85]). Samples of these conditioned coverslips can be
observed using DIC optics for presence/density of bacteria, and coverslips incubated in sterilized
pond water under axenic conditions can be used for comparison.
Competition and Comparative Analysis Experiments
Species composition has been shown to play a role in the loss of cells due to changes in water
current [3,83], as well as the ability of cells to form multi-species aggregates [31,53], and cells may
be induced to form stronger adhesions from the water flow itself. By analyzing the relative loss of
cells in single and mixed populations, we can observe if the species distributions affect the
functional adhesion of particular cell types. For example we could determine if a strongly adherent
cell type secretes material that allows other types of cells in the population to also adhere more
strongly, or whether multiple species stimulate stronger adhesion in each other. As with the
characterization studies, we would test populations with mixtures of dual, triple, and quadruple
species.

We can also test the changes in cellular mucilage secretions by performing simple colorimetric
or fluorescent staining procedures with lectins, alcian blue, or ruthenium red (as in [132]). In such
tests, the substrate surfaces will be removed from the flume after being exposed to water flow and
the cells stained with the desired reagent (e.g. 0.2 % w/v Ruthenium Red or 1% w/v Alcian Blue).
After incubation and destaining, the surface will be examined under DIC or fluorescent optics. This
will allow us to determine the type of mucilage material secreted by the cells under different water
flow regimens and species distributions. By comparing the staining patterns with cells unexposed
to water flow, we will be able to determine whether or not the stress of water flow, or the species
composition of the population, significantly change the mucilage material secreted by the cells.
This information on mucilage composition/abundance can then be analyzed for any correlations
with the rate of cell detachment.

We also plan to determine relative rates of cell immigration onto substrates. In this case,
water flow will be initiated prior to layering any cells onto the platform surface. Diatoms will then
be inoculated into the flowing water. For short-term experiments, 5-25 ml of pond water
containing predetermined concentrations of diatoms will be inoculated into the water just upstream
of the platform. The cells will be added using a pipet mounted onto a boom stand and placed into
the water upstream of the platform. The cells will be released using a peristaltic pump mounted
onto the pipet to ensure reproducible release of cells. Trials of pipet release of diatoms into petri
plates will be used to assess the fraction of cells within the pipet that typically remain adhered to the
pipet walls and are not released into the water stream. After the release of cells into the water, the
number of cells that settle and adhere onto the platform can then be observed by videomicroscope
and counted. For longer-term evaluation, a continuously running flume can be inoculated with a
large concentration of diatoms (approximately 107 cells, enough for a final concentration in the
flume water of about 1000 cells/ml) and allowed to incubate (in the presence of illumination for cell
growth) for several days to several weeks. The number of diatoms settling and adhering onto the
platform surface during this time can then be measured. Changes in cell concentrations due to cell
growth can be determined by taking samples of the flume water each day and measuring the relative
concentrations of each cell type present in the flowing water. To test for differential adhesion onto
surfaces, platforms can be made that are composed of two or more surfaces (e.g. half metal, half
glass), so that the relative number and species distribution colonizing onto each of the different
surfaces can be determined. Such experiments will allow us to compare the emigration/immigration
abilities of diatom species with different surfaces, and find the effective range of water speeds
within which each of these species can remain adhered.

Analysis of Cell/Substratum Contact Sites

Our previous work has demonstrated dramatic differences in the adhesion of different diatom
species, but did not resolve the direct cause of these differences. That is, differences in adhesion
can result from differences in adhesive strength of mucilage material, cohesion of mucilage strands,
or sloughing rate of the strands from the cells. In order to resolve this issue, the number and
location of cell/substratum contact sites occurring during cell movement must be determined. We
plan to measure contact sites directly using reflection interference contrast microscopy.

Reflection interference contrast (RIC) microscopy is a light microscopic technique that allows
the determination of cell sites that are in closest contact with the substratum. RIC has been used to
accurately determine adhesion sites for a number of cell types including moving amoebae,
fibroblasts, bacteria, and carcinoma cells in culture [e.g. 33,46,47]. The technique uses epi-
fluorescent light to illuminate the specimen on the side of the coverslip to which it is adhered.
Using the proper filters, RIC generates phase-dependent interference between the epi-illuminated
light reflecting off of the coverslip/medium boundary, and the light reflected off the surface of the
cell [100,107]. Thus, the further away the cell is from the coverslip, the more the phase difference
between the two sets of light. Such a system results in an image of a gray or light colored cell with
dark spots in the areas of closest cell contact with the substratum.

We plan to exploit this method of microscopy by analyzing motile cells in several ways: 1)
measuring the number of contact sites per cell generated by different diatom species during
movement; 2) analyzing the location of these contact sites (e.g. are they generated at one location
in the cell and then translocated down the raphe and removed); 3) determining the alteration in cell
contact sites as a cell changes direction (e.g. are old sites sloughed off as new sites are generated
which move in the opposite direction, or do the same contact sites remain and change course); and
4) determining the changes in contact site characteristics in the presence of changes in
environmental conditions or species compositions in the population.

The analysis of cellular contact sites will enable us to directly answer several questions:
– How many contact sites are present in different types of motile diatoms? Our previous analysis has
shown considerable differences in adhesion between different types of motile diatoms, and using RIC, we will
be able to directly determine the number of contact sites generated by these different cell types. For example,
we will be able to determine if strongly adhering species such as Stauroneis have more contact sites, or sites
that persist longer, than that of the more weakly adhering Craticula.
– What is the pattern of contact sites in cells such as Pinnularia, in which the valve is broadly linear,
but the path shape is strongly curved? What is the pattern of contact sites in a cell such as Surirella in
which the valve/raphe shape is curved? With RIC we will be able to determine the location of cell contact
sites relative to the valve and raphe, and better determine how such path orientations are generated.
– What is the difference in the distribution pattern of cell/substratum contacts between the raphe and
non-raphe valves of a monoraphid diatom such as Achnanthes? How do these differences correlate with the
measured adhesion of the two sides?
– How does light affect cell adhesion? Previous work has shown how light stimulation at the tips of
cells can alter cell direction [22]. Using RIC, we will be able to determine if light stimulated reversal of cell
direction is driven by reversal of already formed contact sites, loss of particular contact sites, or generation
of new sites.

Correlation of Lab and Field Studies

While the majority of the studies outlined in this proposal are based on work with cultured
algal cells under controlled lab conditions, we also recognize the importance of undertaking studies
that connect the results of the experimental work with natural diatom behavior. To accomplish
this, we propose several stages of field related investigations both concurrently and subsequently to
the experiments proposed in this grant application.
First, we intend to extend our sample collection from our original source pond in order to
correlate changes in diatom distribution over both time and position within the pond. Over the
three year course of this proposal we plan to take sets of samples from our source pond four times
each year (Jan, April, July, October), with 6 sediment samples taken at each time period. These
samples will include two from the area immediately adjacent to the outflow of approx. 6-12 in
depth, two samples from a nearby area of the pond with slower water flow of the same depth, and
two from a deeper more mid-pond area about 12-24 in depth; the two at each site will be replicates
for comparison of collection variability. These samples will be collected, fixed, and analyzed for
relative abundance of the main diatom species under study. At each collection site the rate of water
flow will be measured with a flow meter, and the light intensity at both the water surface and
underlying sediment surface will be recorded. In addition, two samples of top water will be taken
and fixed for analysis of silicate, total nitrogen, nitrate, and phosphorus concentrations in the water.
Some of each water sample will be glass filtered on site to determine the total vs. dissolved
concentrations of each of the elements/nutrients. The pH and temperature of the pond will also be
taken on site. Analysis of these samples will allow us to determine: 1) if there are natural
differences in diatom distributions between high water flow and low water flow sites in the pond; 2)
if there are natural differences in diatom distributions between sites at different depths in the pond;
3) if the relative distributions of the four diatom species changes during the course of the year; 4) if
there are any changes in species distributions which correlate with changes in water chemistry and
temperature; and 5) whether these changes in spatial distribution in the pond correlate with the
species-specific adhesion and motility characteristics and sensitivities we observe in the lab studies.
Based on the results of both our lab and initial field studies, I also plan to work with
colleagues both at DePaul and at other nearby institutions to determine local pond or stream sites
with similar types of diatom distributions. These sites will then become objects of future
investigations, carried out subsequent to those proposed in this grant, in which we plan to
investigate diatom behaviors in natural settings. These experiments will include, for example,
placing cleaned tiles or surfaces (as in 84,108,111) in several areas of the same stream that have
different flow rates. Each of these tiles can then be inoculated with a sample of diatoms from the
same single or multi-species culture, allowed to incubate for several hours, and then analyzed for
the distribution of diatoms remaining as compared to that present in the initial population. Similar
experiments can be performed by placing the tiles in areas of similar stream velocity but different
light exposures (e.g. open, partially shaded, or fully shaded regions) and then comparing the
diatoms remaining on each of the tiles. The cells on the inoculated tiles can also be compared to
the types of diatoms present initially at each of the stream sites, or with the populations colonizing
cleaned tiles that are left at the same site but with no inoculation from diatom cultures. In addition,
stream conditions can be manipulated (e.g. blocks placed upstream to regulate water flow or
shading placed over areas of the stream) to further modify the diatoms' environmental conditions.
These experiments will allow us to make more direct comparison to the behaviors of diatoms in lab
settings vs. their behavior in natural settings.

Significance and Utility of Proposed Project

The information gleaned from the above experiments will significantly increase our
understanding of diatom motile behavior, and will begin to address several types of ecologically
important questions, including:
• What environmental factors (e.g. light, pH, nutrients, temperature, substrate surface composition) most
strongly affect diatom motile behaviors in both single and multi species populations?
• In what way do the characteristics of movement for individual species allow them to successfully
compete for limited resources? Do the motile characteristics of species change when placed in mixed
populations with diatoms of different speeds, adhesive strength, or other characteristics?
• In what way do cell behaviors correspond to the differences in the distribution and activity of cell
contact sites? In what way do the movement of cell contact sites correlate with differences in cell
shape or structure (e.g. with the raphe structure)?
This project is clearly feasible. The PI of this project is well acquainted with the protocols
necessary for isolating, culturing, growing, and handling diatoms, and has worked previously on
mitosis, intracellular motility, reproduction, and physiological responses in diatoms [9,14,15,19,20].
The PI has also used computer-assisted video microscopy to analyze motility [11,12,16], and is
therefore well prepared to carry out all of the experiments outlined in this project. Additionally,
this project can easily be broken up into smaller components that can be performed by
undergraduate research assistants, so that they can be responsible for self-contained sets of
experiments, while still being a part of an overall research project. In addition, this project should
allow the development of more assays that can rapidly assess and compare the vitality of diatom
populations, and quickly determine the relative stress on diatom populations due to alterations in
ecological conditions (e.g. acidification, toxins, increased UV irradiation).

In summary, our plan to experimentally investigate the behaviors and physiological

responses of diatoms should form a strong foundation for understanding diatom activity and for
future studies investigating success in natural settings. This approach has been shown to be useful
in integrating laboratory and field studies [29], and should provide a program that both enhances
our understanding of diatoms as well as one that helps carry out the major educational and societal
objectives of NSF.
 I-2. RUI Impact Statement

Directly involving undergraduates in laboratory experimentation generates their enthusiasm

and engages their minds like no other activity can. I continue to believe that it is incumbent upon all
scientists to encourage and foster such opportunities. Over the past five years I have worked with
over 20 undergraduates in my lab (see below), most of whom had the opportunity to become
directly involved with their own experiments. Through their research, along with related scientific
discussions at weekly lab meetings and/or national meetings, I have attempted to foster an exciting
environment where students are encouraged to question, explore and ponder. I encourage an
atmosphere where the students are constantly questioning - asking what they are doing, why they
are doing it, and how to devise the next set of new experiments. This atmosphere of scientific
enthusiasm is one of the reasons I am committed to the development of undergraduate research
opportunities at DePaul. It is only through ongoing research endeavors, made possible through
grants such as the RUI program, that such undergraduate research opportunities are available on a
steady and continual basis.

It is clear that many of the current problems in our world will require innovative solutions
by people with a sufficient amount of technological and scientific education. For many college
students, it is the ability to observe and participate directly in scientific research that provides the
context for their science courses. Lectures can provide students with the language of science, but
without active participation in the process of science, they will never learn to speak the language.
Through active undergraduate research programs, universities such as DePaul that have a major
focus on education can help to achieve the goal whereby both science and education can truly
become integrated.

The project outlined in this proposal will provide an excellent opportunity for students who
have never before considered science to observe, participate, and become excited about answering
new questions and solving new problems. Most aspects of diatom motility are still virtually
unknown. As such, students working on this problem will not only have the stimulation of direct
participation in scientific inquiry, but will also have the excitement of making a direct and possibly
significant contribution to our understanding of a group of ecologically important organisms. Some
of my students have already made such contributions, and are authors on published manuscripts,
abstracts, or manuscripts in preparation. I believe it is this attribute of science, the thrill of
discovering something that no one else in the world knows, that can truly generate the enthusiasm
and excitement in students. Given the current apprehensions about science funding, it is likely that
only through this personal excitement that students will seriously consider science as a personally
fulfilling career. Only by re-initiating the student's sense of curiosity can we show that research and
education are part of the same process, and that a career in the sciences can lead to an enjoyable
lifetime of accomplishment, learning, and exploration.

The Biology Department at DePaul is a fairly young department. Six of the nine full time
faculty have been hired within the past ten years, with three of them hired within the last three
years. We expect to be replacing two more of the faculty positions within the next two years. As
such, our department contains a large percentage of recently trained, research oriented, faculty who
are excited about both research and teaching. The enthusiasm is further heightened by our new
Environmental Sciences/Biology building, which has increased the space for our teaching and
research labs. Nonetheless, the number of undergraduate majors in our Biology program (approx.
200), along with the institutional commitment of a strong liberal arts education, continues to place
heavy constraints on time and departmental resources for research. These constraints have become
particularly evident in the last year, as the University is mid-way through the implementation of a
new general education program that includes freshman seminar specialty courses and laboratory
science courses for all DePaul undergraduates. Such new general education courses, while useful
in promoting scientific principles to students, have put increased burdens on the faculty time and
departmental resources allocated to research. Therefore, if the excitement of research
opportunities for Biology undergraduates at DePaul is to be maintained, it will be necessary to
obtain funding from outside sources.
The previous RUI grant has clearly had an impact on our undergraduate research environment
at DePaul. Below I have listed the DePaul undergraduates who have worked in my lab over the
past four years, along with their current status (if known) and publication &/or ASCB meeting
attendance also noted.
(Current Students in Bold)
(* = co-authors on abstracts or manuscripts)
(‡ = attended annual American Society for Cell Biology Meeting)

Sarah Corradino - Undergraduate Student - Transferred

Joshua Crea - Current DePaul Undergraduate
Nicholas Disparti*‡ - Division Manager at Leica Microscopes Inc.
Brian Hesler‡ - Recent DePaul graduate
Karie Jeisel - Former DePaul Undergraduate
Melanie Jopek - Current DePaul Undergraduate
Bill Kosmala - Medical Doctor
Matt Lazzarotto*‡ - Newspaper Reporter
Tonette Love - Former DePaul Undergraduate
Jim McGuire*‡ - Former DePaul Undergraduate and Master's Student
Joey Mucha‡ - Former DePaul Undergraduate
Eric Nelson - Abbot Laboratories
Alex Nesterov - Former Undergraduate Student - Transferred
Aimee Norris* - Veterinary School
Moira Silverman - Current DePaul Undergraduate
Christine Skoczylas‡ - In Univ. of Chicago Neurobiology Program
Jamie Vaeth‡ - Recent DePaul graduate
Mark Vaselakos - Osteopathic Medical Doctor
Dan Weber‡ - Entering Dental School
Bernadeta Wibosono - Current DePaul Undergraduate
Troy Woodard - Entering Medical School
Ayesha Worsham‡ - Recent DePaul graduate

In addition, I also have had one high school student, Meghan O’Connor, who worked in the lab this
past summer (1999).
 Our department graduates 20-40 undergraduate majors per year, many of whom go on to
advanced degrees in Pharmacy, Medicine, Dentistry, and other allied health fields, as well as several
each year who enter Biology Ph.D. programs. We also educate students from allied fields such as
Environmental Science, Biochemistry, and Biology Education. With the funding of this proposal,
the department will be able to continue its tradition of providing a high quality, broadly based
background in Biology, and further advance the opportunities for undergraduates like those above
to actively engage in scientific research. The current lack of an RUI grant for my lab has already
begun to have an impact. Last year, for the first time in several years, no students accompanied me
to the annual Cell Biology meeting. This was partially due to the lack of any RUI funding to aid
student travel.

This proposal will also provide the funding for equipment that is needed for the further
development of modern cell biology at DePaul. Equipment purchased from the previous RUI grant
has already been used by several of the other faculty members in the department. The larger pieces
of equipment requested in this proposal (inverted microscope, video dissecting microscope head)
will similarly be available for use by other faculty members and thereby serve students with a wide
variety of interests. Such joint usage of resources attests to the close relationship among DePaul
Biology faculty, and how improvement of equipment resources for one lab benefits the entire
department.

In addition, the equipment requested furthers my ability to film a number of cellular and
microscopic phenomena for educational purposes. Over the last three years, using the equipment
from the last RUI grant, I have recorded a number of video sequences of diatom (and other cell)
motility which has been used for demonstration in General Biology, Cell Biology, Cell Motility, and
Phycology courses. The equipment has also been used to make instructional videos for a local
elementary school. The requested instruments will further our ability to film and edit cellular
processes for placement on the Internet. We are already in the process of developing a site for our
diatom research that we hope in the future will include a number of QuickTime video sequences on
diatom movement.

In summary, the past NSF-RUI grant has had a significant impact on the Biology program at
DePaul, as well as the overall science community here, in several ways:

• The equipment purchased (e.g. time-lapse video recorder, temperature controlled stage) has
aided several research labs with their work, and has allowed us to film several types of
processes that we were previously unable to record such as fish, sea urchin, and frog
development. It has also allowed us to make demonstration videos for several classes.

• The salaries allowed me to fund a number of undergraduates on a part-time basis, several of

whom have been co-authors on manuscripts or abstracts, as well as assist the travel of several
undergraduates to the annual meeting of the American Society for Cell Biology.

• The grant allowed me the access of a full-time technician, a position that has been instrumental
in the ability to train the undergraduates, and have a continuous research program going. The
technicians have also provided unique insights during discussions of research at our weekly
lab meetings, and provided strong role models for the undergraduates.
 The RUI grant as proposed, would allow us to continue and expand this strong science
environment at DePaul, and continue to encourage students into careers of science and technology.
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69. Lind J.L, vanVliet C.J., Bacic A. and Wetherbee R. (1996) Characterization of molecules responsible
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77. Ohtsuka T. (1998) Variation in diatom community structure among habitats within a morphological
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78. Pahlow M., Riebesell U., and Wolf-Gladrow D.A. (1997) Impact of cell shape and chain formation on
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80. Patterson D.M. (1986) The migratory behavior of diatom assemblages in a laboratory tidal micro-
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83. Peterson C.G. (1987) Influences of flow regime on development and dessication response of lotic
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84. Peterson C.G. and Hoagland K.D. (1990) Effects of wind-induced turbulence and algal mat development
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86. Pickett-Heaps J.D. (1983) Valve morphogenesis and the microtubule center in three species of the
diatom Nitzschia. J. Phycol. 19, 269-281.

87. Pickett-Heaps J.D. (1991) Post-mitotic cellular reorganisation in the diatom Cymatopleura solea: The
role of microtubules and the microtubule center. Cell Motil. Cytoskel. 18, 279-292.

88. Pickett-Heaps J.D., Cohn S., Schmid A-M. and Tippit D.H. (1988) Valve morphogenesis in Surirella
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89. Pickett-Heaps J.D., Hill D.R.A., and Blaze K.L. (1991) Active gliding motility in an araphid marine
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90. Pickett-Heaps J.D., Schmid A.M. and Edgar L.A. (1990) The cell biology and phylogeny of diatom
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91. Pickett-Heaps J.D., Spurck T.P., Cohn S.A., Schoeller A. and Edgar L.A. (1984). Sexual reproduction
in the diatom Navicula cuspidata. 16mm color, sound film. 16 min. Cytographics % Dr. J.D. Pickett-
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92. Pickett-Heaps J.D., Tippit D.H., and Andreozzi J.A. (1979) Cell division in the pennate diatom
Pinnularia. III. The valve and associated cytoplasmic organelles. Biol. Cell. 35, 195-198.

93. Pickett-Heaps J.D., Tippit D.H., and Andreozzi J.A. (1979) Cell division in the pennate diatom
Pinnularia. IV. Valve morphogenesis. Biol. Cell. 35, 199-203.

94. Preston T.M., King C.A., and Hyams J.S. (1990) The Cytoskeleton and Cell Motility. Blackie Pub.,
London.

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599.

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99. Round F.E. and Palmer J.D. (1966) Persistent vertical migration rhythms in benthic microflora. II. Field
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100. Rubbi C.P. (1994) Light Microscopy: Essential Data. John Wiley & Sons, New York.
101. Saxton W.M., Porter M.E., Cohn S.A., Scholey J.M., Raff E.C., and McIntosh J.R. (1988)
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102. Schmid A-M.M. (1980) Valve morphogenesis in diatoms: pattern related filamentous system in pennates
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103. Schmid A-M. M. (1995) Aspects of morphogenesis and function of diatom cell walls with implications
for taxonomy. In Wetherbee, R. Andersen, R.A. & Pickett-Heaps, J.D. [Eds.] The Protistan Cell
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104. Schrader H.-J. (1974) Types of raphe structures in the diatoms. Nova Hedwigia Beih. 45, 195-217.

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9, 126-167.

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Cambridge.

108. Stevenson R.J. (1990) Benthic algal community dynamics in a stream during and after a spate. J. N.
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112. Stevenson R.J., Peterson C.G., Kirschtel D.B., King C.C., and Tuchman N.C. (1991) Density-
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114. Tsien R.W. and Tsien R.Y. (1990) Calcium channels, stores, and oscillations. Ann. Rev. Cell Biol. 6,
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319.

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117. Wagner G. and Klein K. (1981) Mechanism of chloroplast movement in Mougeotia. Protoplasma 109,
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118. Waite A., Gallager S. and Dam H.G. (1997) New measurements of phytoplankton aggregation in a
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119. Wang Y., Lu J., Mollet J-C., Gretz M.R. and Hoagland K.D. (1997) Extracellular matrix assembly in
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120. Webster D.R., Cooksey K.E., and Rubin R.W. (1985) An investigation of the involvement of
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122. Wenderoth K. (1982) Reaktion der Kieselalge Navicula peregrina auf Belichtung verschiedener
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123. Wenderoth K. (1983) Phototaxis bei Desmidiaceen und Diatomeen. Biol. Film #C1496. Institut für
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124. Wenderoth K. (1984) Wirkungsspektrum der Step-down-Reaktion bei Diatomeen. Biol. Film
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125. Weiss G.B. (1974) Cellular Pharmacology of Lanthanum Ann. Rev. Pharmacol. 14, 343-354.

126. Werner D. (Ed.) (1977) The Biology of Diatoms. University of Calif. Press, Berkeley.

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128. Wetherbee R., Lind J.L., Poulsen N.C. and Spurck T.P. (1996) Cell motility in marine raphid diatoms
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129. Williamson R.E. (1992) Cytoplasmic streaming in Characean algae: Mechanism, regulation by Ca2+,
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131. Wordeman L., McDonald K.L., and Cande W.Z. (1986) The distribution of cytoplasmic microtubules
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1059-1069.
 BIOGRAPHICAL SKETCH
A. Vitae
Stanley A. Cohn
DePaul University - Department of Biological Sciences
2325 N. Clifton Ave, Chicago, IL 60614
phone: 773-325-7597 fax: 773-325-7596
e-mail: scohn@condor.depaul.edu
Soc Sec # 521-90-8230

Education
B.S. Chemistry, with honors, California Institute of Technology (Caltech), 1979
Ph.D. Molecular, Cellular, and Developmental Biology, Univ. of Colo. (Boulder), 1986
Thesis: Mechanisms of Mitosis and Valve Morphogenesis in Diatoms
Thesis Advisor: Dr. Jeremy Pickett-Heaps

Positions Held
DePaul University, Department of Biological Sciences
Associate Professor with Tenure 1996-present
Assistant Professor 1989-1996
Postdoctoral Research Associate, with Dr. Jonathan Scholey
National Jewish Center for Immunology and Respiratory Medicine, 1986-1989
Graduate Research Assistant, with Dr. Jeremy Pickett-Heaps
Univ. of Colo. (Boulder), 1981-1986
Teaching Assistant, Intro. to Molecular, Cellular, and Developmental Biology
Univ. of Colo. (Boulder), 1979-1981.

Fellowships, Awards, and Offices

American Cancer Society Postdoctoral Fellowship, 1987-1989
University of Colorado Graduate Fellowship, 1984-85
N.I.H. Graduate Training Grant, 1979-80, 1983-85
Silver Medal, Royal Society of Arts, 1979
Senior Class President, Caltech, 1978-79

Memberships
Fellow of the Royal Society of Arts,
American Society for Cell Biology
American Association for the Advancement of Science (AAAS)
International Society for Diatom Research
Council on Undergraduate Research

Grants Awarded
DePaul University
College of Liberal Arts & Sciences Summer Grant (for summer 1990, 1992, 1994, 1997,
1999)
University Research Council, Competitive Research Grant (1989,1990,1991,1992, 1998,
1999)
Quality of Instruction Council (QIC) Development Grant (1991, 1998)
University Research Council Competitive Research Leave (1995)
Joint Quality of Instruction Council/University Research Council Grant (1995)

National Science Foundation

Grant # IBN-IBN-9407279 - July 1994 to June 1997 - Physiology and Ecology of Diatom
Motility - total award $210,000
B. Five Most Relevant Publications (Undergraduate Authors with asterisk):
Cohn, S.A. and Weitzell, R.E. Jr. (1996). Ecological considerations of diatom cell motility: I.
Characterization of motility and adhesion in four diatom species. J. Phycol. 32: 928-939.
Cohn, S.A. and Disparti, N.C.* (1994). Environmental factors influencing diatom cell motility.
J. Phycol. 30: 818-828.
Cohn, S.A., Spurck, T.P., Pickett-Heaps, J.D. and Edgar, L.E. (1989). Perizonium and initial
valve formation in the diatom Navicula cuspidata (Bacillariophyceae). J. Phycol. 25: 15-
26.
Cohn, S.A., Nash, J.* and Pickett-Heaps, J.D. (1989). The effect of drugs on diatom valve
morphogenesis. Protoplasma 149: 130-143.
Cohn, S.A. and Pickett-Heaps, J.D. (1988). The effects of colchicine and dinitrophenol on the
in vivo rates of anaphase A and B in the diatom Surirella. Eur. J. Cell Biol. 46: 523-530.

Other Significant Publications:

Cohn, S.A., Saxton, W.M., Lye, R.J., and Scholey, J.M. (1993) Chapter 5: Analyzing
Microtubule Motors in Real Time. In Meth. Cell. Biol. Vol .39 [J.M. Scholey Ed.],
Academic Press, NY, Pp 75-88.
Cohn, S.A. (1990) The mechanochemistry of kinesin: a review. Mol. Chem. Neuropath. 12:83-
94.
Cohn, S.A., Ingold, A.L. and Scholey, J.M. (1989). Quantitative analysis of sea urchin egg
kinesin-driven microtubule motility. J. Biol. Chem. 264: 4290-4297.
Pickett-Heaps, J.D., Cohn, S., Schmid, A-M. and Tippit, D.H. (1988). Valve morphogenesis in
Surirella (Bacillariophyceae). J. Phycol. 24: 35-49.
Cohn, S.A., Ingold, A.L. and Scholey, J.M. (1987). Correlation between the ATPase and
microtubule translocating activities of sea urchin egg kinesin. Nature 328: 160-163.

C. Collaborators:
Dr. Nancy Tuchman, Loyola University Chicago (collaborator on flume experiments)
Dr. Donat Häder, Institut fur Botanik und Pharmazeutische Biologie, Germany (submitting a
chapter in a volume he is editing)
D. Graduate Students:
Graduate Students Advised (Total = 12):
Dissertation Committee: Lydia Armstrong, PhD, University of Denver, 1989
Thesis Committee: Shylaja Muthyala, MS, DePaul University, 1990
Ankita Chitre, MS, DePaul University, 1991
Jolie Machota, MS, DePaul University, 1993
John Sikora, MS, DePaul University, 1994
Mary McCarthy, MS, DePaul University 1994
Margaret Liotta, MS, DePaul University, 1998
Tim Laurie, MS, DePaul University, 1998
Heather Walczak, MS, DePaul University, 1999

Thesis Advisor: Devry Spreitzer, MS, DePaul University, 1995 (Co-advisor)

Sam Sciortino, Current MS Student DePaul University
Jason Weiss, Current MS Student DePaul University
David Zelner, Current MS Student DePaul University

E. Graduate/Postdoctoral Advisors:
Dr. J. R. McIntosh, University of Colorado, Boulder (Dissertation Committee- 2nd reader)
Dr. Jeremy D. Pickett-Heaps, University of Melbourne, AUSTRALIA (Graduate Advisor)
Dr. Jonathan M. Scholey, University of California, Davis (Postdoctoral Advisor)
 SUMMARY YEAR 1
PROPOSAL BUDGET FOR NSF USE ONLY
ORGANIZATION PROPOSAL NO. DURATION (months)
DePaul University Proposed Granted
PRINCIPAL INVESTIGATOR / PROJECT DIRECTOR AWARD NO.
Stanley A Cohn
NSF Funded Funds Funds
A. SENIOR PERSONNEL: PI/PD, Co-PI’s, Faculty and Other Senior Associates Person-mos. Requested By granted by NSF
(List each separately with title, A.7. show number in brackets) CAL ACAD SUMR proposer (if different)

1. Stanley A Cohn - Associate Professor 0.00 0.00 2.00 $ 11,889 $

2.
3.
4.
5.
6. ( 0 ) OTHERS (LIST INDIVIDUALLY ON BUDGET JUSTIFICATION PAGE) 0.00 0.00 0.00 0
7. ( 1 ) TOTAL SENIOR PERSONNEL (1 - 6) 0.00 0.00 2.00 11,889
B. OTHER PERSONNEL (SHOW NUMBERS IN BRACKETS)
1. ( 0 ) POST DOCTORAL ASSOCIATES 0.00 0.00 0.00 0
2. ( 1 ) OTHER PROFESSIONALS (TECHNICIAN, PROGRAMMER, ETC.) 12.00 0.00 0.00 24,000
3. ( 0 ) GRADUATE STUDENTS 0
4. ( 2 ) UNDERGRADUATE STUDENTS 6,000
5. ( 0 ) SECRETARIAL - CLERICAL (IF CHARGED DIRECTLY) 0
6. ( 0 ) OTHER 0
TOTAL SALARIES AND WAGES (A + B) 41,889
C. FRINGE BENEFITS (IF CHARGED AS DIRECT COSTS) 8,329
TOTAL SALARIES, WAGES AND FRINGE BENEFITS (A + B + C) 50,218
D. EQUIPMENT (LIST ITEM AND DOLLAR AMOUNT FOR EACH ITEM EXCEEDING $5,000.)
Inverted Microscope $ 25,000
Shutter/Aperture equipment 1,500

TOTAL EQUIPMENT 26,500

E. TRAVEL 1. DOMESTIC (INCL. CANADA, MEXICO AND U.S. POSSESSIONS) 2,500
2. FOREIGN 0

F. PARTICIPANT SUPPORT COSTS

1. STIPENDS $ 0
2. TRAVEL 0
3. SUBSISTENCE 0
4. OTHER 0
TOTAL NUMBER OF PARTICIPANTS ( 0) TOTAL PARTICIPANT COSTS 0
G. OTHER DIRECT COSTS
1. MATERIALS AND SUPPLIES 7,000
2. PUBLICATION COSTS/DOCUMENTATION/DISSEMINATION 500
3. CONSULTANT SERVICES 0
4. COMPUTER SERVICES 0
5. SUBAWARDS 0
6. OTHER 50
TOTAL OTHER DIRECT COSTS 7,550
H. TOTAL DIRECT COSTS (A THROUGH G) 86,768
I. INDIRECT COSTS (F&A)(SPECIFY RATE AND BASE)
% of SWB (Rate: 52.0000, Base: 50218)
TOTAL INDIRECT COSTS (F&A) 26,113
J. TOTAL DIRECT AND INDIRECT COSTS (H + I) 112,881
K. RESIDUAL FUNDS (IF FOR FURTHER SUPPORT OF CURRENT PROJECTS SEE GPG II.D.7.j.) 0
L. AMOUNT OF THIS REQUEST (J) OR (J MINUS K) $ 112,881 $
M. COST SHARING PROPOSED LEVEL $ 25,500 AGREED LEVEL IF DIFFERENT $
PI / PD TYPED NAME & SIGNATURE* DATE FOR NSF USE ONLY
Stanley A Cohn INDIRECT COST RATE VERIFICATION
ORG. REP. TYPED NAME & SIGNATURE* DATE Date Checked Date Of Rate Sheet Initials - ORG

NSF Form 1030 (10/99) Supersedes all previous editions 1 *SIGNATURES REQUIRED ONLY FOR REVISED BUDGET (GPG III.B)
 SUMMARY YEAR 2
PROPOSAL BUDGET FOR NSF USE ONLY
ORGANIZATION PROPOSAL NO. DURATION (months)
DePaul University Proposed Granted
PRINCIPAL INVESTIGATOR / PROJECT DIRECTOR AWARD NO.
Stanley A Cohn
NSF Funded Funds Funds
A. SENIOR PERSONNEL: PI/PD, Co-PI’s, Faculty and Other Senior Associates Person-mos. Requested By granted by NSF
(List each separately with title, A.7. show number in brackets) CAL ACAD SUMR proposer (if different)

1. Stanley A Cohn - none 0.00 0.00 2.00 $ 12,483 $

2.
3.
4.
5.
6. ( 0 ) OTHERS (LIST INDIVIDUALLY ON BUDGET JUSTIFICATION PAGE) 0.00 0.00 0.00 0
7. ( 1 ) TOTAL SENIOR PERSONNEL (1 - 6) 0.00 0.00 2.00 12,483
B. OTHER PERSONNEL (SHOW NUMBERS IN BRACKETS)
1. ( 0 ) POST DOCTORAL ASSOCIATES 0.00 0.00 0.00 0
2. ( 1 ) OTHER PROFESSIONALS (TECHNICIAN, PROGRAMMER, ETC.) 12.00 0.00 0.00 25,200
3. ( 0 ) GRADUATE STUDENTS 0
4. ( 2 ) UNDERGRADUATE STUDENTS 6,000
5. ( 0 ) SECRETARIAL - CLERICAL (IF CHARGED DIRECTLY) 0
6. ( 0 ) OTHER 0
TOTAL SALARIES AND WAGES (A + B) 43,683
C. FRINGE BENEFITS (IF CHARGED AS DIRECT COSTS) 8,722
TOTAL SALARIES, WAGES AND FRINGE BENEFITS (A + B + C) 52,405
D. EQUIPMENT (LIST ITEM AND DOLLAR AMOUNT FOR EACH ITEM EXCEEDING $5,000.)

TOTAL EQUIPMENT 0
E. TRAVEL 1. DOMESTIC (INCL. CANADA, MEXICO AND U.S. POSSESSIONS) 2,500
2. FOREIGN 0

F. PARTICIPANT SUPPORT COSTS

1. STIPENDS $ 0
2. TRAVEL 0
3. SUBSISTENCE 0
4. OTHER 0
TOTAL NUMBER OF PARTICIPANTS ( 0) TOTAL PARTICIPANT COSTS 0
G. OTHER DIRECT COSTS
1. MATERIALS AND SUPPLIES 4,000
2. PUBLICATION COSTS/DOCUMENTATION/DISSEMINATION 500
3. CONSULTANT SERVICES 0
4. COMPUTER SERVICES 0
5. SUBAWARDS 0
6. OTHER 50
TOTAL OTHER DIRECT COSTS 4,550
H. TOTAL DIRECT COSTS (A THROUGH G) 59,455
I. INDIRECT COSTS (F&A)(SPECIFY RATE AND BASE)
% of swb (Rate: 52.0000, Base: 52405)
TOTAL INDIRECT COSTS (F&A) 27,251
J. TOTAL DIRECT AND INDIRECT COSTS (H + I) 86,706
K. RESIDUAL FUNDS (IF FOR FURTHER SUPPORT OF CURRENT PROJECTS SEE GPG II.D.7.j.) 0
L. AMOUNT OF THIS REQUEST (J) OR (J MINUS K) $ 86,706 $
M. COST SHARING PROPOSED LEVEL $ 0 AGREED LEVEL IF DIFFERENT $
PI / PD TYPED NAME & SIGNATURE* DATE FOR NSF USE ONLY
Stanley A Cohn INDIRECT COST RATE VERIFICATION
ORG. REP. TYPED NAME & SIGNATURE* DATE Date Checked Date Of Rate Sheet Initials - ORG

NSF Form 1030 (10/99) Supersedes all previous editions 2 *SIGNATURES REQUIRED ONLY FOR REVISED BUDGET (GPG III.B)
 SUMMARY YEAR 3
PROPOSAL BUDGET FOR NSF USE ONLY
ORGANIZATION PROPOSAL NO. DURATION (months)
DePaul University Proposed Granted
PRINCIPAL INVESTIGATOR / PROJECT DIRECTOR AWARD NO.
Stanley A Cohn
NSF Funded Funds Funds
A. SENIOR PERSONNEL: PI/PD, Co-PI’s, Faculty and Other Senior Associates Person-mos. Requested By granted by NSF
(List each separately with title, A.7. show number in brackets) CAL ACAD SUMR proposer (if different)

1. Stanley A Cohn - none 0.00 0.00 2.00 $ 13,107 $

2.
3.
4.
5.
6. ( 0 ) OTHERS (LIST INDIVIDUALLY ON BUDGET JUSTIFICATION PAGE) 0.00 0.00 0.00 0
7. ( 1 ) TOTAL SENIOR PERSONNEL (1 - 6) 0.00 0.00 2.00 13,107
B. OTHER PERSONNEL (SHOW NUMBERS IN BRACKETS)
1. ( 0 ) POST DOCTORAL ASSOCIATES 0.00 0.00 0.00 0
2. ( 1 ) OTHER PROFESSIONALS (TECHNICIAN, PROGRAMMER, ETC.) 12.00 0.00 0.00 26,460
3. ( 0 ) GRADUATE STUDENTS 0
4. ( 2 ) UNDERGRADUATE STUDENTS 6,000
5. ( 0 ) SECRETARIAL - CLERICAL (IF CHARGED DIRECTLY) 0
6. ( 0 ) OTHER 0
TOTAL SALARIES AND WAGES (A + B) 45,567
C. FRINGE BENEFITS (IF CHARGED AS DIRECT COSTS) 9,135
TOTAL SALARIES, WAGES AND FRINGE BENEFITS (A + B + C) 54,702
D. EQUIPMENT (LIST ITEM AND DOLLAR AMOUNT FOR EACH ITEM EXCEEDING $5,000.)

TOTAL EQUIPMENT 0
E. TRAVEL 1. DOMESTIC (INCL. CANADA, MEXICO AND U.S. POSSESSIONS) 2,500
2. FOREIGN 0

F. PARTICIPANT SUPPORT COSTS

1. STIPENDS $ 0
2. TRAVEL 0
3. SUBSISTENCE 0
4. OTHER 0
TOTAL NUMBER OF PARTICIPANTS ( 0) TOTAL PARTICIPANT COSTS 0
G. OTHER DIRECT COSTS
1. MATERIALS AND SUPPLIES 4,000
2. PUBLICATION COSTS/DOCUMENTATION/DISSEMINATION 500
3. CONSULTANT SERVICES 0
4. COMPUTER SERVICES 0
5. SUBAWARDS 0
6. OTHER 50
TOTAL OTHER DIRECT COSTS 4,550
H. TOTAL DIRECT COSTS (A THROUGH G) 61,752
I. INDIRECT COSTS (F&A)(SPECIFY RATE AND BASE)
% of SWB (Rate: 52.0000, Base: 54702)
TOTAL INDIRECT COSTS (F&A) 28,445
J. TOTAL DIRECT AND INDIRECT COSTS (H + I) 90,197
K. RESIDUAL FUNDS (IF FOR FURTHER SUPPORT OF CURRENT PROJECTS SEE GPG II.D.7.j.) 0
L. AMOUNT OF THIS REQUEST (J) OR (J MINUS K) $ 90,197 $
M. COST SHARING PROPOSED LEVEL $ 0 AGREED LEVEL IF DIFFERENT $
PI / PD TYPED NAME & SIGNATURE* DATE FOR NSF USE ONLY
Stanley A Cohn INDIRECT COST RATE VERIFICATION
ORG. REP. TYPED NAME & SIGNATURE* DATE Date Checked Date Of Rate Sheet Initials - ORG

NSF Form 1030 (10/99) Supersedes all previous editions 3 *SIGNATURES REQUIRED ONLY FOR REVISED BUDGET (GPG III.B)
 SUMMARY PROPOSAL BUDGET COMMENTS - Year 3

** E- Travel
Meeting of the American Society for Cell Biology; PI and one student
travel
expenses
 SUMMARY Cumulative
PROPOSAL BUDGET FOR NSF USE ONLY
ORGANIZATION PROPOSAL NO. DURATION (months)
DePaul University Proposed Granted
PRINCIPAL INVESTIGATOR / PROJECT DIRECTOR AWARD NO.
Stanley A Cohn
NSF Funded Funds Funds
A. SENIOR PERSONNEL: PI/PD, Co-PI’s, Faculty and Other Senior Associates Person-mos. Requested By granted by NSF
(List each separately with title, A.7. show number in brackets) CAL ACAD SUMR proposer (if different)

1. Stanley A Cohn - none 0.00 0.00 6.00 $ 37,479 $

2.
3.
4.
5.
6. ( ) OTHERS (LIST INDIVIDUALLY ON BUDGET JUSTIFICATION PAGE) 0.00 0.00 0.00 0
7. ( 1 ) TOTAL SENIOR PERSONNEL (1 - 6) 0.00 0.00 6.00 37,479
B. OTHER PERSONNEL (SHOW NUMBERS IN BRACKETS)
1. ( 0 ) POST DOCTORAL ASSOCIATES 0.00 0.00 0.00 0
2. ( 3 ) OTHER PROFESSIONALS (TECHNICIAN, PROGRAMMER, ETC.) 36.00 0.00 0.00 75,660
3. ( 0 ) GRADUATE STUDENTS 0
4. ( 6 ) UNDERGRADUATE STUDENTS 18,000
5. ( 0 ) SECRETARIAL - CLERICAL (IF CHARGED DIRECTLY) 0
6. ( 0 ) OTHER 0
TOTAL SALARIES AND WAGES (A + B) 131,139
C. FRINGE BENEFITS (IF CHARGED AS DIRECT COSTS) 26,186
TOTAL SALARIES, WAGES AND FRINGE BENEFITS (A + B + C) 157,325
D. EQUIPMENT (LIST ITEM AND DOLLAR AMOUNT FOR EACH ITEM EXCEEDING $5,000.)
$ 26,500

TOTAL EQUIPMENT 26,500

E. TRAVEL 1. DOMESTIC (INCL. CANADA, MEXICO AND U.S. POSSESSIONS) 7,500
2. FOREIGN 0

F. PARTICIPANT SUPPORT COSTS

1. STIPENDS $ 0
2. TRAVEL 0
3. SUBSISTENCE 0
4. OTHER 0
TOTAL NUMBER OF PARTICIPANTS ( 0) TOTAL PARTICIPANT COSTS 0
G. OTHER DIRECT COSTS
1. MATERIALS AND SUPPLIES 15,000
2. PUBLICATION COSTS/DOCUMENTATION/DISSEMINATION 1,500
3. CONSULTANT SERVICES 0
4. COMPUTER SERVICES 0
5. SUBAWARDS 0
6. OTHER 150
TOTAL OTHER DIRECT COSTS 16,650
H. TOTAL DIRECT COSTS (A THROUGH G) 207,975
I. INDIRECT COSTS (F&A)(SPECIFY RATE AND BASE)

TOTAL INDIRECT COSTS (F&A) 81,809

J. TOTAL DIRECT AND INDIRECT COSTS (H + I) 289,784
K. RESIDUAL FUNDS (IF FOR FURTHER SUPPORT OF CURRENT PROJECTS SEE GPG II.D.7.j.) 0
L. AMOUNT OF THIS REQUEST (J) OR (J MINUS K) $ 289,784 $
M. COST SHARING PROPOSED LEVEL $ 25,500 AGREED LEVEL IF DIFFERENT $
PI / PD TYPED NAME & SIGNATURE* DATE FOR NSF USE ONLY
Stanley A Cohn INDIRECT COST RATE VERIFICATION
ORG. REP. TYPED NAME & SIGNATURE* DATE Date Checked Date Of Rate Sheet Initials - ORG

NSF Form 1030 (10/99) Supersedes all previous editions C*SIGNATURES REQUIRED ONLY FOR REVISED BUDGET (GPG III.B)
 Budget Justification

Personnel:
The salaries requested are for PI summer salary, a full-time technician salary, and salary for two
undergraduate students during the school year [30 weeks at 5 hr/wk during the academic year; 11 weeks at 30 hr/wk
during summer/winter break, at $6.25/hr]. The benefits for the technician position are calculated using DePaul’s
normal rate for full-time positions, 29%. This rate includes the following components: FICA 6.20%; Medicare
1.45%; Retirement 8.00%; Unemployment 0.20%; Medical insurance 6.50%; Dental insurance 0.50%; Disability
insurance 0.35%; Life insurance 0.30%; Tuition 5.5%. Benefits for the PI summer support and the undergraduate
student include only FICA and Medicare, totaling 7.65%, the normal rate for part-time and summer faculty
positions. These positions, as described below, are crucial to the objectives of this proposal.
The presence of a full-time technician in the lab was essential to carrying out the planned work during the
last grant period. During the school year, students have severe time constraints, particularly in terms of the specific
hours they have available to do research. At the same time, my teaching responsibilities also constrain the times I
can be present in the lab (e.g. I am teaching a lab course each quarter during the year, and one quarter each year I am
responsible for teaching 3 hr lectures and 4 three-hour laboratory sections each week). It therefore becomes
imperative that a full-time technician, independent from the school calendar, be available to the undergraduates
working in the lab. In this way, we can efficiently train students in the methods of medium preparation and diatom
culturing, as well as particular experimental protocols. Moreover, a technician in the lab allows the constant
presence of someone who can help answer technical questions of the students in the lab as they run up against
problems during an experiment.
In addition, the technicians I hired with the last grant were recent college graduates trying to determine
whether or not to proceed with a career in science. Exposing them to the excitement that is generated within an
active research lab provided, I believe, exactly the extra impetus needed to spur them on to continue in scientific
endeavors. The two technicians I hired have both gone on to graduate school - one to a M.S. program, and the other
into a graduate Pharmacy (Pharm.D.) program.
Similarly, the requested money for undergraduate students is necessary to achieve the goal of increasing the
scientific interests and capabilities of the students. I have been a vigorous advocate for including undergraduates in
research, and have had over 20 undergraduates working in my lab over the past 6 years (see RUI Statement). Most
undergraduate students at DePaul must work to help pay for their tuition, so it is necessary to financially support
them during the summer if they are to be able to participate in the lab research without suffering the loss of summer
salary. A large portion of DePaul students are minority students (about 32% in the 1998 freshman class), and many
of our students are first generation college students receiving financial aid to attend the University. The funding of
the undergraduate stipend would allow the students to earn summer and school year income to help pay for tuition
while still learning science by directly participating in laboratory investigations. The requested funds would allow
me to hire two students during each year (30 weeks @ 5 hrs/wk during the academic year and 11 weeks @ 30
hrs/wk during the winter/summer academic breaks)

Equipment Required
In order to carry out the proposed experiments, a number of pieces of equipment will need to be purchased:

– Additional Stereo-Microscope Head. We currently have both a brightfield/darkfield stand for the stereo-
microscope (used in photo-accumulation and standard coverslip inversion tests) and a boom stand for the
stereo-microscope head (used for flume and temperature controlled adhesion studies). This requires that the
current microscope head be physically moved and aligned each time we need to run a different experiment.
More importantly, we cannot run both flume and photo-stimulation experiments simultaneously. Since these
are both important assays that we need to run simultaneously in the lab, particularly during the summer, we
need an additional stereo-microscope head and video adapter.

– Additional Computer. We currently have only one computer in our lab capable of running the
speed/distance/path analysis program. The current computer is connected to the Zeiss Axioskop in a separate
microscope room. In order to carry out our analysis of motile characteristics for cells being viewed under other
microscopes (e.g. moving on opaque surfaces, with observation under the dissecting microscope) we need
another computer which can be quickly connected to any of the other microscope set-ups in the lab (i.e. the
 head on the boom stand, the head on the darkfield stand, etc.). We plan to purchase a laptop computer with
video input/output capabilities.

– Reflection Interference Contrast Optics and Inverted Microscope. In order to carry out our experiments on the
analysis of cell contact sites, we will need to have Reflection Interference Contrast optics and an inverted
microscope. The microscope objective for this type of optics has already been purchased by the Biology
Department, but several more lenses and filters are still required. In addition, reflectance interference requires
epi-illumination. Therefore, while the Zeiss Axioskop in the lab (already in use) will be sufficient for tests of
moving cells on inverted coverslips (i.e. analysis of contact sites on cells in the process of losing attachment),
an inverted microscope is required to investigate the standard movement of cells gliding over the top of
surfaces. By having two Zeiss microscopes, the objectives and filters can be used interchangeably between the
two microscopes, minimizing the number of optics needed to be purchased.

– Equipment for environmental control of flume set-up. The flume set-up as currently designed operates only at
room temperature and room light conditions. In order to determine the environmental conditions affecting cell
adhesion as measured by resistance to water current, we need additional light and temperature control
equipment. This will include both an overhead lighting system (an overhead variable fiber optic system for
small spot lighting and quartz bulbs for longer term lighting), and a temperature regulated water pump.

– Equipment for Photophobic response experiments. Currently, the Zeiss Axioskop in the lab is sufficient for
basic epi-illumination. In order to adapt the microscope for performing more controlled tests on light directed
responses, a controlled shutter and adjustable centerable diaphragm with a small spot size is required.

The costs of these items are as follows (prices reflect estimates from the companies in parentheses):

Shutter/Aperture Equipment for Axioskop for controlled irradiations (Uniblitz) $ 1,500

Computer with Video Capabilities $ 3,500
Reflection Interference Contrast Equipment (Zeiss) $ 5,000
Light/Pump System for Flume (Lauda) $ 12,000
Stereo-microscope Head w/ video port and lenses (Leica-Wild)$ 5,000
Inverted Microscope for reflection interference contrast (Zeiss)$ 25,000

Total Equipment Costs $ 52,000

Because these equipment costs are extensive, DePaul University has agreed to help share the costs associated with
the needed equipment of this project by purchasing the upgrade equipment for the compound and stereo
microscopes, the Refrigerated Circulating Pump, the Computer, and the Controlled Light Set-up (middle four items
above). These items have the greatest potential to be shared with other members of the DePaul community in their
research endeavors. Therefore, the breakdown of equipment costs is as follows:

DePaul Equipment Costs $ 25,500

Equipment Costs Requested from NSF $ 26,500

Travel:
The proposal requests the travel funds for the PI and one student to one scientific meeting per year
($1250/meeting/year/person). This will allow the timely dissemination of the project results in an appropriate
forum, as well as detailed discussion of the project with numerous colleagues. In addition, the undergraduate
students are able to help present their own data, as well as get a much better look of the scientific enterprise at work.
Over the past several years (except for the last year, when no funds were available), I have taken at least one student
with me to the annual Cell Biology meetings. It has always been a mutually exciting and rewarding experience, and
allowed me to directly involve the students in the process of scientific discussion.
Other Direct Costs:
The supply money as requested is designed for the normal operating costs of the laboratory. The amount
requested for years two and three (4,000/year) is not excessive given the large amount of consumables, glassware,
plasticware, and biochemicals that are continually used in making large amounts of diatom medium and culturing
the cells. Based on our spending over the past three years, the annual costs are expected to be broken down
approximately as follows:

Biochemicals and Reagents $ 1,250

Glassware/Plasticware $ 1,250
Microscopy Supplies $ 1,000
Culture Supplies $ 500

The additional money requested for supplies in the first year ($7000 total in supplies) includes additional
computer and video material needed to connect the second stereo-microscope head to our measurement and
recording systems. The budget also requests money ($500) for funds to cover the costs of printing/publishing one
journal article per year, along with $50/year for photocopying costs.
 Current and Pending Support
(See GPG Section II.D.8 for guidance on information to include on this form.)
The following information should be provided for each investigator and other senior personnel. Failure to provide this information may delay consideration of this proposal.

Other agencies (including NSF) to which this proposal has been/will be submitted.
Investigator: Stanley Cohn
Support: Current Pending Submission Planned in Near Future *Transfer of Support
Project/Proposal Title: Ecological Conditions Affecting Diatom Motility and
Adhesion

Source of Support: DePaul University - Liberal Arts & Science Summer Grant
Total Award Amount: $ 5,200 Total Award Period Covered: 06/15/99 - 09/15/99
Location of Project: DePaul University
Person-Months Per Year Committed to the Project. Cal: Acad: Sumr: 1.00

Support: Current Pending Submission Planned in Near Future *Transfer of Support

Project/Proposal Title:

Source of Support:
Total Award Amount: $ Total Award Period Covered:
Location of Project:
Person-Months Per Year Committed to the Project. Cal: Acad: Sumr:

Support: Current Pending Submission Planned in Near Future *Transfer of Support

Project/Proposal Title:

Source of Support:
Total Award Amount: $ Total Award Period Covered:
Location of Project:
Person-Months Per Year Committed to the Project. Cal: Acad: Sumr:

Support: Current Pending Submission Planned in Near Future *Transfer of Support

Project/Proposal Title:

Source of Support:
Total Award Amount: $ Total Award Period Covered:
Location of Project:
Person-Months Per Year Committed to the Project. Cal: Acad: Sumr:

Support: Current Pending Submission Planned in Near Future *Transfer of Support

Project/Proposal Title:

Source of Support:
Total Award Amount: $ Total Award Period Covered:
Location of Project:
Person-Months Per Year Committed to the Project. Cal: Acad: Summ:
*If this project has previously been funded by another agency, please list and furnish information for immediately preceding funding period.
NSF Form 1239 (10/99) Page G-1 USE ADDITIONAL SHEETS AS NECESSARY
 H. Facilities and Equipment

The Department of Biological Sciences of DePaul University is currently housed at the

Lincoln Park Campus of DePaul University and has recently moved from the O'Connell Science
Building to the new McGowan Biological and Environmental Sciences building. The laboratory
of Dr. Cohn occupies approximately 750 square feet in the building and already contains several
pieces of equipment to be used for the proposed project. This equipment includes a Zeiss
Axioskop video microscope fitted with DIC, phase and fluorescence optics and connected via a
GenLock device to an Amiga 2000 computer. Additional lab equipment includes a Wild MZ8
Stereomicroscope fitted with a video port, a Sony Camera and Video Printer, a Panasonic time-
lapse video recorder, an S-Video recorder, a G3 400 MHz computer with video input/output and
a DAGE DSP-2000 video processor. Additional equipment includes a refrigerator, freezer,
incubators and facilities for cell culturing, pH meter, balances, an electrically controlled thermal
cooling plate, a temperature controlled slide holder, and acrylic flume apparatus.

The new McGowan building, opened in July 1998, includes controlled environment
rooms, darkroom, cold room, tissue culture room, PCR room, animal care facilities, dishwashing
facilities, and a greenhouse, all available as shared facilities. There is also high-quality deionized
water available in each lab. Shared departmental equipment includes autoclaves, an
ultracentrifuge, scintillation counter, refrigerated low-speed centrifuge, clinical grade phase
microscopes, an osmometer, respiratory gas analyzer, scanning spectrophotometer, a digital video
printer, slide printer, and a digital gel scanner/analysis system. There is also a field site van, jointly
used by Biology and Environmental Science. This equipment is all available for joint use by the
Biology faculty. Secretarial assistance for typing, as well as funds for copying, postage and
telephones, is also provided.

The University is also committed to continually monitoring and upgrading the science
capabilities of the campus. In 1993, DePaul opened a new library building, which is continuing to
increase its science reference facilities and databases, and is set-up for relatively rapid intra-city
acquisition and borrowing from nearby institutions. New Chemistry facilities are planned to be
built adjacent to the McGowan building in 3-5 years, giving a significant upgrade to the Chemistry
Department and its academic program. Moreover, DePaul is moving forward with the planning of
a Food Science Program, which is expected to help foster further growth in the sciences at
DePaul.

The location of DePaul near the center of Chicago provides for excellent intellectual and
collaborative opportunities with scientists from the University of Illinois at Chicago, University of
Chicago, Northwestern University, Loyola University as well as many other scientific and medical
facilities within the city. As part of this intellectual exchange, the Biology department maintains a
seminar series during the school year that brings in speakers from throughout the Chicago and
Illinois region.