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1.
Using the components discussed in class, propose a signal
transduction pathway that would result in the release of
cortical granules during fertilization. From this pathway,
identify three potential causes of CG-related infertility.
Cortical granules: releasing components that form
fertilization envelope
Signal Transduction Pathway Leading up to CG release:
1. Ligand binds to GPLR receptor on egg cell and activates
it
2. L-R complex triggers conformational change, which is
transduced to G-protein
3. G-protein exchange GDP for GTP and becomes activated
a. It also separates into G-alpha and G-beta,gamma
4. G-alpha (bound to GTP) travels along membrane until it
encounters Phospholipase C
5. Once Phospholipase C is activated, it catalyzes the
hydrolysis of PIP2 (phospholipid in the egg cell
membrane) into DAG and IP3
a. DAG will remain in the membrane, while IP3 is
soluble in the cytoplasm
6. IP3 will diffuse into the cytoplasm and will bind to
calcium channels on membrane of Smooth ER, opening
them and releasing calcium into the cytoplasm by
facilitated diffusion
7. Calcium levels in the cytoplasm will go above the basal
level (10-4 mM)
8. Calcium will then bind to Calmodulin, activating it
9. Calmodulin will then bind to Calmodulin-induced
Kinase ,
10.
Calmodulin-induced kinase will the phosphorylate
(using ATP) myosin
a. Myosin : cytoskeletal motor proteins
11.
Activated myosins will carry cortical granules being
formed up to the plasma membrane for release.
a. Cortical granules made in the endomembrane
system (being secreted outside the cell)
12.
Myosins move CG up to the PM, where their internal
contents will be released and form the fertilization
envelope outside the fertilized egg
Causes of Infertility:
Lack or abnormal CG formation
Misfolded G-protein
2.
5.
Outline the events involved in a growth-factor/RTKmediated pathway. Using the example outlined in class,
explain why expression of cyclin/CDK is an appropriate
outcome for such a pathway.
8.
1. Organization:
a. Spatial organization of cellular contents
b. Like cytoplasmic and nuclear (genetic) materials
2. Cell Shape
a. Provides mechanical support to the cell and nucleus
b. Scaffolding that helps generate maintain and
maintain cell shape
3. Motility:
a. Movement of the cell itself (cell crawling)
b. Intracellular movement of structures
4. Cell Division:
a. Manages chromosomes (Microtubules bind to and
help separate them)
b. Cytokinesis (Microfilaments actually divide the cell
itself)
5. Regulation:
a. Transmits mechanical signals from environment
i. Like adhesion molecules, either directly or
through adaptor molecules
b. Intracellular movement of molecules in signaling
12.
Recall the three different elements that make up the
cytoskeleton and describe their main functions.
1. Microtubules:
a. Spatial organization in cytoplasm
b. Intracellular transport (motor proteins)
2. Microfilaments:
a. Forming and maintaining cell shape
b. Cell locomotion (specific to crawling cells)
3. Intermediate Filaments:
a. Mechanical strength
b. Nucleus shape ( and organizing chromosomes)
13.
15.
Microtubule Structure:
a single MT is a hollowed out tube made out of
protofilaments
each MT is 25 nm in diameter (largest of the
elements)
protofilaments made up of dimers of the protein
tubulin
beta tubulin and alpha tubulin form tubulin
heterodimers
tubulin heterodimers come together to form
protofilaments, which come together to form hallow
tube
oriented in a specific polarity
o beta subunits face plus(+) end
o alpha subunits face minus (-) end
Tubulin Heterodimer :
alpha and beta tubulin both bind to nucleotide GTP
only beta tubulin can hydrolyze GTP to form GDP
(GTPase activity) (and can exchange GDP for GTP)
alpha tubulin is permanently bound to GTP
o alpha and beta tubulin held together by non-covalent
interactions
o structural polarity to MT because subunits in each
protofilament all point in the same direction
Cytoplasmic MTs
singlet arrangement of protofilaments
hollow ring comprised of 13 parallel protofilaments
Axonemal MTs
Doublet arrangement
Singlet ring with partial ring of 10 protofilaments
o Triplet arrangement
Singlet ring with 2 partial ring (10 protofilaments
each)
16.
MT Assembly :
Start off with population of tubulin dimers (alpha and beta)
Link together to form short fragments : oligomers
Oligos come together to form longer strands :
protofilament
Protofilaments line up laterally to form sheet of
protofilaments
Protofilament sheet folds in on itself to form hollowed
out tube
Hollowed out tube grows from either by the addition
of free tubulin dimers in the cytoplasm to form fully
functional Microtubule
Growth of MT starts out really slow with free dimers
in the cytoplasm
Growth gets rapid once the dimers start linking to
form protofilaments
17.
Define 'critical concentration' as related to MTs. Explain the
effects of the critical concentration on MT assembly. Explain
how critical concentration is related to the polarized assembly
of MTs
Critical Concentration :
The concentration of free tubulin in the cytoplasm at which
rate of tubulin subunit addition is approximately equal to
the rate of subunit loss
MTs tend to grow when tubulin concentration
exceeds Cc and depolymerize when tubulin
concentration falls below Cc
Overall length change is constant
Reached during the plateau phase
Point where growth slows down dramatically
and subunits and coming off and being added
Cc differs for plus end and minus end of MT, which is
why is there is a difference in rates of growth and
disassembly
Plus end grows much faster than minus end
( elongates faster)
18.
Describe the process of 'dynamic instability' and the role
played by GTP in this process.
MTs are continually changing and are very dynamic, at any
given time, some MTs are growing while others are shrinking
Dynamic Instability:
Model to explain constantly changing MT behavior
In a given population of MTs:
Some polymerize and grow while others are
depolymerizing and shrinking
Polymerization may continue for some undefined
period of time
MT may suddenly shrink rapidly (catastrophe)
Can also shrink partially and recommence
growing (rescue)
Can also completely depolymerize and growth
does not resume
Cc extremely sensitive to environment
immediately surrounding that end of MT
Regulated by GTP Cap:
Free tubulin is in the GTP form
Tublin dimers (bound to GTP) added to the of MT during
polymerization
Eventually GTP within tubulin dimer is hydrolyzed to GDP
Beta subunit hydrolyzes GTP when it is a part of MT
GTP hydrolysis destabilizes MT structure
23.
Describe the role of Tau and how it is correlated with
different neurodegenerative diseases.
Tau: (MAP)
found in axons of nerve cells
binds to axonal MTs
stabilizes MTs
promotes MT polymerization/growth
present at the beginning of development, helping
neuron develop properly and keep it stabilized
induces MTs to start polymerizing and stabilizing to
form long outgrowths
play a role in development of neuron polarity
To regulate activity of Tau, kinase phosphorylates Tau
Once Tau is phosphorylated, it changes its conformation so
it can no longer associate with MTs
No longer binds to MTs, so MT is destabilized and
start to depolymerize
To regulate Tau in the opposite direction:
In order to allow phosphorylated Tau to rebind to M,
action of phosphatases are needed to
dephosphorylate Tau and change conformation back
to a state where it can interact with MT and stabilize
them.
PHF: paired helical filament
Insoluble, stable aggregate of Tau
Role of Tau in Neurodegeneration:
Tau has been implicated in neurodegenerative diseases,
such as Alzhemiers disease
Hyperphosphorylation of Tau disable Tau from being
able to bind to MTs
when it cannot bind, MTs become destabilized and
breakdown
when Tau is phosphorylated, can bind to one another
and form filaments
some sort of signaling occurs, making it
hyperphosphorylated, which forms larger aggregates
and forms PHF
PHF: play a role in breakdown of cell
function
o Cytotoxic filaments , impair functions
of neurons and eventually cause their
Microfilaments
Function:
Underlie the PM and form cell cortex
Stabilize cell shape
Involved in muscle contraction
If a cell can crawl, cell locomotion , which is MFbased motion
MFs involved in cytokinesis
During cell division, when cell splits into two
Cytoplasmic Streaming: mainly plant cells
Cytoplasmic contents move around the cells,
which can function to assist diffusion rates
Structure:
Similar to MTs
7 nm in diameter : smallest of the three
elements
polymers of the protein : actin
2 forms:
G-actin (globular)
o Free form of actin
o Isnt polymerized into a MF
F-actin (Filamentous)
o Polymerized form of actin
o When actin is in filament
25.
Compare and contrast microfilament assembly to that of
MTs.
MFs have polarity, similar to MT: has +/- ends
+ end grows faster than end
G-actin (free actin) bound to ATP
similar to GTP in MTs
ATP is eventually hydrolyzed to ADP in MF polymer (when in
filamentous form)
ATP cap to MF
o Actin can reversibly bind ATP and ADP,
Cytoskeleton linked to PM
Integral membrane proteins associated with cytoskeletal
elements, like MFs
In this way, MFs linked to PM membrane proteins can push
PM in direction to generate cell locomotion
27.
Explain the role of WASP. Explain how a defective WASP can
lead to the symptoms associated with Wiskott-Aldrich
Syndrome.
WASP: Wiskott Aldrich Syndrome Protein
Wherever there is actin, you see WASP , it is associated with MFs
Regulates actin polymerization in Hematopoietic Cells (RBCs)
Hematopoiesis : developmental process by which immature
precursor cells develop into mature blood cells
28.
Describe the potential effects of drugs that disrupt
microfilaments.
Drugs that affect MFs will affect the viability of the cell
- they prevent MFs from being able to polymerize/depolymerize or
branch, affecting viability of the cell
29.
Describe the structure and functions of intermediate
filaments (IFs).
Intermediate Filaments :
- Function:
o Structural support
Tension bearing cables
o Nuclear Lamina Scaffolding
Underly nuclear envelope
Provide structural support for the nucleus
Nuclear lamina : network of Ifs beneath inner
layer of nuclear envelope
o Can also provide organization for chromatin in nucleus
- Structure :
o 8-12 nm in diameter : in between diameters of MTs
and MFs
o all share same structure
o Single Subunit: helical protein (N terminus and C
terminus )
o Dimer: single subunits interact and coil around one
another with other subunits to form dimer
o Staggered Tetramer: 2 dimers interact with eachother in
a staggered, antiparallel way
C terminus of one associated with N terminus of the
other
o IF Subunit: Tetramers interact with one another to form
subunit that will be used to construct IFs
Like tough ropes
o 2 tetramers pack together; form 8 protofilaments
o 8 Protofilaments all wind around one another to give IF:
perfect for absorbing tension
30.
Explain how and why cytoplasmic IFs can be used in
tissue/cell-typing and why nuclear IFs cannot.
Cytoplasmic IF Proteins are Cell/Tissue Specific :
- Many different proteins can be used to make IFs
o Many different IFs can therefore be found in
cytoplasm of cells
31.
Provide an overview of the cytoskeleton. Recognize the
cytoskeleton as a complex, dynamic system with a high level
of interaction taking place among the three elements.
Interactive Cytoskeleton Network:
- all elements are linked together
- no one elements alone could provide shape or strength to
cell
- interact and linked with one another through accessory
proteins
- depend on each other to properly function and support the
cell
o requires large group of accessory proteins linking
filaments to one another and to other cell
components
32.
Describe the different levels of cell movement.
Cell Movement:
- Intracellular Movement:
o refers to movement of contents within the cell
o motor proteins transporting cargo along MTs
o cytoskeleton acts as a track and provides machinery
for movement of contents within the cell
- Movement of Cell Itself :
o Motility Structures:
Cilia and Flagella (MT-based)
Crawling Cells (Cell Locomotion) : MFbased
33.
Recall the types of microtubule-associated motor proteins
and describe in general their overall structural/functional
domains.
Motor Proteins:
- type of accessory protein (MAP)
- have ATP hydrolase activity (bind to and hydrolyze ATP)
- use energy from ATP hydrolysis to transport cargo along
cytoskeleton tracks (MTs or MFs)
- travel in a single direction
o move to + end or end of an elements
- motor proteins only associated with MTs and MFs because
there is no polarity associated with IFs, proteins wouldnt
know what direction to go
o need elements that have directionality
34.
Recall the direction of movement associated with each
motor protein with respect the the +/- ends of MTs.
Kynesin: move towards plus end of MTs
Dyneins : move toward minus end of MTs
-
35.
Explain how the cell contents are organized by the
cytoskeleton. Generate a picture to describe how this
organization changes as the cell grows/develops.
MTs Organize the Cell :
- During cell growth/development, there is a particular
organization for the organelles
o Organelles organized by the MTs
- During cell growth/development
o Motor proteins associated with MTs bind to organelles
They pull organelles out as cell grows
Pull ER outward
Pull Golgi inward
36.
Describe how vesicle-based movement occurs in the cell.
Specifically describe how vesicles are transported between the
components of the endomembrane system.
MT Based Vesicle Transport:
- there is directionality to vesicle transport between different
compartments and to the PM
- if MTs all arranged so all plus end would point toward the
PM, Kynesins would be carrying vesicles to PM to be
secreted (move toward plus end)
- If things are being brought into the cell through
endocytosis, Dyneins would carry vesicles into the
body of the cell
37.
Explain why motor proteins are necessary. Given the
diffusion coefficient constant and the equation t = x2/2D,
calculate the time of diffusion for a given molecule or vesicle.
Motor proteins interact with cytoskeletal elements to produce
motion at the molecular level. They are necessary to help
vesicles and molecules move into and out of the cell . Much
faster than simple diffusion
Time of Diffusion: t=x2 /2D
D for a vesicle=5*10-8
Note: 1 cm= 104 um
Rate of Transport for Motor Proteins : 2 um/sec
- for axonemal dyneins: 14 um/sec
38.
Compare and contrast the structure and function of cilia
and flagella.
MT-Based Cell Motility : Involves AXONEMAL MT
Cilia:
- made up of Axonemal MTs
- number per cell: one to many cilia per cell
- Size (Length): 2-10 um long
- Mechanism of movement: Oar-like motion
Flagella
- made up of axonemal MTs
- Number per cell: one to few flagella per cell
- Size (Length): 10-200 um long
- Mechanism of movement: wave-like motion
Note: Prokaryotes have flagella, but they are
structurally different that eukaryotic flagella (proks do
not have cytoskeletal elements)
40.
Describe the ultrastructural details of cilia and flagella.
Both cilia and flagella made out of Axonemal MTs
- at the base of cilia/flagella is basal body
o basal body similar to centriole
o has triplet MT arrangement
consists of 9 sets of triplet MTs
- basal body contains nucleating MTs that extend out
- as MTs extend, they go through Transition Zone (slightly
different MT arrangement)
- Once MT gets up to axoneme , has 9+2 arrangement
o 9 doublets forming outer ring with a pair of
single MTs inside
- MTs associated with many accessory proteins
42.
Describe the structure and function of the microfilamentassociated motor proteins. How do they compare to MT-based
motor proteins?
Actin = Microfilament
Actin-based Cell Locomotion = Crawling Cells
- for cells that lack cilia/flagella
- based on action of MFs (actin)
- MFs have many different accessory proteins and motor
proteins
o Motor proteins associated with MFs : Myosins
Have domains that bind to MF (actin) that
can hydrolyze ATP
Have linker region in between
Move in a directional way (+/-) ends
MF-associated proteins similar to MT-based motor proteins
- both have directionality
- both have ATPase activity
43.
Describe the general structure and function(s) of the cell
cortex.
Cell Cortex :
- made of MFs
- what gives a cell its shape
- MT determine cell shape as cell goes through
differentiation
- MFs maintain cell shape
- Cell cortex rearranges MFs and gives rise to cell
locomotion
- Meshwork of actin filaments underlying PM
o Also includes spectrin network
Network of proteins linking PM to MFs
44.
Create a detailed model outlining the subcellular events
involved in cell locomotion.
Cell Crawling:
- cell that lacks cilia and flagella
- action of MFs
- includes cell with network of MFs just underneath and
connected to PM
o cell will undergo rearrangements to push cell
forward and retract rear of cell to move forward
- Cell will crawl in direction of stimulus
o Extends PM in that direction, pushing forward due
to action of MFs polymerizing in that direction
o Lamellipodium : extension of PM
- Events:
1. Leading edge extends via polymerization of actin
filaments at its tip (forming lamellipodium)
2. New adhesions, anchored by actin, form on the
undersurface of the lamellipodium
Still adhering at other end of the cell
3. The trailing edge (tail) of the cell detaches, and is
drawn forward by the contraction of the cell body
45.
Describe the arrangement of MFs in a cell during cell
locomotion. Are the MFs arranged the same throughout the
crawling cell?
In the leading edge (lamellipodium)
- actin MFs polymerizing in the direction of movement,
pushing PM in that direction
- accessory proteins linking MFs to PM
o proteins crossing through PM and adhering to
whatever cell is crawling across
Cell Locomotion
- as cell crawls forward, forms initial lamellipodium
o small extensions in lamellipodia called filopodia
46.
List and describe the various levels of organization in a
multicellular organism.
Cell Systems in Multicellular Organisms
- cells interact on different levels
- cells interact with eachother to form tissues
- tissues interact to form organs
- organs interact to form organ systems
- organ systems function together to make sure organism
functions in homeostatic capacity
47.
Define a tissue.
48.
List and describe the structure and function(s) of the four
major tissue types found in animals.
4 major tissue types in animals:
1. Epithelial
Cell layer that lines cavities and surfaces
of body structures
Lies on top of connective tissue
Connected to those tissues by basement
membrane (basal lamina)
49.
Describe the structure and function of endothelial tissue.
Endothelial Tissue:
- specific type of tissue found in lining of
cardiovascular system (lining of BV)
- considered a type of epithelial tissue
o even though they have different embryonic origins
- seen in vasodilation
o sent signals to overlaying smooth muscle cell to
relax muscle and to cause BV to dilate
50.
Tissue Formation
1. Cell Recognition
2. Cell Adhesion
Either direct attachment through
transmembrane adhesion proteins
Or through communication of Extracellular
matrix
Like bone (molecules outside the cell)
Important during development and
embryogenesis
Has to start immediately for cells
undergoing embryogenesis
51.
52.
53.
Differentiate between homophilic and heterophilic
interactions of cell adhesion proteins.
Homophilic Interactions: cell adhesion protein interacts
with identical protein
Heterophilic Interactions: interacts with different
proteins
Cell Adhesion Proteins: all transmembrane proteins
54.
List and describe the four categories of cell adhesion
proteins discussed in class (CAMs, cadherins, selectins,
integrins). Explain how each type of adhesion protein mediates
cell adhesion
Cell Adhesion Proteins
- all are transmembrane proteins
- all have some type of extracellular domain
o interacts with:
another cell adhesion protein
or an extracellular matrix component
- all have cytoplasmic domain
o interacts directly with cytoskeletal elements
o interacts indirectly through adaptor proteins
o may facilitate signaling pathway
1. CAMs (Cell Adhesion Molecules)
Have extracellular domains with loops
Loops generated by disulfide bridges
Must have cysteine residues to generate
disulfide bridges
Interact in a homophilic manner
2. Cadherins (Calcium-dependent adhesion
molecules)
In extracellular domain, have regions that bind
calcium
Helps generate structure
Transmembrane domain: anchors protein to PM
Cytoplasmic domain has cytoskeletal
interaction
Interact in hemophilic manner
P-Cadherin and E-Cadherin are
inversely expressed
Shows hemophilic interaction
3. Selectins (type of lectin, or carbohydrate
binding protein)
Have extracellular domains with different
carbohydrate binding domains
Bind to carbohydrates expressed in
another cell type
Cytoplasmic domain interacts with
cytoskeleton
Different types:
55.
Describe the evidence suggesting that cadherins are
important for preferential cell aggregation during
embryogenesis.
Important during vertebrate embryogenesis
- Helps segregate cells during tissue
formation/differentiation (when expressed in embryonic
cells)
- Cells expressing cadherin preferentially bind in
homophilic manner
o P-Cadherin and E-Cadherin are inversely
expressed
Shows hemophilic interaction
56.
Outline and describe the events involved in leukocyte
extravasation (e.g. What allows a WBC to roll along the
endothelial cells? What stops it from rolling? Why is it leaving
the blood vessel...these are just a few examples of questions).
Leukocyte Endothelial Cell Adhesions:
- leukocytes roll along endothelial cells
o cells adhere to one another
o mediated by selectins
leukocyte: L-selectin
Endothelial Cells: E-selectin and P-selectin
o Signals received by leukocyte (from endothelial
cells)
Cause expression and activation of
integrins
o Signals received by endothelial cells
Endothelila cells express ICAM
o Rolling of leukocyte along endothelial cells
STOPS when integrins on leukocyte adhere
to ICAM on endothelial cells
o Further signaling events cause cell cortex of
leukocyte to rearrange
Action of MFs
Allow WBC to leave BV
o WBC crawls in the direction of stimulus
Like a site of inflammation