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Chapter 1 Introduction to Cell Biology
Outline
Nucleus
1 Nuclear envelope
Chromatin and DNA
Nucleolus
Mitochondria
Double membrane
Mitochondrial (maternal) DNA
“Power House” of the cell
Food converted into energy
Adenosine triphosphate (ATP)
Consumes Oxygen, produces CO2
Cell Organelles
Endoplasmic Reticulum
Site where cell membrane and
exported material is made
Ribosomes (rough)
Make proteins
Smooth ER- lipids
Golgi Apparatus
Receives and modifies
Directs new materials
Lysosomes
Intracellular digestion
Releases nutrients
Breakdown of waste
Cell Organelles
Peroxisomes
Hydrogen Peroxide generated and degraded
Cytosol
Water based gel
Chemical reactions
Cytoskeleton
Filaments
(actin, intermediate and microtubules)
Movement of organelles and cell
Structure/strengthen cell
Vesicles
Material transport
Membrane, ER, Golgi derived vesicles
Organic molecules of Cells
Proteins
Carbohydrates
Lipids
Nucleic acids
Back
II. Techniques in cell biology
MICROSCOPY
1) Light Microscope
makes small objects appear bigger, magnify an image up
to 1500 times its original size.
0.2 um (before 2006), now 10-20nm (Fluorescent LM).
2) Electron Microscope
can achieve magnification up to 1 million times
1) Looking at Cells
under Light Microscope
Some Important Discoveries in the History
of Light Microscopy
1611 Kepler suggests a way of making a compound microscope.
1655 Hooke uses a compound microscope to describe small pores in
sections of cork he calls “cells”.
1674 Leeuwenhoek reports his discovery of protozoa. He sees
bacteria for the first time nine years later.
1833 Brown publishes his microscopic observations of orchids,
clearly describing the cell nucleus.
1838 Schleiden and Schwann propose the cell theory, stating that
the nucleated cell is the unit of structure and function in plants and
animals.
1857 Kolliker describes mitochondria in muscle cells.
1876 Abbé analyzes the effects of diffraction on image formation in
the microscope and shows how to optimize microscope design.
1879 Flemming describes with great clarity chromosome behavior
during mitosis in animals.
1881 Retzius describes many animal tissues with a detail. During the
next two decades, he, Cajal, and other histologists develop staining
methods and lay the foundations of microscopic anatomy.
1882 Koch uses aniline dyes to stain microorganisms and identifies
the bacteria that cause tuberculosis and cholera. In the following
two decades, other bacteriologists, such as Klebs and Pasteur,
identify the causative agents of many other diseases by examining
stained preparations under the microscope.
1886 Zeiss makes a series of lenses, to the design of Abbé, that
enable microscopists to resolve structures at the theoretical limits of
visible light.
1898 Golgi first sees and describes the Golgi apparatus by staining
cells with silver nitrate.
1924 Lacassagne and collaborators develop the first autoradiographic
method to localize radiographic polonium in biological specimens.
1930 Lebedeff designs and builds the first inference microscope. In
1932, Zernicke invents the phase-contrast microscope. These two
developments allow unstained living cells to be seen in detail for the
first time.
1941 Coons uses antibiotics coupled to fluorescent dyes to detect
cellular antigens.
1952 Nomarski devises and patents the system of differential
interference contrast for the light microscope that still bears his
name.
1968 Petran and collaborators make the first confocal microscope.
1981 Allen and Inoué perfect video-enhanced light microscopy.
1984 Agard and Sedat use computer deconvolution to reconstruct
Drosophilia polytene nuclei.
1994 Chalfie and collaborators introduce green fluorescent protein
(GFP) as a marker in microscopy
Nobel Prize 2014
Eric Betzig, Stefan W. Hell and
William E. Moerner
The optical microscope can
now peer into the nanoworld.
The importance can't be
overemphasized: Now,
scientists can see how
proteins in fertilized eggs
divide into embryos, or they
can track proteins involved in
Alzheimer's or Parkinson's Filament structures within a nerve
diseases, the committee said. cell are clearly resolved in a STED
microscopy image (circular inset)
but blurry under conventional LM
Similar methods were also developed in 2006 by
Xiaowei Zhuang of Harvard University (stochastic
optical reconstruction microscopy, or STORM)
X
Light Microscope
Can Resolve
Details 0.2 μm
Apart
Simple upright
light microscope
Interference between light waves
W: wavelength
A: amplitude
Two ways to obtain contrast in light microscopy
contrast-
enhancing
methods:
Phase-contrast
microscope
Differential-
interference
microscope
Phase-contrast
microscope
Differential-
interference
microscope
b)phase-contrast;
c)DIC
Living Cells Are Seen Clearly in a Phase-Contrast or a
Differential-Interference-Contrast Microscope
Video-enhance(contrast) microscopy
Direct
immunofluorescence
technique
Fluorochrome: Such as
rhodamine or fluorescein
Indirect immunofluorescence labeled
Technique.
To study the location of a specific protein within the cell by
using of fluorescent antibody (antigen-antibody couple).
GFP can be used to study dynamic processes as they occur in
a living cell.
Green Fluorescent Protein Can Be Used to Tag
Individual Proteins in Living Cells and Organisms
jellyfish
Roger Yonchien Tsien
Bacterial colony using various GFP and GFP-like proteins (from Tsien lab website)
GFP as a reporter. GFP
gene was joined to a fly
promoter that is active only
in neuron.
GFP tagging talin (an actin-binding protein)
2) Electron Microscope
quick freeze
deep etching
Freeze-Fracture and Freeze-Etch EM Provide
Views of Surfaces Inside the Cell
S=(dx/dt)/2x
=110-13sec.
B. Step-by-step procedure for the purification of organelles by
differential centrifugation.
B. Step-by-step procedure for the purification of organelles by
differential centrifugation.
C. Isolation, purification,
and
fractionation of proteins:
Selective Precipitation
(ammonium sulfate)
Liquid Column Chromatography
Ion-exchange Chromatography
Gel Filtration Chromatography
Affinity Chromatography
Polyacrylamide Gel Electrophoresis
D. Determining Protein-Protein
Interaction
Immunoblot: Western-Blot
Yeast two-hybrid system
The two domains;
Reporter gene: LacZ;
A known “Bait”protein X;
An unknown “Fish”protein Y
E. Localization of a specific protein by
immuno-electron microscopy
Feulgen Staining
MTT assay:
MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay,
first described by Mosmann in 1983, is based on the ability of a
mitochondrial dehydrogenase enzyme from viable cells to cleave the
tetrazolium rings of the pale yellow MTT and form a dark blue
formazan crystals which is largely impermeable to cell membranes, thus
resulting in its accumulation within healthy cells. Solubilisation of the
cells by the addition of a detergent results in the liberation of the crystals
which are solubilized . The number of surviving cells is directly
proportional to the level of the formazan product created. The color can
then be quantified using a simple colorimetric assay. The results can be
read on a multiwell scanning spectrophotometer (ELISA reader).
* Mosmann T. Rapid colorimetric assay for cellular growth and survival: application
to proliferation and cytotoxicity assays. J Immunol Methods. 1983 Dec 16;65(1-2):55-
63.
A. Microspectrophotometry
Determining contents of protein and nucleic acid in the cells.
Instrumentation used in infrared microspectroscopy (IR-MSP) permits the
acquisition of spectra from samples as small as 100 pg (10 –10 g), and as
small as 1 pg for Raman microspectroscopy (RA-MSP). This, in turn,
allows the acquisition of spectral data from objects as small as fractions
of human cells, and of small regions of microtome tissue sections. Since
vibrational spectroscopy is exquisitely sensitive to the biochemical
composition of the sample, and variations therein, it is possible to
monitor metabolic processes in tissue and cells, and to construct spectral
maps based on thousands of IR spectra collected from pixels of tissue.
These images, in turn, reveal information on tissue structure,
distribution of cellular components, metabolic activity and state of
health of cells and tissue.
C. Cell engineering
Cell fusion and cell hybridization
Monoclonal antibody
Transgenic
mice
10 weeks
44g and 29g
Knockout mice
See you !