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Cell Biology

Dr. Hongye Li ( 李宏业 )


Tel:85228470
Email: thyli@jnu.edu.cn
Rm525, Dept of Biotech
Jinan University
Contents
Cell biology studies the basic structure and function of the cell.
By employing modern techniques achieved by physics,
chemistry and molecular biology, the basic life of cell are shown
from different layers ( micrological, sub-micrological and
molecular level ).

Main contents include extracellular matrix, biomembrane


systems and protein sorting, the cytoskeleton systems and cell
motility, the nucleus and gene expression, cellular reproduction
and differentiation, aging and apoptosis, signal transduction,
cancer, cell origins and evolution, techniques in cell and
molecular biology etc.
Textbook
 Essential Cell Biology. Bruce
Alberts et al. New York.
Garland Science, 2010.

Grading
 Class participation 30%
 Final closed-book examination 70%
Chapter 1 Introduction to Cell Biology
Outline

 I. Cell Structure and Organelles

 II. Techniques in cell biology


Life
Begins
With Cells
Structure of
Animal Cells
Bacterium
Cell Organelles

 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

A beam-shearing interference system in which


the reference beam is sheared by a minuscule
amount, generally somewhat less than the
diameter of an Airy disk. The technique produces
a monochromatic shadow-cast image that
effectively displays the gradient of optical paths
for both high and low spatial frequencies present
in the specimen.
A comparison of yeast cells that
were growing on the vaginal epithelium
seen with different types of LM.
a)under bright-field;

b)phase-contrast;

c)DIC
Living Cells Are Seen Clearly in a Phase-Contrast or a
Differential-Interference-Contrast Microscope
Video-enhance(contrast) microscopy

Observing living specimens;


Greatly increase the contrast of an image so that
very small objects become visible.
Tissues Are Usually Fixed and Sectioned for Microscopy
Different components of the cell can be selectively stained
Specific Molecules Can Be Located in Cells by
Fluorescence Microscopy
Antibodies Can Be Used to Detect Specific Molecules
Indirect immuno-cytochemistry
(Multicolor) fluorescent in situ hybridization
mFISH
Confocal microscope

Schematic representation of a (simple) confocal microscope setup


Confocal Microscope Produces Optical Sections by
Excluding Out-of-Focus Light
Three-dimensional reconstruction from confocal
microscope images.
Three-dimensional reconstruction from serial sections
Fluorescence Resonance Energy
Transfer , FRET
* Visualizing Molecules
in Living Cells
Rapidly changing intracellular
ion concentrations can be
measured with light-emitting
indicators
Visualizing intracellular Ca2+ concentrations by
using a fluorescent indicator.
Several Ways of Introducing Membrane-impermeable
Molecules into Cells
Light-induced Activation of “Caged” Precursor Molecules
Facilitates Studies of Intracellular Dynamics
Determining microtubule flux in the mitotic spindle
with caged fluorescein linked to tubulin.
Special Light Microscopes
Fluorescence 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

resolves the Fine Structure of the Cell

1. Transmission Electron Microscopy


2. Scanning Electron Microscopy
3. others
1. Transmission Electron Microscopy

Comparison of the lens


systems of LM and TEM
Biological Specimens Require Special Preparation for EM
Two common chemical fixatives used
for electron microscopy
Biological Specimens Require Special Preparation for Electron Microscope

Thin Sectioning for TEM

The wax sections: 5um;


The Plastic ultrathin-sections for
TEM: 50-100nm

Sections of LM: >5um;


Sections of TEM: <0.1μm
A root-tip cell stained with osmium and other heavy metal ions
Localizing proteins under EM
Negative staining and metal-shadowing

Metal Shadowing allows surface


features to be examined at high
resolution by TEM

Examples of negatively stained and


metal-shadowed specimens.
Electron micrographs of a tobacco
rattle virus after negative staining
with potassium
phosphotungstate(a) or shadow
casting with chromium(b).
Localizing proteins under EM

TGN (trans-Golgi network)


Metal Shadowing Allows
Surface Features to Be
Examined at High Resolution
by Transmission Electron
Microscopy
Molecules Can Be Labeled with Radioisotopes
Figure 9-40 Molecular Biology of the Cell (© Garland Science 2008)
Molecules Can Be Labeled with
Radioisotopes
2. Scanning electron microscope (SEM)
Images of Surfaces Can Be Obtained
The nuclear pore. Rapidly frozen nuclear envelopes were
imaged in a high-resolution SEM
Freeze –Fracture
Replication and
Freeze Etching

quick freeze
deep etching
Freeze-Fracture and Freeze-Etch EM Provide
Views of Surfaces Inside the Cell

The thylakoid membranes from the chloroplast of a plant cell


EM tomography. (7.4 Å).
3. Scanning probe microscope (SPM) –
Scanning tunneling
microscope(STM)
Including: :
Scanning tunneling microscope (STM),
Atom force microscope (AFM),
Magnetic force microscope (MFM),
Friction force microscope(MFM)
Other techniques
1. The Fractionation and analysis for
cell contents
A. The technique of differential centrifugation

S=(dx/dt)/2x
=110-13sec.
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

Immuno-gold electron microscopy


F. Localization of Nucleic Acids:

Fluorescent in situ Hybridization (FISH)


Isotope-labeled probe
or Biotin-labeled probe

Feulgen Staining

G. Enzymatic Amplification of DNA by PCR


2. Protein structure determination
A. X-ray Crystallography
A major technique that has been used to discover the
three-dimensional structure of protein molecules, at
atomic resolution.

B. Nuclear Magnetic Resonance (NMR) Spectroscopy


NMR technique has been used to study the structure of
small protein(MW<200KD) or protein domains.
3. The techniques for cytochemistry
Cytochemistry is a science of localizing chemical components of
cells and organelles on histological sections by using various
techniques.
Developing new techniques:
• enzyme cytochemistry,
• microincineration,
• microspectrophotometry,
• radioautography,
• cryo-techniques,
• X-ray microanalysis
• immunocytochemistry.
Trypan blue exclusion assay:
*KALTENBACH JP, KALTENBACH MH, LYONS WB. Nigrosin as a dye for
differentiating live and dead ascites cells. Exp Cell Res. 1958, 15,112.

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.

*(M. Diem, M. Romeo, S. Boydston-White, M. Miljkovi and C. Matthäus. A decade of


vibrational micro-spectroscopy of human cells and tissue (1994–2004) Analyst, 2004,
129, 880 – 885.)
B. Flow Cytometry
4. Autoradiograph of LM and TEM
5. Cell culture and cell engineering
A. Primary culture cell and subculture cell
B. Cell strain and Cell line
 HeLa, BHK21(baby hamster kidney),
CHO(chinese hamster ovary);
 Two tepes of morphes: fibroblast like cell
and epithelial like cell;
 Protoplast culture and anther culture
 Cell-free system

C. Cell engineering
 Cell fusion and cell hybridization
Monoclonal antibody

The hybridomas techniques (Cesar


Milstein and Georges Kohler 1975)

HAT selective medium: (hypoxanthine;


aminopterin; and thymidine)
The cell with HGPRT growing;
The cell lacking HGPRT don’t growing.
(hypoxanthine- guanine phosphoribosyl
transferase,HGPRT)
D. The technique for the take apart and gather up
of cell, and microscope manipulation
Preparation and reform of karyoplast and cytoplast
Transgenic animals and plants

Transgenic
mice
10 weeks
44g and 29g

Ralph Brinster (University of Pennsylvania) and Richard


Palmiterand (University of Washington ) 1981
Mario Capecchi (Late 1980s) (U Utah)
embryonic stem cells in inner cell mass as
target cells
1/104 cells undergo a process of
homologous recombination.

Knockout mice
See you !