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Cell biology 2014 (revised 20/1-14)
Lecture 1:
“Recommended reading”
Chapter 8 Chapter 9
501-505
571-572
Alberts et al
5th edition
579-589
592-593
604-610
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The tree of life
Microbiology
Microbiology
& Cell biology
(prokaryotes)
Nucleus
Eubacteria
Eukaryotes
Archaea
Cytosol
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Biology
Molecular biology
Cell biology
Organism biology
Met Ser Arg Pro
Nanometers
Micrometers
Millimetres
Meters
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The starting point of cell biology: microscopy
I am seeing atoms
Let's call them cells (1665)
Robert Hooke
(1635 – 1703)
Cellulae, little room
Sliced cork
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Conceptual breakthroughs in cell biology
Mikroskopische Untersuchungen über die Übereinstimmung in
der Struktur und dem Wachsthum der Tiere und der Pflanzen
(1839)
- All organisms consist of one or more cells
- The cell is the basic unit of structure
Die Cellularpathologie (1858)
- All cells arise from preexisting cells
On the Origin of Species by Means of Natural Selection (1859)
- All cells have a common ancestor
Zellsubstanz, Kern und Zelltheilung (1882)
- Chromosome (thread) segregation during mitosis
(i.e. precise partitioning/transport of defined cell structures)
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All eukaryotic cells are in principle very similar
- Organelles
- Cytoskeleton
- Nucleus
- Chromosomes
Key questions in cell biology
• Structure and functions of cellular components
• How do cells communicate?
• Which signals trigger cell cycle entry?
• How is cell duplication coordinated?
• How is one cell split into two?
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Multicellular eukaryotes – not just cells
The extra cellular matrix (ECM) works as a scaffold in metazoans
supporting cells in various ways
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Animal tissues mainly consisting of (different) cells
- Epithelia
Protective covering of surfaces, both outside and inside the body
- Muscle
Force generating cells (contraction)
Animal tissues consisting of cells and ECM
- Connective
• Hard tissues of bone and teeth
• Transparent matrix of the cornea
• Ropelike organization of tendons
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How to study individual animal cells
Primary cell cultures
Secondary culture
Explants
Proliferation
(growth factors)
Complete tissue
section
Only cells
Tumor patient
Immortalization
(e.g. by oncogenes)
Cell line, with indefinite
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proliferative potential
I. How to study the function of a protein in cells
Depletion/mutation of
endogenous protein
Normal
(Control)
Overexpression of protein
(ectopic expression)
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II. How to study the function of a protein in cells
Central dogma of
molecular biology
- Loss-of-function mutations
- Gain-of-function mutations
- Overexpressed (trans)gene
DNA
Transcription
- RNA interference
mRNA
Translation
Protein
-Inhibitory (pharmaceutical) drugs
 new field ”chemical genetics”
RNA interference – depletion of a specific protein
ds short RNA (synthetic or expressed as shRNA)
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RISC
mRNA
Duplex formation
mRNA detroyed
Normal cell
RNAi treated cell
DNA:
mRNA:
mRNA degraded!
Protein:
Already existing proteins
decay over time
Systems for overexpression of a protein
Transient transfection
Stable transfection
(plasmid DNA is not replicated)
(Chromosomal integration)
Plasmid
+ Quick (4 – 6 hours)
High expression level
- Heterogeneous cells
Small amount of
transfectants
drug
resistance
+ Homogenous cell line
Unlimited amount of transfectants
- 4 – 6 week to establish a cell line
Impossible if gene product
causes a cell cycle block
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The development of microscopy
~1900
Zacharias Janssen
(1580 -1638)
The first microscope
Today
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The three principle tasks of microscopy
- Produce a magnified image (magnification)
- Separate the details in the image (resolution)
Resolution: the smallest distance between
two objects at which the two objects can
be seen as separate units
Maximal resolution = l/2
- Render the details visible (contrast)
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Bright field microscopy
Ocular
Objective
Stage
Condenser
Lamp
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Specialized bright field microscopy
Enhances the contrast between intracellular structures
Bright field
Phase contrast
Differential
interference
contrast (DIC)
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Creation of contrast in bright field microscopy
Unstained cell
Stained cell
Classical stains
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Preservation of biological structures by fixation
Process in which cellular structures are preserved
and fixed in position by chemical agents
Glutaraldehyde
Formaldehyde
Extensive protein cross-linking
Alcohols
Protein denaturation
Fixation may introduce structural artifacts
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Shortcoming of bright field microscopy
Okay this was interesting.....
...but where is the protein of interest?
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Raising antibodies against specific proteins
Polyclonal antibodies
Purify antibodies
from the blood
of the animal
Epitope
Protein X
Monoclonal antibody
1.
Take out antibody
producing B cells
Protein X
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+
2.
Molec models. 25.2-antibodies
Fuse with myeloma cell to generate a hybridoma
Detection of specific proteins with antibodies
Primary antibody
Specific to epitope
on protein X
Protein X
Secondary antibody
Specific to the primary
antibody, conjugated
with e.g. a fluorochrome
Protein X
Protein X
The primary antibody (e.g. rabbit) is recognized by
many secondary antibodies (e.g. goat anti-rabbit)
Signal amplification
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Principle behind a fluorochrome
Fluorochrome
Excitation
- Emission
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Fluorochrome # 1
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Fluorochrome # 2
A fluorochrome absorb light of a particular wavelength and
re-emit light of a longer wavelength
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How it works in reality
Emission filter
Filter cube
Excitation filter
Beam splitter
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Electron microscopy (EM)
Maximal resolution = l/2
400
700 nm
Maximal resolution 200 nm
Resolving smaller structures demands something
with a much shorter wavelength
e- + 100 000 V
el= 0.004 nm
Resolution 0.002 nm
(0.1 nm in reality)
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Transmission Electron Microscopy (TEM)
Electron gun
e-
eVacuum!
Very thin section
of a cell stained
with heavy metal
Detector
Supporting
grid
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Scanning Electron Microscopy (SEM)
Visualizing surface features
The specimen is coated with metals to deflect electrons
Sequential scanning
Electron gun
e-
e-
e-
e-
Cell with metal coating
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Different forms of microscopy
Bright field microscopy
cell organelles
large
molecules
Electron microscopy
Fluorescence microscopy
Location of molecules
Different techniques –
different ”windows”
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The fluorescent protein revolution
YFP
DsRed
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GFP
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Aeqourea victoria
Protein X
GFP
Protein X GFP
Transient or stable expression
Detection in either live or fixed cells
Video 02.3-brownian_motion.mov
Video 10.6-FRAP
Visualization of signaling in live cells (NFAT):
Video 12.2-nuclear_import.mov
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