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Transcript
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
1
The tree of life
Microbiology
Microbiology
& Cell biology
(prokaryotes)
Nucleus
Eubacteria
Eukaryotes
Archaea
Cytosol
2
Biology
Molecular biology
Cell biology
Organism biology
Met Ser Arg Pro
Nanometers
Micrometers
Millimetres
Meters
3
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
4
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)
5
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?
6
Multicellular eukaryotes – not just cells
The extra cellular matrix (ECM) works as a scaffold in metazoans
supporting cells in various ways
7
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
8
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
9
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)
10
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)
12
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
13
The development of microscopy
~1900
Zacharias Janssen
(1580 -1638)
The first microscope
Today
14
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)
15
Bright field microscopy
Ocular
Objective
Stage
Condenser
Lamp
16
Specialized bright field microscopy
Enhances the contrast between intracellular structures
Bright field
Phase contrast
Differential
interference
contrast (DIC)
17
Creation of contrast in bright field microscopy
Unstained cell
Stained cell
Classical stains
18
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
19
Shortcoming of bright field microscopy
Okay this was interesting.....
...but where is the protein of interest?
20
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
21
+
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
22
Principle behind a fluorochrome
Fluorochrome
Excitation
- Emission
-
-
-
-
Fluorochrome # 1
-
-
Fluorochrome # 2
A fluorochrome absorb light of a particular wavelength and
re-emit light of a longer wavelength
23
How it works in reality
Emission filter
Filter cube
Excitation filter
Beam splitter
-
-
-
24
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)
25
Transmission Electron Microscopy (TEM)
Electron gun
e-
eVacuum!
Very thin section
of a cell stained
with heavy metal
Detector
Supporting
grid
26
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
27
Different forms of microscopy
Bright field microscopy
cell organelles
large
molecules
Electron microscopy
Fluorescence microscopy
Location of molecules
Different techniques –
different ”windows”
28
The fluorescent protein revolution
YFP
DsRed
-
29
-
GFP
-
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
-