how to study cells
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Lecture 1:
1
Cell biology 2014 (revised 20/1-14)
Alberts et al5th edition
Chapter 8501-505571-572
Chapter 9579-589592-593604-610
“Recommended reading”
Eukaryotes
Archaea
Eubacteria
(prokaryotes)
Cytosol
Nucleus
The tree of life
Microbiology Microbiology & Cell biology
2
Biology
Molecular biology Cell biology Organism biology
Met Ser Arg Pro
Nanometers Micrometers Millimetres Meters3
The starting point of cell biology: microscopy
Robert Hooke(1635 – 1703)
Cellulae, little room
Sliced cork
I am seeing atoms
Let's call them cells (1665)
4
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)
5Conceptual breakthroughs in cell biology
All eukaryotic cells are in principle very similar
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
- Organelles- Cytoskeleton- Nucleus- Chromosomes
Multicellular eukaryotes – not just cellsThe extra cellular matrix (ECM) works as a scaffold in metazoans supporting cells in various ways
7
Animal tissues mainly consisting of (different) cells
- Epithelia
- Muscle
Protective covering of surfaces, both outside and inside the body
8
Force generating cells (contraction)
- Connective
Animal tissues consisting of cells and ECM
• Hard tissues of bone and teeth• Transparent matrix of the cornea• Ropelike organization of tendons
How to study individual animal cellsPrimary cell cultures
Explants
Complete tissue section
Only cells
Secondary culture
Proliferation
Immortalization
Cell line, with indefinite proliferative potentialTumor patient
(growth factors)
(e.g. by oncogenes)
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I. How to study the function of a protein in cellsDepletion/mutation of endogenous protein
Overexpression of protein (ectopic expression)
Normal (Control)
10
Central dogma of molecular biology
DNA
mRNA
Protein
Transcription
Translation
- Loss-of-function mutations- Gain-of-function mutations- Overexpressed (trans)gene
- RNA interference
- Inhibitory (pharmaceutical) drugs new field ”chemical genetics”
II. How to study the function of a protein in cells
RNA interference – depletion of a specific protein
mRNA
ds short RNA (synthetic or expressed as shRNA)RISC
Normal cell RNAi treated cellDNA:
mRNA:
Protein:
mRNA detroyed
mRNA degraded!
Already existing proteinsdecay over time
12
Duplex formation
Systems for overexpression of a protein
+ Quick (4 – 6 hours)
High expression level
- Heterogeneous cells
Small amount of transfectants
Transient transfection(plasmid DNA is not replicated)
- 4 – 6 week to establish a cell line
Impossible if gene product causes a cell cycle block
+ Homogenous cell line
Unlimited amount of transfectants
Plasmiddrugresistance
Stable transfection(Chromosomal integration)
13
The development of microscopy
Zacharias Janssen(1580 -1638)
Today
~1900
The first microscope 14
The three principle tasks of microscopy
- Produce a magnified image (magnification)
- Separate the details in the image (resolution)
- Render the details visible (contrast)
Resolution: the smallest distance between two objects at which the two objects can be seen as separate units Maximal resolution = l/2
15
Bright field microscopy
Ocular
Objective
Lamp
Stage
Condenser
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Specialized bright field microscopyEnhances the contrast between intracellular structures
Differential interferencecontrast (DIC)
Phase contrast
Bright field
17
Classical stains
Stained cellUnstained cell
Creation of contrast in bright field microscopy
18
Preservation of biological structures by fixation
Glutaraldehyde
Extensive protein cross-linking
Formaldehyde Alcohols
Fixation may introduce structural artifacts
Process in which cellular structures are preserved and fixed in position by chemical agents
Protein denaturation
19
Shortcoming of bright field microscopy
...but where is the protein of interest?
Okay this was interesting.....
20
Raising antibodies against specific proteins
Protein X
Polyclonal antibodies
Protein X
Monoclonal antibody
Epitope Purify antibodiesfrom the blood of the animal
Take out antibody producing B cells
Fuse with myeloma cell to generate a hybridoma
+
1.
2.
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Molec models. 25.2-antibodies
Detection of specific proteins with antibodies
Protein X Protein X Protein X
Primary antibody Specific to epitope on protein X
The primary antibody (e.g. rabbit) is recognized by many secondary antibodies (e.g. goat anti-rabbit)
Signal amplification
Secondary antibody Specific to the primary antibody, conjugated with e.g. a fluorochrome
22
Principle behind a fluorochrome
A fluorochrome absorb light of a particular wavelength and re-emit light of a longer wavelength
-
--
-
--
ExcitationEmission
Fluorochrome # 1 Fluorochrome # 2
Fluorochrome
- -23
How it works in reality
-
--
Excitation filter
Emission filter
Beam splitter
Filter cube
-24
Electron microscopy (EM)
Maximal resolution = l/2 400 700 nm
Maximal resolution 200 nm
e- + 100 000 V e-
l= 0.004 nm
Resolution 0.002 nm (0.1 nm in reality)
Resolving smaller structures demands something with a much shorter wavelength
25
Transmission Electron Microscopy (TEM)
Electron gun
Very thin section of a cell stainedwith heavy metal
Detector
Vacuum!
e- e-
Supportinggrid
26
Scanning Electron Microscopy (SEM)
The specimen is coated with metals to deflect electrons
Visualizing surface features
e-e- e-e-
Cell with metal coating
Detector Electron gun
Sequential scanning
27
Different forms of microscopy
Electron microscopy
Fluorescence microscopyBright field microscopycell organelles Location of molecules
largemolecules Different techniques –
different ”windows”28
Protein X GFP
Protein X GFP
Transient or stable expressionDetection in either live or fixed cells
The fluorescent protein revolution
Aeqourea victoria
GFP
YFPDsRed
29
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Video 10.6-FRAP
Visualization of signaling in live cells (NFAT):Video 12.2-nuclear_import.mov
Video 02.3-brownian_motion.mov
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