how to study cells
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Cell biology 2014 (revised 20/1-14). Lecture 1: . How to study cells. “Recommended reading”. Chapter 8 501-505 571-572. Chapter 9 579-589 592-593 604-610. Alberts et al. 5th edition. The tree of life. Microbiology. Microbiology & Cell biology. (prokaryotes). Nucleus. - PowerPoint PPT PresentationTRANSCRIPT
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Lecture 1:
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Cell biology 2014 (revised 20/1-14)
Alberts et al5th edition
Chapter 8501-505571-572
Chapter 9579-589592-593604-610
“Recommended reading”
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Eukaryotes
Archaea
Eubacteria
(prokaryotes)
Cytosol
Nucleus
The tree of life
Microbiology Microbiology & Cell biology
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Biology
Molecular biology Cell biology Organism biology
Met Ser Arg Pro
Nanometers Micrometers Millimetres Meters3
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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)
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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
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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
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Multicellular eukaryotes – not just cellsThe 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
- Muscle
Protective covering of surfaces, both outside and inside the body
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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
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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)
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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
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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
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Duplex formation
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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)
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The development of microscopy
Zacharias Janssen(1580 -1638)
Today
~1900
The first microscope 14
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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
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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
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Classical stains
Stained cellUnstained cell
Creation of contrast in bright field microscopy
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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
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Shortcoming of bright field microscopy
...but where is the protein of interest?
Okay this was interesting.....
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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
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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
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Principle behind a fluorochrome
A fluorochrome absorb light of a particular wavelength and re-emit light of a longer wavelength
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ExcitationEmission
Fluorochrome # 1 Fluorochrome # 2
Fluorochrome
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How it works in reality
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Excitation filter
Emission filter
Beam splitter
Filter cube
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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
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Transmission Electron Microscopy (TEM)
Electron gun
Very thin section of a cell stainedwith heavy metal
Detector
Vacuum!
e- e-
Supportinggrid
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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
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Different forms of microscopy
Electron microscopy
Fluorescence microscopyBright field microscopycell organelles Location of molecules
largemolecules Different techniques –
different ”windows”28
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Protein X GFP
Protein X GFP
Transient or stable expressionDetection in either live or fixed cells
The fluorescent protein revolution
Aeqourea victoria
GFP
YFPDsRed
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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