Lecture 1: 1 Cell biology 2014 (revised 20/1-14) Alberts et al 5th edition Chapter 8 501-505 571-572...

Lecture 1:

1

Cell biology 2014 (revised 20/1-14)

Alberts et al5th edition

Chapter 8

501-505571-572

Chapter 9

579-589592-593604-610

“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)

5

Conceptual 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)

9

I. How to study the function of a protein in cells

Depletion/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

16

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.

21

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

-

--

-

--

Excitation

Emission

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

YFP

DsRed

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