1. Lecture 1: 1 Cell biology 2014 (revised 20/1-14) Alberts et al 5th edition Chapter 8 501-505 571-572 Chapter 9 579-589 592-593 604-610 “Recommended reading”

2. Eukaryotes Archaea Eubacteria (prokaryotes) Cytosol Nucleus The tree of life Microbiology & Cell biology 2

3. Biology Molecular biology Cell biology Organism biology MetSerArgPro NanometersMicrometersMillimetresMeters 3

4. 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

5. 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

6. 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

7. Multicellular eukaryotes – not just cells The extra cellular matrix (ECM) works as a scaffold in metazoans supporting cells in various ways 7

8. 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

9. How to study individual animal cells Primary cell cultures Explants Complete tissue section Only cells Secondary culture Proliferation Immortalization Cell line, with indefinite proliferative potential Tumor patient (growth factors) (e.g. by oncogenes) 9

10. I. How to study the function of a protein in cells Depletion/mutation of endogenous protein Overexpression of protein (ectopic expression) Normal (Control) 10

11. 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

12. RNA interference – depletion of a specific protein mRNA ds short RNA (synthetic or expressed as shRNA) RISC Normal cellRNAi treated cell DNA: mRNA: Protein: mRNA detroyed mRNA degraded! Already existing proteins decay over time 12 Duplex formation

13. 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 Plasmid drug resistance Stable transfection (Chromosomal integration) 13

14. The development of microscopy Zacharias Janssen (1580 -1638) Today ~1900 The first microscope 14

15. 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  2 15

16. Bright field microscopy Ocular Objective Lamp Stage Condenser 16

17. Specialized bright field microscopy Enhances the contrast between intracellular structures Differential interference contrast (DIC) Phase contrast Bright field 17

18. Classical stains Stained cellUnstained cell Creation of contrast in bright field microscopy 18

19. 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

20. Shortcoming of bright field microscopy...but where is the protein of interest? Okay this was interesting..... 20

21. Raising antibodies against specific proteins Protein X Polyclonal antibodies Protein X Monoclonal antibody Epitope Purify antibodies from 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

22. Detection of specific proteins with antibodies 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

23. Principle behind a fluorochrome A fluorochrome absorb light of a particular wavelength and re-emit light of a longer wavelength - - - - - - Excitation Emission Fluorochrome # 1Fluorochrome # 2 Fluorochrome - - 23

24. How it works in reality - - - Excitation filter Emission filter Beam splitter Filter cube - 24

25. Electron microscopy (EM) Maximal resolution  2 400 700 nm Maximal resolution 200 nm e-e- + 100 000 V e-e- = 0.004 nm Resolution 0.002 nm (0.1 nm in reality) Resolving smaller structures demands something with a much shorter wavelength 25

26. Transmission Electron Microscopy (TEM) Electron gun Very thin section of a cell stained with heavy metal Detector Vacuum! e-e- e-e- Supporting grid 26

27. Scanning Electron Microscopy (SEM) The specimen is coated with metals to deflect electrons Visualizing surface features e-e- e-e- e-e- e-e- Cell with metal coating Detector Electron gun Sequential scanning 27

28. Different forms of microscopy Electron microscopy Fluorescence microscopyBright field microscopy cell organellesLocation of molecules large molecules Different techniques – different ”windows” 28

29. Protein XGFP Protein X GFP Transient or stable expression Detection in either live or fixed cells The fluorescent protein revolution Aeqourea victoria GFP YFP DsRed 29 - - - - Video 10.6-FRAP Visualization of signaling in live cells (NFAT): Video 12.2-nuclear_import.mov Video 02.3-brownian_motion.mov