Today’s lecture (Based on Chapter 1): 1. Basic organization of the cell

1. Today’s lecture (Based on Chapter 1): • 1. Basic organization of the cell • 2. The static cell vs the living cell • 3. Experimental approaches in cell physiology

2. http://www.studiodaily.com/main/technique/tprojects/6850.htmlhttp://www.studiodaily.com/main/technique/tprojects/6850.html

3. Experimental Methods • Observation of response, change, adaptation • → fluorescent labelling of cellular structures

4. Blue = nucleus (Hoescht staining of double-stranded DNA) Green = mitochondria (Mitotracker staining of inner membrane)

7. http://www.ucsf.edu/sedat/mito.html

8. Red = actin filaments Green = microtubules Blue = Nucleus

9. Red = actin filaments Green = microtubules

10. Experimental Methods • Observation of response, change, adaptation • → fluorescent reporting of cellular events

11. Visualization of a calcium transient in an isolated cardiomyocyte

13. Visualizing the rotation of the ATP synthase http://www.k2.phys.waseda.ac.jp/F1movies/F1Prop.htm

15. Experimental Methods • Observation of response, change, adaptation • → measurement of protein levels

16. Western Blot

17. Experimental Methods • Observation of response, change, adaptation • measurement of protein levels • → Western blot • → activity measurement

18. Experimental Methods • Observation of response, change, adaptation • measurement of protein levels • → Western blot • → activity measurement • → immunohistochemistry

19. Human Papillomavirus DNA demonstrated by In Situ Hybridisation (pink) in epithelial cells identified by indirect immunofluorescence using antibody against cytokeratin (green)

20. Experimental Methods Observation of response, change, adaptation → measurement of protein localization within the cell

21. Green fluorescent protein

24. Experimental Methods Observation of response, change, adaptation → measurement of protein localization within the cell: GFP fusion proteins

25. Experimental Methods Observation of response, change, adaptation → measurement of mRNA levels

26. Northern Blot

27. cDNA array

28. Experimental Methods Isolated cells

30. Isolated cardiomyocytes (rat)

32. Experimental methods • Experimental manipulation • (a) Physical/chemical

33. Experimental methods • Experimental manipulation • Physical/chemical • genetic

34. Experimental methods • Experimental manipulation • Physical/chemical • genetic • - Gene knockout

35. Saccharomyces cerevisiae (yeast) gene knockout collection

36. A mouse for every gene: the NIH mouse gene knockout project

37. Some shocking information about our genome: Is bigger better? # of genes 30000 20000 14000 5500 fly human yeast worm mouse

38. Don’t bet on the human genome

39. Some shocking information about our genome: Not at all # of genes 30000 30000 20000 14000 5500 fly human yeast worm mouse

40. Experimental manipulation • Physical/chemical • Genetic • Gene ‘knockout’ • Gene expression ‘knockdown’ (RNAi)

41. RNAi Dicer – cuts RNA into short (~20nt) sequences RISC = RNA-Induced Silencing Complex (includes the protein ‘Argonaute’)

42. Experimental manipulation • Physical/chemical • Genetic • Gene ‘knockout’ • Gene expression ‘knockdown’ • Overexpression / Transgenic expression

43. Be careful!

44. Other approaches to studying the molecular physiology of animal cells…

45. Nucleotide and protein databases SwissProt - amino acid sequences of all known proteins NCBI (National Center for Biotechnology Information) – nucleotide databases

46. Strategies for discovery (and cure) using molecular & genetic tools Identify effected individual ↓ Sequence genome ↓ Identify mutation(s) Clone mutant gene Identify mouse homologue (BLAST) ↓ ↓ ↓ Express in cells Make transgenic mouse Knockout mouse gene Observe phenotype (if any) ↓ Develop fetal screening program and correct?

47. http://multimedia.mcb.harvard.edu/media.html

48. For Thursday: Read Chapter 2 on protein regulation