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

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. http://www.studiodaily.com/main/technique/tprojects/6850.html. Experimental Methods Observation of response, change, adaptation

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

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  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)

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

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

  7. Red = actin filaments Green = microtubules

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

  9. Visualization of a calcium transient in an isolated cardiomyocyte

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

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

  12. Western Blot

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

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

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

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

  17. Green fluorescent protein

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

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

  20. Northern Blot

  21. cDNA array

  22. Experimental Methods Isolated cells

  23. Isolated cardiomyocytes (rat)

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

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

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

  27. Saccharomyces cerevisiae (yeast) gene knockout collection

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

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

  30. Don’t bet on the human genome

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

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

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

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

  35. Be careful!

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

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

  38. 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?

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

  40. For Thursday: Read Chapter 2 on protein regulation

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