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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 • 2. The static cell vs the living cell • 3. Experimental approaches in cell physiology
http://www.studiodaily.com/main/technique/tprojects/6850.htmlhttp://www.studiodaily.com/main/technique/tprojects/6850.html
Experimental Methods • Observation of response, change, adaptation • → fluorescent labelling of cellular structures
Blue = nucleus (Hoescht staining of double-stranded DNA) Green = mitochondria (Mitotracker staining of inner membrane)
Red = actin filaments Green = microtubules Blue = Nucleus
Red = actin filaments Green = microtubules
Experimental Methods • Observation of response, change, adaptation • → fluorescent reporting of cellular events
Visualization of a calcium transient in an isolated cardiomyocyte
Visualizing the rotation of the ATP synthase http://www.k2.phys.waseda.ac.jp/F1movies/F1Prop.htm
Experimental Methods • Observation of response, change, adaptation • → measurement of protein levels
Experimental Methods • Observation of response, change, adaptation • measurement of protein levels • → Western blot • → activity measurement
Experimental Methods • Observation of response, change, adaptation • measurement of protein levels • → Western blot • → activity measurement • → immunohistochemistry
Human Papillomavirus DNA demonstrated by In Situ Hybridisation (pink) in epithelial cells identified by indirect immunofluorescence using antibody against cytokeratin (green)
Experimental Methods Observation of response, change, adaptation → measurement of protein localization within the cell
Experimental Methods Observation of response, change, adaptation → measurement of protein localization within the cell: GFP fusion proteins
Experimental Methods Observation of response, change, adaptation → measurement of mRNA levels
Experimental Methods Isolated cells
Experimental methods • Experimental manipulation • (a) Physical/chemical
Experimental methods • Experimental manipulation • Physical/chemical • genetic
Experimental methods • Experimental manipulation • Physical/chemical • genetic • - Gene knockout
Some shocking information about our genome: Is bigger better? # of genes 30000 20000 14000 5500 fly human yeast worm mouse
Some shocking information about our genome: Not at all # of genes 30000 30000 20000 14000 5500 fly human yeast worm mouse
Experimental manipulation • Physical/chemical • Genetic • Gene ‘knockout’ • Gene expression ‘knockdown’ (RNAi)
RNAi Dicer – cuts RNA into short (~20nt) sequences RISC = RNA-Induced Silencing Complex (includes the protein ‘Argonaute’)
Experimental manipulation • Physical/chemical • Genetic • Gene ‘knockout’ • Gene expression ‘knockdown’ • Overexpression / Transgenic expression
Other approaches to studying the molecular physiology of animal cells…
Nucleotide and protein databases SwissProt - amino acid sequences of all known proteins NCBI (National Center for Biotechnology Information) – nucleotide databases
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?
For Thursday: Read Chapter 2 on protein regulation