Download Lecture 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
http://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)
http://www.ucsf.edu/sedat/mito.html
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
Western Blot
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
Green fluorescent protein
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
Northern Blot
cDNA array
Experimental Methods
Isolated cells
Isolated cardiomyocytes (rat)
Experimental methods
1.Experimental manipulation
(a) Physical/chemical
Experimental methods
1.Experimental manipulation
(a) Physical/chemical
(b) genetic
Experimental methods
1.Experimental manipulation
(a) Physical/chemical
(b) genetic
- Gene knockout
Saccharomyces cerevisiae (yeast) gene knockout collection
A mouse for every gene: the NIH mouse gene knockout
project
Some shocking information about our genome:
Is bigger better?
fly
20000
14000
5500
yeast
worm
30000
# of
genes
mouse
human
Don’t bet on the human genome
Some shocking information about our genome:
Not at all
worm
30000
fly
20000
14000
5500
yeast
30000
# of
genes
mouse
human
Experimental manipulation
1. Physical/chemical
2. 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
1. Physical/chemical
2. Genetic
- Gene ‘knockout’
- Gene expression ‘knockdown’
- Overexpression / Transgenic expression
Be careful!
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
↓
↓
Express in cells
Identify mouse homologue (BLAST)
↓
Make transgenic mouse
Knockout mouse gene
Observe phenotype (if any)
↓
Develop fetal screening program and correct?
http://multimedia.mcb.harvard.edu/media.html
For Thursday:
Read Chapter 2 on protein regulation