daw lecture 3
TRANSCRIPT
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MCB 130L Part 2 Lecture 3
Transfection andProtein localization
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Exploring protein function1) Where is it localized in the cell?
2) What is it doing in the cell?
Approaches:a) Make antibodies - immunofluorescence
b) Express the protein in cells with a tag Fuse to GFP
Approaches:a) Reduce protein levels - RNA interference
b) Increase protein levels over - express
c) Express mutant versions
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Exploring protein function1) Where is it localized in the cell?
2) What is it doing in the cell?
Approaches:a) Make antibodies - immunofluorescence
Approaches:a) Reduce protein levels - RNA interference
Transfection!!!!
b) Express the protein in cells with a tag Fuse to GFP
b) Increase protein levels over - express
c) Express mutant versions
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Transfection =
Introduction of DNA into mammalian cells
Gene is transcribed and translated into protein
= expressed
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Direct introduction of the DNA
Electroporation - electric
field temporarily disruptsplasma membrane
Biolistics (gene gun)- fireDNA coated particles intocell
Microinjection
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Infection:Use recombinant viruses to deliver DNA
RetrovirusesAdenoviruses
Virally-mediated introduction of the DNA
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Positively charged carrier moleculesare mixed with the DNA and added tocell culture media:
Calcium Phosphate
DEAE Dextranliposomesmicelles
Carrier-DNA complexes bind to plasmamembrane and are taken up
Carrier-mediated introduction of the DNA
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Types of Transfection
Transient: Expression assayed 24-48
hours post transfection
Stable: Integration of the transfected DNA
into the cell genome - selectable
marker like neomycin resistancerequired
stably transfected cell line
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DNA expression vector transfected:
pCMV/GFP
Insert gene
in herePolyadenylation
site
PolyadenylationsitepUC
Bacterial origin of replication
Forexpressionin cells
Togeneratestable cellline
Foramplificationof theplasmid inbacteria
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PROTEIN X
PROTEIN Y
GFP
GFP
GFP ZPROTEIN
Three ways to make Green fluorescentprotein GFP fusion constructs:
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EXPERIMENT:
Transfect unknown GFP fusion proteinProtein X, Y or Z
Visualize GFP protein fluorescence by fluorescencemicroscopy in living cells
Counter-stain with known marker to compare
localization patterns in living cells = vital stain
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Some Cellular Organelles
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Compartments/organelles examined Protein sequences sufficient for localization Vital stains
Secretory Pathway: Endoplasmic Reticulum
Golgi Complex
Endocytotic Pathway: Endosomes
Mitochondria
Nuclei
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Transport through nuclearporesignal = basic amino acid
stretchesexample: P-P- K-K-K-R-K -V
Nucleus
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Import of proteins into nucleus through nuclear pore
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Nuclear Stain:
Hoechst 33258binds DNA
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Transmembrane transport signalExample: H 2 N-M-L-S-L- R -Q-S-I- R -F-F- K-P-A-A-T- R -T-L-C-S-S-R-Y-L-L
Mitochondria
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Protein being transported across mitochondrial membranes
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Mitochondrial dye =MitoTracker Red
Non-fluorescent untiloxidized
Accumulates inmitochondria and oxidized
Diffuses throughmembranes
Mitotracker
DNA
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ER-Tracker Blue-White
Live bovine pulmonary arteryendothelial cells
Endoplasmic Reticulum marker
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Steve Rogers, U. Illinois
Golgi (ceramide)DNA (Hoechst)
Cultured Epithelial Cells
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Golgi (ceramide)
Lysosomes (LysoTracker)
DNA (Hoechst)
MDCK CellsMadin-Darby Canine KidneyPolarized Epithelial Cells
Molecular Probes, Inc.
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Endocytosis can be divided into 3 categories:
1. Phagocytosis - eating
2. Pinocytosis - sipping
3. Receptor-mediated endocytosis:deliberate uptake of specific molecules
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nuclear envelope
endoplasmic reticulum
lysosome
earlyendosome
lateendosome
Golgi apparatus
cis Golgi
network
trans Golgi
network
Golgistack
CYTOSOL
plasmamembrane
Cellular components of the endocytic pathway
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RECEPTOR-MEDIATED ENDOCYTOSIS occurs through specialmembrane sites coated with the protein CLATHRIN.Receptors interact with clathrin indirectly, through
ADAPTIN proteins.Coated membrane buds that contain clathrin, adaptins, and
receptors bound to their ligands pinch off to form coatedvesicles.
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Iron is carried inblood by the proteinTRANSFERRIN and is taken up intocells by endocytosismediated by theTRANSFERRIN
RECEPTOR
Inside the endosome
Fe3+
is released.Transferrin receptorsthen return to thecell surface, wherethe transferrindissociates
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Rhodamine transferrin
Does the fluorescent green protein co-localize?
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TODAY: Transfect Cells transiently withunknown protein X, Y or Z fused to GFP
In two days: Vital stain with another dye to compare Visualize both GFP and dye in the sameliving cells! by fluorescence microscopy
Where are the unknown proteinslocalized???