mcb 130l part 2 lecture 3 transfection and protein localization
TRANSCRIPT
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MCB 130L Part 2 Lecture 3
Transfection and Protein localization
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Exploring protein function
1) 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
interferenceb) Increase protein levels “over-express”
c) “Express” mutant versions
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Exploring protein function
1) 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 disrupts plasma membrane
Biolistics (gene gun)- fire DNA coated particles into cell
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 molecules are mixed with the DNA and added to cell culture media:
Calcium PhosphateDEAE Dextranliposomesmicelles
Carrier-DNA complexes bind to plasma membrane 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 resistance required
“stably transfected” cell line
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DNA “expression” vector transfected:
pCMV/GFP
CMV
Promoter
Insert genein here
Polyadenylationsite
SV40
PromoterNeomycin
resistance
PolyadenylationsitepUC
Bacterial origin of replicationAmpicillin
resistance
For expression in cells
To generate stable cell line
For amplification of the plasmid in bacteria
GFP
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PROTEIN X
PROTEIN Y
GFP
GFP
GFP ZPROTEIN
Three ways to make Green fluorescent protein “GFP” fusion constructs:
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EXPERIMENT:
Transfect unknown GFP fusion proteinProtein X, Y or Z
Visualize GFP protein fluorescence by fluorescence microscopy 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 ReticulumGolgi Complex
Endocytotic Pathway:Endosomes
Mitochondria
Nuclei
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Transport through nuclear poresignal = basic amino acid stretches example: 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 33258 binds DNA
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Transmembrane transport signalExample: H2N-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 until oxidized
Accumulates in mitochondria and oxidized
Diffuses through membranes
Mitotracker
DNA
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nuclear envelope
endoplasmic reticulum
lysosome
earlyendosome
lateendosome
Golgi apparatus
cisGolginetwork
transGolginetwork
Golgistack
CYTOSOL
plasmamembrane
Cellular components of the secretory and endocytic pathways
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Entry into E.R.:Transmembrane transport signal= hydrophobic amino acid stretches
Example: H2N-M-M-S-F-V-S-L-L-V-G-I-L-F-W-A-T-E-A-E-Q-L-T-K-C-E-V-F-Q
Retention in E.R. lumen:
Signal = K-D-E-L-COOH
Endoplasmic Reticulum
at amino terminus
at carboxy terminus
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ER-Tracker Blue-White
Live bovine pulmonary artery endothelial cells
Endoplasmic Reticulum marker
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Mitotracker Red and ER-blue/white
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Golginucleus
From the ER, secreted and membrane proteins move to the Golgi, a series of membrane-bound compartments found near the nucleus
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BODIPY-TR ceramide
Golgi marker
Ceramide = lipidWhen metabolized, concentrates in the Golgi
Red fluorophore
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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
cisGolginetwork
transGolginetwork
Golgistack
CYTOSOL
plasmamembrane
Cellular components of the endocytic pathway
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Endosomes - pinch off from plasma membrane
Clathrin -coated pits and vesicles
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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 coated
vesicles.
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Iron is carried inblood by the proteinTRANSFERRINand is taken up intocells by endocytosismediated by the TRANSFERRIN
RECEPTOR
Inside the endosomeFe3+ is released.Transferrin receptors then return to thecell surface, wherethe transferrin dissociates
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Rhodamine transferrin
Does the fluorescent green protein co-localize?
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TODAY:•Transfect Cells transiently with unknown 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 same living cells! by fluorescence microscopy
Where are the unknown proteins localized???