transfection and protein localisation
TRANSCRIPT
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Transfection andProtein 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 interference
b) 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 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
C M V
P r o m
o t e r
Insert gene
in here
Polyadenylationsite
S V 4 0
P
r o m o t e r
N e o
m y c i n
r e s i s t a n c e
PolyadenylationsitepUC
Bacterial origin of replication
A m p
i c i l l i n
r e s i s
t a n c e
Forexpressionin cells
Togeneratestable cellline
Foramplificationof theplasmid inbacteria
G F P
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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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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 signal
Example: 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 inmitochondria and oxidized
Diffuses throughmembranes
Mitotracker
DNA
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nuclear envelope
endoplasmic reticulum
lysosome
earlyendosome
lateendosome
Golgi apparatus
cis
Golginetwork
trans
Golginetwork
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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Mitotracker Red and ER-blue/white
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Golginucleus
From the ER, secreted and membrane proteins move to the Golgi, aseries of membrane-bound compartments found near the nucleus
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BODIPY-TR ceramide
Golgi marker
Ceramide = lipid
When metabolized,concentrates in theGolgi
Red fluorophore
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Golgi (ceramide)
Lysosomes (LysoTracker)
DNA (Hoechst)
MDCK CellsMadin-Darby Canine Kidney
Polarized Epithelial Cells
Molecular Probes, Inc.
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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 coatedvesicles.
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Iron is carried inblood by the protein
TRANSFERRINand is taken up intocells by endocytosismediated by the
TRANSFERRINRECEPTOR
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 with unknownprotein 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???