MCB 130L Part 2 Lecture 3

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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?
Approaches a) Make antibodies -
immunofluorescence
b) Express the protein in cells with a tag ?
Fuse to GFP
2) What is it doing in the cell?
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?
Approaches a) Make antibodies -
immunofluorescence
b) Express the protein in cells with a tag ?
Fuse to GFP
2) What is it doing in the cell?
Approaches a) Reduce protein levels - RNA
interference
b) Increase protein levels over-express c)
Express mutant versions
Transfection!!!!
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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 Microinjectio
n
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Virally-mediated introduction of the DNA
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Carrier-mediated introduction of the DNA
Positively charged carrier molecules are mixed
with the DNA and added to cell culture
media Calcium Phosphate DEAE
Dextran liposomes micelles
Carrier-DNA complexes bind to plasma membrane and
are taken up
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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
Insert gene in here
For expression in cells
Polyadenylation site
GFP
CMV Promoter
SV40 Promoter
To generate stable cell line
pCMV/GFP
Ampicillin resistance
Neomycin resistance
For amplification of the plasmid in bacteria
Polyadenylation site
pUC
Bacterial origin of replication
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Three ways to make Green fluorescent protein
GFP fusion constructs
PROTEIN X
GFP
PROTEIN Y
GFP
GFP
Z
PROTEIN
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EXPERIMENT
Transfect unknown GFP fusion protein Protein 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

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Nucleus
Transport through nuclear pore signal basic
amino acid stretches example P-P-K-K-K-R-K-V
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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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Mitochondria
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
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Protein being transported across mitochondrial
membranes
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Mitochondrial dye MitoTracker Red
Diffuses through membranes
Non-fluorescent until oxidized
Accumulates in mitochondria and oxidized
Mitotracker
DNA
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Cellular components of the secretory and
endocytic pathways
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Endoplasmic Reticulum
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
at amino terminus
Retention in E.R. lumen Signal K-D-E-L-COOH
at carboxy terminus
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Endoplasmic Reticulum marker
ER-Tracker Blue-White
Live bovine pulmonary artery endothelial cells
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Mitotracker Red and ER-blue/white
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From the ER, secreted and membrane proteins move
to the Golgi, a series of membrane-bound
compartments found near the nucleus
nucleus
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Golgi marker
BODIPY-TR ceramide
Ceramide lipid When metabolized, concentrates
in the Golgi
Red fluorophore
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Cultured Epithelial Cells
DNA (Hoechst)
Golgi (ceramide)
Steve Rogers, U. Illinois
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MDCK Cells Madin-Darby Canine Kidney Polarized
Epithelial Cells
DNA (Hoechst)
Golgi (ceramide)
Lysosomes (LysoTracker)
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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Cellular components of the endocytic pathway
lysosome
plasma membrane
late endosome
nuclear envelope
endoplasmic reticulum
early endosome
CYTOSOL
Golgi apparatus
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Endosomes - pinch off from plasma membrane
Clathrin -coated pits and vesicles
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RECEPTOR-MEDIATED ENDOCYTOSIS occurs through
special membrane 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 in blood by the
protein TRANSFERRIN and is taken up into cells by
endocytosis mediated by the TRANSFERRIN
RECEPTOR
Inside the endosome Fe3 is released. Transferrin
receptors then return to the cell surface,
where the 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???