Transcript DAW Lecture 3 PPT

MCB 130L Part 2 Lecture 3
Transfection and
Protein localization
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
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!!!!
Transfection =
Introduction of DNA into mammalian cells
Gene is transcribed and translated into protein
= “expressed”
Direct introduction of the DNA
Electroporation - electric
field temporarily disrupts
plasma membrane
Biolistics (gene gun)- fire
DNA coated particles into
cell
Microinjection
Virally-mediated introduction of the DNA
Infection:
Use recombinant viruses to deliver DNA
Retroviruses
Adenoviruses
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
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
DNA “expression” vector transfected:
For
expression
in cells
Insert gene
in here
Polyadenylation
site
To
generate
stable cell
line
pCMV/GFP
For
amplification
of the
plasmid in
bacteria
pUC
Polyadenylation
site
Bacterial origin of replication
Three ways to make Green fluorescent
protein “GFP” fusion constructs:
PROTEIN X
GFP
PROTEIN
GFP
PROTEIN Y
GFP
Z
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”
Some Cellular Organelles
•Compartments/organelles examined
•Protein sequences sufficient for localization
•Vital stains
Nuclei
Mitochondria
Secretory Pathway:
Endoplasmic Reticulum
Golgi Complex
Endocytotic Pathway:
Endosomes
Nucleus
Transport through nuclear
pore
signal = basic amino acid
stretches
example: P-P-K-K-K-R-K-V
Import of proteins into nucleus through nuclear pore
Nuclear Stain:
Hoechst 33258
binds DNA
Mitochondria
Transmembrane transport signal
Example: H2N-M-L-S-L-R-Q-S-I-R-F-F-KP-A-A-T-R-T-L-C-S-S-R-Y-L-L
Protein being transported across mitochondrial membranes
Mitochondrial dye =
MitoTracker Red
Diffuses through
membranes
Non-fluorescent until
oxidized
Accumulates in
mitochondria and oxidized
Mitotracker
DNA
Cellular components of the secretory and endocytic pathways
lysosome
plasma
membrane
late
endosome
nuclear envelope
endoplasmic reticulum
early
endosome
CYTOSOL
cis
Golgi
trans
Golgi
stack
Golgi
network
network
Golgi apparatus
Endoplasmic Reticulum
Entry into E.R.:
Transmembrane transport signal
= hydrophobic amino acid stretches
at amino terminus
Example: H2N-M-M-S-F-V-S-L-L-V-G-I-LF-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
at carboxy terminus
Endoplasmic Reticulum marker
ER-Tracker Blue-White
Live bovine pulmonary artery
endothelial cells
Mitotracker Red and ERblue/white
From the ER, secreted and membrane proteins move to the Golgi, a
series of membrane-bound compartments found near the nucleus
nucleus
Golgi
Golgi marker
BODIPY-TR ceramide
Ceramide = lipid
When metabolized,
concentrates in the
Golgi
Red fluorophore
Cultured Epithelial Cells
DNA (Hoechst)
Golgi (ceramide)
Steve Rogers, U. Illinois
MDCK Cells
Madin-Darby Canine Kidney
Polarized Epithelial Cells
DNA (Hoechst)
Golgi (ceramide)
Lysosomes (LysoTracker)
Molecular Probes, Inc.
Endocytosis can be divided into 3 categories:
1. Phagocytosis - “eating”
2. Pinocytosis - “sipping”
3. Receptor-mediated endocytosis:
deliberate uptake of specific molecules
Cellular components of the endocytic pathway
lysosome
plasma
membrane
late
endosome
nuclear envelope
endoplasmic reticulum
early
endosome
CYTOSOL
cis
Golgi
trans
Golgi
stack
Golgi
network
network
Golgi apparatus
Endosomes - pinch off from plasma membrane
Clathrin -coated pits and vesicles
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.
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
Rhodamine transferrin
Does the fluorescent green protein co-localize?
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???