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:

Insert gene
For
in here
expression
in cells GFP
Polyadenylation
V r site
CM ote
r om
P

Pr
SV ote
om
40 r
To
pCMV/GFP generate
stable cell
Amp ance
resis

res
Ne
line

i
o
s
icilli
t

m
t a
ycin
nce
For
n

amplification
of the
plasmid in Polyadenylation
bacteria pUC site
   
Bacterial origin of replication
Three ways to make Green fluorescent
protein “GFP” fusion constructs:

PROTEIN X GFP

GFP PROTEIN Y

PROTEIN 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-K-
P-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
late membrane
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-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
at carboxy terminus
   
Endoplasmic Reticulum marker
ER-Tracker Blue-White

Live bovine pulmonary artery

endothelial cells

   
 Mitotracker Red and ER-
blue/white

   
From the ER, secreted and membrane proteins move to the Golgi, a
series of membrane-bound compartments found near the nucleus

Golgi
nucleus

   
 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
late membrane
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???