Transfection And Protein Localisation

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 in here

For expression in cells

GFP

pCMV/GFP

pUC  

Ne o m ycin res i s t a nce

 

n icilli Amp ance t resis

For amplification of the plasmid in bacteria

40 r SV ote om Pr

V r CM ote om r P

Polyadenylation site

To generate stable cell line

Polyadenylation site

Bacterial origin of replication

Three ways to make Green fluorescent protein “GFP” fusion constructs:

GFP

PROTEIN X

GFP PROTEIN

 

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 network

 

Golgi stack

trans Golgi 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

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 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???