MCB 130L Part 2 Lecture 3

Transfection and Protein localization

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

interferenceb) Increase protein levels “over-express”

c) “Express” mutant versions

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

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

Infection:Use recombinant viruses to deliver DNA

RetrovirusesAdenoviruses

Virally-mediated introduction of the DNA

Positively charged carrier molecules are mixed with the DNA and added to cell culture media:

Calcium PhosphateDEAE Dextranliposomesmicelles

Carrier-DNA complexes bind to plasma membrane and are taken up

Carrier-mediated introduction of the DNA

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:

pCMV/GFP

CMV

Promoter

Insert genein here

Polyadenylationsite

SV40

PromoterNeomycin

resistance

PolyadenylationsitepUC

Bacterial origin of replicationAmpicillin

resistance

For expression in cells

To generate stable cell line

For amplification of the plasmid in bacteria

GFP

PROTEIN X

PROTEIN Y

GFP

GFP

GFP ZPROTEIN

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

EXPERIMENT:

Transfect unknown GFP fusion proteinProtein 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

Secretory Pathway:Endoplasmic ReticulumGolgi Complex

Endocytotic Pathway:Endosomes

Mitochondria

Nuclei

Transport through nuclear poresignal = basic amino acid stretches example: P-P-K-K-K-R-K-V

Nucleus

Import of proteins into nucleus through nuclear pore

Nuclear Stain:

Hoechst 33258 binds DNA

Transmembrane transport signalExample: 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

Protein being transported across mitochondrial membranes

Mitochondrial dye = MitoTracker Red

Non-fluorescent until oxidized

Accumulates in mitochondria and oxidized

Diffuses through membranes

Mitotracker

DNA

nuclear envelope

endoplasmic reticulum

lysosome

earlyendosome

lateendosome

Golgi apparatus

cisGolginetwork

transGolginetwork

Golgistack

CYTOSOL

plasmamembrane

Cellular components of the secretory and endocytic pathways

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

ER-Tracker Blue-White

Live bovine pulmonary artery endothelial cells

Endoplasmic Reticulum marker

Mitotracker Red and ER-blue/white

Golginucleus

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

BODIPY-TR ceramide

Golgi marker

Ceramide = lipidWhen metabolized, concentrates in the Golgi

Red fluorophore

Steve Rogers, U. Illinois

Golgi (ceramide)

DNA (Hoechst)

Cultured Epithelial Cells

Golgi (ceramide)

Lysosomes (LysoTracker)

DNA (Hoechst)

MDCK CellsMadin-Darby Canine KidneyPolarized Epithelial Cells

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

nuclear envelope

endoplasmic reticulum

lysosome

earlyendosome

lateendosome

Golgi apparatus

cisGolginetwork

transGolginetwork

Golgistack

CYTOSOL

plasmamembrane

Cellular components of the endocytic pathway

Endosomes - pinch off from plasma membrane

Clathrin -coated pits and vesicles

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 coated

vesicles.

Iron is carried inblood by the proteinTRANSFERRINand is taken up intocells by endocytosismediated by the TRANSFERRIN

RECEPTOR

Inside the endosomeFe3+ is released.Transferrin receptors then return to thecell surface, wherethe 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???