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 interference b) 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 Retroviruses Adenoviruses Virally-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 Carrier-mediated introduction of the DNA

Types of Transfection Transient: Expression assayed 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 gene in here Polyadenylation site SV40 Promoter Neomycin resistance Polyadenylation site pUC Bacterial origin of replication Ampicillin resistance For expression in cells To generate stable cell line For amplification of the plasmid in bacteria GFP

PROTEIN X PROTEIN Y GFP Z PROTEIN Three ways to make Green fluorescent protein “GFP” fusion constructs:

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 Secretory Pathway: Endoplasmic Reticulum Golgi Complex Endocytotic Pathway: Endosomes Mitochondria Nuclei

Transport through nuclear pore signal = 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 binds DNA

Transmembrane transport signal Example: H 2 N-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 early endosome late endosome Golgi apparatus cis Golgi network trans Golgi network Golgi stack CYTOSOL plasma membrane Cellular components of the secretory and endocytic pathways

Entry into E.R.: Transmembrane transport signal = hydrophobic amino acid stretches Example:H 2 N-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

Golgi nucleus 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 = lipid When 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 Cells Madin-Darby Canine Kidney Polarized 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 early endosome late endosome Golgi apparatus cis Golgi network trans Golgi network Golgi stack CYTOSOL plasma membrane Cellular components of the endocytic pathway

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 Fe 3+ 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???