MCB 130 L Part 2 Lecture 3 Transfection

MCB 130 L 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: Insert gene in here For expression in cells p. UC Ne o m ycin r e s i s tanc e p. CMV/GFP 40 r SV ote om n icilli Amp ance st resi For amplification of the plasmid in bacteria Polyadenylation site Pr V r CM ote om r P GFP To generate stable cell line Polyadenylation site Bacterial origin of replication

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

Protein being transported across mitochondrial membranes

Mitochondrial dye = Mito. Tracker 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: 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 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

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 (Lyso. Tracker) 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 network Golgi stack trans Golgi 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 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? ? ?