MCB 130L Part 2 Lecture 3 Transfection and Protein localization

1. MCB 130L Part 2 Lecture 3 Transfection and Protein localization

2. 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

3. 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!!!!

4. Transfection = Introduction of DNA into mammalian cells Gene is transcribed and translated into protein = “expressed”

5. Direct introduction of the DNA Electroporation - electric field temporarily disrupts plasma membrane Biolistics (gene gun)- fire DNA coated particles into cell Microinjection

6. Infection: Use recombinant viruses to deliver DNA Retroviruses Adenoviruses Virally-mediated introduction of the DNA

7. 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

8. 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

9. DNA “expression” vector transfected: Insert gene in here For expression in cells Polyadenylation site GFP CMV Promoter SV40 Promoter To generate stable cell line pCMV/GFP Ampicillin resistance Neomycin resistance For amplification of the plasmid in bacteria Polyadenylation site pUC Bacterial origin of replication

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

11. 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 inliving cells = “vital stain”

12. Some Cellular Organelles

13. Nuclei Mitochondria Secretory Pathway: Endoplasmic Reticulum Golgi Complex Endocytotic Pathway: Endosomes • Compartments/organelles examined • Protein sequences sufficient for localization • Vital stains

14. Nucleus Transport through nuclear pore signal = basic amino acid stretches example: P-P-K-K-K-R-K-V

15. Import of proteins into nucleus through nuclear pore

16. Nuclear Stain: Hoechst 33258 binds DNA

17. 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

18. Protein being transported across mitochondrial membranes

19. Mitochondrial dye = MitoTracker Red Diffuses through membranes Non-fluorescent until oxidized Accumulates in mitochondria and oxidized Mitotracker DNA

20. lysosome plasma membrane late endosome nuclear envelope endoplasmic reticulum early endosome CYTOSOL cis Golgi network Golgi stack trans Golgi network Golgi apparatus Cellular components of the secretory and endocytic pathways

21. Endoplasmic Reticulum 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 at amino terminus Retention in E.R. lumen: Signal = K-D-E-L-COOH at carboxy terminus

22. Endoplasmic Reticulum marker ER-Tracker Blue-White Live bovine pulmonary artery endothelial cells

23. Mitotracker Red and ER-blue/white

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

25. Golgi marker BODIPY-TR ceramide Ceramide = lipid When metabolized, concentrates in the Golgi Red fluorophore

26. Cultured Epithelial Cells DNA (Hoechst) Golgi (ceramide) Steve Rogers, U. Illinois

27. MDCK Cells Madin-Darby Canine Kidney Polarized Epithelial Cells DNA (Hoechst) Golgi (ceramide) Lysosomes (LysoTracker) Molecular Probes, Inc.

28. Endocytosis can be divided into 3 categories: 1. Phagocytosis - “eating” 2. Pinocytosis - “sipping” 3. Receptor-mediated endocytosis: deliberate uptake of specific molecules

29. cis Golgi network Golgi stack trans Golgi network Cellular components of the endocytic pathway lysosome plasma membrane late endosome nuclear envelope endoplasmic reticulum early endosome CYTOSOL Golgi apparatus

30. Endosomes - pinch off from plasma membrane Clathrin -coated pits and vesicles

31. 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.

32. 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

33. Rhodamine transferrin Does the fluorescent green protein co-localize?

34. 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 dyein the same living cells! by fluorescence microscopy Where are the unknown proteins localized???