1. Lab 5A and 5B Overview Investigating protein sorting signals using cloning, transfection, GFP-fusion proteins, and vital stains for cellular compartments 1. Protein sorting and membrane trafficking - or - How cells deliver things to the right place 2. Fluorescent proteins are critical tools in Cell biology Last Week 3. Transfection and transgene expression - or - How we get DNA into cells to express “designer” genes 4. Fluorescent markers for different compartments of the secretory and endocytic pathways This Week

2. Cloning vector for expressing GFP fusion proteins in mammalian cells (constructed in bacteria) GFPProtein X Protein YGFP in ZProteGFP Fusion proteins are usually introduced into cells as DNA constructs pCMV: Strong, constitutive promoter Neomycin: Selectable marker for mammalian cells BGH pA: Polyadenylation sequence Ampicillin: Selectable marker for bacterial cells pUC: Origin of replication for bacterial cells

3. The negatively-charged phosphate backbone of DNA must be neutralized by positively charged counterions to allow transport across the plasma membrane. TRANSFECTION - from trans, meaning “across” DNA is a large, charged molecule that normally doesn’t cross cell membranes… so we have to use tricks to get it into cells

4. Principle of transfection with Effectene ® reagent + Enhancer Outline of transfection protocol (Qiagen)

5. There are many points along the way where transfection efficiency can be compromised Proton sponge hypothesis: sequestration of cations by DNA leads to the osmotic swelling and rupture of endosomes, releasing DNA vector into the cytoplasm

6. Only a fraction of treated cells will be successfully transfected, and thus the expression of the transgene will be quite VARIABLE - here GFP is used as a marker of transfection

7. Cloning vector for expressing GFP fusion proteins in mammalian cells (constructed in bacteria) GFPProtein X Protein YGFP in ZProteGFP Fusion proteins are usually introduced into cells as DNA constructs pCMV: Strong, constitutive promoter Neomycin Resistance Gene: Selectable marker For mammalian cells BGH pA: Polyadenylation sequence Ampicillin: Selectable marker for bacterial cells pUC: Origin of replication for bacterial cells

8. Alternative strategies to ectopically express genes/siRNAs Retroviral Vectors -High Efficiency -Stable DNA integration -Replication Incompetent -Level 2 Bio-Safety TransfectRetrovirus Electroporation -High Efficiency -Many Cells/even intact tissues -Cell Fusion -Loss of intracellular components

9. Gene Gun -Applicable to many tissues -Penetrates Mitochondria/Chloroplasts -Shallow penetration of particles -Cell damage Microinjection -No selection process -DNA delivery accurately controlled -Minimal perturbation of cells -Technically difficult/few cells Alternative strategies to ectopically express genes/siRNAs

10. 4 transfected constructs Z Y X U 5 vital dye counterstains Golgi Endosome ER Mitochondria Nucleus

11. Ceramide is a LIPID that gets trapped after modification in the Golgi apparatus LabelExEm BODIPY- TR GreenRed Fluorophore BODIPY ® -TR Ceramide (Molecular Probes)

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

13. Iron is carried in blood by the protein TRANSFERRIN and is taken up into cells by endocytosis mediated by the TRANSFERRIN RECEPTOR.

14. EXEM Red Green Rhodamine-labeled TRANSFERRIN protein can be used to track receptor-mediated endocytosis

15. MitoTracker Red CM-H 2 XRos “… the reduced versions of these probes do not fluoresce until they enter an actively respiring cell, where they are oxidized to the fluorescent mitochondrion-selective probe and then sequestered in the mitochondria.” MOLECULAR PROBES handbook EXEM Red Green

16. Image from Nikon Cultured Lung Epithelial Cells Mitochondria (MitoTracker) DNA (DAPI) Actin (Phalloidin)

17. “ ER-Tracker Blue-White DPX is a highly selective and photostable stain for the ER in live cells… Staining at low concentrations does not appear to be toxic to cells.” (MOLECULAR PROBES Handbook) ER-Tracker Blue-White DPX EXEM Blue UV

18. Image from Invitrogen ER (ER-Tracker) Cultured Endothelial Cells

19. 4 transfected constructs Z Y X U 5 vital dye counterstains Golgi Endosome ER Mitochondria Nucleus 20 Data Sets

20. Potential Challenges Encountered in this Week’s Lab Photobleaching Autofluorescence - increases as cells die Bleed-through between different filter sets Nonspecific labeling of organelles Data Management

21. wavelength exem intensity excitation and emission filters Fluorescence Microscopy Stokes’ shift Fluorophore (or “Fluorochrome”) Excitation maximum Emission maximum Fluorescein490520 Rhodamine550580 DAPI or Hoechst345455

22. Fluorescence wavelength filters must be designed to match the excitation/emission spectra of the fluorophores you plan to use.

23. Bodipy-TR ceramide Red Channel

24. Autofluorescence of dying cells GFP GFP Channel

25. Simultaneous localization of cellular components To be useful, a “counterstain” should fluoresce at a wavelength different from GFP - e.g. RED or BLUE Mitochondria (MitoTracker) Lysosomes (Lyso-Tracker) N “Colocalization” can help to establish that two molecules are in the same place at the same time. If the location of one is known, it can reveal the location of a less well- characterized component. Mitochondria (MitoTracker) Colocalization GFP Fusion Protein -Beech et al. Science (2000) -Invitrogen