Published on February 8, 2008
Slide2: What was the biological question addressed? What major claims do the authors make? What are the major results that the authors have obtained? Basic Method: Basic Method Cells Co-transfect with gene of interest (gene X) plus CD20 cDNA Use flow cytometry to restrict analysis to only transfected cells Use flow cytometry to determine cell size and cell cycle status Gene X cDNA CD20 cDNA CD20 Y Slide4: Optical Design PMT 1 PMT 2 PMT 5 PMT 4 Dichroic Filters Bandpass Filters Laser Flow cell PMT 3 Scatter Sensor Sample Slide5: Optics - Forward Scatter Channel When a laser light source is used, the amount of light scattered in the forward direction (along the same axis that the laser light is traveling) is detected in the forward scatter channel The intensity of forward scatter is proportional to the size and shape of cells (or other particles) Slide6: Forward Angle Light Scatter Purdue University Cytometry Laboratories Slide7: Optics - Side Scatter Channel When a laser light source is used, the amount of light scattered to the side (perpendicular to the axis that the laser light is traveling) is detected in the side or 90o scatter channel The intensity of side scatter is proportional to the optical homogeneity of cells (or other particles) Slide8: 90 Degree Light Scatter Purdue University Cytometry Laboratories DNA Probes: DNA Probes DNA in cells can be stained with a fluorescent dye DNA probes like Propidium Iodide are STOICHIOMETRIC –the number of molecules of probe bound is directly proportional to the amount of DNA in the cell Can measure DNA in individual cells and determine the frequency of cells with a particular DNA content Cell cycle status of the population The Cell Cycle: The Cell Cycle G1 M G2 S G0 Quiescent cells DNA Histogram: DNA Histogram G0-G1 S G2-M Fluorescence Intensity # of Events 2n 4n Effect of cell cycle blockade on cell size: Effect of cell cycle blockade on cell size Fig. 1 Fibroblasts Rapamycin: Rapamycin Isolated from Easter Island soil sample Streptomyces hygroscopicus In the local lingo Easter Island = Rapa Nui Antifungal TOR – target of rapamycin found in yeast Rapamycin forms a complex with FKBP12 which binds to and inhibits TOR Everybody has TOR. In mammals mTOR Potent immunosuppresant Rapamycin/TOR: Rapamycin/TOR Yeast under favorable growth conditions upregulate: Ribosome biogenesis Translation initiation Nutrient import Yeast starved for nutrients, depleted of TOR, or treated with Rapamycin: Downregulate general protein synthesis Upregulate macroautophagy Activate stress responsive transcription factors TOR: TOR Regulates translation via phosphorylation of S6K1 and 4E-BP S6 kinase phosphorylates the S6 ribosomal protein. Also has other substrates Enhances translation but mechanism is not clear eIF4E binding proteins: 4E-BP: eIF4E binding proteins: 4E-BP Insulin AA (Leucine) Cell Proliferation Apoptosis Effect of cell cycle blockade on cell size: Effect of cell cycle blockade on cell size Fig. 1 Fibroblasts Slide19: Osteosarcoma cell line Asynchronous Treated with Rap A, B. 72 hours p<.002 C Cell growth D Cell cycle E. S6K1 activity ↓ 4EBP phosphorylation ↓ F Size decreas most pronounced in G1 G Protein/cell ↓ Slide20: Diane C. Fingar et al. Genes Dev. 2002; 16: 1472-1487 Figure 2. Rapamycin and LY294002 treatment of cycling U2OS cells reduces cell size and inhibits proliferation and cell cycle progression Different cell lines Slide21: Fig. 3 RR-mTOR rapamycin resistant mTOR cant bind RAP/FKBP12 complex KD – kinase dead RR/KD – kinase dead and rap resistant U2OS cells Slide22: eIF4E overexpression partially rescues the Rap induced decrease in cell size eIF4E levels are normally limiting so if overexpressed, blocks 4EBP1 suppression AA-4EBP1 Phosphorylation site defective mutant binds constitutively to the eIF4E-Cap complex and inhibits Cap-dependent translation Fig. 5 Other data: Other data Overexpression of S6K1 and eIF4E increase cell size in a co-operative manner Overexpression of the AA-4EBP1 constitutively active mutant decreases cell size Authors’ conclusions: Authors’ conclusions Cell cycle progression and cell growth are separable and distinct processes in mammals as in other organisms mTOR is the mechanism by which rapamycin reduces cell size The responsible downstream targets are eIF4E and S6K1 Slide25: Diane C. Fingar et al. Genes Dev. 2002; 16: 1472-1487 Figure 7. Model depicting the role of mTOR signaling in control of cell size through its downstream targets S6K1 and 4EBP1/eIF4E Slide26: Did the experiments presented justify the claims that were made? What major unanswered questions were raised by this work? What experiments need to be done to answer such questions? Slide28: Holtz, et al. Cell 123(4):569-80 (2005)
http://www.microbio.uab.edu/ptrm/bedwell06/FingarPaper.ppt. PowerPoint Presentation. mTOR Raptor AMPK TGFb type I, II receptors S32,36 S536 Rapamycin ...