What was the biological question addressed? What major claims do the authors make?

2. What was the biological question addressed? • What major claims do the authors make? • What are the major results that the authors have obtained?

3. X X 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

4. Optical Design PMT 5 PMT 4 Sample PMT 3 Dichroic Filters Flow cell PMT 2 Scatter PMT 1 Laser Sensor Bandpass Filters

5. 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)

6. Laser FALS Sensor Forward Angle Light Scatter Purdue University Cytometry Laboratories

7. 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)

8. Laser FALS Sensor 90LS Sensor 90 Degree Light Scatter Purdue University Cytometry Laboratories

9. 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

10. The Cell Cycle M G2 G1 Quiescent cells G0 S

11. DNA Histogram 2n 4n G0-G1 G2-M S # of Events Fluorescence Intensity

12. Effect of cell cycle blockade on cell size Fibroblasts Fig. 1

14. 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

15. 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

16. 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

17. eIF4E binding proteins: 4E-BP Apoptosis Insulin AA (Leucine) Cell Proliferation

18. Effect of cell cycle blockade on cell size Fibroblasts Fig. 1

19. 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 ↓

20. Figure 2. Rapamycin and LY294002 treatment of cycling U2OS cells reduces cell size and inhibits proliferation and cell cycle progression Diane C. Fingar et al. Genes Dev. 2002; 16: 1472-1487 Different cell lines

21. U2OS cells RR-mTOR rapamycin resistant mTOR cant bind RAP/FKBP12 complex KD – kinase dead RR/KD – kinase dead and rap resistant Fig. 3

22. 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

23. 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

24. 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

25. Figure 7. Model depicting the role of mTOR signaling in control of cell size through its downstream targets S6K1 and 4EBP1/eIF4E Diane C. Fingar et al. Genes Dev. 2002; 16: 1472-1487

26. 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?

28. Holtz, et al. Cell 123(4):569-80 (2005)