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Caenhorhabditis elegans 1. ~ 1000 cells, small, easy to use for genetics 2. Entire lineage and nerve system mapped. 3. 3 day life cycle, easy to use for genetics. 4. hermaphrodite Lineage by the John Sulston The story of the lineage work Upper Panel: Nomarski photomicrograph of one gonad arm of an adult C. elegans hermaphrodite. The distal portion of the gonad arm is up; the proximal portion is down. Lower Panel: Nomarski photomicrograph of an adult C. elegans male gonad. The distal portion of the testis is above; the proximal region is below. Using vulval development as a model system Vulva: laying eggs mating Reproductive system in C. elegans Three steps of vulval development 1, precursor cells dorsal gonad P1 cell migration hox genes P12 2, vulval induction Cell signaling Cell fate Lineage 3, morphogenesis cell division, fusion, migration, etc. E E V V V E Judith Kimble: AC WT 3° 3° 2° 1° gonad 2° 3° 3° 3° X - AC 3° 3° 3° 3° Indicating: A: AC is required for vulval induction B: AC may send a signal to induce vulval cells C: Both. Paul Sternberg: AC WT 3° 3° VPC ablation 1°/2° Indicating: 1°/2° 2° 1° 2° X X X X X X 3° 1°/2° anchor cell inductive signal E E V V Wild type E E E signal V E 100% induction E E E Vulvaless 0% V V V Multivulva V 200% V V pathway function signal pathway function - use genetics to get the major players in the pathway Earlier direct screens for vulval induction mutations Mutagen Vulvaless wild type Multivulva lin-1 lin-3 let-23 EGF EGFR Horvitz and Sulston 1980, Genetics Ferguson and Horvitz 1985, Genetics Ferguson et al. 1987. Nature Aroian et al. 1991. Nature Hill and Sternberg 1992. Nature Vulval induction Identification of Ras Mammal: identified through oncogenic mutations in 1980 Ras homolog in yeast was identified in 1985. Ras function in development was first identified in 1990 (C. elegans) GDP Exchange reaction GTP RAS GDP RAS GTP Inactive target protein Active state Pi Hydrolysis reaction Supressors of lin-15 mutation EMS lin-15(-); ; X(-) Vulvaless lin-15(-), Muv X = EGFR, RAS and others. Han and Sternberg 1990, Cell; Beitel et al. 1990, Nature; Aroian et al. 1990 Nature Vote: A. B. lin-15 X X Vulval induction lin-15 Vulval induction Relationship between RAS and EGFR lin-15 EGFR RAS Vulval induction EGFR(lf) Vulvaless RAS (gf) Multivulva EGFR(lf) + RAS (gf) Multivulva Vote: A. RAS B. EGFR EGFR Vulval induction RAS Vulval induction Pathway in early 1992 EGFR RAS Strategy 1: moving up from Ras signal Exchange reaction GDP GTP RAS GDP RAS GTP Inactive Active state Pi signal Hydrolysis reaction target protein QuickTime™ and a GIF decompressor are needed to see this picture. QuickTime™ and a GIF decompressor are needed to see this picture. First strike: discovery of GTPase activating protein (GAP) 1. Questions addressed RAS:GDP GTPase of Ras In vitro In vivo RAS:GTP Pi weak strong t 1/2 = 30 min t 1/2 < 1 min A: Something in cells can stimulate the GTPase activity B: Something in intro inhibits the GTPase activity The student and PI were mammalian biochemists. What system will they likely chose to identify the “something”? A: Mammalian cells. B. Drosophila. C. Xenopus oocyte D. Yeast. Approach: Using Xenopus oocytes - ease to manipulate due to large size - good assay for biological activity, oocyte maturation. Does GTP or GDP bind to injected Ras? In vivo WT Ras mostly bind to GDP Oncogenic Ras mostly to GTP In vitro GTPase activity: speed in vivo T 1/2 = 2-3 min for wild type. >1000 min for Asp12 WT vs. Oncogenes WT Gly 12 RAS:GDP Pi Oncogenes Val 12 Asp 12 RAS:GTP GTPase activity RAS:GDP Pi RAS:GTP X GTPase activity What causes the difference between in vitro and in vivo? Add cytoplasmic factor to in vitro > 200 fold difference Trahey and McCormick Science Oct 1987 QuickTime™ and a GIF decompressor are needed to see this picture. How is Ras regulated by the signal? EGFR Exchange reaction GDP GTP RAS GDP RAS GTP Inactive Active state Pi EGFR GAP target protein Cell 1990. It is all GAP Kaplan DR, Morrison DK, Wong G, McCormick F, Williams LT. PDGF beta-receptor stimulates tyrosine phosphorylation of GAP and association of GAP with a signaling complex. Cell. 1990 61:125-33. Receptor GAP Ras Target Doug Lowy: not so fast, GNEF is more important EGFR Exchange reaction GDP GTP RAS GDP RAS GTP Inactive Active state Pi EGFR GAP target protein Exchange GDP GTP RAS GDP RAS GTP Inactive GAP Model 1. EGFR GNEF Model 2. EGFR GAP RAS RAS Active state Pi Ras (His116) mutant cause GDP to GTP exchange 10 x faster but its sensitivity to GAP is the same. If model 2 is right, GAP activity determines the GTPRas/GDPRas ratio add EGF, activity should dramatically increase. (A. Yes, B. No) If model 1 is right, His 116 already already cause the exchange 10 X fast Adding EGF would have a small effect. (A. Yes, B. No) Results: model 1 should be right. Zhang et al. 1991. Science The End