Inositol lipid signalling

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Inositol lipid signalling by Bob Michel

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Inositol lipid signalling

  1. 1. Who am I and why am I here? Long-time collaborator of Geoff Brown’s • Helped with some EM pictures • • Helped with analysis of the relationship between proliferation and differentiation during haematopoiesis • • • • • • Variant cell lines from the human promyelocyte line HL60. Leuk Res. 1982 6:491-8. Cell proliferation and CD11b expression are controlled independently during HL60 cell differentiation initiated by 1,25 alpha-dihydroxyvitamin D3 or all-trans-retinoic acid. Exp Cell Res. 2001 266:126-34. HL60 cells halted in G1 or S phase differentiate normally. Exp Cell Res. 2002 15;281:2838. Cell differentiation and proliferation - simultaneous but independent? Exp Cell Res. 2003 10;291:282-8. The sequential determination model of hematopoiesis. Trends Immunol. 2007 28:442-8. Versatility and nuances of the architecture of haematopoiesis - Implications for the nature of leukaemia. Leuk Res. 2012 36:14-22. Tried to see how inositol phosphates might be implicated in myeloid differentiation. We obtained some interesting but still unexplained results. • • • • Inositol lipids and phosphates in the regulation of the growth and differentiation of haemopoietic and other cells. Phil Trans R Soc B. 1990 327:193-207. Changes in the levels of inositol lipids and phosphates during the differentiation of HL60 promyelocytic cells towards neutrophils or monocytes. Proc Biol Sci. 1991 245:193201. Comparison of the levels of inositol metabolites in transformed haemopoietic cells and their normal counterparts. Biochem J. 1993 289:667-73. Intracellular concentrations of Ins, GroPIns and InsP5 increase during haemopoietic cell differentiation. Biochim Biophys Acta. 1994 1222:101-8.
  2. 2. What are inositols? Myo-inositol and others myo H • There are 9 isomeric inositols (hexahydroxycyclohexanes). They are small, sugar-like (C6H12O6), and very stable • Nature mainly uses myo-inositol (Ins) - but some others too (neo, scyllo, D-chiro, muco, epi) – s denote OH inversion cf. Ins
  3. 3. Direct synthesis supplies only myoinositol • Ins is made by a single short route from glucose 6-phosphate: • myo-inositol 3-phosphate synthase (MIPS, above) plus Ins monophosphatase (InsPase) • All MIPSs are related
  4. 4. Ins and Ins lipids: 1850-2000 • 1850-1942 – Ins discovered and structure determined. • 1930-50s - Ins in mycobacterial lipids, and then in plant and animal lipids (Anderson & Roberts, Klenk, Faure & Morelec-Coulon). • 1940s – Ins in brain lipids: some lipid has an Ins:P ratio of ~2 (Folch). • 1950s – PtdIns turnover increases when secretory tissues stimulated (Hokins) - but why? • 1960s – Three lipids: PtdIns, PtdIns4P and PtdIns(4,5)P2 (Ballou). • Mid-1980s – PLC/Ins(1,4,5)P3/DAG signalling pathway established (we thought we had ‘finished’). • Since 1988 – more lipids, and many more functions – notably in signalling (PI3K) and membrane trafficking.
  5. 5. ‘Stabilising’ solutes (all kingdoms) Mycothiol (actinobacteria) PtdIns (eukaryotes, few bacteria) PtdInsPs (eukaryotes) InsPPs (eukaryotes) InsPns (eukaryotes) Ins Ins Sphingolipids (eukaryotes) PtdIns anchors for proteins/cbtes (eukaryotes) Ins S’lipid anchors for proteins/cbtes (eukaryotes)
  6. 6. The final (?) phosphoinositide complement: PtdIns, 3 x PtdInsP, 3 x PtdInsP2, PtdInsP3 1996 1988-93 1996
  7. 7. Synthesis and degradation now defined
  8. 8. 1930 Since 1953, polyphosphoinositides 1940 1950 1960 1970 (PPIn) have garnered many functions! PtdIns (fast turnoverHokins) PtdIns4P, PtdIns(4,5)P2  Membrane structure Cell Signalling 1980 1990 2000 PtdIns3P PtdIns(3,4)P2 PtdIns(3,4,5)P3 PtdIns5P, PtdIns(3,5)P2 Cytoskeletal Regulation Membrane trafficking
  9. 9. Polyphosphoinositide locations and functions – McCrea & de Camilli, Physiology (Bethesda), 2009, 24, 8-16 - PtdIns is in all membranes (made in ER)
  10. 10. Major functions in Eukaryotes • • • • PtdIns - abundant membrane lipid PtdIns4P - membrane traffic regulator PtdIns5P – emerging . . . . PtdIns3P and PtdIns(3,5)P2 - membrane traffic regulators – and more (later) • PtdIns(4,5)P2 - target of phospholipase C action and Type I PI3K action; cytoskeletal and ion channel regulator; needed for exocytosis • PtdIns(3,4,5)P3 (from PtdIns(4,5)P2) signalling, late in eukaryote diversification.
  11. 11. Finally, focus on PtdIns(3,5)P2 Enlarged vacuole MVB sorting defects fab1D phenotypes Slow growth Defective at raised vacuole temperature acidification Mutants of Fab1 (PtdIns3P 5kinase): faults caused by failure to make PtdIns(3,5)P2.
  12. 12. Dictyostelium discoideum PIPkIII • Gene DDB0204693, same domain structure as other PIPkIIIs. • PIPkIII disrupted by insertion of blasticidin resistance construct, checked by sequencing around insertion points. Some behaviours remain normal • Chemotaxis • Sporulation • Spore germination
  13. 13. PIPkIII- cells grown in suspension are more vacuolated than normal Ax3–wildtype RI-Random insert PIPkIII-1/2 PIPkIII disrupted
  14. 14. Hypo-osmotic stress transiently enhances cell vacuolation
  15. 15. Fab1/PIKfyve is under complex regulation, still ill-understood.
  16. 16. Since the mid-2000s PtdIns(3,5)P2 has gathered ever more functions!
  17. 17. Protein traffic to MVB AMPA receptor endocytosis [99,100] Virus assembly & release Effector? Ent3/Ent5 Endolysosomal Ca2+ release EIAV matrix protein Protein targetting to apical PM Endolysosomal Na+ exit TRPML/ mucolipin channels Two-pore ion channels: TPC1/2 etc Effector? Transporter exocytosis/ activation: GLUT4, EEAT4, CFTR, etc PtdIns(3,5)P2 Effector? Effector? Yeast/stomatal vacuole acidification Effector? Raptor Effector? Melanosome assembly Gene reg. by Tup1/Cti6 Formin II Atg18 Membrane fission/ vesicle recycling Autophagy ‘Stress’ regulation of mTORC1 Actin-driven vesicle traffic to plasma membrane
  18. 18. . . . . and the TLR-stimulated and apilimodinhibited differentiation of TH17 lymphocytes. Some PtdIns(3,5)P2 reviews. Efe et al. Curr Opin Cell Biol. 2005 17, 402-8. Michell et al. Trends Biochem Sci. 2006 31, 52-63. Dove et al. Biochem J. 2009 419, 1-13. Botelho Traffic 2011 13, 1-8 Michell FEBS J 2013 in press. doi: 10.1111/febs.12452. Takasuga & Sasaki J Biochem. 2013 154, 211-218.

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