Who am I and why am I here? Long-time collaborator of Geoff Brown’s

• Helped with some EM pictures• Variant cell lines from the human promyelocyte line HL60. Leuk Res. 1982 6:491-8.

• Helped with analysis of the relationship between proliferation and differentiation during haematopoiesis • 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;281:28-38.

• 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 245:193-201.

• 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.

What are inositols? Myo-inositol and others

• 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

H

myo

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

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.

Ins

‘Stabilising’ solutes

(all kingdoms)

PtdIns(eukaryotes,few bacteria)

PtdInsPs(eukaryotes)

Ins Sphingolipids(eukaryotes)

PtdIns anchorsfor proteins/cbtes

(eukaryotes)

Ins S’lipid anchorsfor proteins/cbtes

(eukaryotes)

Mycothiol(actino-

bacteria)

InsPns(eukaryotes)

InsPPs(eukaryotes)

The final (?) phosphoinositide complement: PtdIns, 3 x PtdInsP, 3 x

PtdInsP2, PtdInsP3

1988-93

1996

1996

Synthesis and degradation now defined

Since 1953, polyphosphoinositides (PPIn)

have garnered many functions!

CellSignalling

Membranetrafficking

Membranestructure

1930

1940

1950

1960

1970

1980

1990

2000

PtdIns(fast turnover-Hokins)

PtdIns4P,

PtdIns(4,5)P2

PtdIns3PPtdIns(3,4)P2

PtdIns(3,4,5)P3PtdIns5P,PtdIns(3,5)P2

CytoskeletalRegulation

Polyphosphoinositide locations and functions – McCrea & de Camilli, Physiology (Bethesda),

2009, 24, 8-16- PtdIns is in all membranes (made in ER)

• 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.

Major functions in Eukaryotes

fab1D phenotypes

Defective vacuole

acidification

Slow growth at

raised temperatur

e

MVB sorting defects

Enlarged

vacuoleMutants of

Fab1 (PtdIns3P 5-

kinase): faults caused by failure to

make PtdIns(3,5)P2.

Finally, focus on PtdIns(3,5)P2

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

PIPkIII- cells grown in suspension are more vacuolated

than normal

Ax3–wildtype RI-Random PIPkIII-1/2 insert PIPkIII disrupted

Hypo-osmotic stress transiently enhances cell

vacuolation

Fab1/PIKfyve is under complex regulation, still ill-understood.

Since the mid-2000s PtdIns(3,5)P2 has

gathered ever more functions!

Membrane fission/vesicle

recycling

Formin II

Actin-driven vesicle traffic to

plasma membrane

Yeast/stomatal vacuole

acidification

Raptor

‘Stress’ regulation

of mTORC1

Transporter exo-cytosis/ activa-

tion: GLUT4, EEAT4, CFTR,

etc

Two-pore ionchannels:TPC1/2 etc

Endo-lysosomal

Na+ exit

EIAV matrixprotein

Virus assembly& release

AMPA receptorendocytosis

[99,100]

Autophagy

Protein trafficto MVB

TRPML/mucolipinchannels

Endolysosomal

Ca2+ release

Ent3/Ent5

Atg18

Effector?

Protein targetting to

apical PM

PtdIns(3,5)P2

Gene reg. by Tup1/Cti6

Effector?

Effector?

Effector?

Effector?

Effector?

Melanosome assembly

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, 1-8Michell FEBS J 2013 in press. doi: 10.1111/febs.12452. Takasuga & Sasaki J Biochem. 2013 154, 211-218.

. . . . and the TLR-stimulated and apilimod-inhibited differentiation of TH17 lymphocytes.