Inositol lipid signalling by Bob Michel

1. Who am I and why am I here?
Long-time collaborator of Geoff Brown’s
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Helped with some EM pictures
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Helped with analysis of the relationship between proliferation and differentiation during
haematopoiesis
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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.
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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. 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. 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. 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. ‘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. The final (?) phosphoinositide complement:
PtdIns, 3 x PtdInsP, 3 x PtdInsP2, PtdInsP3
1996
1988-93
1996

7. Synthesis and
degradation now
defined

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. Polyphosphoinositide locations and functions – McCrea &
de Camilli, Physiology (Bethesda), 2009, 24, 8-16
- PtdIns is in all membranes (made in ER)

10. Major functions in Eukaryotes
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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. 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. 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. PIPkIII- cells grown in suspension
are more vacuolated than normal
Ax3–wildtype
RI-Random
insert
PIPkIII-1/2
PIPkIII disrupted

14. Hypo-osmotic stress transiently
enhances cell vacuolation

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

16. Since the mid-2000s
PtdIns(3,5)P2 has gathered
ever more functions!

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. . . . . 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.