Continuous phosphatidylinositol metabolism is required for cleavage of crane fly spermatocytes

Abstract

Successful cleavage of animal cells requires co-ordinated regulation of the actomyosin contractile ring and cleavage furrow ingression. Data from a variety of systems implicate phosphoinositol lipids and calcium release as potential regulators of this fundamental process. Here we examine the requirement for various steps of the phosphatidylinositol (PtdIns) cycle in dividing crane fly (Nephrotoma suturalis) spermatocytes. PtdIns cycle inhibitors were added to living cells after cleavage furrows formed and began to ingress. Inhibitors known to block PtdIns recycling (lithium), PtdIns phosphorylation (wortmannin, LY294002) or phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] hydrolysis [U73122 (U7)] all stopped or slowed furrowing. The effect of these drugs on cytokinesis was quite rapid (within 0-4 minutes), so continuous metabolism of PtdIns appears to be required for continued cleavage furrow ingression. U7 caused cleavage furrow regression concomitant with depletion of F-actin from the contractile ring, whereas the other inhibitors caused neither regression nor depletion of F-actin. That U7 depletes furrow-associated actin seems counterintuitive, as inhibition of phospholipase C would be expected to increase cellular levels of PtdIns(4,5)P2 and hence increase actin polymerization. Our confocal images suggest, however, that F-actin might accumulate at the poles of U7-treated cells, consistent with the idea that PtdIns(4,5)P2 hydrolysis may be required for actin filaments formed at the poles to participate in contractile ring assembly at the furrow.

TheDrosophilaphosphatidylinositol transfer protein encoded byvibratoris essential to maintain cleavage-furrow ingression in cytokinesis

“…The hydrolysis of PtdIns(4,5)P 2 to inositol (1,4,5)-triphosphate [Ins(1,4,5)P 3 ] by PLC is similarly required to maintain the furrow (Emoto et al, 2005;Field et al, 2005b;Wong et al, 2005). Together, these findings are consistent with inhibitor studies in crane fly spermatocytes that suggest the requirement of continuous PtdIns metabolism for cytokinesis (Saul et al, 2004). A precise assessment of comparative functions of different phosphorylated forms of PtdIns in cytokinesis will require a more detailed analysis of furrow behaviour following the downregulation of other steps in the pathway, by using comparable technical approaches.…”

“…the PtdIns-cycling pathway has been described for cytokinesis of crane fly spermatocytes in a study using chemical inhibitors (Saul et al, 2004). Moreover, genetic studies have implicated the two kinases that successively phosphorylate PtdIns: in Drosophila the phosphatidylinositol 4-kinase (PI-4 kinase) four wheel drive (fwd) (Brill et al, 2000), and in S. pombe the phosphatidylinositol-4-phosphate 5-kinase [PI(4)P-5 kinase] (Cullen et al, 2000;Zhang et al, 2000), to produce PtdIns(4,5)P 2 as being required for cytokinesis.…”

A Global, Myosin Light Chain Kinase-dependent Increase in Myosin II Contractility Accompanies the Metaphase–Anaphase Transition in Sea Urchin Eggs

“…Calcium has been shown to be required for cytokinesis in several embryonic cell types (Fluck et al, 1991;Miller et al, 1993;Chang and Meng, 1995;Muto et al, 1996;Webb et al, 1997;Saul et al, 2004;Wong et al, 2005), and myosin activation (Shuster and Burgess, 2002a;Stack et al, 2006) as well as membrane addition (Shuster and Burgess, 2002a) are candidate downstream effectors. Imaging of calcium dynamics in dividing L. pictus eggs revealed that there is a mobilization of calcium at the metaphase-anaphase transition that is necessary for cytokinesis (Groigno and Whitaker, 1998), raising the possibility that this calcium transient may partially serve as the "timer" for the activation of myosin II.…”

Polyploidization and Cancer

“…296 Hydrolysis of PtdIns(4,5)P2 by phosphlipasec(PLC) yields inositol trisphosphate (IP 3 ), which stimulates calcium release from internal stores. Inhibitors of PLC can interfere with cytokinesis, 297 which can in some cases be rescued by addition of calcium. 298 Alternatively, PtdIns(4,5)P2 may play a direct role in recruiting membrane proteins required for stability of the furrow.…”