Phospholipase C-related catalytically inactive protein regulates cytokinesis by protecting phosphatidylinositol 4,5-bisphosphate from metabolism in the cleavage furrow

Satoshi Asano,Keiju Kamijo,Takashi Kanematsu,Kozue Hamao,Yosuke Yamawaki,Mitsuki Nishimoto,Yasuka Ikura,Masato Hirata

doi:10.1038/s41598-019-49156-3

Abstract

Cytokinesis is initiated by the formation and ingression of the cleavage furrow. Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] accumulation followed by RhoA translocation to the cleavage furrow are prerequisites for cytokinesis progression. Here, we investigated whether phospholipase C (PLC)-related catalytically inactive protein (PRIP), a metabolic modulator of PI(4,5)P2, regulates PI(4,5)P2-mediated cytokinesis. We found that PRIP localised to the cleavage furrow during cytokinesis. Moreover, HeLa cells with silenced PRIP displayed abnormal cytokinesis. Importantly, PI(4,5)P2 accumulation at the cleavage furrow, as well as the localisation of RhoA and phospho-myosin II regulatory light chain to the cleavage furrow, were reduced in PRIP-silenced cells. The overexpression of oculocerebrorenal syndrome of Lowe-1 (OCRL1), a phosphatidylinositol-5-phosphatase, in cells decreased PI(4,5)P2 levels during early cytokinesis and resulted in cytokinesis abnormalities. However, these abnormal cytokinesis phenotypes were ameliorated by the co-expression of PRIP but not by co-expression of a PI(4,5)P2-unbound PRIP mutant. Collectively, our results indicate that PRIP is a component at the cleavage furrow that maintains PI(4,5)P2 metabolism and regulates RhoA-dependent progression of cytokinesis. Thus, we propose that PRIP regulates phosphoinositide metabolism correctively and mediates normal cytokinesis progression.

Highlights

Cytokinesis is the final step of cell division, in which a parent cell divides into two daughter cells by the formation and ingression of a cleavage furrow at the plasma membrane after chromosome segregation
We first investigated the localisation of PLC-related catalytically inactive protein (PRIP) during cytokinesis using HeLa and HEK293 cells transiently transfected with PRIP1 or PRIP2
Both enhanced green fluorescent protein (EGFP)-PRIP1 and EGFP-PRIP2 signals in HeLa cells clearly localised to the cleavage furrow, where phosphorylated myosin II regulatory light chain (MRLC) signal was observed (Fig. 1a)

Summary

Introduction

Cytokinesis is the final step of cell division, in which a parent cell divides into two daughter cells by the formation and ingression of a cleavage furrow at the plasma membrane after chromosome segregation. PI(4,5)P2 levels in the plasma membrane are regulated by a variety of metabolic enzymes such as phospholipase C (PLC), phosphoinositide kinases, www.nature.com/scientificreports/. Properly maintaining PI(4,5)P2 levels at the cleavage furrow is crucial for normal cytokinesis progression[6]. Cleavage furrow progression requires the activation of the small GTPase RhoA, which requires epithelial cell transforming sequence 2 (ECT2), a Rho GDP/GTP exchange factor (RhoGEF)[7]. The direct binding of Rho-GEF and RhoA to PI(4,5)P2 in the cleavage furrow could be an important process in cytokinesis progression. Contractile force is generated by non-muscle myosin II, which is regulated by RhoA/ Rho-associated protein kinase (ROCK) signalling. Amino acid R40 in PLCδ PH is a critical amino acid residue for Ins(1,4,5)P3 binding, and PRIP1 PH(R134Q) mutant (R134 corresponds to R40 in PLCδ PH) fails to bind with Ins(1,4,5)P3 or PI(4,5)P223,26

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Conclusion