The unexpected role of Drosophila OCRL during cytokinesis

Abstract

Inositides are intrinsic components of cell membranes that regulate a wide variety of cellular functions. PtdIns(4,5)P_2, one of the most abundant phosphoinositides, is restricted at the plasma membrane where it regulates numerous functions including cell division. We have recently established that the Drosophila inositol 5-phosphatase, dOCRL, is essential for cytokinesis, the last step of cell division (Ben El Kadhi et al. 2011).⁸ We demonstrated that dOCRL is required for the dephosphorylation of PtdIns(4,5)P₂ at the surface of endosomes, resulting in the restriction of this phosphoinositide to the cell cortex during cytokinesis. dOCRL is the Drosophila ortholog of human OCRL1, a PtdIns(4,5)P₂ phosphatase mutated in the X-linked disorder oculocerebrorenal Lowe syndrome. Here, we discuss the relevance of our findings with reference to the role of human OCRL1 in non-pathological and pathological conditions.

Keywords: :

• phosphoinositides
• PtdIns(4,5)P2
• cytokinesis
• cell division
• OCRL1
• Lowe syndrome
• intracellular trafficking

This article refers to:

Phosphoinositides are intrinsic components of cell membranes that regulate a wide variety of cellular functions.^1,2 The phosphoinositide family is composed of seven members that are synthesized through the phosphorylation of the 3-, 4- and/or 5-position of the inositol ring. Phosphoinositides are produced at cellular membranes and influence a number of processes including cell survival, vesicular trafficking, cytoskeletal reorganization, cell migration and cell division. Phosphoinositides bind and regulate a plethora of proteins at the interface of the cytosol and cellular membranes. The spatial compartmentalization of phosphoinositides is regulated by the action of specific kinases, phosphatases and phospholipases. This integrated signaling network controls phosphoinositide segregation on different cellular membranes and contributes to create specialized sub-membrane domains with specific biological functions.³ For instance, during cell division, PtdIns(4,5)P₂ accumulates at the cleavage furrow where it plays essential roles during cytokinesis.^4-7

We have recently reported an unexpected role of the Drosophila PtdIns(4,5)P₂ phosphatase OCRL (dOCRL) during cytkinesis.⁸ Importantly, mutations in the OCRL1 gene, the human ortholog of dOCRL, are the cause of the Oculocerebrorenal syndrome of Lowe.⁹ This rare X-linked genetic disease is characterized by mental retardation, congenital cataract and renal dysfunction. The OCRL1 gene encodes for two splice isoforms (OCRL1a and OCRL1b) that comprise a central inositol 5-phosphatase domain, a poorly characterized ASH domain and a catalytically inactive Rho-GAP domain.¹⁰ In addition, OCRL1 isoforms were shown to bind with different affinity to clathrin. In vitro, the preferential substrate of OCRL1 is the phosphate at the 5- position of PtdIns(4,5)P₂.¹¹ Impairment of the inositol 5-phosphatase activity of OCRL1 is at the basis of the molecular dysfunctions causing the Lowe syndrome.¹² While the functions of OCRL1 have been subject to intense investigations, they still remain poorly understood. Difficulties in OCRL1 studies rely on the fact that cells also express INPP5B, an OCRL1 paralog that was shown to fulfill similar functions.¹³ Drosophila expresses only one OCRL ortholog, dOCRL, and we have recently demonstrated that its PtdIns(4,5)P₂ phosphatase activity was important to establish PtdIns(4,5)P₂ homeostasis, to regulate vesicular trafficking and to ensure the fidelity of cytokinesis.⁸

OCRL Proteins Regulate Homeostasis of PtdIns(4,5)P₂

Similarly to its human ortholog, dOCRL associates with the membrane of several class of endosomes.^8,14 dOCRL insures that PtdIns(4,5)P₂ pools are principally restricted at the plasma membrane by dephosphorylating this phosphoinositide on endomembranes. When dOCRL is knocked-down by RNAi, Drosophila cells in culture abnormally accumulate PtdIns(4,5)P₂ at the surface of giant endocytic vacuoles.⁸ Interestingly, it has been recently reported that OCRL1 also regulates PtdIns(4,5)P₂ levels on endosomes of human cells. Similarly to what we observed in Drosophila, HeLa cells RNAi-depleted for OCRL1, present abnormal, enlarged endosomes enriched in PtdIns(4,5)P₂.¹⁵ Therefore, regulation of PtdIns(4,5)P₂ homeostasis and control of endosomal morphology by OCRL proteins is a general mechanism conserved across evolution. In addition, the function of OCRL proteins in the establishment of PtdIns(4,5)P₂ homeostasis is likely to participate to the underlying causes of the Lowe syndrome since cells from patient suffering from this disease have been shown to present elevated levels of PtdIns(4,5)P₂.¹⁶

OCRL Proteins and Vesicular Trafficking

We established that dOCRL does not preferentially associate with one specific endosomal compartment, and is found at the surface of early, late and recycling endosomes. How dOCRL is targeted to these endosomes is not currently understood. However, two-hybrid experiments have revealed that its human ortholog, OCRL1, interacts with 16 members of the rab protein family, which regulate membrane trafficking.¹⁷ Furthermore, it has been shown that rab5 and rab6 directly stimulate the inositol 5-phosphatase activity of OCRL1.¹⁸ Therefore, it is tempting to speculate that rab proteins regulate OCRL proteins recruitment at the surface of endosomes to control homeostasis of PtdIns(4,5)P₂. Inactivation of OCRL proteins in Drosophila and in human, leads to a strong disorganization of the endocytic compartments with the apparition of enlarged endosomes.^8,15 In Drosophila, these large endocytic vacuoles appear to be the result of an unregulated fusion of early, late and recycling endosomes. The molecular explanation of this defect is still unclear but our observations suggest that the abnormal accumulation of PtdIns(4,5)P₂ on endocytic compartments, disrupt the phosphoinositide signature of each family of endosomes¹⁹ and trigger homotypic fusion of these undefined endosomes.

OCRL Proteins and Cytokinesis

We found that when dOCRL is depleted by dsRNA, up to 40% of Drosophila cells fails cytokinesis and become multinucleated.⁸ This cytokinesis failure is characterized by an abortive cleavage furrow that still forms but regresses rapidly and does not successively separate the two daughter cells. This phenotype is directly linked to the deregulation of PtdIns(4,5)P2 homeostasis: during cytokinesis, the abnormal accumulation of PtdIns(4,5)P2 on endomembrane mis-targets essential components of the cytokinetic ring such as rhoA, actin, myosin and anillin. These components are recruited on endomembranes, at the expense of the cleavage furrow, and cannot establish a stable, efficient, cortical cytokinetic ring. These observations brought into light the essential role of PtdIns(4,5)P2 as one of the major spatial cue that secures cleavage furrow positioning and stability during cytokinesis. The important role of dOCRL during cytokinesis appears to be conserved in human. RNAi depletion of OCRL1 in human cells delays abscission of the intercellular bridge connecting the two daughter cells after cytokinesis.²⁰ In human cells, rab35 recruit OCRL1 at the intercellular bridge where it locally dephosphorylates PtdIns(4,5)P2 and modifies lipid and actin composition. This remodeling of the intercellular bridge was proved to be necessary to the subsequent step of abscission. Accordingly, intercellular bridge of cells from Lowe syndrome patient show an abnormal accumulation of PtdIns(4,5)P2 and actin that delays cell abscission. While OCRL proteins are important for cytokinesis in Drosophila and in mammals, their inactivation does not trigger similar cell division defects. One possible explanation is that human cells also express INPP5B that could partially substitute for OCRL1 function during cell division. Interestingly, we have found that human INPP5B could partially rescue dOCRL function during cytokinesis in Drosophila cells (unpublished results). These two studies have identified a novel and unexpected role of OCRL proteins during cell division.^8,20 Drosophila which expresses only one ortholog of OCRL1 appears as a powerful and promising model to further dissect in vivo the different functions of proteins of the OCRL family.

Acknowledgments

K.B.E.K. is a recipient of IRIC, UdeM and Canadian Institutes for Health Research scholarships. G.E. is recipient of a Tier II Canada Research Chair in Vesicular Transport and Cell Signaling. S.C. is the recipient of a Fonds de la Recherche en Santé du Québec Junior 1. This work was supported by grants from the Canadian Institutes for Health Research (S.C.: MOP-89877, G.E.: MOP-114899) and from the Cancer Research Society (G.E.).