dOCRL maintains immune cell quiescence by regulating endosomal traffic

Abstract

Lowe Syndrome is a developmental disorder characterized by eye, kidney, and neurological pathologies, and is caused by mutations in the phosphatidylinositol-5-phosphatase OCRL. OCRL plays diverse roles in endocytic and endolysosomal trafficking, cytokinesis, and ciliogenesis, but it is unclear which of these cellular functions underlie specific patient symptoms. Here, we show that mutation of Drosophila OCRL causes cell-autonomous activation of hemocytes, which are macrophage-like cells of the innate immune system. Among many cell biological defects that we identified in docrl mutant hemocytes, we pinpointed the cause of innate immune cell activation to reduced Rab11-dependent recycling traffic and concomitantly increased Rab7-dependent late endosome traffic. Loss of docrl amplifies multiple immune-relevant signals, including Toll, Jun kinase, and STAT, and leads to Rab11-sensitive mis-sorting and excessive secretion of the Toll ligand Spåtzle. Thus, docrl regulation of endosomal traffic maintains hemocytes in a poised, but quiescent state, suggesting mechanisms by which endosomal misregulation of signaling may contribute to symptoms of Lowe syndrome.

IPIP27 Coordinates PtdIns(4,5)P2 Homeostasis for Successful Cytokinesis

“…PtdIns(4,5)P 2 is a strong promoter of actin assembly, and we detected strong actin enrichment on the vacuolar membranes (Figure 2B), where it colocalized with GFP-Tubby (Figure 2C). Although depletion of dIPIP or dOCRL caused enlargement of endosomal compartments, consistent with a role for these proteins in the endocytic pathway (Figures S2A–S2C) [31, 32], the larger vacuoles are largely devoid of endosome markers (Figures S2D and S2E). We believe it most likely that the vacuoles derive from endosomes, but, due to their altered phosphoinositide composition, with ectopically accumulated PtdIns(4,5)P 2 , they have lost their endosomal identity [39].…”

“…The C-terminal region of dIPIP also contains a conserved PxxP motif ( 151 PxPPPRR 157 ) that in mammalian IPIP27A binds the SH3 domain of pacsin 2 [35], and a putative clathrin-binding site at its C terminus that is not present in mammalian IPIP27. Localization experiments in Drosophila S2 cells indicated the presence of dIPIP on cytoplasmic puncta that likely correspond to endocytic compartments, where it colocalizes with dOCRL (Figure 1C; see also Figure 7C) [32]. IPIP27 interaction and co-localization with OCRL is therefore conserved in Drosophila melanogaster .

Figure 1dIPIP Interacts with dOCRL and Is Required for Cytokinesis(A) Schematic of human IPIP27A and IPIP27B and Drosophila dIPIP.(B) Pull-down using GST- dIPIP wild-type (WT) or F&H mutant (F267A) and Drosophila S2 cell lysate.

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“…In cytokinesis of mammalian cells, OCRL1 is recruited to the intercellular bridge by Rab35, where it hydrolyzes PtdIns(4,5)P 2 to promote actin dissolution and abscission [ 14 ]. In contrast to mammals, which also express the OCRL1 paralog INPP5B that may partially compensate for loss of OCRL1 [ 30 ], Drosophila have only a single enzyme, Drosophila ortholog of OCRL (dOCRL) [ 31 , 32 ]. Depletion of dOCRL from cultured Drosophila cells results in a more dramatic cytokinesis phenotype, with failure at the ingression stage, resulting in binucleation [ 31 ].…”

PTEN reduces endosomal PtdIns(4,5)P2 in a phosphatase-independent manner via a PLC pathway

“…We and others have previously reported that depletion of OCRL1 or depletion of dOCRL, its Drosophila melanogaster orthologue, causes several characteristic phenotypes: abnormal accumulation of PtdIns(4,5)P 2 on endosomes, disorganization of the endocytic compartments, and cytokinetic defects (Ungewickell et al, 2004; Choudhury et al, 2005; Erdmann et al, 2007; Ben El Kadhi et al, 2011, 2012; Dambournet et al, 2011; Vicinanza et al, 2011; Nández et al, 2014; Cauvin et al, 2016; De Leo et al, 2016; Del Signore et al, 2017; Carim et al, 2019). In control dividing cells, PtdIns(4,5)P 2 concentrates at the cortical equator (Emoto et al, 2005; Field et al, 2005; Roubinet et al, 2011) and recruits the cytokinetic machinery that allows subsequent cytokinesis (Ben El Kadhi et al, 2011; Liu et al, 2012; Cauvin and Echard, 2015).…”

A genome engineering resource to uncover principles of cellular organization and tissue architecture by lipid signaling

“…Protein levels of Actin are used as the loading control. OCRL antibody used here has been described in Del Signore et al, 2017 . ( C ) Immunolabeling of pericardial nephrocytes from 3rd instar larvae to examine the distribution of the OCRL protein.…”