Multiple ciliary localization signals control INPP5E ciliary targeting

Abstract

Primary cilia are sensory membrane protrusions whose dysfunction causes ciliopathies. INPP5E is a ciliary phosphoinositide phosphatase mutated in ciliopathies like Joubert syndrome. INPP5E regulates numerous ciliary functions, but how it accumulates in cilia remains poorly understood. Herein, we show INPP5E ciliary targeting requires its folded catalytic domain and is controlled by four conserved ciliary localization signals (CLSs): LLxPIR motif (CLS1), W383 (CLS2), FDRxLYL motif (CLS3) and CaaX box (CLS4). We answer two long-standing questions in the field. First, partial CLS1-CLS4 redundancy explains why CLS4 is dispensable for ciliary targeting. Second, the essential need for CLS2 clarifies why CLS3-CLS4 are together insufficient for ciliary accumulation. Furthermore, we reveal that some Joubert syndrome mutations perturb INPP5E ciliary targeting, and clarify how each CLS works: (i) CLS4 recruits PDE6D, RPGR and ARL13B, (ii) CLS2-CLS3 regulate association to TULP3, ARL13B, and CEP164, and (iii) CLS1 and CLS4 cooperate in ATG16L1 binding. Altogether, we shed light on the mechanisms of INPP5E ciliary targeting, revealing a complexity without known parallels among ciliary cargoes.

Non-catalytic role of phosphoinositide 3-kinase in mesenchymal cell migration through non-canonical induction of p85β/AP2-mediated endocytosis

“…To gain insights into the cellular mechanisms of iSH2-mediated endocytosis, we first examined in cellulo interaction between iSH2 and AP2 by applying an inducible co-recruitment assay 50 , 51 (Supplementary Fig. 6a ).…”

Non-catalytic role of phosphoinositide 3-kinase in mesenchymal cell migration through non-canonical induction of p85β/AP-2-mediated endocytosis

“…To understand molecular mechanisms of iSH2-mediated endocytosis, we examined possible association between iSH2 and AP-2 by applying an inducible co-recruitment assay 48, 49 (Extended Data Fig. 6a).…”

EVC-EVC2 complex stability and ciliary targeting are regulated by modification with ubiquitin and SUMO

“…The monoclonal mouse anti-EVC antibody used for the interactomics experiments has been described elsewhere ( Pacheco et al, 2012 ). Mouse monoclonal antibodies against acetylated alpha-tubulin (Sigma, T7451), alpha-tubulin (Proteintech, 66031-1-Ig), gamma-tubulin (Santa Cruz, sc-17787), and EGFP (Proteintech, 66002-1-Ig) were used as described previously ( Cilleros-Rodriguez et al, 2022 ), as was the case for rabbit polyclonal antibodies against Myc epitope (Proteintech, 16286-1-AP) and EGFP (Proteintech, 50430-2-AP), as well as for the Chromotek GFP-Trap_MA beads (Proteintech, gtma), and for all secondary antibodies. Other antibodies used here include: mouse anti-Flag (Sigma, F1804, IF: 1:200, WB: 1:2000), mouse anti-Flag (Proteintech, 66008-3-Ig, IF: 1:200, WB: 1:2000), mouse anti-ARL13B (Proteintech, 66739-1-Ig, IF: 1:100), mouse anti-V5 (Thermofisher, MA5-15253, WB: 1:1000), rabbit anti-human EVC (Sigma, HPA016046, WB: 1:1000), rabbit anti-USP7 (Santa Cruz, sc-30164, WB: 1:500), rabbit anti-AHI1 (Proteintech, 22045-1-AP, IF: 1:50), rabbit anti-MKS1 (Proteintech, 16206-1-AP, IF: 1:50), rabbit anti-ubiquitin (Proteintech, 10201-2-AP, WB: 1:1000), rabbit anti-SUMO2/3 (Proteintech, 11251-1-AP, WB: 1:1000), and rat anti-HA (Proteintech, 7c9, WB: 1:1000).…”

“…The resulting plasmids, therefore, express (EVC/EVC2)-Flag-(Ub/SUMO3), but not EGFP, whose CDS is now after the introduced stop codon. For CRISPR targeting of human EVC , the following sgRNA-coding sequences were cloned into pSpCas9(BB)-2A-Puro (PX459) V2.0 (Addgene #62988), as previously described: sgEVC1: cgg​cct​gca​aga​gcg​acg​cg; sgEVC2: cag​ccg​cgc​gtc​gct​ctt​gc; sgEVC3: ctt​tgg​ctt​ggc​tgc​cgc​gc; sgEVC4: gtg​ctg​ctg​ggc​gcc​gcg​ct ( Barbeito et al, 2021 ; Cilleros-Rodriguez et al, 2022 ). Site-directed mutagenesis was performed by overlap extension PCR as reported previously ( Barbeito et al, 2021 ; Cilleros-Rodriguez et al, 2022 ).…”

“…HEK293T were Frontiers in Cell and Developmental Biology frontiersin.org transfected using the calcium phosphate method and lysed 40-48 h later. IMCD3 and hTERT-RPE1 cells were reverse transfected using JetPrime (Polyplus-transfection) and fixed 48 h later for cilia analysis, as reported previously (Cilleros-Rodriguez et al, 2022). Generation of EVC-null HEK293T cell lines by CRISPR was performed as previously described (Barbeito et al, 2021;Cilleros-Rodriguez et al, 2022), using the plasmids described below.…”