Interaction of INPP5E with ARL13B is essential for its ciliary membrane retention but dispensable for its ciliary entry

Abstract

Compositions of proteins and lipids within cilia and on the ciliary membrane are maintained to be distinct from those of the cytoplasm and plasma membrane, respectively, by the presence of the ciliary gate. INPP5E is a phosphoinositide 5-phosphatase that is localized on the ciliary membrane by anchorage via its C-terminal prenyl moiety. In addition, the ciliary membrane localization of INPP5E is determined by the small GTPase ARL13B. However, it remained unclear as to how ARL13B participates in the localization of INPP5E. We here show that wild-type INPP5E, INPP5E(WT), in ARL13B-knockout cells and an INPP5E mutant defective in ARL13B binding, INPP5E(ΔCTS), in control cells were unable to show steady-state localization on the ciliary membrane. However, not only INPP5E(WT) but also INPP5E(ΔCTS) was able to rescue the abnormal localization of ciliary proteins in INPP5E-knockout cells. Analysis using the chemically induced dimerization system demonstrated that INPP5E(WT) in ARL13B-knockout cells and INPP5E(ΔCTS) in control cells were able to enter cilia, but neither was retained on the ciliary membrane due to the lack of the INPP5E–ARL13B interaction. Thus, our data demonstrate that binding of INPP5E to ARL13B is essential for its steady-state localization on the ciliary membrane but is dispensable for its entry into cilia.

Superresolution Microscopy Reveals Distinct Phosphoinositide Subdomains Within the Cilia Transition Zone

“…We directly compared GFP-INPP5E localization in live hTERT-RPE1 cells with cells fixed using a standard 4% PFA method followed by 0.1% Triton X-100 permeabilization. In the majority of live cells GFP-INPP5E was concentrated at the cilia base with lower levels in the axoneme, whereas 4% PFA fixation induced an almost exclusive axoneme distribution (Figures 7C,D), similar to that reported previously (Bielas et al, 2009;Jacoby et al, 2009;Humbert et al, 2012;Plotnikova et al, 2015;Conduit et al, 2017;Dyson et al, 2017;Qiu et al, 2021 Graph shows the lateral diameter between the highest intensity points of the pAKT(S473), CEP164 or TCTN1 puncta perpendicular to the plane of the axoneme. Bars represent mean ± SEM, n = 3 independent experiments, ≥30 cilia imaged per experiment and all cilia with two distinct pAKT(S473), CEP164 or TCTN1 puncta measured, statistical significance was determined using one-way ANOVA (p < 0.0001) followed by Tukey's post hoc test, ***p < 0.001, ****p < 0.0001.…”

“…INPP5E is a significant regulator of transition zone PIs, however, all reports to date have shown endogenous and recombinant INPP5E localize to the ciliary axoneme away from its substrates (Bielas et al, 2009;Jacoby et al, 2009;Humbert et al, 2012;Plotnikova et al, 2015;Conduit et al, 2017;Dyson et al, 2017;Qiu et al, 2021). We previously showed that in a minority of cells INPP5E also partially co-localized with TCTN1 at the transition zone (Dyson et al, 2017).…”

“…Inpp5e-null cells show elevated transition zone PI(4,5)P 2 and PI(3,4,5)P 3 signals and defective transition zone function (Dyson et al, 2017). Multiple studies have reported INPP5E localizes to the ciliary axoneme, a distribution that is disrupted by the MORM syndrome mutation (Jacoby et al, 2009;Garcia-Gonzalo et al, 2011;Humbert et al, 2012;Roberson et al, 2015;Slaats et al, 2016;Conduit et al, 2017;Dyson et al, 2017;Goetz et al, 2017;Qiu et al, 2021). INPP5E localization to the axoneme is dependent on a functional transition zone and a network of the cilia associated proteins ARL13B, PDE6D and CEP164 (Garcia-Gonzalo et al, 2011;Humbert et al, 2012;Roberson et al, 2015;Slaats et al, 2016;Goetz et al, 2017;Qiu et al, 2021).…”

The ARF GAPs ELMOD1 and ELMOD3 act at the Golgi and Cilia to Regulate Ciliogenesis and Ciliary Protein Traffic

“…As a C-terminal isoprenylated protein, INPP5E traffics throughout the cell as cargo of the PDE6D transporter and is deposited onto membranes upon either ARL2 or ARL3 binding to PDE6D (86,104,106,113,120–122). Thus, the absence of INPP5E from cilia might be explained by the lack of ARL3, if required for dislocation from PDE6D, or lack of retention offered by binding to ARL13B (81,82). While staining ELMOD1/3 KO lines for INPP5E, we noticed an increase in INPP5E staining intensity in serum starved cells (24 hrs) that co-localized with the Golgi marker GM130 (Fig.…”

“…Together, they regulate export of G protein coupled receptors from cilia (61,79,80). ARL13B interacts with INPP5E, a PIP 5'-phosphatase, and PDE6D (aka PDE6δ or Pr/BPδ), a transporter of prenylated cargos that includes INPP5E (81–83). ARL13B possesses GEF activity for ARL3, which in turn can bind directly to PDE6D resulting in the release of its cargo (84–87).…”