Abstract
Primary cilia are evolutionary conserved microtubule-based organelles that protrude from the surface of most mammalian cells. Phosphoinositides (PI) are membrane-associated signaling lipids that regulate numerous cellular events via the recruitment of lipid-binding effectors. The temporal and spatial membrane distribution of phosphoinositides is regulated by phosphoinositide kinases and phosphatases. Recently phosphoinositide signaling and turnover has been observed at primary cilia. However, the precise localization of the phosphoinositides to specific ciliary subdomains remains undefined. Here we use superresolution microscopy (2D stimulated emission depletion microscopy) to map phosphoinositide distribution at the cilia transition zone. PI(3,4,5)P3 and PI(4,5)P2 localized to distinct subregions of the transition zone in a ring-shape at the inner transition zone membrane. Interestingly, the PI(3,4,5)P3 subdomain was more distal within the transition zone relative to PtdIns(4,5)P2. The phosphoinositide effector kinase pAKT(S473) localized in close proximity to these phosphoinositides. The inositol polyphosphate 5-phosphatase, INPP5E, degrades transition zone phosphoinositides, however, studies of fixed cells have reported recombinant INPP5E localizes to the ciliary axoneme, distant from its substrates. Notably, here using live cell imaging and optimized fixation/permeabilization protocols INPP5E was found concentrated at the cilia base, in a distribution characteristic of the transition zone in a ring-shaped domain of similar dimensions to the phosphoinositides. Collectively, this superresolution map places the phosphoinositides in situ with the transition zone proteins and reveals that INPP5E also likely localizes to a subdomain of the transition zone membrane, where it is optimally situated to control local phosphoinositide metabolism.
Highlights
The small hair-like sensory organelle the primary cilium is a critical regulator of cell biology (Bangs and Anderson, 2017)
We have shown PI(4,5)P2 and PI(3,4,phosphatidylinositol 3 (5)P3) localize to the cilia transition zone (Conduit et al, 2017; Dyson et al, 2017), their precise localization within the complex architecture created by the transition zone protein components is unknown
This is in part due to the resolution of confocal microscopy, which has been used for all studies of ciliary PIs to date (Chavez et al, 2015; Garcia-Gonzalo et al, 2015; Park et al, 2015; Conduit et al, 2017; Dyson et al, 2017) (reviewed in Conduit and Vanhaesebroeck (2020)) and is limited to ∼250 nm, approximately the same size as the diameter of the transition zone (Yang et al, 2015). 2D stimulated emission depletion (STED) microscopy is a superresolution imaging technique that reaches 50 nm resolution for cells (Hell and Wichmann, 1994; Klar et al, 2000; Donnert et al, 2007), making it ideal to resolve the transition zone architecture
Summary
The small hair-like sensory organelle the primary cilium is a critical regulator of cell biology (Bangs and Anderson, 2017). The transition zone is the region at the base of the axoneme distal to the basal body which acts a gate governing the entry and exit of molecules to the cilium (Gonçalves and Pelletier, 2017). This zone consists of three multi-protein complexes MKS, NPHP and CEP290 that form part of the ciliary diffusion barrier (Chih et al, 2011; Garcia-Gonzalo et al, 2011; Sang et al, 2011), the molecular mechanisms of barrier function remain incompletely understood. These complexes consist of transmembrane, membrane-associated and cytosolic proteins which are dependent upon each other for localization to the transition zone
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