NESPRIN1 FORMS ROOTLETIN-LIKE CYTOSKELETAL STRUCTURES IN ROD AND CONE PHOTORECEPTORS

Abstract

Purpose : Nesprins are spectrin repeats-containing proteins that associate with the nuclear envelope (NE) through their evolutionary-conserved C-terminal KASH (Klarsicht/Anc1/Syne1 homology) domain. Their canonical function is to dock the nucleus to molecular motors and cytoskeletal components during nuclear migration and anchorage. We recently identified CNS-specific isoforms of Nesprin1 that are unable to localize at the NE because they lack a KASH domain (KASH-LESS isoforms). Accordingly, besides the canonical localization of Nesprin1 at the NE of a subset of retinal cell types, we routinely observed a noncanonical Nesprin1 immunoreactivity in inner segments and outer nuclear layer of mouse retinas. We report that this noncanonical Nesprin1 immunoreactivity corresponds to the localization of a KASH-LESS isoform of Nesprin1.

Methods : Nesprin1 expression was knocked out in rods by breeding mice carrying floxed alleles of Nesprin1 (Nes1^f/f) with Rho-Cre mice. ^RhoNes1^Δ/Δ and ^RhoNes1^Δ/WT eyes were processed for immunofluorescence microscopy and co-labeled with antibodies against Nesprin1 and various cytoskeletal markers of rods and cones. Conditional expression of an EGFP-tagged KASH domain in the mouse retina was driven by a Chx10-Cre strain.

Results : A 120 kDa isoform of Nesprin1 forms long filamentous structures that span the inner segments of photoreceptors and colocalize with rootletin. In rods, this structure extends from basal bodies to the outer limiting membrane (OLM). In cones, this structure further extends across the OLM to form a capping structure that wraps the apical side of cone nuclei. In the outer nuclear layer, Nesprin1 forms shorter filaments that associate in a KASH-dependent manner with a subdomain of the NE of rods. Rootletin is recruited at that location by these short Nesprin1 filaments.

Conclusions : We describe two novel Nesprin1-containing structures that are intimately associated with the rootletin network of photoreceptors. The biological relevance of these noncanonical Nesprin1-based structures to photoreceptor homeostasis and physiology is currently being investigated.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.

 

A noncanonical isoform of Nesprin1 (green) forms long filamentous structures (arrows) that span the inner segment of photoreceptors (Tg: individually-labelled rod photoreceptors).