LINC Complexes Mediate the Positioning of Cone Photoreceptor Nuclei in Mouse Retina

David Razafsky,P J Stewart-Hutchinson,Didier Hodzic,Alexander Markov,Nathaniel Blecher,Sang-Chul Nam

doi:10.1371/journal.pone.0047180

Abstract

It has long been observed that many neuronal types position their nuclei within restricted cytoplasmic boundaries. A striking example is the apical localization of cone photoreceptors nuclei at the outer edge of the outer nuclear layer of mammalian retinas. Yet, little is known about how such nuclear spatial confinement is achieved and further maintained. Linkers of the Nucleoskeleton to the Cytoskeleton (LINC complexes) consist of evolutionary-conserved macromolecular assemblies that span the nuclear envelope to connect the nucleus with the peripheral cytoskeleton. Here, we applied a new transgenic strategy to disrupt LINC complexes either in cones or rods. In adult cones, we observed a drastic nuclear mislocalization on the basal side of the ONL that affected cone terminals overall architecture. We further provide evidence that this phenotype may stem from the inability of cone precursor nuclei to migrate towards the apical side of the outer nuclear layer during early postnatal retinal development. By contrast, disruption of LINC complexes within rod photoreceptors, whose nuclei are scattered across the outer nuclear layer, had no effect on the positioning of their nuclei thereby emphasizing differential requirements for LINC complexes by different neuronal types. We further show that Sun1, a component of LINC complexes, but not A-type lamins, which interact with LINC complexes at the nuclear envelope, participate in cone nuclei positioning. This study provides key mechanistic aspects underlying the well-known spatial confinement of cone nuclei as well as a new mouse model to evaluate the pathological relevance of nuclear mispositioning.

Highlights

Many CNS tissues display a laminar organization that consists in various number of nuclear layers separated by synaptic zones
Single KO mice of either Sun proteins or Nesprins do not show any apparent developmental defects thereby emphasizing the redundant function of multiple Sun and Nesprin encoding genes during mammalian CNS development. To overcome these limitations and bypass the potential contribution of cell nonautonomous phenotypes associated to KO approaches, we developed a new mouse model allowing for the spatiotemporal disruption of LINC complexes and applied this transgenic strategy to examine the mechanisms of nuclear positioning within mouse photoreceptor cells
We raised transgenic mice harboring a genetic cassette (Fig. 1B) consisting in the KASH domain of mouse Nesprin2 fused to EGFP (EGFP-KASH2) cloned downstream from a LoxPflanked open reading frame encoding b-galactosidase fused to a V5 epitope (LacZ/V5) [27]

Summary

Introduction

Many CNS tissues display a laminar organization that consists in various number of nuclear layers separated by synaptic zones. Cone photoreceptors provide spectacular examples of polarized nuclear positioning Their nuclei invariably localize on the apical side of the ONL while their axons extend across the thickness of the ONL to establish synaptic contact with second order neurons within the OPL [1,2]. One can wonder whether this specific nuclear positioning has any functional relevance since, by comparison, rod photoreceptors do not require any particular spatial confinement of their nuclei to function. Answering this question first requires the identification of molecular mechanisms underlying the establishment and maintenance of nuclear spatial confinement

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Results

Conclusion