PPP1R35 is a novel centrosomal protein that regulates centriole length in concert with the microcephaly protein RTTN.

Andrew Michael Sydor,Estelle Laurent,Helen Liu,Cristina Rovelli,Brian Raught,Etienne Coyaud,Vito Mennella

doi:10.7554/elife.37846

Abstract

Centrosome structure, function, and number are finely regulated at the cellular level to ensure normal mammalian development. Here, we characterize PPP1R35 as a novel bona fide centrosomal protein and demonstrate that it is critical for centriole elongation. Using quantitative super-resolution microscopy mapping and live-cell imaging we show that PPP1R35 is a resident centrosomal protein located in the proximal lumen above the cartwheel, a region of the centriole that has eluded detailed characterization. Loss of PPP1R35 function results in decreased centrosome number and shortened centrioles that lack centriolar distal and microtubule wall associated proteins required for centriole elongation. We further demonstrate that PPP1R35 acts downstream of, and forms a complex with, RTTN, a microcephaly protein required for distal centriole elongation. Altogether, our study identifies a novel step in the centriole elongation pathway centered on PPP1R35 and elucidates downstream partners of the microcephaly protein RTTN.

Highlights

The centrosome is a membrane-less organelle whose major role is to organize, orient, and regulate the site of microtubule formation
To confirm that GFP-PPP1R35 localization is consistent with the endogenous protein, we imaged U2OS cells labeled with antibodies against PPP1R35 and g-tubulin by confocal microscopy (Figure 1d) and cells labeled with antibodies against PPP1R35 and CETN1 by 3D structured illumination microscopy (3DSIM), and observed co-localization (Figure 1e)
Since PPP1R35 forms a complex with the microcephaly protein RTTN and this protein has been previously linked to centriole elongation, where its loss resulted in shortened centrioles (Chen et al, 2017), we investigated whether PPP1R35 knockdown results in diminished centriole length

Summary

Introduction

The centrosome is a membrane-less organelle whose major role is to organize, orient, and regulate the site of microtubule formation. The Drosophila RTTN homolog, Ana, was demonstrated to be a centrosomal component critical for maintaining the structural integrity of centrioles (Stevens et al, 2009), whereas human RTTN, localized near the centriolar cartwheel, has been shown to be dispensable for initial centriole assembly, but critical for formation of a full-length centriole (Chen et al, 2017). It remains unclear what factors are downstream of RTTN and how they promote the elongation and stabilization of the centriole once the cartwheel is formed. We describe a novel centriolar component essential for centriole formation and identify a new mechanistic step downstream of RTTN in the pathway to reach a fully elongated centriole and functional centrosome

Results

15 TRIM26

Discussion

Materials and methods