A molecular mechanism of mitotic centrosome assembly in Drosophila.

Paul T Conduit,James Holder,Catarina C Vicente,Jennifer H Richens,Alan Wainman,Carly I Dix,Ian M Dobbie,Lothar Schermelleh,Jordan W Raff,Metta B Pratt,Zsofia A Novak

doi:10.7554/elife.03399

Paul T Conduit, James Holder + Show 9 more

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https://doi.org/10.7554/elife.03399

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Abstract

Centrosomes comprise a pair of centrioles surrounded by pericentriolar material (PCM). The PCM expands dramatically as cells enter mitosis, but it is unclear how this occurs. In this study, we show that the centriole protein Asl initiates the recruitment of DSpd-2 and Cnn to mother centrioles; both proteins then assemble into co-dependent scaffold-like structures that spread outwards from the mother centriole and recruit most, if not all, other PCM components. In the absence of either DSpd-2 or Cnn, mitotic PCM assembly is diminished; in the absence of both proteins, it appears to be abolished. We show that DSpd-2 helps incorporate Cnn into the PCM and that Cnn then helps maintain DSpd-2 within the PCM, creating a positive feedback loop that promotes robust PCM expansion around the mother centriole during mitosis. These observations suggest a surprisingly simple mechanism of mitotic PCM assembly in flies.

Highlights

Centrosomes help regulate many cell processes, including cell shape, cell polarity, and cell division (Doxsey et al, 2005; Bettencourt-Dias and Glover, 2007), and centrosome defects have been implicated in several human pathologies (Nigg and Raff, 2009; Zyss and Gergely, 2009)
We selected the nine proteins, including Cnn, which have been most strongly implicated in the pericentriolar material (PCM) recruitment in flies: Asl-GFP, AurA-GFP, GFP-Cnn, DGp71WD-GFP, D-PLP-GFP, DSas-4-GFP, DSpd-2-GFP, γ-tubulin-GFP, and Polo-GFP (Mennella et al, 2013)
Asl-GFP and DSas-4-GFP are known to be closely associated with centrioles (Fu and Glover, 2012; Mennella et al, 2012), and their fusions were tightly localized in the centre of the PCM; they exhibited a fluorescence intensity profile similar to that of sub-resolution (170 nm) beads (Figure 1A), indicating that their true distribution was below the resolution of our microscope system

Summary

Introduction

Centrosomes help regulate many cell processes, including cell shape, cell polarity, and cell division (Doxsey et al, 2005; Bettencourt-Dias and Glover, 2007), and centrosome defects have been implicated in several human pathologies (Nigg and Raff, 2009; Zyss and Gergely, 2009). Centrosomes are the major microtubule (MT)-organising centres (MTOCs) in many animal cells. They form when centrioles assemble a matrix of pericentriolar material (PCM) around themselves. Several hundred proteins are concentrated in the PCM, including many MT-organising proteins, cell-cycle regulators, and checkpoint and signalling proteins (Müller et al, 2010); the centrosome appears to function as an important co-ordination centre in the cell (Doxsey et al, 2005). These include (1) centriole-associated proteins, such as ‘Asl/Cep152’ (Bonaccorsi et al, 1998; Varmark et al, 2007; Dzhindzhev et al, 2010) and ‘Sas-4/CPAP’ (Cho et al, 2006; Gopalakrishnan et al, 2011), (2) proteins that have a centriole-associated fraction and a fraction that spreads out into the PCM, such as ‘Pericentrin/D-PLP’ (Martinez-Campos et al, 2004; Zimmerman et al, 2004; Fu and Glover, 2012; Lawo et al, 2012; Mennella et al, 2012) and ‘DSpd-2/Cep192’ (Pelletier et al, 2004; Dix and Raff, 2007; GomezFerreria et al, 2007; Giansanti et al, 2008; Zhu et al, 2008; Joukov et al, 2010; Decker et al, 2011; Joukov et al, 2014), (3) proteins that reside in the PCM, such as ‘Cnn/Cdk5Rap2’ (Megraw et al, 1999; Lucas and Raff, 2007; Fong et al, 2008; Barr et al, 2010; Conduit et al, 2010), ‘DGp71WD/NEDD1’ (Haren et al, 2006, 2009; Lüders et al, 2006; Manning et al, 2010), and ‘γ-tubulin’ (Sunkel et al, 1995; Hannak et al, 2002), and (4) mitotic protein kinases, such as ‘Polo/Plk1’

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