A FRET-based study reveals site-specific regulation of spindle position checkpoint proteins at yeast centrosomes.

Yuliya Gryaznova,Gislene Pereira,Gabriele Malengo,Victor Sourjik,Ayse Koca Caydasi

doi:10.7554/elife.14029

Yuliya Gryaznova, Gislene Pereira + Show 3 more

Open Access

https://doi.org/10.7554/elife.14029

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Journal: eLife	Publication Date: May 9, 2016
Citations: 55	License type: CC BY 4.0

Affiliation: DKFZ-ZMBH Alliance, Heidelberg University

Abstract

The spindle position checkpoint (SPOC) is a spindle pole body (SPB, equivalent of mammalian centrosome) associated surveillance mechanism that halts mitotic exit upon spindle mis-orientation. Here, we monitored the interaction between SPB proteins and the SPOC component Bfa1 by FRET microscopy. We show that Bfa1 binds to the scaffold-protein Nud1 and the γ-tubulin receptor Spc72. Spindle misalignment specifically disrupts Bfa1-Spc72 interaction by a mechanism that requires the 14-3-3-family protein Bmh1 and the MARK/PAR-kinase Kin4. Dissociation of Bfa1 from Spc72 prevents the inhibitory phosphorylation of Bfa1 by the polo-like kinase Cdc5. We propose Spc72 as a regulatory hub that coordinates the activity of Kin4 and Cdc5 towards Bfa1. In addition, analysis of spc72∆ cells shows that a mitotic-exit-promoting dominant signal, which is triggered upon elongation of the spindle into the bud, overrides the SPOC. Our data reinforce the importance of daughter-cell-associated factors and centrosome-based regulations in mitotic exit and SPOC control.

Highlights

Alongside their canonical role as microtubule organizing centers, centrosomes of metazoans or spindle pole bodies (SPBs, the functional equivalent of the centrosome) of fungi modulate eukaryotic cell division by serving as signaling centers (Arquint et al, 2014; Fu et al, 2015)
In contrast to the conventional FRET detection, which is based on acceptor emission measurements, the acceptor photobleaching FRET monitors the increase in donor fluorescence upon photobleaching of the acceptor (Figure 1—figure supplement 1) (Llopis et al, 2000; Wouters and Bastiaens, 2001)
The proportionate increase in the donor fluorescence intensity after photobleaching of the acceptor directly yields the apparent FRET efficiency (EFRET) (Karpova et al, 2003; Kentner and Sourjik, 2009), which is further corrected for the unspecific signal observed in the donor-only sample (Figure 1—figure supplement 1B–D)

Summary

Introduction

Alongside their canonical role as microtubule organizing centers, centrosomes of metazoans or spindle pole bodies (SPBs, the functional equivalent of the centrosome) of fungi modulate eukaryotic cell division by serving as signaling centers (Arquint et al, 2014; Fu et al, 2015). The SPB is associated with components of two linked pathways: the mitotic exit network (MEN) and the spindle position checkpoint (SPOC). The MEN drives mitotic exit (transition from M-G1 phase) after extension of the anaphase spindle into the daughter cell body. The SPOC is a surveillance mechanism that monitors orientation of the mitotic spindle. The SPOC prevents M-G1 transition when the spindle fails to align along the mother to daughter axis and so is unable to deliver one nucleus into the daughter cell (Bloecher et al, 2000; Muhua et al, 1998; Pereira et al, 2000; Wang et al, 2000; Yeh et al, 1995).

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