Different spindle checkpoint proteins monitor microtubule attachment and tension at kinetochores inDrosophilacells

Abstract

The spindle assembly checkpoint detects errors in kinetochore attachment to the spindle including insufficient microtubule occupancy and absence of tension across bi-oriented kinetochore pairs. Here, we analyse how the kinetochore localization of the Drosophila spindle checkpoint proteins Bub1, Mad2, Bub3 and BubR1, behave in response to alterations in microtubule binding or tension. To analyse the behaviour in the absence of tension, we treated S2 cells with low doses of taxol to disrupt microtubule dynamics and tension, but not kinetochore-microtubule occupancy. Under these conditions, we found that Mad2 and Bub1 do not accumulate at metaphase kinetochores whereas BubR1 does. Consistently, in mono-oriented chromosomes, both kinetochores accumulate BubR1 whereas Bub1 and Mad2 only localize at the unattached kinetochore. To study the effect of tension we analysed the kinetochore localization of spindle checkpoint proteins in relation to tension-sensitive kinetochore phosphorylation recognised by the 3F3/2 antibody. Using detergent-extracted S2 cells as a system in which kinetochore phosphorylation can be easily manipulated, we observed that BubR1 and Bub3 accumulation at kinetochores is dependent on the presence of phosphorylated 3F3/2 epitopes. However, Bub1 and Mad2 localize at kinetochores regardless of the 3F3/2 phosphorylation state. Altogether, our results suggest that spindle checkpoint proteins sense distinct aspects of kinetochore interaction with the spindle, with Mad2 and Bub1 monitoring microtubule occupancy while BubR1 and Bub3 monitor tension across attached kinetochores.

Core Exosome-independent Roles for Rrp6 in Cell Cycle Progression

“…As a cellular marker for checkpoint activation, we visualized BubR1, a checkpoint protein that localizes to kinetochores in cells that are responding to the SAC (Logarinho et al, 2004). We did not observe a change in BubR1 recruitment frequency under any condition where dRrp6 was depleted compared with mock-depleted cells (Supplemental Figure S6A).…”

“…Cy2-(1:200), Texas Red-(1:200), and Cy5-(1:800) conjugated secondary antibodies were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA). Antibodies to Aurora A (Giet et al, 2002) and BubR1 (Logarinho et al, 2004) were used as described previously. Antibodies to Mmp1 (1:500; monoclonal Ab 5H7b11) and TrioB (1:50; monoclonal Ab 9.4A) were obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the National Institute of Child Health and Human Development and maintained by the University of Iowa.…”

Temporal regulation of the first mitosis in Xenopus and mouse embryos

“…The SAC is a mechanism regulating cell division in many organisms such as yeast (Chen et al, 1999), insects (Logarinho et al, 2004), nematodes (Encalada et al, 2005), amphibians (Gorbsky, 1997), or mammals (Skoufias et al, 2001). It also acts in meiotically dividing germ cells (Homer, 2006;Homer et al, 2005;Marston and Amon, 2004;Tsurumi et al, 2004).…”

Molecular insights into mechanisms regulating faithful chromosome separation in female meiosis

“…As a protein kinase, Bub1 can bind and phosphorylate Bub3, 53 and be sensitive to tension across the centromere. [54][55][56] Bub1 accumulates on kinetochores in the absence of tension, 44,47,48,54,57 as a platform for other spindle checkpoint proteins. 58 Bub1 is also reported to control centromeric cohesin and loss of Bub1 function causes checkpoint impairment.…”