Synchronizing chromosome segregation by flux-dependent force equalization at kinetochores

Abstract

The synchronous movement of chromosomes during anaphase ensures their correct inheritance in every cell division. This reflects the uniformity of spindle forces acting on chromosomes and their simultaneous entry into anaphase. Although anaphase onset is controlled by the spindle assembly checkpoint, it remains unknown how spindle forces are uniformly distributed among different chromosomes. In this paper, we show that tension uniformity at metaphase kinetochores and subsequent anaphase synchrony in Drosophila S2 cells are promoted by spindle microtubule flux. These results can be explained by a mechanical model of the spindle where microtubule poleward translocation events associated with flux reflect relaxation of the kinetochore–microtubule interface, which accounts for the redistribution and convergence of kinetochore tensions in a timescale comparable to typical metaphase duration. As predicted by the model, experimental acceleration of mitosis precludes tension equalization and anaphase synchrony. We propose that flux-dependent equalization of kinetochore tensions ensures a timely and uniform maturation of kinetochore–microtubule interfaces necessary for error-free and coordinated segregation of chromosomes in anaphase.

The perpetual movements of anaphase

“…The classic examples of the former include plants [55] and insect spermatocytes [56]. Drosophila embryos, S2 cells, Xenopus oocyte extract spindles, newt lung cells, mouse, pig and human cells are all examples of the latter [49,50,[57][58][59][60][61][62][63][64][65]. Indeed, in these systems microtubule minus-end depolymerization is turned off or significantly attenuated during anaphase [54,65,66].…”

Drosophila S2 Cells as a Model System to Investigate Mitotic Spindle Dynamics, Architecture, and Function

“…This has been particularly successful in the study of the molecular mechanism regulating kinetochore microtubule dynamics (Maiato et al, 2005), where laser microsurgery of K-fibers in S2 cells stably expressing GFP-α-tubulin generates a reproducible essay characterized by K-fiber growth from their kinetochore-associated end at near flux rates (Fig. 5A) (Maiato et al, 2004a;Matos et al, 2009). Another useful application is the laser-mediated ablation of centrosomes, which allows one to investigate the molecular basis of acentrosomal spindle formation in animal somatic cells, as well as to dissect how acentrosomal spindles are maintained by ablating centrosomes after spindle assembly (Fig.…”

“…However, microtubules, including those attached at kinetochores, remain dynamic and can be recycled due to turnover (Gorbsky and Borisy, 1989;Zhai et al, 1995) and poleward flux (Mitchison, 1989). This requires a labile interface that enables microtubules to slip and eventually detach from the kinetochore in response to a poleward force, whose origin remains controversial (Cameron et al, 2006;Dumont and Mitchison, 2009;Ganem et al, 2005;Matos et al, 2009;Miyamoto et al, 2004;Rogers et al, 2004). The importance of a labile kinetochore-microtubule interface is reflected in the capacity to correct mistakes inherent to the stochastic nature of mitotic spindle assembly and the interaction between microtubules and chromosomes (Bakhoum et al, 2009b;Ganem et al, 2005;Matos et al, 2009).…”

“…This requires a labile interface that enables microtubules to slip and eventually detach from the kinetochore in response to a poleward force, whose origin remains controversial (Cameron et al, 2006;Dumont and Mitchison, 2009;Ganem et al, 2005;Matos et al, 2009;Miyamoto et al, 2004;Rogers et al, 2004). The importance of a labile kinetochore-microtubule interface is reflected in the capacity to correct mistakes inherent to the stochastic nature of mitotic spindle assembly and the interaction between microtubules and chromosomes (Bakhoum et al, 2009b;Ganem et al, 2005;Matos et al, 2009). In fact, increasing the stability of kinetochore-microtubule attachments in a way that errors could more difficultly be corrected is, by itself, sufficient to induce chromosomal instability in once stable diploid cells (Bakhoum et al, 2009a).…”

“…Additionally, Drosophila culture cells have a low chromosome number (typically between 4-12) and details of mitotic spindle morphogenesis can be easily observed at the light microscopy (LM) level by the use of available stable cell lines expressing fluorescent components of the mitotic apparatus (Mahoney et al, 2006;Maiato et al, 2004a). Finally, Drosophila culture cells are amenable for live cell microscopy and micromanipulation techniques, such as Fluorescent Speckle Microscopy (FSM) and laser microsurgery (Maiato et al, 2005;Maiato et al, 2004a;Matos et al, 2009). In the following sections we describe in detail some of the state-of-the-art methodologies currently in practice in our laboratory for the study and micromanipulation of microtubule organization, dynamics and function during mitosis in live Drosophila culture cells.…”