Kinetochore-driven formation of kinetochore fibers contributes to spindle assembly during animal mitosis

Abstract

It is now clear that a centrosome-independent pathway for mitotic spindle assembly exists even in cells that normally possess centrosomes. The question remains, however, whether this pathway only activates when centrosome activity is compromised, or whether it contributes to spindle morphogenesis during a normal mitosis. Here, we show that many of the kinetochore fibers (K-fibers) in centrosomal Drosophila S2 cells are formed by the kinetochores. Initially, kinetochore-formed K-fibers are not oriented toward a spindle pole but, as they grow, their minus ends are captured by astral microtubules (MTs) and transported poleward through a dynein-dependent mechanism. This poleward transport results in chromosome bi-orientation and congression. Furthermore, when individual K-fibers are severed by laser microsurgery, they regrow from the kinetochore outward via MT plus-end polymerization at the kinetochore. Thus, even in the presence of centrosomes, the formation of some K-fibers is initiated by the kinetochores. However, centrosomes facilitate the proper orientation of K-fibers toward spindle poles by integrating them into a common spindle.

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

“…In fact, in vertebrate cells, K-fibers were observed forming in association with chromosomes without a direct connection to the centrosome or chromatin (Khodjakov et al, 2003;O'Connell et al, 2009). In agreement, studies in live Drosophila S2 cells reported that unattached kinetochores that are not facing a centrosome are able to form microtubules de novo, and that K-fiber growth likely occurs by microtubule addition at their kinetochore-associated end (Maiato et al, 2004a).…”

“…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.…”

“…Of particular interest for high-throughput genome-wide screenings, specific gene silencing can be easily achieved by RNA interference (RNAi) and commercial dsRNA libraries are available (Goshima et al, 2007). 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).…”

“…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.…”

The perpetual movements of anaphase

“…Importantly, "flux" was subsequently shown to require ATP hydrolysis but was insensitive to vanadate (which at micromolar concentrations inhibits dynein but not kinesin ATPase activity in vitro), suggesting the involvement of motors of the kinesin family [48]. As we know more about "flux" we begin to realize that this process may actually involve two independent components: the active depolymerization of spindle microtubules at their minus-ends, which may occur in the absence of any detectable poleward sliding of microtubules [49,50] and the poleward sliding of microtubules, including k-fibers, which may occur in the absence of any detectable depolymerization at their minus-ends [51][52][53]. Several works in many organisms and cell types have shown that, as a rule, the velocity at which microtubules depolymerize at their minus-ends is equal or slower than that of chromosome movement to the poles [54].…”