Different local curvature developments as a function of distance from MT wall are consistent with the model that some adjacent PFs adhere close to the wall (group 2)

Different local curvature developments as a function of distance from MT wall are consistent with the model that some adjacent PFs adhere close to the wall (group 2). F. a site where MTs can either add or lose tubulin subunits as chromosomes move, so it is significant for normal mitosis (Rieder and Salmon, 1998). Identification of the proteins that provide these functions is now well advanced in budding yeast (Westermann et al., 2007) and higher eukaryotes (Cheeseman and Desai, 2008;Liu et al., 2006). Several motor enzymes contribute to chromosome segregation, but MT depolymerization in vitro, even in the absence of soluble nucleotides, can mimic chromosome-to-pole motion in vivo (Coue et al., 1991;Koshland et al., 1988). Moreover, minus-end directed motors are dispensable for poleward chromosome motion in yeasts (Grishchuk and McIntosh, 2006;Tanaka et al., 2007), suggesting that the root of chromosome movement lies in MT dynamics, not motor activity. MT shortening can generate force because tubulin dynamics are associated with GTP hydrolysis. Tubulin-bound GTP is hydrolyzed shortly after polymerization, so most of the MT wall is GDP-tubulin. Surprisingly, GDP-tubulin will not polymerize, probably because its shape does not fit the MT lattice (Wang and Nogales, 2005). Assembled GDP-tubulin is therefore strained by interactions with its neighbors in the MT wall. PF-03814735 This strain is relieved during depolymerization when strands of tubulin dimers, called protofilaments (PFs), become flared at the MT end (Mandelkow et al., 1991); presumably this PF shape reveals the minimum energy configuration of GDP tubulin, whereas GTP tubulin PFs are comparatively straight (Chretien et al., 1995;Muller-Reichert et al., 1998). Thus, the morphology of a MT end in vitro reflects its polymerization state. PF bending during MT shortening has been proposed to do mechanical work (Koshland et al., 1988). Indeed, microbeads coupled to MTs by static links, e.g. a MAP or a biotin-streptavidin bond, experience a brief tug during PF bending (Grishchuk et al., 2005). Cargo that is bound to MTs by either motor enzymes (Lombillo et al., 1995b) or an encircling protein complex (Westermann et al., 2006) will move processively during MT shortening in vitro, even without ATP. Thus, if kinetochores were harnessed to MTs with the right couplers, the energy liberated by tubulin depolymerization could drive chromosome-to-pole motion (Efremov et al., 2007;Hill, 1985;Molodtsov et al., 2005). This raises the question of how kinetochores take advantage of the depolymerization machinery to facilitate chromosome segregation. Kinetochore structure has been studied for years, but most kinetochores are so small that informative early work has been done either by immuno light microscopy or electron microscopy. Recent light microscopy has localized tagged kinetochore components along the spindle PF-03814735 axis with almost nanometer precision (Joglekar et al., 2008;Schittenhelm et al., 2007), but electron microscopy has defined the image of kinetochores that most scientists consider. With this method, vertebrate chromosomes show kinetochore-associated MTs (KMTs) penetrating a darkly staining outer plate (Rieder and Salmon, 1998), which has commonly been interpreted as the principal MT-chromosome connection. Electron tomography of well-preserved kinetochores in PtK1cells has refined this description as a meshwork of fibers that connect adjacent KMTs to one another and to nearby chromatin (Dong et al., 2007). PFs at the ends of many KMTs flare as they penetrate this plate and approach the chromatin (VandenBeldt et al., 2006). The fraction of KMTs with flared ends appears greater PF-03814735 during anaphase than metaphase, perhaps a reflection of increased MT depolymerization in anaphase. However, ~70% of metaphase Mouse monoclonal to KRT15 KMTs have flared ends, which poses an enigma: why do so many KMTs appear to be depolymerizing when their length is, on average, constant? Is KMT PF flare a simple reflection of dynamic state, as in vitro, or do other factors affect PF curvature in vivo? To address these questions and to develop a more complete picture of the kinetochore-MT interface, we have used electron tomography to study the 3D structure of individual PFs at the plus ends of MTs from mitotic PtK1cells, obtaining quantitative information about the curvature of PFs from several kinds of MTs. Approximately half of the PFs at KMT plus ends curve differently from those on either polymerizing or depolymerizing MTs in vitro. Our data suggest that these shapes do not result simply from MT PF-03814735 dynamics but from associations between PFs and kinetochore-associated fibrils. These fibrils appear to impede PF bending, suggesting a mechanism for converting the energy of MT depolymerization into chromosome movement. This hypothesis is.

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