A selective disruption of the mouse CENP-E gene was generated to test how this kinetochore-associated, kinesin-like protein contributes to chromosome segregation. The removal of CENP-E in primary cells produced spindles in which some metaphase chromosomes lay juxtaposed to a spindle pole, despite the absence of microtubules stably bound to their kinetochores. Most CENP-E-free chromosomes moved to the spindle equator, but their kinetochores bound only half the normal number of microtubules. Deletion of CENP-E in embryos led to early developmental arrest. Selective deletion of CENP-E in liver revealed that tissue regeneration after chemical damage was accompanied by aberrant mitoses marked by chromosome missegregation. CENP-E is thus essential for the maintenance of chromosomal stability through efficient stabilization of microtubule capture at kinetochores.

This study uses electron tomography linked to a variety of other EM methods to provide an integrated view of the flagellar pocket and basal body area of the African trypanosome procyclic trypomastigote. We reveal the pocket as an asymmetric membranous `balloon' with two boundary structures. One of these – the collar – defines the flagellum exit point. The other defines the entry point of the flagellum into the pocket and consists of both an internal transitional fibre array and an external membrane collarette. A novel set of nine radial fibres is described in the basal body proximal zone. The pocket asymmetry is invariably correlated with the position of the probasal body and Golgi. The neck region, just distal to the flagellum exit site, is a specialised area of membrane associated with the start of the flagellum attachment zone and signifies the point where a special set of four microtubules, nucleated close to the basal bodies, joins the subpellicular array. The neck region is also associated with the single Golgi apparatus of the cell. The flagellar exit point interrupts the subpellicular microtubule array with discrete endings of microtubules at the posterior side. Overall, our studies reveal a highly organised, yet dynamic, area of cytoplasm and will be informative in understanding its function.

These observations suggest that myosin-II along with actin crosslinkers establish local cortical tension and elasticity, allowing for contractility independent of a circumferential cytoskeletal array. Furthermore, myosin-II and actin crosslinkers may influence each other as they modulate the dynamics and mechanics of cell-shape change.

Summary Kinetochores of mitotic chromosomes are coupled to spindle microtubules in ways that allow the energy from tubulin dynamics to drive chromosome motion. Most kinetochore-associated microtubule ends display curving “protofilaments,” strands of tubulin dimers that bend away from the microtubule axis. Both a kinetochore “plate” and an encircling, ring-shaped protein complex have been proposed to link protofilament bending to poleward chromosome motion. Here we show by electron tomography that slender fibrils connect curved protofilaments directly to the inner kinetochore. Fibril-protofilament associations correlate with a local straightening of the flared protofilaments. Theoretical analysis reveals that protofilament-fibril connections would be efficient couplers for chromosome motion, and experimental work on two very different kinetochore components suggests that filamentous proteins can couple shortening microtubules to cargo movements. These analyses define a ring-independent mechanism for harnessing microtubule dynamics directly to chromosome movement.

The fission yeast Schizosaccharomyces pombe divides by medial fission through the use of an actomyosin contractile ring. Precisely at the end of anaphase, the ring begins to constrict and the septum forms. Proper coordination of cell division with mitosis is crucial to ensure proper segregation of chromosomes to daughter cells. The Sid2p kinase is one of several proteins that function as part of a novel signaling pathway required for initiation of medial ring constriction and septation. Here, we show that Sid2p is a component of the spindle pole body at all stages of the cell cycle and localizes transiently to the cell division site during medial ring constriction and septation. A medial ring and an intact microtubule cytoskeleton are required for the localization of Sid2p to the division site. We have established an in vitro assay for measuring Sid2p kinase activity, and found that Sid2p kinase activity peaks during medial ring constriction and septation. Both Sid2p localization to the division site and activity depend on the function of all of the other septation initiation genes: cdc7, cdc11, cdc14, sid1, spg1, and sid4. Thus, Sid2p, a component of the spindle pole body, by virtue of its transient localization to the division site, appears to determine the timing of ring constriction and septum delivery in response to activating signals from other Sid gene products.