Dual pathway spindle assembly increases both the speed and the fidelity of mitosis

Roughly half of all animal somatic cell spindles assemble by the classical prophase pathway, in which the centrosomes separate ahead of nuclear envelope breakdown (NEBD). The remainder assemble by the prometaphase pathway, in which the centrosomes separate following NEBD. Why cells use dual pathway spindle assembly is unclear. Here, by examining the timing of NEBD relative to the onset of Eg5-mEGFP loading to centrosomes, we show that a time window of 9.2 ± 2.9 min is available for Eg5-driven prophase centrosome separation ahead of NEBD, and that those cells that succeed in separating their centrosomes within this window subsequently show .3-fold fewer chromosome segregation errors and a somewhat faster mitosis. A longer time window would allow more cells to complete prophase centrosome separation and further reduce segregation errors, but at the expense of a slower mitosis. Our data reveal dual pathway mitosis in a new light, as a substantive strategy that increases both the speed and the fidelity of mitosis

Long-range cooperative binding of kinesin to a microtubule in the presence of ATP

Interaction of kinesin-coated latex beads with a single microtubule (MT) was directly observed by fluorescence microscopy. In the presence of ATP, binding of a kinesin bead to the MT facilitated the subsequent binding of other kinesin beads to an adjacent region on the MT that extended for micrometers in length. This cooperative binding was not observed in the presence of ADP or 5′-adenylylimidodiphosphate (AMP-PNP), where binding along the MT was random. Cooperative binding also was induced by an engineered, heterodimeric kinesin, WT/E236A, that could hydrolyze ATP, yet remained fixed on the MT in the presence of ATP. Relative to the stationary WT/E236A kinesin on a MT, wild-type kinesin bound preferentially in close proximity, but was biased to the plus-end direction. These results suggest that kinesin binding and ATP hydrolysis may cause a long-range state transition in the MT, increasing its affinity for kinesin toward its plus end. Thus, our study highlights the active involvement of MTs in kinesin motility

Dynamic Behavior of Primary Cilia During Cellular Migration

Dermal papilla cells (DPCs) play pivotal roles in hair follicle development and repetitive hair growth cycles, in which cellular migration is deeply involved. We previously suggested that primary cilia of DPCs regulate the hair follicle physiologies via intercellular signal transduction. However, the role of the primary cilia in cell migration has yet to be addressed. Here, immunofluorescence microscopic observations revealed that primary cilia of DPCs predominantly protruded toward the backward direction in the initial phase of cellular migration, while the organelle of non-migrating cells oriented randomly. In addition, the average length of the primary cilia became shorter upon the onset of the active motion, compared to those in multicellular spheroids. Moreover, the appearance frequency was dramatically decreased in migrating cells. Similar results were obtained using human dermal fibroblasts and articular synoviocytes. Collectively, our current findings suggest that those fibroblastic cells set the primary cilia to the backward direction in the initial phase of migration, along with the tuning of the length and appearance frequency of the sensory organelle. These regulations should be essential for proper cellular migration in tissue developments and wound-healing processes

The Dynamic Cell Walking, hopping, diffusing and braking modes of kinesin-5

Abstract It is clear that the main cellular mission of the molecular motor kinesin-5 (known as Eg5 in vertebrates) is to cross-link antiparallel microtubules and to slide them apart, thus playing a critical role during bipolar spindle formation. Nonetheless, important questions about the cell biological and biophysical mechanisms of Eg5 remain unanswered. With the 20th 'birthday' of Eg5 approaching, we discuss recent insights into the in vitro and in vivo functions of Eg5, in the context of our own recent work

Single-headed mode of kinesin-5

In most organisms, kinesin-5 motors are essential for mitosis and meiosis, where they crosslink and slide apart the antiparallel microtubule half-spindles. Recently, it was shown using single-molecule optical trapping that a truncated, double-headed human kinesin-5 dimer can step processively along microtubules. However, processivity is limited (~8 steps) with little coordination between the heads, raising the possibility that kinesin-5 motors might also be able to move by a nonprocessive mechanism. To investigate this, we engineered single-headed kinesin-5 dimers. We show that a set of these single-headed Eg5 dimers drive microtubule sliding at about 90% of wild-type velocity, indicating that Eg5 can slide microtubules by a mechanism in which one head of each Eg5 head-pair is effectively redundant. On the basis of this, we propose a muscle-like model for Eg5-driven microtubule sliding in spindles in which most force-generating events are single-headed interactions and alternate-heads processivity is rare