NuMA is required for proper spindle assembly and chromosome alignment in prometaphase

To circumvent these technical problems, we performed siRNA experiments in which we depleted the majority of NuMA in human cultured cells. Depleted mitotic cells show a prolonged duration of prometaphase, with spindle pole defects and with unattached, unaligned chromosomes.Our data confirm that NuMA is important for spindle pole formation, and for cohesion of centrosome-derived microtubules with the bulk of spindle microtubules. Our findings of NuMA-dependent defects in chromosome alignment suggest that NuMA is involved in stabilizing kinetochore fibres.NuMA, the protein of the 'Nucleus and Mitotic Apparatus', is a structural protein in vertebrates of approximately 230 kDa. It localizes to the nucleus during interphase, and accumulates at the spindle poles during mitosis [1]. NuMA has been implicated in the formation of the mitotic spindle, in particular in focusing the spindle poles [2]. Moreover, in recent years it has been shown that part of NuMA localizes to the cell cortex during mitosis where it interacts with the protein LGN/pins [3,4]. It has been suggested that cortical NuMA participates in spindle orientation, a role that has also been attributed to related proteins in Drosophila and Caenorhabditis elegans, termed Mud or LIN-5, respectively [4-9]. So far, the majority of experiments that tested the role of vertebrate NuMA relied on methods such as antibody microinjection, overexpression of NuMA mutants, or depletion of NuMA from cytoplasmic extracts [10-22]. The cumulative evidence from these experiments pointed towards a function of NuMA in crosslinking microtubules at the spindle poles, enabling the formation and maintenance of the bipolar spindle apparatus. The shortcomings of these experiments were that they could not distinguish between a direct effect on NuMA function, and an indirect effect on interacting proteins: 1) Antibodies are large proteins; therefore, upon microinjection they might sterically hinder the function of neighbouring proteins that