Using Microscopy to Examine Mechanisms of Cell Division

The accurate segregation of chromosomes during cell division requires the assembly of a microtubule-based mitotic spindle. In the research presented here, I examined two aspects of mitotic spindle morphogenesis. First, I looked at how chromosomes attach to microtubules in mitosis. The capture of microtubule plus-ends by kinetochores (the site of chromosome-microtubule attachment) has been described previously. However, recent data suggests that another mechanism must also contribute to this essential process. Here, with the aid of real-time confocal microscopy, I present direct evidence for an additional chromosome-spindle attachment mechanism in which the minus-ends of microtubules attached to chromosomes are captured by spindle microtubules and incorporated into the spindle. I also show that this process depends on the microtubule cross-linking MAP (microtubule associated protein), NuMA. Second, I examined the molecular basis of bipolar spindle formation: in particular, contributions of the non-motor MAP TPX2 to spindle structure and function in mammalian cells. My live cell recordings revealed that in cells depleted of TPX2, multipolar spindles formed by progressive spindle pole fragmentation. Consistent with structural role for TPX2 in the spindle, I found that while normal bipolar spindles resistant to collapse to monopolar spindles after inhibition of the mitotic motor Eg5, multipolar spindles that formed in the absence of TPX2 were not. In addition, I found that microtubule nucleation and growth during spindle assembly is not dependent on TPX2, and that cells form robust kinetochore microtubule bundles after TPX2 knockdown. These data do not suggest a direct role of TPX2 in the establishment of chromosome-microtubule attachments. However, my results shed new light on the spindle defects in cells without T P X 2 and confirm an important role for T P X 2 in the structural stability of the mitotic spindle