Spindle Positioning by Cortical Pulling Forces

SummaryProper spatial control of the cell division plane is essential to any developing organism. In most cell types, the relative size of the two daughter cells is determined by the position of the mitotic spindle within the geometry of the mother cell. We review the underlying mechanisms responsible for positioning of the mitotic spindle, both in cases where the spindle is placed in the center of the cell and in cases where the spindle is placed away from the center of the cell. We discuss the idea that cortical pulling forces are sufficient to provide a general mechanism for spindle positioning within symmetrically and asymmetrically dividing cells

Building a spindle of the correct length in human cells requires the interaction between TPX2 and Aurora A

To assemble mitotic spindles, cells nucleate microtubules from a variety of sources including chromosomes and centrosomes. We know little about how the regulation of microtubule nucleation contributes to spindle bipolarity and spindle size. The Aurora A kinase activator TPX2 is required for microtubule nucleation from chromosomes as well as for spindle bipolarity. We use bacterial artificial chromosome–based recombineering to introduce point mutants that block the interaction between TPX2 and Aurora A into human cells. TPX2 mutants have very short spindles but, surprisingly, are still bipolar and segregate chromosomes. Examination of microtubule nucleation during spindle assembly shows that microtubules fail to nucleate from chromosomes. Thus, chromosome nucleation is not essential for bipolarity during human cell mitosis when centrosomes are present. Rather, chromosome nucleation is involved in spindle pole separation and setting spindle length. A second Aurora A–independent function of TPX2 is required to bipolarize spindles

Correct spindle elongation at the metaphase/anaphase transition is an APC-dependent event in budding yeast

At the metaphase to anaphase transition, chromosome segregation is initiated by the splitting of sister chromatids. Subsequently, spindles elongate, separating the sister chromosomes into two sets. Here, we investigate the cell cycle requirements for spindle elongation in budding yeast using mutants affecting sister chromatid cohesion or DNA replication. We show that separation of sister chromatids is not sufficient for proper spindle integrity during elongation. Rather, successful spindle elongation and stability require both sister chromatid separation and anaphase-promoting complex activation. Spindle integrity during elongation is dependent on proteolysis of the securin Pds1 but not on the activity of the separase Esp1. Our data suggest that stabilization of the elongating spindle at the metaphase to anaphase transition involves Pds1-dependent targets other than Esp1

Salt-dependent rheology and surface tension of protein condensates using optical traps

An increasing number of proteins with intrinsically disordered domains have been shown to phase separate in buffer to form liquid-like phases. These protein condensates serve as simple models for the investigation of the more complex membrane-less organelles in cells. To understand the function of such proteins in cells, the material properties of the condensates they form are important. However, these material properties are not well understood. Here, we develop a novel method based on optical traps to study the frequency-dependent rheology and the surface tension of PGL-3 condensates as a function of salt concentration. We find that PGL-3 droplets are predominantly viscous but also exhibit elastic properties. As the salt concentration is reduced, their elastic modulus, viscosity and surface tension increase. Our findings show that salt concentration has a strong influence on the rheology and dynamics of protein condensates suggesting an important role of electrostatic interactions for their material properties.Comment: 5 pages, 3 figures, 1 supplemen