Physical determinants of bipolar mitotic spindle assembly and stability in fission yeast.

Mitotic spindles use an elegant bipolar architecture to segregate duplicated chromosomes with high fidelity. Bipolar spindles form from a monopolar initial condition; this is the most fundamental construction problem that the spindle must solve. Microtubules, motors, and cross-linkers are important for bipolarity, but the mechanisms necessary and sufficient for spindle assembly remain unknown. We describe a physical model that exhibits de novo bipolar spindle formation. We began with physical properties of fission-yeast spindle pole body size and microtubule number, kinesin-5 motors, kinesin-14 motors, and passive cross-linkers. Our model results agree quantitatively with our experiments in fission yeast, thereby establishing a minimal system with which to interrogate collective self-assembly. By varying the features of our model, we identify a set of functions essential for the generation and stability of spindle bipolarity. When kinesin-5 motors are present, their bidirectionality is essential, but spindles can form in the presence of passive cross-linkers alone. We also identify characteristic failed states of spindle assembly-the persistent monopole, X spindle, separated asters, and short spindle, which are avoided by the creation and maintenance of antiparallel microtubule overlaps. Our model can guide the identification of new, multifaceted strategies to induce mitotic catastrophes; these would constitute novel strategies for cancer chemotherapy.

Contributions of Microtubule Dynamic Instability and Rotational Diffusion to Kinetochore Capture.

Microtubule dynamic instability allows search and capture of kinetochores during spindle formation, an important process for accurate chromosome segregation during cell division. Recent work has found that microtubule rotational diffusion about minus-end attachment points contributes to kinetochore capture in fission yeast, but the relative contributions of dynamic instability and rotational diffusion are not well understood. We have developed a biophysical model of kinetochore capture in small fission-yeast nuclei using hybrid Brownian dynamics/kinetic Monte Carlo simulation techniques. With this model, we have studied the importance of dynamic instability and microtubule rotational diffusion for kinetochore capture, both to the lateral surface of a microtubule and at or near its end. Over a range of biologically relevant parameters, microtubule rotational diffusion decreased capture time, but made a relatively small contribution compared to dynamic instability. At most, rotational diffusion reduced capture time by 25%. Our results suggest that while microtubule rotational diffusion can speed up kinetochore capture, it is unlikely to be the dominant physical mechanism for typical conditions in fission yeast. In addition, we found that when microtubules undergo dynamic instability, lateral captures predominate even in the absence of rotational diffusion. Counterintuitively, adding rotational diffusion to a dynamic microtubule increases the probability of end-on capture.

Microscopic origins of anisotropic active stress in motor-driven nematic liquid crystals.

The cytoskeleton, despite comprising relatively few building blocks, drives an impressive variety of cellular phenomena ranging from cell division to motility. These building blocks include filaments, motor proteins, and static crosslinkers. Outside of cells, these same components can form novel materials exhibiting active flows and nonequilibrium contraction or extension. While dipolar extensile or contractile active stresses are common in nematic motor-filament systems, their microscopic origin remains unclear. Here we study a minimal physical model of filaments, crosslinking motors, and static crosslinkers to dissect the microscopic mechanisms of stress generation in a two-dimensional system of orientationally aligned rods. We demonstrate the essential role of filament steric interactions which have not previously been considered to significantly contribute to active stresses. With this insight, we are able to tune contractile or extensile behavior through the control of motor-driven filament sliding and crosslinking. This work provides a roadmap for engineering stresses in active liquid crystals. The mechanisms we study may help explain why flowing nematic motor-filament mixtures are extensile while gelled systems are contractile.