Wave-like oscillations of clamped microtubules driven by collective dynein transport.

Microtubules (MTs) are observed to move and buckle driven by ATP-dependent molecular motors in both mitotic and interphasic eukaryotic cells as well as in specialized structures such as flagella and cilia with a stereotypical geometry. In previous work, clamped MTs driven by a few kinesin motors were seen to buckle and occasionally flap in what was referred to as flagella-like motion. Theoretical models of active-filament dynamics and a following force have predicted that, with sufficient force and binding-unbinding, such clamped filaments should spontaneously undergo periodic buckling oscillations. However, a systematic experimental test of the theory and reconciliation to a model was lacking. Here, we have engineered a minimal system of MTs clamped at their plus ends and transported by a sheet of dynein motors that demonstrate the emergence of spontaneous traveling-wave oscillations along single filaments. The frequencies of tip oscillations are in the millihertz range and are statistically indistinguishable in the onset and recovery phases. We develop a 2D computational model of clamped MTs binding and unbinding stochastically to motors in a "gliding-assay" geometry. The simulated MTs oscillate with a frequency comparable to experiment. The model predicts the effect of MT length and motor density on qualitative transitions between distinct phases of flapping, regular oscillations, and looping. We develop an effective "order parameter" based on the relative deflection along the filament and orthogonal to it. The transitions predicted in simulations are validated by experimental data. These results demonstrate a role for geometry, MT buckling, and collective molecular motor activity in the emergence of oscillatory dynamics.

Collective dynein transport of the nucleus by pulling on astral microtubules during Saccharomyces cerevisiae mitosis.

Positioning the nucleus at the bud neck during Saccharomyces cerevisiae mitosis involves pulling forces of cytoplasmic dynein localized in the daughter cell. Although genetic analysis has revealed a complex network positioning the nucleus, quantification of the forces acting on the nucleus and the number of dyneins driving the process has remained difficult. To better understand the collective forces involved in nuclear positioning, we compare a model of dyneins-driven microtubule (MT) pulling, MT pushing, and cytoplasmic drag to experiments. During S. cerevisiae mitosis, MTs interacting with the cortex nucleated by the daughter spindle pole body (SPB) (SPB-D) are longer than the mother SPB (SPB-M), increasing further during spindle elongation in anaphase. Interphasic SPB mobility is effectively diffusive, while the mitotic mobility is directed. By optimizing a computational model of the mobility of the nucleus due to diffusion and MTs pushing at the cell membrane to experiment, we estimate the viscosity governing the drag force on nuclei during positioning. A force balance model of mitotic SPB mobility compared to experimental mobility suggests that even one or two dynein dimers are sufficient to move the nucleus in the bud neck. Using stochastic computer simulations of a budding cell, we find that punctate dynein localization can generate sufficient force to reel in the nucleus to the bud neck. Compared to uniform motor localization, puncta involve fewer motors suggesting a functional role for motor clustering. Stochastic simulations also suggest that a higher number of force generators than predicted by force balance may be required to ensure the robustness of spindle positioning.

Aster swarming by symmetry breaking of cortical dynein transport and coupling kinesins.

Microtubule (MT) radial arrays or asters establish the internal topology of a cell by interacting with organelles and molecular motors. We proceed to understand the general pattern forming potential of aster-motor systems using a computational model of multiple MT asters interacting with motors in cellular confinement. In this model dynein motors are attached to the cell cortex and plus-ended motors resembling kinesin-5 diffuse in the cell interior. The introduction of 'noise' in the form of MT length fluctuations spontaneously results in the emergence of coordinated, achiral vortex-like rotation of asters. The coherence and persistence of rotation require a threshold density of both cortical dyneins and coupling kinesins, while the onset is diffusion-limited with relation to the cortical dynein mobility. The coordinated rotational motion emerges due to the resolution of a 'tug-of-war' of multiple cortical dynein motors bound to MTs of the same aster by 'noise' in the form of MT dynamic instability. This transient symmetry breaking is amplified by local coupling by kinesin-5 complexes. The lack of widespread aster rotation across cell types suggests that biophysical mechanisms that suppress such intrinsic dynamics may have evolved. This model is analogous to more general models of locally coupled self-propelled particles (SPP) that spontaneously undergo collective transport in the presence of 'noise' that have been invoked to explain swarming in birds and fish. However, the aster-motor system is distinct from SPP models with regard to the particle density and 'noise' dependence, providing a set of experimentally testable predictions for a novel sub-cellular pattern forming system.

Collective effects of yeast cytoplasmic dynein based microtubule transport.

Teams of cortically anchored dyneins pulling at microtubules (MTs) are known to be essential for aster, spindle and nuclear positioning during cell division and fertilization. While the single-molecule basis of dynein processivity is now better understood, the effect of increasing numbers of motors on transport is not clear. Here, we examine the collective transport properties of a Saccharomyces cerevisiae cytoplasmic dynein fragment, widely used as a minimal model, by a combination of quantitative MT gliding assays and stochastic simulations. We find both MT lengths and motor densities qualitatively affect the degree of randomness of MT transport. However, the directionality and velocity of MTs increase above a threshold number of motors (N) interacting with a filament. To better understand this behavior, we simulate a gliding assay based on a model of uniformly distributed immobilized motors transporting semi-flexible MTs. Each dynein dimer is modeled as an effective stochastic stepper with asymmetric force dependent detachment dynamics, based on single-molecule experiments. Simulations predict increasing numbers of motors (N) result in a threshold dependent transition in directionality and transport velocity and a monotonic decrease in effective diffusivity. Thus both experiment and theory show qualitative agreement in the emergence of coordination in transport above a threshold number of motor heads. We hypothesize that the phase-transition like property of this dynein could play a role in vivo during yeast mitosis, when this dynein localizes to the cortex and pulls astral MTs of increasing length, resulting in correct positioning and orientation of the nucleus at the bud-neck.

A Motor-Gradient and Clustering Model of the Centripetal Motility of MTOCs in Meiosis I of Mouse Oocytes.

Asters nucleated by Microtubule (MT) organizing centers (MTOCs) converge on chromosomes during spindle assembly in mouse oocytes undergoing meiosis I. Time-lapse imaging suggests that this centripetal motion is driven by a biased 'search-and-capture' mechanism. Here, we develop a model of a random walk in a drift field to test the nature of the bias and the spatio-temporal dynamics of the search process. The model is used to optimize the spatial field of drift in simulations, by comparison to experimental motility statistics. In a second step, this optimized gradient is used to determine the location of immobilized dynein motors and MT polymerization parameters, since these are hypothesized to generate the gradient of forces needed to move MTOCs. We compare these scenarios to self-organized mechanisms by which asters have been hypothesized to find the cell-center- MT pushing at the cell-boundary and clustering motor complexes. By minimizing the error between simulation outputs and experiments, we find a model of "pulling" by a gradient of dynein motors alone can drive the centripetal motility. Interestingly, models of passive MT based "pushing" at the cortex, clustering by cross-linking motors and MT-dynamic instability gradients alone, by themselves do not result in the observed motility. The model predicts the sensitivity of the results to motor density and stall force, but not MTs per aster. A hybrid model combining a chromatin-centered immobilized dynein gradient, diffusible minus-end directed clustering motors and pushing at the cell cortex, is required to comprehensively explain the available data. The model makes experimentally testable predictions of a spatial bias and self-organized mechanisms by which MT asters can find the center of a large cell.