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Abstract The microtubule cytoskeleton is a major structural element inside cells that directs self‐organization using microtubule‐associated proteins and motors. It has been shown that finite‐sized, spindle‐like microtubule organizations, called “tactoids,” can form in vitro spontaneously from mixtures of tubulin and the antiparallel crosslinker, MAP65, from the MAP65/PRC1/Ase family. Here, we probe the ability of MAP65 to form tactoids as a function of the ionic strength of the buffer to attempt to break the electrostatic interactions binding MAP65 to microtubules and inter‐MAP65 binding. We observe that, with increasing monovalent salts, the organizations change from finite tactoids to unbounded length bundles, yet the MAP65 binding and crosslinking appear to stay intact. We further explore the effects of ionic strength on the dissociation constant of MAP65 using both microtubule pelleting and single‐molecule binding assays. We find that salt can reduce the binding, yet salt never negates it. Instead, we believe that the salt is affecting the ability of the MAP65 to form phase‐separated droplets, which cause the nucleation and growth of tactoids, as recently demonstrated. 

Intracellular transport of cargoes in the cell is essential for the organization and functioning cells, especially those that are large and elongated. The cytoskeletal networks inside large cells can be highly complex, and this cytoskeletal organization can have impacts on the distance and trajectories of travel. Here, we experimentally created microtubule networks with varying mesh sizes and examined the ability of kinesin-driven quantum dot cargoes to traverse the network. Using the experimental data, we deduced parameters for cargo detachment at intersections and away from intersections, allowing us to create an analytical theory for the run length as a function of mesh size. We also used these parameters to perform simulations of cargoes along paths extracted from the experimental networks. We find excellent agreement between the trends in run length, displacement, and trajectory persistence length comparing the experimental and simulated trajectories. 

During anaphase, antiparallel–overlapping midzone microtubules elongate and form bundles, contributing to chromosome segregation and the location of contractile ring formation. Midzone microtubules are dynamic in early but not late anaphase; however, the kinetics and mechanisms of stabilization are incompletely understood. Using photoactivation of cells expressing PA-EGFP-α-tubulin we find that immediately after anaphase onset, a single highly dynamic population of midzone microtubules is present; as anaphase progresses, both dynamic and stable populations of midzone microtubules coexist. By mid-cytokinesis, only static, non-dynamic microtubules are detected. The velocity of microtubule sliding also de-creases as anaphase progresses, becoming undetectable by late anaphase. Following depletion of PRC1, midzone microtubules remain highly dynamic in anaphase and fail to form static arrays in telophase despite furrowing. Cells depleted of Kif4a contain elongated PRC1overlap zones and fail to form static arrays in telophase. Cells blocked in cytokinesis form short PRC1 overlap zones that do not coalesce laterally; these cells also fail to form static arrays in telophase. Together, our results demonstrate that dynamic turnover and sliding of midzone microtubules is gradually reduced during anaphase and that the final transition to astatic array in telophase requires both lateral and longitudinal compaction of PRC1 containing overlap zones. 

The cytoskeleton is a major focus of physical studies to understand organization inside cells given its primary role in cell motility, cell division, and cell mechanics. Recently, protein condensation has been shown to be another major intracellular organizational strategy. Here, we report that the microtubule crosslinking proteins, MAP65-1 and PRC1, can form phase separated condensates at physiological salt and temperature without additional crowding agents in vitro. The size of the droplets depends on the concentration of protein. MAP65 condensates are liquid at first and can gelate over time. We show that these condensates can nucleate and grow microtubule bundles that form asters, regardless of the viscoelasticity of the condensate. The droplet size directly controls the number of projections in the microtubule asters, demonstrating that the MAP65 concentration can control the organization of microtubules. When gel-like droplets nucleate and grow asters from a shell of tubulin at the surface, the microtubules are able to re-fluidize the MAP65 condensate, returning the MAP65 molecules to solution. This work implies that there is an interplay between condensate formation from microtubule-associated proteins, microtubule organization, and condensate dissolution that could be important for the dynamics of intracellular organization. 

ABSTRACT During anaphase, a microtubule-containing structure called the midzone forms between the segregating chromosomes. The midzone is composed of an antiparallel array of microtubules and numerous microtubule-associated proteins that contribute to midzone formation and function. In many cells, the midzone is an important source of signals that specify the location of contractile ring assembly and constriction. The midzone also contributes to the events of anaphase by generating forces that impact chromosome segregation and spindle elongation; some midzone components contribute to both processes. The results of recent experiments have increased our understanding of the importance of the midzone, a microtubule array that has often been overlooked. This Journal of Cell Science at a Glance article will review, and illustrate on the accompanying poster, the organization, formation and dynamics of the midzone, and discuss open questions for future research.