Spontaneous self-organization of active matter has been demonstrated in a number of biological systems including bacteria, cells, and cytoskeletal filaments. Cytoskeletal filaments act as active polar rods when they are propelled along a glass surface via motor proteins. Actin has previously been shown to display polar or nematic ordering, whereas microtubules have been shown to create large vortices. For the first time, we combine both the actin and microtubule gliding into a composite active system. In the absence of actin filaments, microtubule filament organization transitions from isotropic to nematic to polar as a function of filament density. We find that the presence of a crowder, methylcellulose, is essential for this transition. In the absence of microtubules, actin transitions from isotropic to nematic. In combination, microtubules are affected by the presence of actin and the overall density of the filaments, becoming entrained with the nematic alignment of actin. Actin filaments are not as affected by the presence of microtubules. These results serve as first step in exploring the rich emergent behavior that can result from composite active matter system with tunable particle properties, self-propulsion speeds, and interparticle interactions.

Microtubule self-organization is an important physical process underlying a number of essential cellular functions including cell division. In cell division, the dominant organization is the mitotic spindle, a football-shaped microtubule organization. We are interested in the underlying fundamental principles behind the self-organization of the spindle shape. Prior biological works have hypothesized that motor proteins are required for the proper formation of the spindle. Many of these motor proteins are also microtubule-crosslinkers, so it is unclear if the important aspect is the motor activity or the crosslinking. In this study, we seek to address this question by examining the self-organization of microtubules using crosslinkers alone. We use a minimal system composed of tubulin, an antiparallel microtubule-crosslinking protein, and a crowding agent to explore the phase space of organizations as a function of tubulin and crosslinker concentration. We find that the concentration of the antiparallel crosslinker, MAP65, has a significant effect on the organization and resulted in spindle-like organizations at relatively low concentration without the need for motor activity. Surprisingly, the length of the microtubules only moderately affects the equilibrium organization. We characterize both the shape and dynamics of these spindle-like organizations. We find that they are birefringent homogeneous tactoids. The microtubules have slow mobility, but the crosslinkers have fast mobility within the tactoids. These structures represent a first step in the recapitulation of self-organized spindles of microtubules that can be used as initial structures for further biophysical and active matter studies relevant to the biological process of cell division.

Microtubules are essential cellular structures, which are the basis for the mitotic spindle. We show that microtubule polymerization in the presence of a crosslinker results in spindle-like assemblies.

Correlating the location of subcellular structures with dynamic cellular behaviors is difficult when working with organisms that lack the molecular genetic tools needed for expressing fluorescent protein fusions. Here, we describe a protocol for fixing, permeabilizing, and staining cells in a single step while imaging on a microscope. In contrast to traditional, multi‐step fixing and staining protocols that take hours, the protocol outlined here achieves satisfactory staining within minutes. This approach takes advantage of well‐characterized small molecules that stain specific subcellular structures, including nuclei, mitochondria, and actin networks. Direct visualization of the entire process allows for rapid optimization of cell fixation and staining, as well as straightforward identification of fixation artifacts. Moreover, live imaging prior to fixation reveals the dynamic history of cellular features, making it particularly useful for model systems without the capacity for expressing fluorescent protein fusions. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Fixing, permeabilizing, and staining mammalian cells in one step on the microscope

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-EGF-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 midcytokinesis, only static, non-dynamic microtubules are detected. The velocity of microtubule sliding also decreases 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 zones of PRC1 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 a static array in telophase requires both lateral and longitudinal compaction of PRC1 containing overlap zones.

The microtubule cytoskeleton is essential for various biological processes such as the intracellular distribution of molecules and organelles, cell morphogenesis, chromosome segregation, and specification of the location of contractile ring formation. Distinct cell types contain microtubules with different extents of stability. For example, microtubules in neurons are highly stabilized to support organelle (or vesicular) transport over large distances, and microtubules in motile cells are more dynamic. In some cases, such as the mitotic spindle, both dynamic and stable microtubules coexist. Alteration of microtubule stability is connected to disease states, making understanding microtubule stability an important area of research. Methods to measure microtubule stability in mammalian cells are described here. Together, these approaches allow microtubule stability to be measured qualitatively or semiquantitatively following staining for post‐translational modifications of tubulin or treating cells with microtubule destabilizing agents such as nocodazole. Microtubule stability can also be measured quantitatively by performing fluorescence recovery after photobleaching or fluorescence photoactivation of tubulin in live cells. These methods should be helpful for those seeking to understand microtubule dynamics and stabilization. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Fixing and staining cells for tubulin post‐translational modifications Basic Protocol 2: Evaluating microtubule stability following treatment with nocodazole in live or fixed cells Basic Protocol 3: Measurement of microtubule dynamic turnover by quantification of fluorescence recovery after photobleaching Basic Protocol 4: Measurement of microtubule dynamic turnover by quantification of dissipation of fluorescence after photoactivation

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 decreases 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 PRC1 overlap 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 a static array in telophase requires both lateral and longitudinal compaction of PRC1 containing overlap zones.