Antiparallel Crosslinker Research Articles

Ionic strength alters crosslinker-driven self-organization of microtubules.

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.

The effect of ionic strength in microtubule tactoid formation

Cell division via mitosis is one of the most important biological processes to sustain life. Microtubules, along with their associated proteins, crosslinkers, and enzymes, play a crucial role in the formation of the mitotic spindle. Previous studies on self-organization of microtubules showed that, in presence of polymer crowding agents, microtubules self-organize into bundles. Introducing certain concentrations of an antiparallel crosslinker, MAP65, in the system resulted in the bundles coalescing into homogenous, birefringent tactoids.

Actin Controls the Dynamics and Microtubule Crosslinkers Tune Co-localization in Crosslinked Composite Actin-microtubule Networks

The cytoskeleton is a complex, active biomaterial system with the ability to control strain, localize objects in space and time, and self-organize and self-repair. Recent advances in composite networks of actin filaments and microtubules are better replicating cellular systems and promoting new avenues for biomaterials research in composite materials. Each type of filament comes with a host of accessory proteins and enzymes that can enable a wide array of activities. Here, we combine actin and microtubule filaments in the presence of increasing crosslinkers. For actin, we use permanent biotin-streptavidin crosslinkers to bundle and strengthen the actin network. For microtubules, we use the antiparallel crosslinker, MAP65. The presence of high concentrations of MAP65 causes actin to co-localize with microtubules. We find that the mobility of filaments in the network is dominated by the degree of crosslinking by actin filaments. The effect of microtubule crosslinking only affects filament mobility at intermediate actin crosslinking. This work demonstrates that actin filaments serve as the component that controls network dynamics, whereas microtubules have more control over network organization and localization.

The Tail of XCTK2 Contains Two Distinct Microtubule Binding Domains

Molecular motors organize the microtubules (MTs) that make up the mitotic spindle, and defects in motor function can cause improper spindle assembly that leads to chromosome mis-segregation. The Kinesin-14 family of molecular motors facilitates spindle pole focusing by cross-linking and sliding anti-parallel and parallel MTs through their motor and tail domains. How the tail contributes to these functions is unclear. Kinesin-14 tail MT binding is inhibited by importin α/β association through a nuclear localization signal (NLS). To understand how importin α/β association regulates the cross-linking and sliding activity of Kinesin-14s, we visualized Xenopus XCTK2 cross-linking and sliding in vitro. Surprisingly, importin α/β preferentially inhibited anti-parallel cross-linking and sliding. These results suggest that the tail may contain two different MT binding domains (MBDs) that recognize the anti-parallel and parallel MT orientations. To test this hypothesis, we created a deletion series of proteins from the N-terminus and C-terminus of the tail and tested them for MT binding. We identified two MBDs, which we call MBD1 and MBD2, that had MT binding abilities similar to the full-length tail. YPet-tagged MBD1, which contains the NLS, displayed robust FRET with importin α-CyPet and importin β, indicating tight association; whereas, MBD2 did not. In the presence of importin α/β, MBD1 was also completely inhibited from binding to MTs. These results are consistent with the model that MBD1 recognizes the anti-parallel conformation, and MBD2 recognizes the parallel conformation. We are currently generating site-specific mutations within MBD1 and MBD2 to inhibit MT binding so that we can demonstrate separation of cross-linking activities in the full-length protein. Once we identify the mutations that disrupt anti-parallel or parallel MT cross-linking, we will test how these modes of cross-linking and sliding contribute to spindle assembly.

Self-organization of spindle-like microtubule structures.

Microtubule self-organization is an essential physical process underlying several essential cellular functions, including cell division. In cell division, the dominant arrangement is the mitotic spindle, a football-shaped microtubule-based machine responsible for separating the chromosomes. We are interested in the underlying fundamental principles behind the self-organization of the spindle shape. Prior biological works have hypothesized that motor proteins control the proper formation of the spindle. Many of these motor proteins are also microtubule-crosslinkers, so it is unclear if the critical 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 arrangements at relatively low concentration without the need for motor activity. Surprisingly, the length of the microtubules only moderately affects the equilibrium phase. 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.

Complexes Made between Microtubules, Motor Proteins, and Antiparallel Crosslinkers Produce Dynamic Interactions

Microtubules constitute the rigid, yet dynamic, network of the cytoskeleton, and are responsible for creating tracks for cargo transport. Important processes of the cell, such as cell division, cell motility, cell homeostasis, and cell signaling are driven by interactions between microtubules, motor proteins, and microtubule associated proteins. Using in vitro reconstitution of microtubules bundled by MAP65, an antiparallel crosslinker, in kinesin gliding assays, we seek to explore how complex, active biological networks self-organize. We systematically changed the concentration of kinesin, MAP65, and microtubules and observed dynamic motility activities of the condensed states. Networks of microtubules with low MAP65 concentrations can be pulled apart completely by kinesin motors. In contrast, we observe inhibition of motility in high crosslinker concentrations. Cell-like networks that can mimic types of cell motility are observed with intermediate MAP65 concentrations. We quantified the motion by tracking the edge of the condensed state over time and find that it spreads with a velocity proportional to the amount of crosslinker in the system. When the microtubule concentration is varied, we find an interesting regime where the velocity is negative, indicative of contraction, which is rarely observed for microtubule-kinesin active matter systems.

Structural Basis for Microtubule Binding by the Anti-Parallel Microtubule Crosslinker PRC1 (Protein Regulator of Cytokinesis-1)

Successful cytokinesis is essential for maintaining genome stability. The central spindle, an array of antiparallel microtubules formed prior to cytokinesis, is required for complete furrow ingression to form two daughter cells. Members of the conserved MAP65 family (Ase1, AtMAP-65, PRC1) play a key role in organizing microtubules in the central spindle. Invitro studies indicate that members of this protein family can crosslink microtubules with an anti-parallel orientation bias. We have combined structural methods and single molecule fluorescence to understand the structural basis for biased crosslinking by the human MAP65 protein PRC1.We describe the crystal structure of the conserved domain of PRC1. This domain is capable of binding microtubules (Kd ∼ 6 uM), but shows no sequence similarity to other known microtubule binding proteins. This domain has a spectrin fold, a motif ubiquitous in proteins interacting with the actin cytoskeleton, but not seen previously in microtubule binding proteins. Cryo-EM and helical image analysis yielded a 3D map of PRC1 bound to microtubules. Docking the crystal structure into the EM map gives us a structural view of PRC1-microtubule interactions.PRC1 also has a proteolytically sensitive Lys/Arg rich domain that is thought to regulate microtubule binding. Single-molecule assays reveal that the presence of this domain allows the protein to undergo long diffusional sliding on microtubules. Constructs in which this domain is truncated show reduced interaction-lifetime and crosslinking efficiency.These results suggests that the two domains act together to achieve biased anti-parallel crosslinking. We propose a model where oriented binding and polarity bias is achieved by the spectrin domain and the positively charged flexible domain enhances the probability of biased crosslinking by increasing the dwell time of PRC1 on microtubules via a diffusive binding mechanism.