Cellular structures are established and maintained through a dynamic interplay between assembly and regulatory processes. Self-organization of molecular components provides a variety of possible spatial structures: the regulatory machinery chooses the most appropriate to express a given cellular function. Here we study the extent and the characteristics of self-organization using microtubules and molecular motors as a model system. These components are known to participate in the formation of many cellular structures, such as the dynamic asters found in mitotic and meiotic spindles. Purified motors and microtubules have previously been observed to form asters in vitro. We have reproduced this result with a simple system consisting solely of multi-headed constructs of the motor protein kinesin and stabilized microtubules. We show that dynamic asters can also be obtained from a homogeneous solution of tubulin and motors. By varying the relative concentrations of the components, we obtain a variety of self-organized structures. Further, by studying this process in a constrained geometry of micro-fabricated glass chambers, we demonstrate that the same final structure can be reached through different assembly 'pathways.

In eukaryotic cells, microtubules and their associated motor proteins can be organized into various large-scale patterns. Using a simplified experimental system combined with computer simulations, we examined how the concentrations and kinetic parameters of the motors contribute to their collective behavior. We observed self-organization of generic steady-state structures such as asters, vortices, and a network of interconnected poles. We identified parameter combinations that determine the generation of each of these structures. In general, this approach may become useful for correlating the morphogenetic phenomena taking place in a biological system with the biophysical characteristics of its constituents.

The microtubule cytoskeleton is essential to cell morphogenesis. Growing microtubule plus ends have emerged as dynamic regulatory sites in which specialized proteins, called plus-end-binding proteins (+TIPs), bind and regulate the proper functioning of microtubules. However, the molecular mechanism of plus-end association by +TIPs and their ability to track the growing end are not well understood. Here we report the in vitro reconstitution of a minimal plus-end tracking system consisting of the three fission yeast proteins Mal3, Tip1 and the kinesin Tea2. Using time-lapse total internal reflection fluorescence microscopy, we show that the EB1 homologue Mal3 has an enhanced affinity for growing microtubule end structures as opposed to the microtubule lattice. This allows it to track growing microtubule ends autonomously by an end recognition mechanism. In addition, Mal3 acts as a factor that mediates loading of the processive motor Tea2 and its cargo, the Clip170 homologue Tip1, onto the microtubule lattice. The interaction of all three proteins is required for the selective tracking of growing microtubule plus ends by both Tea2 and Tip1. Our results dissect the collective interactions of the constituents of this plus-end tracking system and show how these interactions lead to the emergence of its dynamic behaviour. We expect that such in vitro reconstitutions will also be essential for the mechanistic dissection of other plus-end tracking systems.

During cell division, microtubules are arranged in a large bipolar structure, the mitotic spindle, to segregate the duplicated chromosomes. Antiparallel microtubule overlaps in the spindle center are essential for establishing bipolarity and maintaining spindle stability throughout mitosis. In anaphase, this antiparallel microtubule array is tightly bundled forming the midzone, which serves as a hub for the recruitment of proteins essential for late mitotic events. The molecular mechanism of midzone formation and the control of its size are not understood. Using an in vitro reconstitution approach, we show here that PRC1 autonomously bundles antiparallel microtubules and recruits Xklp1, a kinesin-4, selectively to overlapping antiparallel microtubules. The processive motor Xklp1 controls overlap size by overlap length-dependent microtubule growth inhibition. Our results mechanistically explain how the two conserved, essential midzone proteins PRC1 and Xklp1 cooperate to constitute a minimal protein module capable of dynamically organizing the core structure of the central anaphase spindle.

SummaryGrowing microtubule ends serve as transient binding platforms for essential proteins that regulate microtubule dynamics and their interactions with cellular substructures. End-binding proteins (EBs) autonomously recognize an extended region at growing microtubule ends with unknown structural characteristics and then recruit other factors to the dynamic end structure. Using cryo-electron microscopy, subnanometer single-particle reconstruction, and fluorescence imaging, we present a pseudoatomic model of how the calponin homology (CH) domain of the fission yeast EB Mal3 binds to the end regions of growing microtubules. The Mal3 CH domain bridges protofilaments except at the microtubule seam. By binding close to the exchangeable GTP-binding site, the CH domain is ideally positioned to sense the microtubule's nucleotide state. The same microtubule-end region is also a stabilizing structural cap protecting the microtubule from depolymerization. This insight supports a common structural link between two important biological phenomena, microtubule dynamic instability and end tracking.