Self-Organization of Minimal Anaphase Spindle Midzone Bundles.

In anaphase spindles, antiparallel microtubules associate to form tight midzone bundles, as required for functional spindle architecture and correct chromosome segregation. Several proteins selectively bind to these overlaps to control cytokinesis. How midzone bundles assemble is poorly understood. Here, using an in vitro reconstitution approach, we demonstrate that minimal midzone bundles can reliably self-organize in solution from dynamic microtubules, the microtubule crosslinker PRC1, and the motor protein KIF4A. The length of the central antiparallel overlaps in these microtubule bundles is similar to that observed in cells and is controlled by the PRC1/KIF4A ratio. Experiments and computer simulations demonstrate that minimal midzone bundle formation results from promoting antiparallel microtubule crosslinking, stopping microtubule plus-end dynamicity, and motor-driven midzone compaction and alignment. The robustness of this process suggests that a similar self-organization mechanism may contribute to the reorganization of the spindle architecture during the metaphase to anaphase transition in cells.

Micropattern-guided assembly of overlapping pairs of dynamic microtubules.

Interactions between antiparallel microtubules are essential for the organization of spindles in dividing cells. The ability to form immobilized antiparallel microtubule pairs in vitro, combined with the ability to image them via TIRF microscopy, permits detailed biochemical characterization of microtubule cross-linking proteins and their effects on microtubule dynamics. Here, we describe methods for chemical micropatterning of microtubule seeds on glass surfaces in configurations that specifically promote the formation of antiparallel microtubule overlaps in vitro. We demonstrate that this assay is especially well suited for reconstitution of minimal midzone overlaps stabilized by the antiparallel microtubule cross-linking protein PRC1 and its binding partners. The micropatterning method is suitable for use with a broad range of proteins, and the assay is generally applicable to any microtubule cross-linking protein.

Seeded microtubule growth for cryoelectron microscopy of end-binding proteins.

End-binding proteins (EBs) have the ability to autonomously track the ends of growing microtubules, where they recruit several proteins that control various aspects of microtubule cytoskeleton organization and function. The structural nature of the binding site recognized by EBs at growing microtubule ends has been a subject of debate. Recently, a fluorescence microscopy assay used for the study of dynamic end tracking in vitro was adapted for cryoelectron microscopy (cryo-EM). In combination with single-particle reconstruction methods, this modified assay was used to produce the first subnanometer-resolution model of how the microtubule-binding domain of EBs binds to microtubules grown in the presence of GTPγS. A GTPγS microtubule can be considered a static mimic of the transiently existing binding region of EBs at a microtubule end growing in the presence of GTP. Here we describe in detail the procedure used to generate these samples. It relies on the polymerization of microtubules from preformed stabilized and quantum dot-labeled microtubule seeds. This allows the cryo-EM analysis of proteins bound to paclitaxel-free microtubules. It provides freedom for using different GTP analogues during microtubule elongation independent of their nucleation properties. This assay could also be useful for the cryo-EM analysis of other microtubule-associated proteins.

New insights into genotype-phenotype correlations for the doublecortin-related lissencephaly spectrum.

X-linked isolated lissencephaly sequence and subcortical band heterotopia are allelic human disorders associated with mutations of doublecortin (DCX), giving both familial and sporadic forms. DCX encodes a microtubule-associated protein involved in neuronal migration during brain development. Structural data show that mutations can fall either in surface residues, likely to impair partner interactions, or in buried residues, likely to impair protein stability. Despite the progress in understanding the molecular basis of these disorders, the prognosis value of the location and impact of individual DCX mutations has largely remained unclear. To clarify this point, we investigated a cohort of 180 patients who were referred with the agyria-pachygyria subcortical band heterotopia spectrum. DCX mutations were identified in 136 individuals. Analysis of the parents' DNA revealed the de novo occurrence of DCX mutations in 76 cases [62 of 70 females screened (88.5%) and 14 of 60 males screened (23%)], whereas in the remaining cases, mutations were inherited from asymptomatic (n = 14) or symptomatic mothers (n = 11). This represents 100% of families screened. Female patients with DCX mutation demonstrated three degrees of clinical-radiological severity: a severe form with a thick band (n = 54), a milder form (n = 24) with either an anterior thin or an intermediate thickness band and asymptomatic carrier females (n = 14) with normal magnetic resonance imaging results. A higher proportion of nonsense and frameshift mutations were identified in patients with de novo mutations. An analysis of predicted effects of missense mutations showed that those destabilizing the structure of the protein were often associated with more severe phenotypes. We identified several severe- and mild-effect mutations affecting surface residues and observed that the substituted amino acid is also critical in determining severity. Recurrent mutations representing 34.5% of all DCX mutations often lead to similar phenotypes, for example, either severe in sporadic subcortical band heterotopia owing to Arg186 mutations or milder in familial cases owing to Arg196 mutations. Taken as a whole, these observations demonstrate that DCX-related disorders are clinically heterogeneous, with severe sporadic and milder familial subcortical band heterotopia, each associated with specific DCX mutations. There is a clear influence of the individual mutated residue and the substituted amino acid in determining phenotype severity.

End-binding proteins and Ase1/PRC1 define local functionality of structurally distinct parts of the microtubule cytoskeleton.

The microtubule cytoskeleton is crucial for the intracellular organization of eukaryotic cells. It is a dynamic scaffold that has to perform a variety of very different functions. This multitasking is achieved through the activity of numerous microtubule-associated proteins. Two prominent classes of proteins are central to the selective recognition of distinct transiently existing structural features of the microtubule cytoskeleton. They define local functionality through tightly regulated protein recruitment. Here we summarize the recent developments in elucidating the molecular mechanism underlying the action of microtubule end-binding proteins (EBs) and antiparallel microtubule crosslinkers of the Ase1/PRC1 family that represent the core of these two recruitment modules. Despite their fundamentally different activities, these conserved families share several common features.

Molecular basis for specific regulation of neuronal kinesin-3 motors by doublecortin family proteins.

Doublecortin (Dcx) defines a growing family of microtubule (MT)-associated proteins (MAPs) involved in neuronal migration and process outgrowth. We show that Dcx is essential for the function of Kif1a, a kinesin-3 motor protein that traffics synaptic vesicles. Neurons lacking Dcx and/or its structurally conserved paralogue, doublecortin-like kinase 1 (Dclk1), show impaired Kif1a-mediated transport of Vamp2, a cargo of Kif1a, with decreased run length. Human disease-associated mutations in Dcx's linker sequence (e.g., W146C, K174E) alter Kif1a/Vamp2 transport by disrupting Dcx/Kif1a interactions without affecting Dcx MT binding. Dcx specifically enhances binding of the ADP-bound Kif1a motor domain to MTs. Cryo-electron microscopy and subnanometer-resolution image reconstruction reveal the kinesin-dependent conformational variability of MT-bound Dcx and suggest a model for MAP-motor crosstalk on MTs. Alteration of kinesin run length by MAPs represents a previously undiscovered mode of control of kinesin transport and provides a mechanism for regulation of MT-based transport by local signals.

EBs recognize a nucleotide-dependent structural cap at growing microtubule ends.

Growing 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.

Snapshots of kinesin motors on microtubule tracks.

Kinesin motors couple ATP hydrolysis to movement along microtubules, which act both as tracks and as activators of kinesin ATPase activity. Cryo-electron microscopy and image processing enables generation of three-dimensional snapshots of kinesin motors on their tracks at different stages of their ATPase cycle, and can reveal their motor mechanisms at secondary structure resolution. Here, we describe in detail the methods and conditions employed in our lab to prepare high-quality frozen-hydrated samples, which yield structural insights into kinesin motor mechanisms.

Template-free 13-protofilament microtubule-MAP assembly visualized at 8 A resolution.

Microtubule-associated proteins (MAPs) are essential for regulating and organizing cellular microtubules (MTs). However, our mechanistic understanding of MAP function is limited by a lack of detailed structural information. Using cryo-electron microscopy and single particle algorithms, we solved the 8 Å structure of doublecortin (DCX)-stabilized MTs. Because of DCX's unusual ability to specifically nucleate and stabilize 13-protofilament MTs, our reconstruction provides unprecedented insight into the structure of MTs with an in vivo architecture, and in the absence of a stabilizing drug. DCX specifically recognizes the corner of four tubulin dimers, a binding mode ideally suited to stabilizing both lateral and longitudinal lattice contacts. A striking consequence of this is that DCX does not bind the MT seam. DCX binding on the MT surface indirectly stabilizes conserved tubulin-tubulin lateral contacts in the MT lumen, operating independently of the nucleotide bound to tubulin. DCX's exquisite binding selectivity uncovers important insights into regulation of cellular MTs.