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

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

Structural studies of the Doublecortin family of MAPs

Doublecortin (DCX) is a microtubule (MT)-stabilizing protein essential for neuronal migration during human brain development. Missense mutations in DCX cause severe brain defects. This implies that the many other MT-stabilizing proteins in neurons cannot compensate for DCX function. To understand the unusual properties of DCX, we expressed the recombinant human DCX in Sf9 cells and undertook structural characterization of its interaction with MTs using cryo-electron microscopy. DCX specifically nucleates 13-protofilament (13-pf) MTs, the architecture of human MTs in vivo. Cryo-electron tomography (cryo-ET) of DCX-nucleated MTs showed that they are primarily built from B-lattice contacts interrupted by a single discontinuity, the seam. Because of this asymmetry, we used single-particle reconstruction and determined the 8 Å structure of DCX-stabilized 13-pf MTs in the absence of a stabilizing drug. The DCX-binding site, at the corner of four tubulin dimers, is ideally suited to stabilize both lateral and longitudinal tubulin lattice contacts. Its precise geometry suggests that DCX is sensitive to the angle between pfs, and thereby provides insight into the specificity of DCX for 13-pf MT architecture. DCX’s precise interaction at the corner of four tubulin dimers also means that DCX does not bind the MT seam. Our work has provided mechanistic insight into the evolutionarily conserved DCX family of MT-stabilizing proteins and also into more general regulatory mechanisms of the MT cytoskeleton

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

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

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.National Institutes of Health (U.S.) (Grant NIH-P30-HD-18655)National Institutes of Health (U.S.) (Grant 2T32NS007473-11)National Institutes of Health (U.S.) (Grant 2T32NS007484-11)National Institutes of Health (U.S.) (Grant 1F32D070549-01)National Science Foundation (U.S.) (CAREER Award)National Institutes of Health (U.S.) (Grant 5R21NS063185-02