Novel roles for BicD in pronuclear fusion and meiosis II progression via localization of the CHC/TACC/Msps complex to MII spindles

ABSTRACTVertebrate Clathrin heavy chain (Chc) plays a moonlighting function during mitosis. Chc forms a complex with TACC3 (Transforming Acidic Coiled Coil 3) and ch-TOG (colonic hepatic tumor overexpressed gene) at the spindle microtubules, forming inter microtubule bridges that stabilize the K-fibers. Since Drosophila Chc is a cargo of the dynein adaptor Bicaudal-D (BicD), we investigated whether BicD regulates Clathrin function at the spindle. We found that BicD localizes, like Chc, to centrosomes and spindles during mitosis and meiosis II, and that Chc interacts with Drosophila TACC (D-TACC). Using deGradFP to reduce the activity of BicD in mature eggs and very young embryos, we uncovered a novel function of BicD in meiosis II. The affected meiosis II products underwent abnormal rounds of additional replications and failed to carry out pronuclear fusion. Pointing to a mechanism, we found that the localization of Clathrin/D-TACC/Minispindles (Msps, homolog of ch-TOG) to the meiosis II spindles was impaired upon BicD knockdown. Furthermore, the meiotic products showed abnormal staining for PH3 and reduced recruitment of spindle assembly checkpoint (SAC) components. Altogether, our results support the notion that BicD performs a key activity in assembling the meiotic spindle apparatus. This function of BicD seems conserved in evolution because C. elegans embryos with reduced activities of these genes developed comparable phenotypes.

Microtubule polymerase and processive plus-end tracking functions originate from distinct features within TOG domain arrays

XMAP215/Stu2/Alp14 accelerates tubulin polymerization while processively tracking microtubule (MT) plus ends via tumor overexpressed gene (TOG) domain arrays. It remains poorly understood how these functions arise from tubulin recruitment, mediated by the distinct TOG1 and TOG2 domains, or the assembly of these arrays into large square complexes. Here, we describe a relationship between MT plus-end tracking and polymerase functions revealing their distinct origin within TOG arrays. We study Alp14 mutants designed based on structural models, with defects in either tubulin recruitment or self-organization. Using in vivo live imaging in fission yeast and in vitro MT dynamics assays, we show that tubulins recruited by TOG1 and TOG2 serve concerted, yet distinct, roles in MT plus-end tracking and polymerase functions. TOG1 is critical for processive plus-end tracking, whereas TOG2 is critical for accelerating tubulin polymerization. Inactivating interfaces that stabilize square complexes lead to defects in both processive MT plus-end tracking and polymerase. Our studies suggest that a dynamic cycle between square and unfurled TOG array states gives rise to processive polymerase activity at MT plus ends.

Structural basis of tubulin recruitment and assembly by microtubule polymerases with tumor overexpressed gene (TOG) domain arrays

XMAP215/Stu2/Alp14 proteins accelerate microtubule plus-end polymerization by recruiting tubulins via arrays of tumor overexpressed gene (TOG) domains, yet their mechanism remains unknown. Here, we describe the biochemical and structural basis for TOG arrays in recruiting and polymerizing tubulins. Alp14 binds four tubulins via dimeric TOG1-TOG2 subunits, in which each domain exhibits a distinct exchange rate for tubulin. X-ray structures revealed square-shaped assemblies composed of pseudo-dimeric TOG1-TOG2 subunits assembled head-to-tail, positioning four unpolymerized tubulins in a polarized wheel-like configuration. Crosslinking and electron microscopy show Alp14-tubulin forms square assemblies in solution, and inactivating their interfaces destabilize this organization without influencing tubulin binding. An X-ray structure determined using approach to modulate tubulin polymerization revealed an unfurled assembly, in which TOG1-TOG2 uniquely bind to two polymerized tubulins. Our findings suggest a new microtubule polymerase model in which TOG arrays recruit tubulins by forming square assemblies that then unfurl, facilitating their concerted polymerization into protofilaments.

Structural Basis of Tubulin Recruitment and Assembly by Tumor Overexpressed Gene (TOG) domain array Microtubule Polymerases

XMAP215/Stu2/Alp14 proteins accelerate microtubule plus-end polymerization by recruiting tubulins via arrays of Tumor Overexpressed Gene (TOG) domains. The underlying mechanism of these arrays as microtubule polymerases remains unknown. Here, we describe the biochemical and structural basis for TOG domain arrays in recruiting and polymerizing tubulins. Alp14 binds four tubulins via dimeric TOG1-TOG2 arrays, each with distinct exchange rates. X-ray structures reveal pseudo-dimeric square-shaped assemblies in which four TOG domains position four unpolymerized tubulins in a polarized wheel-like configuration. Crosslinking confirms square assemblies form in solution, and inactivation of their interfaces destabilizes square organizations without influencing tubulin binding. Using an approach to modulate tubulin polymerization, we determined a X-ray structure showing an unfurled assembly in which TOG1 and TOG2 uniquely bind two polymerized tubulins. Our findings suggest a new microtubule polymerase model in which TOG arrays recruit tubulins by forming square assemblies, which then unfurl facilitating their concerted polymerization into protofilaments.

Roles for tubulin recruitment and self-organization by TOG domain arrays in Microtubule plus-end tracking and polymerase

The XMAP215/Stu2/Alp14 microtubule polymerases utilize Tumor Overexpressed Gene (TOG) domain arrays to accelerate microtubule plus-end polymerization. Structural studies suggest a microtubule polymerase model in which TOG arrays recruit four αβ-tubulins, forming large square assemblies; an array of TOG1 and TOG2 domains may then unfurl from the square state to polymerize two αβ-tubulins into protofilaments at microtubule ends. Here, we test this model using two biochemically characterized classes of fission yeast Alp14 mutants. Using in vitro reconstitution and in vivo live cell imaging, we show that αβ-tubulins recruited by TOG1 and TOG2 domains serve non-additive roles in microtubule plus-end tracking and polymerase activities. Alp14 mutants with inactivated square assembly interfaces have defects in processive plus-end tracking and poor microtubule polymerase, indicating a functional role for square assemblies in processive tracking. These studies provide functional insights into how TOG1 and TOG2 domain arrays recruit tubulins and promote polymerase at microtubule plus ends.