Ancestral roles of the Fam20C family of secreted protein kinases revealed in C. elegans.

Fam20C is a secreted protein kinase mutated in Raine syndrome, a human skeletal disorder. In vertebrates, bone and enamel proteins are major Fam20C substrates. However, Fam20 kinases are conserved in invertebrates lacking bone and enamel, suggesting other ancestral functions. We show that FAMK-1, the Caenorhabditis elegans Fam20C orthologue, contributes to fertility, embryogenesis, and development. These functions are not fulfilled when FAMK-1 is retained in the early secretory pathway. During embryogenesis, FAMK-1 maintains intercellular partitions and prevents multinucleation; notably, temperature elevation or lowering cortical stiffness reduces requirement for FAMK-1 in these contexts. FAMK-1 is expressed in multiple adult tissues that undergo repeated mechanical strain, and selective expression in the spermatheca restores fertility. Informatic, biochemical, and functional analysis implicate lectins as FAMK-1 substrates. These findings suggest that FAMK-1 phosphorylation of substrates, including lectins, in the late secretory pathway is important in embryonic and tissue contexts where cells are subjected to mechanical strain

Mechanisms mitigating problems with multiple kinetochores on one microtubule in early mitosis

Proper chromosome segregation in mitosis relies on correct kinetochore interaction with spindle microtubules. In early mitosis, each kinetochore usually interacts with the lateral side of each microtubule and is subsequently tethered at the microtubule end. However, since eukaryotic cells carry multiple chromosomes, multiple kinetochores could occasionally interact with a single microtubule. The consequence of this is unknown. Here we find that, although two kinetochores (two pairs of sister kinetochores) can interact with the lateral side of one microtubule, only one kinetochore can form sustained attachment to the microtubule end in budding yeast. This leads to detachment of the other kinetochore from the microtubule end or its proximity. Intriguingly, in this context, kinetochore sliding along a microtubule towards a spindle pole delays and diminishes discernible kinetochore detachment. This effect expedites collection of the entire set of kinetochores to a spindle pole. We propose that cells are equipped with the kinetochore sliding mechanism to mitigate the problem with multiple kinetochores on one microtubule in early mitosis

Molecular mechanisms facilitating the initial kinetochore encounter with spindle microtubules

The initial kinetochore encounter with a spindle microtubule (kinetochore capture) is one of the rate-limiting steps in establishing proper kinetochore–microtubule interaction during mitosis. Kinetochore capture is facilitated by multiple factors such as microtubule extension in various directions, kinetochore diffusion and microtubule pivoting. In addition, kinetochores generate short microtubules, which subsequently interact with a spindle microtubule. Kinetochore-derived microtubules may facilitate kinetochore capture, but their contribution is elusive. Here we find that Stu1 recruits Stu2 to budding yeast kinetochores, which promotes microtubule generation there. By removing Stu2 specifically from kinetochores, we show that kinetochore-derived microtubules shorten the half-life of non-captured kinetochores from 48–49 to 28–34 seconds. Using computational simulation we find that multiple factors facilitate kinetochore capture, redundantly or synergistically. In particular, kinetochore-derived microtubules play important roles both by making a significant contribution on their own and by synergistically enhancing the effects of KT diffusion and MT pivoting. Our study reveals fundamental mechanisms facilitating the initial kinetochore encounter with spindle microtubules

Spindly is required for rapid migration of human cells

Dynein is the sole processive minus-end-directed microtubule motor found in animals. It has roles in cell division, membrane trafficking, and cell migration. Together with dynactin, dynein regulates centrosomal orientation to establish and maintain cell polarity, controls focal adhesion turnover and anchors microtubules at the leading edge. In higher eukaryotes, dynein/dynactin requires additional components such as Bicaudal D to form an active motor complex and for regulating its cellular localization. Spindly is a protein that targets dynein/dynactin to kinetochores in mitosis and can activate its motility in vitro However, no role for Spindly in interphase dynein/dynactin function has been found. We show that Spindly binds to the cell cortex and microtubule tips and colocalizes with dynein/dynactin at the leading edge of migrating U2OS cells and primary fibroblasts. U2OS cells that lack Spindly migrated slower in 2D than control cells, although centrosome polarization appeared to happen properly in the absence of Spindly. Re-expression of Spindly rescues migration, but the expression of a mutant, which is defective for dynactin binding, failed to rescue this defect. Taken together, these data demonstrate that Spindly plays an important role in mediating a subset of dynein/dynactin's function in cell migration

Dynamic partitioning of mitotic kinesin-5 cross-linkers between microtubule-bound and freely diffusing states

The dynamic behavior of homotetrameric kinesin-5 during mitosis is poorly understood. Kinesin-5 may function only by binding, cross-linking, and sliding adjacent spindle microtubules (MTs), or, alternatively, it may bind to a stable “spindle matrix” to generate mitotic movements. We created transgenic Drosophila melanogaster expressing fluorescent kinesin-5, KLP61F-GFP, in a klp61f mutant background, where it rescues mitosis and viability. KLP61F-GFP localizes to interpolar MT bundles, half spindles, and asters, and is enriched around spindle poles. In fluorescence recovery after photobleaching experiments, KLP61F-GFP displays dynamic mobility similar to tubulin, which is inconsistent with a substantial static pool of kinesin-5. The data conform to a reaction–diffusion model in which most KLP61F is bound to spindle MTs, with the remainder diffusing freely. KLP61F appears to transiently bind MTs, moving short distances along them before detaching. Thus, kinesin-5 motors can function by cross-linking and sliding adjacent spindle MTs without the need for a static spindle matrix

Quantitative analysis of an anaphase B switch: predicted role for a microtubule catastrophe gradient

Anaphase B in Drosophila embryos is initiated by the inhibition of microtubule (MT) depolymerization at spindle poles, which allows outwardly sliding interpolar (ip) MTs to drive pole–pole separation. Using fluorescence recovery after photobleaching, we observed that MTs throughout the preanaphase B spindle are very dynamic and display complete recovery of fluorescence, but during anaphase B, MTs proximal to the poles stabilize and therefore display lower recovery than those elsewhere. Fluorescence microscopy of the MT tip tracker EB1 revealed that growing MT plus ends localize throughout the preanaphase B spindle but concentrate in the overlap region of interpolar MTs (ipMTs) at anaphase B onset. None of these changes occurred in the presence of nondegradable cyclin B. Modeling suggests that they depend on the establishment of a spatial gradient of MT plus-end catastrophe frequencies, decreasing toward the equator. The resulting redistribution of ipMT plus ends to the overlap zone, together with the suppression of minus-end depolymerization at the poles, could constitute a mechanical switch that initiates spindle elongation

BUB-1 and CENP-C recruit PLK-1 to control chromosome alignment and segregation during meiosis I in C. elegans oocytes

Phosphorylation is a key post-translational modification that is utilised in many biological processes for the rapid and reversible regulation of protein localisation and activity. Polo-like kinase 1 (PLK-1) is essential for both mitotic and meiotic cell divisions, with key functions being conserved in eukaryotes. The roles and regulation of PLK-1 during mitosis have been well characterised. However, the discrete roles and regulation of PLK-1 during meiosis have remained obscure. Here, we used Caenorhabditis elegans (C. elegans) oocytes to show that PLK-1 plays distinct roles in meiotic spindle assembly and/or stability, chromosome alignment and segregation, and polar body extrusion during meiosis I. Furthermore, by a combination of live imaging and biochemical analysis we identified the chromosomal recruitment mechanisms of PLK-1 during C. elegans oocyte meiosis. The spindle assembly checkpoint kinase BUB-1 directly recruits PLK-1 to the kinetochore and midbivalent while the chromosome arm population of PLK-1 depends on a direct interaction with the centromeric-associated protein CENP-CHCP-4. We found that perturbing both BUB-1 and CENP-CHCP-4 recruitment of PLK-1 leads to severe meiotic defects, resulting in highly aneuploid oocytes. Overall, our results shed light on the roles played by PLK-1 during oocyte meiosis and provide a mechanistic understanding of PLK-1 targeting to meiotic chromosomes.</p

The kinetochore-microtubule coupling machinery is repurposed in sensory nervous system morphogenesis

Dynamic coupling of microtubule ends to kinetochores, built on the centromeres of chromosomes, directs chromosome segregation during cell division. Here, we report that the evolutionarily ancient kinetochore-microtubule&nbsp;coupling machine, the KMN (Knl1/Mis12/Ndc80-complex) network, plays a critical role in neuronal morphogenesis. We show that the KMN network concentrates in microtubule-rich dendrites of developing sensory neurons that collectively extend in a multicellular morphogenetic event that occurs during C.&nbsp;elegans embryogenesis. Post-mitotic degradation of KMN components in sensory neurons disrupts dendritic extension, leading to patterning and functional defects in the sensory nervous system. Structure-guided mutations revealed that the molecular interface that couples kinetochores to spindle microtubules also functions in neuronal development. These results identify a cell-division-independent function for the chromosome-segregation machinery and define a microtubule-coupling-dependent event in sensory nervous system morphogenesis

BUB-1 targets PP2A:B56 to regulate chromosome congression during meiosis I in C. elegans oocytes

Protein Phosphatase 2A (PP2A) is a heterotrimer composed of scaffolding (A), catalytic (C), and regulatory (B) subunits. PP2A complexes with B56 subunits are targeted by Shugoshin and BUBR1 to protect centromeric cohesion and stabilise kinetochore-microtubule attachments in yeast and mouse meiosis. In Caenorhabditis elegans, the closest BUBR1 orthologue lacks the B56-interaction domain and Shugoshin is not required for meiotic segregation. Therefore, the role of PP2A in C. elegans female meiosis is unknown. We report that PP2A is essential for meiotic spindle assembly and chromosome dynamics during C. elegans female meiosis. BUB-1 is the main chromosome-targeting factor for B56 subunits during prometaphase I. BUB-1 recruits PP2A:B56 to the chromosomes via a newly identified LxxIxE motif in a phosphorylation-dependent manner, and this recruitment is important for proper chromosome congression. Our results highlight a novel mechanism for B56 recruitment, essential for recruiting a pool of PP2A involved in chromosome congression during meiosis I

Molecular mechanism of dynein recruitment to kinetochores by the Rod-Zw10-Zwilch complex and Spindly

The molecular motor dynein concentrates at the kinetochore region of mitotic chromosomes in animals to accelerate spindle microtubule capture and to control spindle checkpoint signaling. In this study, we describe the molecular mechanism used by the Rod-Zw10-Zwilch complex and the adaptor Spindly to recruit dynein to kinetochores in Caenorhabditis elegans embryos and human cells. We show that Rod's N-terminal beta-propeller and the associated Zwilch subunit bind Spindly's C-terminal domain, and we identify a specific Zwilch mutant that abrogates Spindly and dynein recruitment in vivo and Spindly binding to a Rod beta-propeller-Zwilch complex in vitro. Spindly's N-terminal coiled-coil uses distinct motifs to bind dynein light intermediate chain and the pointed-end complex of dynactin. Mutations in these motifs inhibit assembly of a dynein-dynactin-Spindly complex, and a null mutant of the dynactin pointed-end subunit p27 prevents kinetochore recruitment of dynein-dynactin without affecting other mitotic functions of the motor. Conservation of Spindly-like motifs in adaptors involved in intracellular transport suggests a common mechanism for linking dynein to cargo.This work was supported by a European Research Council Starting Grant (Dyneinome 338410) and a European Molecular Biology Organization Installation Grant to R. Gassmann. This work was also supported by funding from the Fundacao para a Ciencia e a Tecnologia to R. Gassmann (IF/01015/2013/CP1157/CT0006), C. Pereira (SFRH_BPD_95648_2013), and D.J. Barbosa (SFRH_BPD_101898_2014). Some C. elegans strains were provided by the Caenorhabditis Genetics Center, which is funded by the National Institutes of Health Office of Research Infrastructure Programs (P40 OD010440)