Non-canonical role for the BAF complex subunit DPF3 in mitosis and ciliogenesis.

DPF3, along with other subunits, is a well-known component of the BAF chromatin remodeling complex, which plays a key role in regulating chromatin remodeling activity and gene expression. Here, we elucidated a non-canonical localization and role for DPF3. We showed that DPF3 dynamically localizes to the centriolar satellites in interphase and to the centrosome, spindle midzone and bridging fiber area, and midbodies during mitosis. Loss of DPF3 causes kinetochore fiber instability, unstable kinetochore-microtubule attachment and defects in chromosome alignment, resulting in altered mitotic progression, cell death and genomic instability. In addition, we also demonstrated that DPF3 localizes to centriolar satellites at the base of primary cilia and is required for ciliogenesis by regulating axoneme extension. Taken together, these findings uncover a moonlighting dual function for DPF3 during mitosis and ciliogenesis.

Branched microtubule nucleation and dynein transport organize RanGTP asters in Xenopus laevis egg extract.

Chromosome segregation relies on the correct assembly of a bipolar spindle. Spindle pole self-organization requires dynein-dependent microtubule (MT) transport along other MTs. However, during M-phase RanGTP triggers MT nucleation and branching generating polarized arrays with nonastral organization in which MT minus ends are linked to the sides of other MTs. This raises the question of how branched-MT nucleation and dynein-mediated transport cooperate to organize the spindle poles. Here, we used RanGTP-dependent MT aster formation in Xenopus laevis (X. laevis) egg extract to study the interplay between these two seemingly conflicting organizing principles. Using temporally controlled perturbations of MT nucleation and dynein activity, we found that branched MTs are not static but instead dynamically redistribute over time as poles self-organize. Our experimental data together with computer simulations suggest a model where dynein together with dynactin and NuMA directly pulls and move branched MT minus ends toward other MT minus ends.

MCRS1 modulates the heterogeneity of microtubule minus-end morphologies in mitotic spindles.

Faithful chromosome segregation requires the assembly of a bipolar spindle, consisting of two antiparallel microtubule (MT) arrays having most of their minus ends focused at the spindle poles and their plus ends overlapping in the spindle midzone. Spindle assembly, chromosome alignment, and segregation require highly dynamic MTs. The plus ends of MTs have been extensively investigated but their minus-end structure remains poorly characterized. Here, we used large-scale electron tomography to study the morphology of the MT minus ends in three dimensionally reconstructed metaphase spindles in HeLa cells. In contrast to the homogeneous open morphology of the MT plus ends at the kinetochores, we found that MT minus ends are heterogeneous, showing either open or closed morphologies. Silencing the minus end-specific stabilizer, MCRS1 increased the proportion of open MT minus ends. Altogether, these data suggest a correlation between the morphology and the dynamic state of the MT ends. Taking this heterogeneity of the MT minus-end morphologies into account, our work indicates an unsynchronized behavior of MTs at the spindle poles, thus laying the groundwork for further studies on the complexity of MT dynamics regulation.

Chromosome segregation fidelity requires microtubule polyglutamylation by the cancer downregulated enzyme TTLL11.

Regulation of microtubule (MT) dynamics is key for mitotic spindle assembly and faithful chromosome segregation. Here we show that polyglutamylation, a still understudied post-translational modification of spindle MTs, is essential to define their dynamics within the range required for error-free chromosome segregation. We identify TTLL11 as an enzyme driving MT polyglutamylation in mitosis and show that reducing TTLL11 levels in human cells or zebrafish embryos compromises chromosome segregation fidelity and impairs early embryonic development. Our data reveal a mechanism to ensure genome stability in normal cells that is compromised in cancer cells that systematically downregulate TTLL11. Our data suggest a direct link between MT dynamics regulation, MT polyglutamylation and two salient features of tumour cells, aneuploidy and chromosome instability (CIN).

NEDD1-S411 phosphorylation plays a critical function in the coordination of microtubule nucleation during mitosis.

During mitosis, spindle assembly relies on centrosomal and acentrosomal microtubule nucleation pathways that all require the γ-Tubulin Ring Complex (γ-TuRC) and its adaptor protein NEDD1. The activity of these different pathways needs to be coordinated to ensure bipolar spindle assembly ( Cavazza et al., 2016) but the underlying mechanism is still unclear. Previous studies have identified three sites in NEDD1 (S377, S405 and S411) that when phosphorylated drive MT nucleation at the centrosomes, around the chromosomes and on pre-existing MTs respectively ( Lüders et al., 2006; Pinyol et al., 2013; Sdelci et al., 2012). Here we aimed at getting additional insights into the mechanism that coordinates the different MT nucleation pathways in dividing cells using a collection of HeLa stable inducible cell lines expressing NEDD1 phospho-variants at these three sites and Xenopus egg extracts. Our results provide further support for the essential role of phosphorylation at the three residues. Moreover, we directly demonstrate that S411 phosphorylation is essential for MT branching using TIRF microscopy in Xenopus egg extracts and we show that it plays a crucial role in ensuring the balance between centrosome and chromosome-dependent MT nucleation required for bipolar spindle assembly in mitotic cells.

The human sperm basal body is a complex centrosome important for embryo preimplantation development.

The mechanism of conversion of the human sperm basal body to a centrosome after fertilization, and its role in supporting human early embryogenesis, has not been directly addressed so far. Using proteomics and immunofluorescence studies, we show here that the human zygote inherits a basal body enriched with centrosomal proteins from the sperm, establishing the first functional centrosome of the new organism. Injection of human sperm tails containing the basal body into human oocytes followed by parthenogenetic activation, showed that the centrosome contributes to the robustness of the early cell divisions, increasing the probability of parthenotes reaching the compaction stage. In the absence of the sperm-derived centrosome, pericentriolar material (PCM) components stored in the oocyte can form de novo structures after genome activation, suggesting a tight PCM expression control in zygotes. Our results reveal that the sperm basal body is a complex organelle which converts to a centrosome after fertilization, ensuring the early steps of embryogenesis and successful compaction. However, more experiments are needed to elucidate the exact molecular mechanisms of centrosome inheritance in humans.

Autosomal recessive variants in TUBGCP2 alter the γ-tubulin ring complex leading to neurodevelopmental disease.

Microtubules help building the cytoskeleton of neurons and other cells. Several components of the gamma-tubulin (γ-tubulin) complex have been previously reported in human neurodevelopmental diseases. We describe two siblings from a consanguineous Turkish family with dysmorphic features, developmental delay, brain malformation, and epilepsy carrying a homozygous mutation (p.Glu311Lys) in TUBGCP2 encoding the γ-tubulin complex 2 (GCP2) protein. This variant is predicted to disrupt the electrostatic interaction of GCP2 with GCP3. In primary fibroblasts carrying the variant, we observed a faint delocalization of γ-tubulin during the cell cycle but normal GCP2 protein levels. Through mass spectrometry, we observed dysregulation of multiple proteins involved in the assembly and organization of the cytoskeleton and the extracellular matrix, controlling cellular adhesion and of proteins crucial for neuronal homeostasis including axon guidance. In summary, our functional and proteomic studies link TUBGCP2 and the γ-tubulin complex to the development of the central nervous system in humans.

DnaJB6 is a RanGTP-regulated protein required for microtubule organization during mitosis.

Bipolar spindle organization is essential for the faithful segregation of chromosomes during cell division. This organization relies on the collective activities of motor proteins. The minus-end-directed dynein motor complex generates spindle inward forces and plays a major role in spindle pole focusing. The dynactin complex regulates many dynein functions, increasing its processivity and force production. Here, we show that DnaJB6 is a novel RanGTP-regulated protein. It interacts with the dynactin subunit p150Glued (also known as DCTN1) in a RanGTP-dependent manner specifically in M-phase, and promotes spindle pole focusing and dynein force generation. Our data suggest a novel mechanism by which RanGTP regulates dynein activity during M-phase.

Microtubule nucleation during central spindle assembly requires NEDD1 phosphorylation on serine 405 by Aurora A.

During mitosis, the cell sequentially constructs two microtubule-based spindles to ensure faithful segregation of chromosomes. A bipolar spindle first pulls apart the sister chromatids, then a central spindle further separates them away. Although the assembly of the first spindle is well described, the assembly of the second remains poorly understood. We report here that the inhibition of Aurora A leads to an absence of the central spindle resulting from a lack of nucleation of microtubules in the midzone. In the absence of Aurora A, the HURP (also known as DLGAP5) and NEDD1 proteins that are involved in nucleation of microtubules fail to concentrate in the midzone. HURP is an effector of RanGTP, whereas NEDD1 serves as an anchor for the γ-tubulin ring complex (γTURC). Interestingly, Aurora A phosphorylates HURP and NEDD1 during assembly of the initial bipolar spindle. We show here that the expression of a NEDD1 isoform mimicking phosphorylation by Aurora A is sufficient to restore microtubule nucleation in the midzone under conditions of Aurora A inhibition. These results reveal a new control mechanism of microtubule nucleation by Aurora A during assembly of the central spindle.

Insights of the tubulin code in gametes and embryos: from basic research to potential clinical applications in humans†.

Microtubules are intracellular filaments that define in space and in time a large number of essential cellular functions such as cell division, morphology and motility, intracellular transport and flagella and cilia assembly. They are therefore essential for spermatozoon and oocyte maturation and function, and for embryo development. The dynamic and functional properties of the microtubules are in large part defined by various classes of interacting proteins including MAPs (microtubule associated proteins), microtubule-dependent motors, and severing and modifying enzymes. Multiple mechanisms regulate these interactions. One of them is defined by the high diversity of the microtubules themselves generated by the combination of different tubulin isotypes and by several tubulin post-translational modifications (PTMs). This generates a so-called tubulin code that finely regulates the specific set of proteins that associates with a given microtubule thereby defining the properties and functions of the network. Here we provide an in depth review of the current knowledge on the tubulin isotypes and PTMs in spermatozoa, oocytes, and preimplantation embryos in various model systems and in the human species. We focus on functional implications of the tubulin code for cytoskeletal function, particularly in the field of human reproduction and development, with special emphasis on gamete quality and infertility. Finally, we discuss some of the knowledge gaps and propose future research directions.

Functional Analysis of Human Pathological Semen Samples in an Oocyte Cytoplasmic Ex Vivo System.

Human fertilization and embryo development involve a wide range of critical processes that determine the successful development of a new organism. Although Assisted Reproduction Technologies (ART) may help solve infertility problems associated to severe male factor, the live birth rate is still low. A high proportion of ART failures occurs before implantation. Understanding the causes for these failures has been difficult due to technical and ethical limitations. Diagnostic procedures on human spermatozoa in particular have been limited to morphology and swimming behaviours while other functional requirements during early development have not been addressed due to the lack of suitable assays. Here, we have established a quantitative system based on the use of Xenopus egg extracts and human spermatozoa. This system provides novel possibilities for the functional characterization of human spermatozoa. Using clinical data we show that indeed this approach offers a set of complementary data for the functional evaluation of spermatozoa from patients.

Proteomic Profiling of Microtubule Self-organization in M-phase.

Microtubules (MTs) and associated proteins can self-organize into complex structures such as the bipolar spindle, a process in which RanGTP plays a major role. Addition of RanGTP to M-phase Xenopus egg extracts promotes the nucleation and self-organization of MTs into asters and bipolar-like structures in the absence of centrosomes or chromosomes. We show here that the complex proteome of these RanGTP-induced MT assemblies is similar to that of mitotic spindles. Using proteomic profiling we show that MT self-organization in the M-phase cytoplasm involves the non-linear and non-stoichiometric recruitment of proteins from specific functional groups. Our study provides for the first time a temporal understanding of the protein dynamics driving MT self-organization in M-phase.

Nek9 Phosphorylation Defines a New Role for TPX2 in Eg5-Dependent Centrosome Separation before Nuclear Envelope Breakdown.

Centrosomes [1, 2] play a central role during spindle assembly in most animal cells [3]. In early mitosis, they organize two symmetrical microtubule arrays that upon separation define the two poles of the forming spindle. Centrosome separation is tightly regulated [4, 5], occurring through partially redundant mechanisms that rely on the action of microtubule-based dynein and kinesin motors and the actomyosin system [6]. While centrosomes can separate in prophase or in prometaphase after nuclear envelope breakdown (NEBD), prophase centrosome separation optimizes spindle assembly and minimizes the occurrence of abnormal chromosome attachments that could end in aneuploidy [7, 8]. Prophase centrosome separation relies on the activity of Eg5/KIF11, a mitotic kinesin [9] that accumulates around centrosomes in early mitosis under the control of CDK1 and the Nek9/Nek6/7 kinase module [10-17]. Here, we show that Eg5 localization and centrosome separation in prophase depend on the nuclear microtubule-associated protein TPX2 [18], a pool of which localizes to the centrosomes before NEBD. This localization involves RHAMM/HMMR [19] and the kinase Nek9 [20], which phosphorylates TPX2 nuclear localization signal (NLS) preventing its interaction with importin and nuclear import. The pool of centrosomal TPX2 in prophase has a critical role for both microtubule aster organization and Eg5 localization, and thereby for centrosome separation. Our results uncover an unsuspected role for TPX2 before NEBD and define a novel regulatory mechanism for centrosome separation in prophase. They furthermore suggest NLS phosphorylation as a novel regulatory mechanism for spindle assembly factors controlled by the importin/Ran system.

Role of Kif15 and its novel mitotic partner KBP in K-fiber dynamics and chromosome alignment.

Faithful segregation of the genetic material during the cell cycle is key for the continuation of life. Central to this process is the assembly of a bipolar spindle that aligns the chromosomes and segregates them to the two daughter cells. Spindle bipolarity is strongly dependent on the activity of the homotetrameric kinesin Eg5. However, another kinesin, Kif15, also provides forces needed to separate the spindle poles during prometaphase and to maintain spindle bipolarity at metaphase. Here we identify KBP as a specific interaction partner of Kif15 in mitosis. We show that KBP promotes the localization of Kif15 to the spindle equator close to the chromosomes. Both Kif15 and KBP are required for the alignment of all the chromosomes to the metaphase plate and the assembly of stable kinetochore fibers of the correct length. Taken together our data uncover a novel role for Kif15 in complex with KBP during mitosis.

The C-terminal domain of TPX2 is made of alpha-helical tandem repeats.

BACKGROUND: TPX2 (Targeting Protein for Xklp2) is essential for spindle assembly, activation of the mitotic kinase Aurora A and for triggering microtubule nucleation. Homologs of TPX2 in Chordata and plants were previously identified. Currently, proteins of the TPX2 family have little structural information and only small parts are covered by defined protein domains. METHODS: We have used computational sequence analyses and structural predictions of proteins of the TPX2 family, supported with Circular Dichroism (CD) measurements. RESULTS: Here, we report our finding that the C-terminal domain of TPX2, which is responsible of its microtubule nucleation capacity and is conserved in all members of the family, is actually formed by tandem repeats, covering well above 2/3 of the protein. We propose that this region forms a flexible solenoid involved in protein-protein interactions. Structural prediction and molecular modeling, combined with Circular Dichroism (CD) measurements reveal a predominant alpha-helical content. Furthermore, we identify full length homologs in fungi and shorter homologs with a different domain organization in diptera (including a paralogous expansion in Drosophila). CONCLUSIONS: Our results, represent the first computational and biophysical analysis of the TPX2 proteins family and help understand the structure and evolution of this conserved protein family to direct future structural studies.

The sequential activation of the mitotic microtubule assembly pathways favors bipolar spindle formation.

Centrosome maturation is the process by which the duplicated centrosomes recruit pericentriolar components and increase their microtubule nucleation activity before mitosis. The role of this process in cells entering mitosis has been mostly related to the separation of the duplicated centrosomes and thereby to the assembly of a bipolar spindle. However, spindles can form without centrosomes. In fact, all cells, whether they have centrosomes or not, rely on chromatin-driven microtubule assembly to form a spindle. To test whether the sequential activation of these microtubule assembly pathways, defined by centrosome maturation and nuclear envelope breakdown, plays any role in spindle assembly, we combined experiments in tissue culture cells and Xenopus laevis egg extracts with a mathematical model. We found that interfering with the sequential activation of the microtubule assembly pathways compromises bipolar spindle assembly in tissue culture cells but not in X. laevis egg extracts. Our data suggest a novel function for centrosome maturation that determines the contribution of the chromosomal microtubule assembly pathway and favors bipolar spindle formation in most animal cells in which tubulin is in limiting amounts.

Allosteric inhibition of Aurora-A kinase by a synthetic vNAR domain.

The vast majority of clinically approved protein kinase inhibitors target the ATP-binding pocket directly. Consequently, many inhibitors have broad selectivity profiles and most have significant off-target effects. Allosteric inhibitors are generally more selective, but are difficult to identify because allosteric binding sites are often unknown or poorly characterized. Aurora-A is activated through binding of TPX2 to an allosteric site on the kinase catalytic domain, and this knowledge could be exploited to generate an inhibitor. Here, we generated an allosteric inhibitor of Aurora-A kinase based on a synthetic, vNAR single domain scaffold, vNAR-D01. Biochemical studies and a crystal structure of the Aurora-A/vNAR-D01 complex show that the vNAR domain overlaps with the TPX2 binding site. In contrast with the binding of TPX2, which stabilizes an active conformation of the kinase, binding of the vNAR domain stabilizes an inactive conformation, in which the αC-helix is distorted, the canonical Lys-Glu salt bridge is broken and the regulatory (R-) spine is disrupted by an additional hydrophobic side chain from the activation loop. These studies illustrate how single domain antibodies can be used to characterize the regulatory mechanisms of kinases and provide a rational basis for structure-guided design of allosteric Aurora-A kinase inhibitors.

From meiosis to mitosis - the sperm centrosome defines the kinetics of spindle assembly after fertilization in Xenopus.

Bipolar spindle assembly in the vertebrate oocyte relies on a self-organization chromosome-dependent pathway. Upon fertilization, the male gamete provides a centrosome, and the first and subsequent embryonic divisions occur in the presence of duplicated centrosomes that act as dominant microtubule organizing centres (MTOCs). The transition from meiosis to embryonic mitosis involves a necessary adaptation to integrate the dominant chromosome-dependent pathway with the centrosomes to form the bipolar spindle. Here, we took advantage of the Xenopus laevis egg extract system to mimic in vitro the assembly of the first embryonic spindle and investigate the respective contributions of the centrosome and the chromosome-dependent pathway to the kinetics of the spindle bipolarization. We found that centrosomes control the transition from the meiotic to the mitotic spindle assembly mechanism. By defining the kinetics of spindle bipolarization, the centrosomes ensure their own positioning to each spindle pole and thereby their essential correct inheritance to the two first daughter cells of the embryo for the development of a healthy organism.