A Force-Induced Directional Switch of a Molecular Motor Enables Parallel Microtubule Bundle Formation.

Microtubule-organizing centers (MTOCs) nucleate microtubules that can grow autonomously in any direction. To generate bundles of parallel microtubules originating from a single MTOC, the growth of multiple microtubules needs to coordinated, but the underlying mechanism is unknown. Here, we show that a conserved two-component system consisting of the plus-end tracker EB1 and the minus-end-directed molecular motor Kinesin-14 is sufficient to promote parallel microtubule growth. The underlying mechanism relies on the ability of Kinesin-14 to guide growing plus ends along existing microtubules. The generality of this finding is supported by yeast, Drosophila, and human EB1/Kinesin-14 pairs. We demonstrate that plus-end guiding involves a directional switch of the motor due to a force applied via a growing microtubule end. The described mechanism can account for the generation of parallel microtubule networks required for a broad range of cellular functions such as spindle assembly or cell polarization.

A phylogenetic profiling approach identifies novel ciliogenesis genes in Drosophila and C. elegans.

Cilia are cellular projections that perform sensory and motile functions in eukaryotic cells. A defining feature of cilia is that they are evolutionarily ancient, yet not universally conserved. In this study, we have used the resulting presence and absence pattern in the genomes of diverse eukaryotes to identify a set of 386 human genes associated with cilium assembly or motility. Comprehensive tissue-specific RNAi in Drosophila and mutant analysis in C. elegans revealed signature ciliary defects for 70-80% of novel genes, a percentage similar to that for known genes within the cluster. Further characterization identified different phenotypic classes, including a set of genes related to the cartwheel component Bld10/CEP135 and two highly conserved regulators of cilium biogenesis. We propose this dataset defines the core set of genes required for cilium assembly and motility across eukaryotes and presents a valuable resource for future studies of cilium biology and associated disorders.

Correction: A modified TurboID approach identifies tissue-specific centriolar components in C. elegans.

[This corrects the article DOI: 10.1371/journal.pgen.1010150.].

A modified TurboID approach identifies tissue-specific centriolar components in C. elegans.

Proximity-dependent labeling approaches such as BioID have been a great boon to studies of protein-protein interactions in the context of cytoskeletal structures such as centrosomes which are poorly amenable to traditional biochemical approaches like immunoprecipitation and tandem affinity purification. Yet, these methods have so far not been applied extensively to invertebrate experimental models such as C. elegans given the long labeling times required for the original promiscuous biotin ligase variant BirA*. Here, we show that the recently developed variant TurboID successfully probes the interactomes of both stably associated (SPD-5) and dynamically localized (PLK-1) centrosomal components. We further develop an indirect proximity labeling method employing a GFP nanobody-TurboID fusion, which allows the identification of protein interactors in a tissue-specific manner in the context of the whole animal. Critically, this approach utilizes available endogenous GFP fusions, avoiding the need to generate multiple additional strains for each target protein and the potential complications associated with overexpressing the protein from transgenes. Using this method, we identify homologs of two highly conserved centriolar components, Cep97 and BLD10/Cep135, which are present in various somatic tissues of the worm. Surprisingly, neither protein is expressed in early embryos, likely explaining why these proteins have escaped attention until now. Our work expands the experimental repertoire for C. elegans and opens the door for further studies of tissue-specific variation in centrosome architecture.

The hydrolethalus syndrome protein HYLS-1 links core centriole structure to cilia formation.

Centrioles are subcellular organelles composed of a ninefold symmetric microtubule array that perform two important functions: (1) They build centrosomes that organize the microtubule cytoskeleton, and (2) they template cilia, microtubule-based projections with sensory and motile functions. We identified HYLS-1, a widely conserved protein, based on its direct interaction with the core centriolar protein SAS-4. HYLS-1 localization to centrioles requires SAS-4 and, like SAS-4, HYLS-1 is stably incorporated into the outer centriole wall. Unlike SAS-4, HYLS-1 is dispensable for centriole assembly and centrosome function in cell division. Instead, HYLS-1 plays an essential role in cilia formation that is conserved between Caenorhabditis elegans and vertebrates. A single amino acid change in human HYLS1 leads to a perinatal lethal disorder termed hydrolethalus syndrome, and we show that this mutation impairs HYLS-1 function in ciliogenesis. HYLS-1 is required for the apical targeting/anchoring of centrioles at the plasma membrane but not for the intraflagellar transport-dependent extension of the ciliary axoneme. These findings classify hydrolethalus syndrome as a severe human ciliopathy and shed light on the dual functionality of centrioles, defining the first stably incorporated centriolar protein that is not required for centriole assembly but instead confers on centrioles the capacity to initiate ciliogenesis.

SAS-6 coiled-coil structure and interaction with SAS-5 suggest a regulatory mechanism in C. elegans centriole assembly.

The centriole is a conserved microtubule-based organelle essential for both centrosome formation and cilium biogenesis. Five conserved proteins for centriole duplication have been identified. Two of them, SAS-5 and SAS-6, physically interact with each other and are codependent for their targeting to procentrioles. However, it remains unclear how these two proteins interact at the molecular level. Here, we demonstrate that the short SAS-5 C-terminal domain (residues 390-404) specifically binds to a narrow central region (residues 275-288) of the SAS-6 coiled coil. This was supported by the crystal structure of the SAS-6 coiled-coil domain (CCD), which, together with mutagenesis studies, indicated that the association is mediated by synergistic hydrophobic and electrostatic interactions. The crystal structure also shows a periodic charge pattern along the SAS-6 CCD, which gives rise to an anti-parallel tetramer. Overall, our findings establish the molecular basis of the specific interaction between SAS-5 and SAS-6, and suggest that both proteins individually adopt an oligomeric conformation that is disrupted upon the formation of the hetero-complex to facilitate the correct assembly of the nine-fold symmetric centriole.

Maternal inheritance of functional centrioles in two parthenogenetic nematodes.

Centrioles are the core constituent of centrosomes, microtubule-organizing centers involved in directing mitotic spindle assembly and chromosome segregation in animal cells. In sexually reproducing species, centrioles degenerate during oogenesis and female meiosis is usually acentrosomal. Centrioles are retained during male meiosis and, in most species, are reintroduced with the sperm during fertilization, restoring centriole numbers in embryos. In contrast, the presence, origin, and function of centrioles in parthenogenetic species is unknown. We found that centrioles are maternally inherited in two species of asexual parthenogenetic nematodes and identified two different strategies for maternal inheritance evolved in the two species. In Rhabditophanes diutinus, centrioles organize the poles of the meiotic spindle and are inherited by both the polar body and embryo. In Disploscapter pachys, the two pairs of centrioles remain close together and are inherited by the embryo only. Our results suggest that maternally-inherited centrioles organize the embryonic spindle poles and act as a symmetry-breaking cue to induce embryo polarization. Thus, in these parthenogenetic nematodes, centrioles are maternally-inherited and functionally replace their sperm-inherited counterparts in sexually reproducing species.

A microtubule-independent role for centrosomes and aurora a in nuclear envelope breakdown.

Aurora A kinase localizes to centrosomes and is required for centrosome maturation and spindle assembly. Here we describe a microtubule-independent role for Aurora A and centrosomes in nuclear envelope breakdown (NEBD) during the first mitotic division of the C. elegans embryo. Aurora A depletion does not alter the onset or kinetics of chromosome condensation, but dramatically lengthens the interval between the completion of condensation and NEBD. Inhibiting centrosome assembly by other means also lengthens this interval, albeit to a lesser extent than Aurora A depletion. By contrast, centrosomally nucleated microtubules and the nuclear envelope-associated motor dynein are not required for timely NEBD. These results indicate that mitotic centrosomes generate a diffusible factor, which we propose is activated Aurora A, that promotes NEBD. A positive feedback loop, in which an Aurora A-dependent increase in centrosome size promotes Aurora A activation, may temporally couple centrosome maturation to NEBD during mitotic entry.

Inhibition of centrosome protein assembly leads to p53-dependent exit from the cell cycle.

Previous evidence has indicated that an intact centrosome is essential for cell cycle progress and that elimination of the centrosome or depletion of individual centrosome proteins prevents the entry into S phase. To investigate the molecular mechanisms of centrosome-dependent cell cycle progress, we performed RNA silencing experiments of two centrosome-associated proteins, pericentriolar material 1 (PCM-1) and pericentrin, in primary human fibroblasts. We found that cells depleted of PCM-1 or pericentrin show lower levels of markers for S phase and cell proliferation, including cyclin A, Ki-67, proliferating cell nuclear antigen, minichromosome maintenance deficient 3, and phosphorylated retinoblastoma protein. Also, the percentage of cells undergoing DNA replication was reduced by >50%. At the same time, levels of p53 and p21 increased in these cells, and cells were predisposed to undergo senescence. Conversely, depletion of centrosome proteins in cells lacking p53 did not cause any cell cycle arrest. Inhibition of p38 mitogen-activated protein kinase rescued cell cycle activity after centrosome protein depletion, indicating that p53 is activated by the p38 stress pathway.

An acentriolar centrosome at the C. elegans ciliary base.

In animal cells, the functions of the microtubule cytoskeleton are coordinated by centriole-based centrosomes via γ-tubulin complexes embedded in the pericentriolar material or PCM.1 PCM assembly has been best studied in the context of mitosis, where centriolar SPD-2 recruits PLK-1, which in turn phosphorylates key scaffolding components like SPD-5 and CNN to promote expansion of the PCM polymer.2-4 To what extent these mechanisms apply to centrosomes in interphase or in differentiated cells remains unclear.5 Here, we examine a novel type of centrosome found at the ciliary base of C. elegans sensory neurons, which we show plays important roles in neuronal morphogenesis, cellular trafficking, and ciliogenesis. These centrosomes display similar dynamic behavior to canonical, mitotic centrosomes, with a stable PCM scaffold and dynamically localized client proteins. Unusually, however, they are not organized by centrioles, which degenerate early in terminal differentiation.6 Yet, PCM not only persists but continues to grow with key scaffolding proteins including SPD-5 expressed under control of the RFX transcription factor DAF-19. This assembly occurs in the absence of the mitotic regulators SPD-2, AIR-1 and PLK-1, but requires tethering by PCMD-1, a protein which also plays a role in the initial, interphase recruitment of PCM in early embryos.7 These results argue for distinct mechanisms for mitotic and non-mitotic PCM assembly, with only the former requiring PLK-1 phosphorylation to drive rapid expansion of the scaffold polymer.

Cep97 Is Required for Centriole Structural Integrity and Cilia Formation in Drosophila.

Centrioles are highly elaborate microtubule-based structures responsible for the formation of centrosomes and cilia. Despite considerable variation across species and tissues within any given tissue, their size is essentially constant [1, 2]. While the diameter of the centriole cylinder is set by the dimensions of the inner scaffolding structure of the cartwheel [3], how centriole length is set so precisely and stably maintained over many cell divisions is not well understood. Cep97 and CP110 are conserved proteins that localize to the distal end of centrioles and have been reported to limit centriole elongation in vertebrates [4, 5]. Here, we examine Cep97 function in Drosophila melanogaster. We show that Cep97 is essential for formation of full-length centrioles in multiple tissues of the fly. We further identify the microtubule deacetylase Sirt2 as a Cep97 interactor. Deletion of Sirt2 likewise affects centriole size. Interestingly, so does deletion of the acetylase Atat1, indicating that loss of stabilizing acetyl marks impairs centriole integrity. Cep97 and CP110 were originally identified as inhibitors of cilia formation in vertebrate cultured cells [6], and loss of CP110 is a widely used marker of basal body maturation. In contrast, in Drosophila, Cep97 appears to be only transiently removed from basal bodies and loss of Cep97 strongly impairs ciliogenesis. Collectively, our results support a model whereby Cep97 functions as part of a protective cap that acts together with the microtubule acetylation machinery to maintain centriole stability, essential for proper function in cilium biogenesis.

Functional Architecture of Deleterious Genetic Variants in the Genome of a Wrangel Island Mammoth.

Woolly mammoths were among the most abundant cold-adapted species during the Pleistocene. Their once-large populations went extinct in two waves, an end-Pleistocene extinction of continental populations followed by the mid-Holocene extinction of relict populations on St. Paul Island ∼5,600 years ago and Wrangel Island ∼4,000 years ago. Wrangel Island mammoths experienced an episode of rapid demographic decline coincident with their isolation, leading to a small population, reduced genetic diversity, and the fixation of putatively deleterious alleles, but the functional consequences of these processes are unclear. Here, we show that a Wrangel Island mammoth genome had many putative deleterious mutations that are predicted to cause diverse behavioral and developmental defects. Resurrection and functional characterization of several genes from the Wrangel Island mammoth carrying putatively deleterious substitutions identified both loss and gain of function mutations in genes associated with developmental defects (HYLS1), oligozoospermia and reduced male fertility (NKD1), diabetes (NEUROG3), and the ability to detect floral scents (OR5A1). These data suggest that at least one Wrangel Island mammoth may have suffered adverse consequences from reduced population size and isolation.

Centriole assembly requires both centriolar and pericentriolar material proteins.

Centrioles organize pericentriolar material to form centrosomes and also template the formation of cilia. Despite the importance of centrioles in dividing and differentiated cells, their assembly remains poorly understood at a molecular level. Here, we develop a fluorescence microscopy-based assay for centriole assembly in the 1-cell stage C. elegans embryo. We use this assay to characterize SAS-6, a centriolar protein that we identified based on its requirement for centrosome duplication. We show that SAS-6, a member of a conserved metazoan protein family, is specifically required for new centriole assembly, a result we confirm by electron microscopy. We further use the centriole assembly assay to examine the roles of three pericentriolar material proteins: SPD-5, the kinase aurora-A, and gamma-tubulin. Our results suggest that the pericentriolar material promotes daughter centriole formation by concentrating gamma-tubulin around the parent centriole. Thus, both centriolar and pericentriolar material proteins contribute to centriole assembly.

Assembly of centrosomal proteins and microtubule organization depends on PCM-1.

The protein PCM-1 localizes to cytoplasmic granules known as "centriolar satellites" that are partly enriched around the centrosome. We inhibited PCM-1 function using a variety of approaches: microinjection of antibodies into cultured cells, overexpression of a PCM-1 deletion mutant, and specific depletion of PCM-1 by siRNA. All approaches led to reduced targeting of centrin, pericentrin, and ninein to the centrosome. Similar effects were seen upon inhibition of dynactin by dynamitin, and after prolonged treatment of cells with the microtubule inhibitor nocodazole. Inhibition or depletion of PCM-1 function further disrupted the radial organization of microtubules without affecting microtubule nucleation. Loss of microtubule organization was also observed after centrin or ninein depletion. Our data suggest that PCM-1-containing centriolar satellites are involved in the microtubule- and dynactin-dependent recruitment of proteins to the centrosome, of which centrin and ninein are required for interphase microtubule organization.

Differential Requirements for Centrioles in Mitotic Centrosome Growth and Maintenance.

Centrosomes, the predominant sites of microtubule nucleation and anchorage, coordinate spindle assembly and cell division in animal cells. At the onset of mitosis, centrioles accumulate microtubule-organizing pericentriolar material (PCM) in a process termed centrosome maturation. To what extent centrosome maturation depends on the continued activity of mitotic regulators or the presence of centrioles has hitherto been unclear. Using the C. elegans early embryo, we show that PCM expansion requires the Polo-like kinase PLK-1 and CEP192 (SPD-2 in C. elegans), but not its upstream regulator Aurora A (AIR-1), while maintenance of the PCM polymer depends exclusively on PLK-1. SPD-2 and PLK-1 are highly concentrated at centrioles. Unexpectedly, laser microsurgery reveals that while centrioles are required for PCM recruitment and centrosome structural integrity they are dispensable for PCM maintenance. We propose a model whereby centrioles promote centrosome maturation by recruiting PLK-1, but subsequent maintenance occurs via PLK-1 acting directly within the PCM.

UFD-2 is an adaptor-assisted E3 ligase targeting unfolded proteins.

Muscle development requires the coordinated activities of specific protein folding and degradation factors. UFD-2, a U-box ubiquitin ligase, has been reported to play a central role in this orchestra regulating the myosin chaperone UNC-45. Here, we apply an integrative in vitro and in vivo approach to delineate the substrate-targeting mechanism of UFD-2 and elucidate its distinct mechanistic features as an E3/E4 enzyme. Using Caenorhabditis elegans as model system, we demonstrate that UFD-2 is not regulating the protein levels of UNC-45 in muscle cells, but rather shows the characteristic properties of a bona fide E3 ligase involved in protein quality control. Our data demonstrate that UFD-2 preferentially targets unfolded protein segments. Moreover, the UNC-45 chaperone can serve as an adaptor protein of UFD-2 to poly-ubiquitinate unfolded myosin, pointing to a possible role of the UFD-2/UNC-45 pair in maintaining proteostasis in muscle cells.

Transient and Partial Nuclear Lamina Disruption Promotes Chromosome Movement in Early Meiotic Prophase.

Meiotic chromosome movement is important for the pairwise alignment of homologous chromosomes, which is required for correct chromosome segregation. Movement is driven by cytoplasmic forces, transmitted to chromosome ends by nuclear membrane-spanning proteins. In animal cells, lamins form a prominent scaffold at the nuclear periphery, yet the role lamins play in meiotic chromosome movement is unclear. We show that chromosome movement correlates with reduced lamin association with the nuclear rim, which requires lamin phosphorylation at sites analogous to those that open lamina network crosslinks in mitosis. Failure to remodel the lamina results in delayed meiotic entry, altered chromatin organization, unpaired or interlocked chromosomes, and slowed chromosome movement. The remodeling kinases are delivered to lamins via chromosome ends coupled to the nuclear envelope, potentially enabling crosstalk between the lamina and chromosomal events. Thus, opening the lamina network plays a role in modulating contacts between chromosomes and the nuclear periphery during meiosis.

The minus end in sight.

Microtubules are intrinsically polar structures. A consequence of this polarity is that the two ends of the microtubule polymer exhibit different properties. The more dynamic plus ends and the mechanisms that regulate their behavior have been the focus of much recent attention. Here, we concentrate on the dynamics and regulation of minus ends, which play distinct but equally critical roles in microtubule function. In the first part of this review, we compare the in vitro and in vivo behavior of microtubules from a minus end perspective. This comparison suggests that cells possess conserved mechanisms to specifically inhibit minus end polymerization, and perhaps also to actively promote depolymerization. In the second part, we focus on the spatial positioning of minus ends, which is achieved by localized microtubule nucleation, minus end capping and minus end anchoring as well as by motor-dependent sorting. These mechanisms are used in different biological contexts to generate the diversity of organized microtubule arrays in cells.

Centrioles initiate cilia assembly but are dispensable for maturation and maintenance in C. elegans.

Cilia are cellular projections that assemble on centriole-derived basal bodies. While cilia assembly is absolutely dependent on centrioles, it is not known to what extent they contribute to downstream events. The nematode C. elegans provides a unique opportunity to address this question, as centrioles do not persist at the base of mature cilia. Using fluorescence microscopy and electron tomography, we find that centrioles degenerate early during ciliogenesis. The transition zone and axoneme are not completely formed at this time, indicating that cilia maturation does not depend on intact centrioles. The hydrolethalus syndrome protein HYLS-1 is the only centriolar protein known to remain at the base of mature cilia and is required for intraflagellar transport trafficking. Surprisingly, targeted degradation of HYLS-1 after initiation of ciliogenesis does not affect ciliary structures. Taken together, our results indicate that while centrioles are essential to initiate cilia formation, they are dispensable for cilia maturation and maintenance.

The hydrolethalus syndrome protein HYLS-1 regulates formation of the ciliary gate.

Transition fibres (TFs), together with the transition zone (TZ), are basal ciliary structures thought to be crucial for cilium biogenesis and function by acting as a ciliary gate to regulate selective protein entry and exit. Here we demonstrate that the centriolar and basal body protein HYLS-1, the C. elegans orthologue of hydrolethalus syndrome protein 1, is required for TF formation, TZ organization and ciliary gating. Loss of HYLS-1 compromises the docking and entry of intraflagellar transport (IFT) particles, ciliary gating for both membrane and soluble proteins, and axoneme assembly. Additional depletion of the TF component DYF-19 in hyls-1 mutants further exacerbates TZ anomalies and completely abrogates ciliogenesis. Our data support an important role for HYLS-1 and TFs in establishment of the ciliary gate and underline the importance of selective protein entry for cilia assembly.