Orchestrating vesicle transport, ESCRTs and kinase surveillance during abscission.

During the final stage of cell division, the future daughter cells are physically separated through abscission. This process requires coordination of many molecular machines, including endocytic and secretory vesicle trafficking proteins as well as ESCRT (endosomal sorting complex required for transport) proteins, that mediate a complex series of events to culminate in the final separation of daughter cells. Abscission is coordinated with other cellular processes (for example, nuclear pore reassembly) through mitotic kinases such as Aurora B and Polo-like kinase 1, which act as master regulators to ensure proper progression of abscission.

A last-minute rescue of trapped chromatin.

Chromosome segregation and cytokinesis must be tightly coordinated to ensure that chromosomes are accurately partitioned between dividing cells. In this issue, Steigemann et al. (2009) report that Aurora B kinase promotes proper chromosome segregation by delaying abscission when chromatin is trapped between dividing cells.

New frontiers: discovering cilia-independent functions of cilia proteins.

In most vertebrates, mitotic spindles and primary cilia arise from a common origin, the centrosome. In non-cycling cells, the centrosome is the template for primary cilia assembly and, thus, is crucial for their associated sensory and signaling functions. During mitosis, the duplicated centrosomes mature into spindle poles, which orchestrate mitotic spindle assembly, chromosome segregation, and orientation of the cell division axis. Intriguingly, both cilia and spindle poles are centrosome-based, functionally distinct structures that require the action of microtubule-mediated, motor-driven transport for their assembly. Cilia proteins have been found at non-cilia sites, where they have distinct functions, illustrating a diverse and growing list of cellular processes and structures that utilize cilia proteins for crucial functions. In this review, we discuss cilia-independent functions of cilia proteins and re-evaluate their potential contributions to "cilia" disorders.

The kinetochore protein, CENPF, is mutated in human ciliopathy and microcephaly phenotypes.

BACKGROUND: Mutations in microtubule-regulating genes are associated with disorders of neuronal migration and microcephaly. Regulation of centriole length has been shown to underlie the pathogenesis of certain ciliopathy phenotypes. Using a next-generation sequencing approach, we identified mutations in a novel centriolar disease gene in a kindred with an embryonic lethal ciliopathy phenotype and in a patient with primary microcephaly. METHODS AND RESULTS: Whole exome sequencing data from a non-consanguineous Caucasian kindred exhibiting mid-gestation lethality and ciliopathic malformations revealed two novel non-synonymous variants in CENPF, a microtubule-regulating gene. All four affected fetuses showed segregation for two mutated alleles [IVS5-2A>C, predicted to abolish the consensus splice-acceptor site from exon 6; c.1744G>T, p.E582X]. In a second unrelated patient exhibiting microcephaly, we identified two CENPF mutations [c.1744G>T, p.E582X; c.8692 C>T, p.R2898X] by whole exome sequencing. We found that CENP-F colocalised with Ninein at the subdistal appendages of the mother centriole in mouse inner medullary collecting duct cells. Intraflagellar transport protein-88 (IFT-88) colocalised with CENP-F along the ciliary axonemes of renal epithelial cells in age-matched control human fetuses but did not in truncated cilia of mutant CENPF kidneys. Pairwise co-immunoprecipitation assays of mitotic and serum-starved HEKT293 cells confirmed that IFT88 precipitates with endogenous CENP-F. CONCLUSIONS: Our data identify CENPF as a new centriolar disease gene implicated in severe human ciliopathy and microcephaly related phenotypes. CENP-F has a novel putative function in ciliogenesis and cortical neurogenesis.

Characterization of the human NEK7 interactome suggests catalytic and regulatory properties distinct from those of NEK6.

Human NEK7 is a regulator of cell division and plays an important role in growth and survival of mammalian cells. Human NEK6 and NEK7 are closely related, consisting of a conserved C-terminal catalytic domain and a nonconserved and disordered N-terminal regulatory domain, crucial to mediate the interactions with their respective proteins. Here, in order to better understand NEK7 cellular functions, we characterize the NEK7 interactome by two screening approaches: one using a yeast two-hybrid system and the other based on immunoprecipitation followed by mass spectrometry analysis. These approaches led to the identification of 61 NEK7 interactors that contribute to a variety of biological processes, including cell division. Combining additional interaction and phosphorylation assays from yeast two-hybrid screens, we validated CC2D1A, TUBB2B, MNAT1, and NEK9 proteins as potential NEK7 interactors and substrates. Notably, endogenous RGS2, TUBB, MNAT1, NEK9, and PLEKHA8 localized with NEK7 at key sites throughout the cell cycle, especially during mitosis and cytokinesis. Furthermore, we obtained evidence that the closely related kinases NEK6 and NEK7 do not share common interactors, with the exception of NEK9, and display different modes of protein interaction, depending on their N- and C-terminal regions, in distinct fashions. In summary, our work shows for the first time a comprehensive NEK7 interactome that, combined with functional in vitro and in vivo assays, suggests that NEK7 is a multifunctional kinase acting in different cellular processes in concert with cell division signaling and independently of NEK6.

Loss of centrosome integrity induces p38-p53-p21-dependent G1-S arrest.

Centrosomes organize the microtubule cytoskeleton for both interphase and mitotic functions. They are implicated in cell-cycle progression but the mechanism is unknown. Here, we show that depletion of 14 out of 15 centrosome proteins arrests human diploid cells in G1 with reduced Cdk2-cyclin A activity and that expression of a centrosome-disrupting dominant-negative construct gives similar results. Cell-cycle arrest is always accompanied by defects in centrosome structure and function (for example, duplication and primary cilia assembly). The arrest occurs from within G1, excluding contributions from mitosis and cytokinesis. The arrest requires p38, p53 and p21, and is preceded by p38-dependent activation and centrosomal recruitment of p53. p53-deficient cells fail to arrest, leading to centrosome and spindle dysfunction and aneuploidy. We propose that loss of centrosome integrity activates a checkpoint that inhibits G1-S progression. This model satisfies the definition of a checkpoint in having three elements: a perturbation that is sensed, a transducer (p53) and a receiver (p21).

Dynein light intermediate chain 1 is required for progress through the spindle assembly checkpoint.

The spindle assembly checkpoint monitors microtubule attachment to kinetochores and tension across sister kinetochores to ensure accurate division of chromosomes between daughter cells. Cytoplasmic dynein functions in the checkpoint, apparently by moving critical checkpoint components off kinetochores. The dynein subunit required for this function is unknown. Here we show that human cells depleted of dynein light intermediate chain 1 (LIC1) delay in metaphase with increased interkinetochore distances; dynein remains intact, localised and functional. The checkpoint proteins Mad1/2 and Zw10 localise to kinetochores under full tension, whereas BubR1 is diminished at kinetochores. Metaphase delay and increased interkinetochore distances are suppressed by depletion of Mad1, Mad2 or BubR1 or by re-expression of wtLIC1 or a Cdk1 site phosphomimetic LIC1 mutant, but not Cdk1-phosphorylation-deficient LIC1. When the checkpoint is activated by microtubule depolymerisation, Mad1/2 and BubR1 localise to kinetochores. We conclude that a Cdk1 phosphorylated form of LIC1 is required to remove Mad1/2 and Zw10 but not BubR1 from kinetochores during spindle assembly checkpoint silencing.

Mutations and aneuploidy: co-conspirators in cancer?

The role of intragenic point mutations in human cancer is well established. However, the contribution of massive genomic changes collectively known as aneuploidy is less certain. Recent experimental work suggests that aneuploidy is required for sporadic carcinogenesis in mice and that it may collaborate with intragenic mutations during tumorigenesis. The genomic plasticity afforded by aneuploidy could facilitate emergence of protumorigenic gene dosage changes and accelerate accumulation of oncogenes and loss of tumor suppressor genes. These new findings force us to rethink the pathogenesis of carcinoma in ways that have significant implications for diagnosis and therapy.

Centrosome control of the cell cycle.

Early observations of centrosomes, made a century ago, revealed a tiny dark structure surrounded by a radial array of cytoplasmic fibers. We now know that the fibers are microtubules and that the dark organelles are centrosomes that mediate functions far beyond the more conventional role of microtubule organization. More recent evidence demonstrates that the centrosome serves as a scaffold for anchoring an extensive number of regulatory proteins. Among these are cell-cycle regulators whose association with the centrosome is an essential step in cell-cycle control. Such studies show that the centrosome is required for several cell-cycle transitions, including G(1) to S-phase, G(2) to mitosis and metaphase to anaphase. In this review (which is part of the Chromosome Segregation and Aneuploidy series), we discuss recent data that provide the most direct links between centrosomes and cell-cycle progression.

Chromatin remodeling proteins interact with pericentrin to regulate centrosome integrity.

Pericentrin is an integral centrosomal component that anchors regulatory and structural molecules to centrosomes. In a yeast two-hybrid screen with pericentrin we identified chromodomain helicase DNA-binding protein 4 (CHD4/Mi2beta). CHD4 is part of the multiprotein nucleosome remodeling deacetylase (NuRD) complex. We show that many NuRD components interacted with pericentrin by coimmunoprecipitation and that they localized to centrosomes and midbodies. Overexpression of the pericentrin-binding domain of CHD4 or another family member (CHD3) dissociated pericentrin from centrosomes. Depletion of CHD3, but not CHD4, by RNA interference dissociated pericentrin and gamma-tubulin from centrosomes. Microtubule nucleation/organization, cell morphology, and nuclear centration were disrupted in CHD3-depleted cells. Spindles were disorganized, the majority showing a prometaphase-like configuration. Time-lapse imaging revealed mitotic failure before chromosome segregation and cytokinesis failure. We conclude that pericentrin forms complexes with CHD3 and CHD4, but a distinct CHD3-pericentrin complex is required for centrosomal anchoring of pericentrin/gamma-tubulin and for centrosome integrity.

Pericentrin forms a complex with intraflagellar transport proteins and polycystin-2 and is required for primary cilia assembly.

Primary cilia are nonmotile microtubule structures that assemble from basal bodies by a process called intraflagellar transport (IFT) and are associated with several human diseases. Here, we show that the centrosome protein pericentrin (Pcnt) colocalizes with IFT proteins to the base of primary and motile cilia. Immunogold electron microscopy demonstrates that Pcnt is on or near basal bodies at the base of cilia. Pcnt depletion by RNA interference disrupts basal body localization of IFT proteins and the cation channel polycystin-2 (PC2), and inhibits primary cilia assembly in human epithelial cells. Conversely, silencing of IFT20 mislocalizes Pcnt from basal bodies and inhibits primary cilia assembly. Pcnt is found in spermatocyte IFT fractions, and IFT proteins are found in isolated centrosome fractions. Pcnt antibodies coimmunoprecipitate IFT proteins and PC2 from several cell lines and tissues. We conclude that Pcnt, IFTs, and PC2 form a complex in vertebrate cells that is required for assembly of primary cilia and possibly motile cilia and flagella.

A novel human protein of the maternal centriole is required for the final stages of cytokinesis and entry into S phase.

Centrosomes nucleate microtubules and contribute to mitotic spindle organization and function. They also participate in cytokinesis and cell cycle progression in ways that are poorly understood. Here we describe a novel human protein called centriolin that localizes to the maternal centriole and functions in both cytokinesis and cell cycle progression. Centriolin silencing induces cytokinesis failure by a novel mechanism whereby cells remain interconnected by long intercellular bridges. Most cells continue to cycle, reenter mitosis, and form multicellular syncytia. Some ultimately divide or undergo apoptosis specifically during the protracted period of cytokinesis. At later times, viable cells arrest in G1/G0. The cytokinesis activity is localized to a centriolin domain that shares homology with Nud1p and Cdc11p, budding and fission yeast proteins that anchor regulatory pathways involved in progression through the late stages of mitosis. The Nud1p-like domain of centriolin binds Bub2p, another component of the budding yeast pathway. We conclude that centriolin is required for a late stage of vertebrate cytokinesis, perhaps the final cell cleavage event, and plays a role in progression into S phase.

Survivin modulates microtubule dynamics and nucleation throughout the cell cycle.

Survivin is a member of the chromosomal passenger complex implicated in kinetochore attachment, bipolar spindle formation, and cytokinesis. However, the mechanism by which survivin modulates these processes is unknown. Here, we show by time-lapse imaging of cells expressing either green fluorescent protein (GFP)-alpha-tubulin or the microtubule plus-end binding protein GFP-EB1 that depletion of survivin by small interfering RNAs (siRNAs) increased both the number of microtubules nucleated by centrosomes and the incidence of microtubule catastrophe, the transition from microtubule growth to shrinking. In contrast, survivin overexpression reduced centrosomal microtubule nucleation and suppressed both microtubule dynamics in mitotic spindles and bidirectional growth of microtubules in midbodies during cytokinesis. siRNA depletion or pharmacologic inhibition of another chromosomal passenger protein Aurora B, had no effect on microtubule dynamics or nucleation in interphase or mitotic cells even though mitosis was impaired. We propose a model in which survivin modulates several mitotic events, including spindle and interphase microtubule organization, the spindle assembly checkpoint and cytokinesis through its ability to modulate microtubule nucleation and dynamics. This pathway may affect the microtubule-dependent generation of aneuploidy and defects in cell polarity in cancer cells, where survivin is commonly up-regulated.

Viral Infection or IFN-α Alters Mitotic Spindle Orientation by Modulating Pericentrin Levels.

Congenital microcephaly occurs in utero during Zika virus (ZIKV) infection. The single-gene disorder, Majewski osteodysplastic primordial dwarfism type II (MOPDII), also leads to microcephaly and is concomitant with a decrease in the centrosomal protein, pericentrin (PCNT). This protein is a known contributor of mitotic spindle misorientation and ultimately, microcephaly. Similar to MOPDII, either viral infection or interferon (IFN)-α exposure reduced PCNT levels at the mitotic spindle poles. We unexpectedly found that infection of cells with any one of a diverse set of viruses, such as ZIKV, dengue virus, cytomegalovirus, influenza A virus, or hepatitis B virus, or treatment of cells with the anti-viral cytokine, IFN-α, produced mitotic spindle misorientation. These findings demonstrate a related mechanism for the development of microcephaly in viral infection, the host's antiviral IFN response, and primordial dwarfism.

New dimensions of asymmetric division in vertebrates.

Traditionally, we imagine that cell division gives rise to two identical daughter cells. Nevertheless, all cell divisions, to some degree, display asymmetry. Asymmetric cell division is defined as the generation of two daughter cells with different physical content and/or developmental potential. Several organelles and cellular components including the centrosome, non-coding RNA, chromatin, and recycling endosomes are involved in the process of asymmetric cell division. Disruption of this important process is known to induce profound defects in development, the immune response, regeneration of tissues, aging, and cancer. Here, we discuss recent advances that expand our understanding of the mechanisms and consequences of asymmetric cell division in vertebrate organisms.

PCNT point mutations and familial intracranial aneurysms.

OBJECTIVE: To identify novel genes involved in the etiology of intracranial aneurysms (IAs) or subarachnoid hemorrhages (SAHs) using whole-exome sequencing. METHODS: We performed whole-exome sequencing in 13 individuals from 3 families with an autosomal dominant IA/SAH inheritance pattern to look for candidate genes for disease. In addition, we sequenced PCNT exon 38 in a further 161 idiopathic patients with IA/SAH to find additional carriers of potential pathogenic variants. RESULTS: We identified 2 different variants in exon 38 from the PCNT gene shared between affected members from 2 different families with either IA or SAH (p.R2728C and p.V2811L). One hundred sixty-four samples with either SAH or IA were Sanger sequenced for the PCNT exon 38. Five additional missense mutations were identified. We also found a second p.V2811L carrier in a family with a history of neurovascular diseases. CONCLUSION: The PCNT gene encodes a protein that is involved in the process of microtubule nucleation and organization in interphase and mitosis. Biallelic loss-of-function mutations in PCNT cause a form of primordial dwarfism (microcephalic osteodysplastic primordial dwarfism type II), and ≈50% of these patients will develop neurovascular abnormalities, including IAs and SAHs. In addition, a complete Pcnt knockout mouse model (Pcnt -/-) published previously showed general vascular abnormalities, including intracranial hemorrhage. The variants in our families lie in the highly conserved PCNT protein-protein interaction domain, making PCNT a highly plausible candidate gene in cerebrovascular disease.

Mitosis-specific anchoring of gamma tubulin complexes by pericentrin controls spindle organization and mitotic entry.

Microtubule nucleation is the best known function of centrosomes. Centrosomal microtubule nucleation is mediated primarily by gamma tubulin ring complexes (gamma TuRCs). However, little is known about the molecules that anchor these complexes to centrosomes. In this study, we show that the centrosomal coiled-coil protein pericentrin anchors gamma TuRCs at spindle poles through an interaction with gamma tubulin complex proteins 2 and 3 (GCP2/3). Pericentrin silencing by small interfering RNAs in somatic cells disrupted gamma tubulin localization and spindle organization in mitosis but had no effect on gamma tubulin localization or microtubule organization in interphase cells. Similarly, overexpression of the GCP2/3 binding domain of pericentrin disrupted the endogenous pericentrin-gamma TuRC interaction and perturbed astral microtubules and spindle bipolarity. When added to Xenopus mitotic extracts, this domain uncoupled gamma TuRCs from centrosomes, inhibited microtubule aster assembly, and induced rapid disassembly of preassembled asters. All phenotypes were significantly reduced in a pericentrin mutant with diminished GCP2/3 binding and were specific for mitotic centrosomal asters as we observed little effect on interphase asters or on asters assembled by the Ran-mediated centrosome-independent pathway. Additionally, pericentrin silencing or overexpression induced G2/antephase arrest followed by apoptosis in many but not all cell types. We conclude that pericentrin anchoring of gamma tubulin complexes at centrosomes in mitotic cells is required for proper spindle organization and that loss of this anchoring mechanism elicits a checkpoint response that prevents mitotic entry and triggers apoptotic cell death.

The Drosophila kinesin-like protein KLP67A is essential for mitotic and male meiotic spindle assembly.

We have performed a mutational analysis together with RNA interference to determine the role of the kinesin-like protein KLP67A in Drosophila cell division. During both mitosis and male meiosis, Klp67A mutations cause an increase in MT length and disrupt discrete aspects of spindle assembly, as well as cytokinesis. Mutant cells exhibit greatly enlarged metaphase spindle as a result of excessive MT polymerization. The analysis of both living and fixed cells also shows perturbations in centrosome separation, chromosome segregation, and central spindle assembly. These data demonstrate that the MT plus end-directed motor KLP67A is essential for spindle assembly during mitosis and male meiosis and suggest that the regulation of MT plus-end polymerization is a key determinant of spindle architecture throughout cell division.

Centrosome abnormalities and chromosome instability occur together in pre-invasive carcinomas.

Centrosomes play critical roles in processes that ensure proper segregation of chromosomes and maintain the genetic stability of human cells. They contribute to mitotic spindle organization and regulate aspects of cytokinesis and cell cycle progression. We and others have shown that centrosomes are abnormal in most aggressive carcinomas. Moreover, centrosome defects have been implicated in chromosome instability and loss of cell cycle control in invasive carcinoma. Others have suggested that centrosome defects only occur late in tumorigenesis and may not contribute to early steps of tumor development. To address this issue, we examined pre-invasive human carcinoma in situ lesions for centrosome defects and chromosome instability. We found that a significant fraction of precursor lesions to some of the most common human cancers had centrosome defects, including in situ carcinomas of the uterine cervix, prostate, and female breast. Moreover, centrosome defects occurred together with mitotic spindle defects, chromosome instability, and high cytologic grade. Because most pre-invasive lesions are not uniformly mutant for p53, the development of centrosome defects does not appear to require abrogation of p53 function. Our findings demonstrate that centrosome defects occur concurrently with chromosome instability and cytologic changes in the earliest identifiable step in human cancer. Our results suggest that centrosome defects may contribute to the earliest stages of cancer development through the generation of chromosome instability. This, together with ongoing structural changes in chromosomes, could accelerate accumulation of alleles carrying pro-oncogenic mutations and loss of alleles containing wild-type tumor suppressor genes and thus accelerate the genomic changes characteristic of carcinoma, the most prevalent human cancer.

The Centrosome, a Multitalented Renaissance Organelle.

The centrosome acts as a microtubule-organizing center (MTOC) from the G1 to G2 phases of the cell cycle; it can mature into a spindle pole during mitosis and/or transition into a cilium by elongating microtubules (MTs) from the basal body on cell differentiation or cell cycle arrest. New studies hint that the centrosome functions in more than MT organization. For instance, it has recently been shown that a specific substructure of the centrosome-the mother centriole appendages-are required for the recycling of endosomes back to the plasma membrane. This alone could have important implications for a renaissance in our understanding of the development of primary cilia, endosome recycling, and the immune response. Here, we review newly identified roles for the centrosome in directing membrane traffic, the immunological synapse, and the stress response.