Human microcephaly ASPM protein is a spindle pole-focusing factor that functions redundantly with CDK5RAP2.

Microcephaly-associated mutations disrupt microtubule-dependent WDR62 translocation from the Golgi complex to the mitotic spindle poles, impair mitotic progression, and alter neurogenic trajectories in patient induced Pluripotent Stem Cell-derived 2D and 3D models of human neurodevelopment.

The displacement of the mitotic spindle to one side of a cell is important for many cells to divide unequally. While recent progress has begun to unveil some of the molecular mechanisms of mitotic spindle displacement, far less is known about how spindle displacement is precisely timed. A conserved mitotic progression mechanism is known to time events in dividing cells, although this has never been linked to spindle displacement. This mechanism involves the anaphase-promoting complex (APC), its activator Cdc20/Fizzy, its degradation target cyclin, and cyclin-dependent kinase (CDK). Here we show that these components comprise a previously unrecognized timer for spindle displacement. In the Caenorhabditis elegans zygote, mitotic spindle displacement begins at a precise time, soon after chromosomes congress to the metaphase plate. We found that reducing the function of the proteasome, the APC, or Cdc20/Fizzy delayed spindle displacement. Conversely, inactivating CDK in prometaphase caused the spindle to displace early. The consequence of experimentally unlinking spindle displacement from this timing mechanism was the premature displacement of incompletely assembled components of the mitotic spindle. We conclude that in this system, asymmetric positioning of the mitotic spindle is normally delayed for a short time until the APC inactivates CDK, and that this delay ensures that the spindle does not begin to move until it is fully assembled. To our knowledge, this is the first demonstration that mitotic progression times spindle displacement in the asymmetric division of an animal cell. We speculate that this link between the cell cycle and asymmetric cell division might be evolutionarily conserved, because the mitotic spindle is displaced at a similar stage of mitosis during asymmetric cell divisions in diverse systems.

The diverse populations of microtubule polymers in cells are functionally distinguished by different posttranslational modifications, including polyglutamylation. Polyglutamylation is enriched on subsets of microtubules including those found in the centrioles, mitotic spindle, and cilia. However, whether this modification alters intrinsic microtubule dynamics or affects extrinsic associations with specific interacting partners remains to be determined. Here we identify the microtubule-binding protein centriole and spindle-associated protein (CSAP), which colocalizes with polyglutamylated tubulin to centrioles, spindle microtubules, and cilia in human tissue culture cells. Reducing tubulin polyglutamylation prevents CSAP localization to both spindle and cilia microtubules. In zebrafish, CSAP is required for normal brain development and proper left-right asymmetry, defects that are qualitatively similar to those reported previously for depletion of polyglutamylation-conjugating enzymes. We also find that CSAP is required for proper cilia beating. Our work supports a model in which polyglutamylation can target selected microtubule-associated proteins, such as CSAP, to microtubule subpopulations, providing specific functional capabilities to these populations.

Kinetochore Orientation during Meiosis Is Controlled by Aurora B and the Monopolin Complex

Involvement of the telomeric protein Pin2/TRF1 in the regulation of the mitotic spindle

In the presence of unaligned chromosomes or other spindle abnormalities, the mitotic spindle checkpoint blocks both chromatid separation at anaphase and exit from mitosis. The spindle checkpoint is sustained by signaling from kinetochores that are not properly attached to microtubules of the mitotic spindle. Normally, when chromosomes align at the spindle equator at metaphase, microtubule-kinetochore interactions act to extinguish checkpoint signaling. The cessation of the checkpoint signal activates the Anaphase-Promoting Complex/Cyclosome (APC/C), an E3 ubiquitin ligase. The APC/C ubiquitylates the mitotic effectors, securin and cyclin B, which are then degraded by the proteasome. Securin and cyclin B degradation initiates chromatid separation at anaphase and triggers mitotic exit. We have found that long-term arrest of Hela cells with their chromosomes aligned at the metaphase plate results in chromatid separation without mitotic exit. Recently, we and others have characterized C13orf3/Ska3, a newly discovered component of the Ska (Spindle and kinetochore-associated) protein complex. We found that in mitosis Ska3 protein accumulates at kinetochores reaching maximal levels at metaphase. Upon anaphase onset, kinetochore-associated Ska3 levels decline and are eventually lost at telophase. We demonstrated that cells depleted of Ska3 by RNAi achieve metaphase alignment but fail to silence the spindle checkpoint. During this mitotic arrest with chromosomes aligned at the metaphase plate, kinetochores retain robust microtubule attachments. Kinetochores within these Ska3-depleted cells accumulate abnormally high levels of the mitotic spindle checkpoint protein, Bub1, presumably accounting for the inability to silence the checkpoint. Under extended metaphase arrest, the chromatids of individual chromosomes eventually separate, but cells remain arrested in mitosis. To test if other means of inducing metaphase arrest would also lead to chromatid separation, we treated cells in mitosis with the proteasome inhibitor, MG132. Again, after several hours at metaphase, chromatids separated. We speculate that for cells arrested at metaphase, continual pulling forces of the kinetochores on the mitotic spindle eventually overcomes the cohesin complex that holds chromatids together. This “cohesin slippage” results in abnormal chromatid separation for cells still in mid-mitosis. This response reflects a novel terminal phenotype for cells arrested in mitosis with an intact mitotic spindle. (Supported by the NIGMS and the McCasland Foundation.) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3868.

Cells with blocked microtubule polymerization are delayed in mitosis, but eventually manage to proliferate despite substantial chromosome missegregation. While several studies have analyzed the first cell division after microtubule depolymerization, we have asked how cells cope long-term with microtubule impairment. We allowed 24 clonal populations of yeast cells with beta-tubulin mutations preventing proper microtubule polymerization, to evolve for ˜150 generations. At the end of the laboratory evolution experiment, cells had regained the ability to form microtubules and were less sensitive to microtubule-depolymerizing drugs. Whole-genome sequencing identified recurrently mutated genes, in particular for tubulins and kinesins, as well as pervasive duplication of chromosome VIII. Recreating these mutations and chromosome VIII disomy prior to evolution confirmed that they allow cells to compensate for the original mutation in beta-tubulin. Most of the identified mutations did not abolish function, but rather restored microtubule functionality. Analysis of the temporal order of resistance development in independent populations repeatedly revealed the same series of events: disomy of chromosome VIII followed by a single additional adaptive mutation in either tubulins or kinesins. Since tubulins are highly conserved among eukaryotes, our results have implications for understanding resistance to microtubule-targeting drugs widely used in cancer therapy.

Amino acid transporters play a vital role in cellular homeostasis by maintaining protein synthesis. L-type amino acid transporter 1 (LAT1/SLC7A5/CD98lc) is a major transporter of large neutral amino acids in cancer cells because of its predominant expression. Although amino acid restriction with various amino acid analog treatments is known to induce mitotic defects, the involvement of amino acid transporters in cell division remains unclear. In this study, we identified that LAT1 is responsible for mitotic progression in a transport activity-independent manner. LAT1 knockdown activates the spindle assembly checkpoint, leading to a delay in metaphase. LAT1 maintains proper spindle orientation with confinement of the lateral cortex localization of the NuMA protein, which mediates the pulling force against the mitotic spindle toward the lateral cortex. Unexpectedly, JPH203, an inhibitor of LAT1 amino acid transport activity, does not affect mitotic progression. Moreover, the transport activity-deficient LAT1 mutant maintains the proper spindle orientation and mitotic progression. LAT1 forms a heterodimer with CD98 (SLC3A2/CD98hc) both in interphase and mitosis. Although CD98 knockdown decreases the plasma membrane localization of LAT1, it does not affect mitotic progression. LAT1 is localized to the Golgi and ER not only at the plasma membrane in interphase, and promotes Golgi unlinking during the mitotic entry, leading to centrosome maturation. These results suggest that LAT1 supports mitotic progression in an amino acid transport activity-independent manner and that Golgi-localized LAT1 is important for mitotic progression through the acceleration of Golgi unlinking and centrosome maturation. These findings reveal a novel LAT1 function in mitosis.

Recent work has underscored the importance of membrane trafficking events during cytokinesis. For example, targeted membrane secretion occurs at the cleavage furrow in animal cells, and proteins that regulate endocytosis also influence the process of cytokinesis. Nonetheless, the prevailing dogma is that endosomal membrane trafficking ceases during mitosis and resumes after cell division is complete. In this study, we have characterized endocytic membrane trafficking events that occur during mammalian cell cytokinesis. We have found that, although endocytosis ceases during the early stages of mitosis, it resumes during late mitosis in a temporally and spatially regulated pattern as cells progress from anaphase to cytokinesis. Using fixed and live cell imaging, we have found that, during cleavage furrow ingression, vesicles are internalized from the polar region and subsequently trafficked to the midbody area during later stages of cytokinesis. In addition, we have demonstrated that cytokinesis is inhibited when clathrin-mediated endocytosis is blocked using a series of dominant negative mutants. In contrast to previous thought, we conclude that endocytosis resumes during the later stages of mitosis, before cytokinesis is completed. Furthermore, based on our findings, we propose that the proper regulation of endosomal membrane traffic is necessary for the successful completion of cytokinesis.

Superhelical simian virus 40 DNA migrates more rapidly during electrophoresis in agarose gels than covalently closed DNA free of superhelical turns (relaxed DNA). The difference in electrophoretic mobility between superhelical and relaxed DNA was used to monitor the activity of a protein from human tissue culture cells which converts superhelical DNA into relaxed DNA. Purified DNA-relaxing protein removes both negative and positive superhelical turns and acts in a catalytic manner. The relaxation of DNA proceeds in a stepwise fashion and DNA intermediates with decreasing numbers of superhelical turns are seen during the course of the reaction.

Numb, an intrinsic Notch inhibitor and cell fate determinant, has been shown to possess many functions in proper cell division. These include its role in endocytosis, a tumor suppressor role via regulation of p53 stability, and asymmetric self renewal during progenitor cell division. Studies have shown that a proper localization of fully functional Plk1 is required for mitotic entry, progression and exit. Plk1 has been shown to play a critical role in bi-polar spindle formation and maturation during mitosis. It has been shown that chemical inhibition as well as genetic knockdown of Plk1 results in cells forming a mono-polar spindle phenotype surrounded by DNA rather than a bi-polar arrangement on either side of the metaphase plate. This severe mitotic phenotype results in a G2/M cell cycle arrest and mitotic catastrophe. Also, milder inhibition of Plk1 has been shown to result in errors in bi-polar spindle formation including multi-polar phenotypes or incomplete microtubule attachment and division. Some of these less pronounced phenotypes may not prevent cell division and may contribute to cellular aneuploidy and cancer. In this study, employing melanoma cell lines A375 and HS294T, we demonstrated that Numb and Plk1 interact as shown by co-immunoprecipitation and immunofluorescent assays. In addition, we found that both Plk1 and Numb co-localize to the spindle poles in metaphase. Further, using lentiviral shRNA knockdown of Plk1 or Numb, we also found that loss of Plk1 or Numb dysregulates the localization of the other. Most striking was the pronounced mislocalization of Plk1 from the spindle poles. Although, the majority of metaphase cells were still bi-polar, there was a marked increase in mislocalized γ-tubulin in some instances. Additionally, in the cells with two defined spindle poles, γ-tubulin still appears to be less pronounced at strictly the spindle pole with an apparent indistinct staining surrounding the poles indicating that Numb may not be directly required for γ-tubulin localization to the spindles but the misregulated Plk1 localization slows or prevents spindle pole maturation and γ-tubulin recruitment. Conversely, the differing phenotypes of γ-tubulin localization may also be explained by a redundant role of Numb-like, though this is yet to be explored. Overall, these data suggested that Numb may possess tumor suppressor functions by regulating proper Plk1 localization and proper spindle pole maturation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1062.

The direction in which a cell divides is determined by the orientation of its mitotic spindle at metaphase. Spindle orientation is therefore important for a wide range of developmental processes, ranging from germline stem cell division to epithelial tissue homeostasis and regeneration. In multiple cell types in multiple animals, spindle orientation is controlled by a conserved biological machine that mediates a pulling force on astral microtubules. Restricting the localization of this machine to only specific regions of the cortex can thus determine how the mitotic spindle is oriented. As we review here, recent findings based on studies in tunicate, worm, fly and vertebrate cells have revealed that the mechanisms for mediating this restriction are surprisingly diverse.

Autosomal recessive primary microcephaly (MCPH) delineates a genetically heterogeneous and rare subgroup of congenital microcephalies characterized by a pronounced reduction of brain volume at birth and intellectual disability.1,2 Genetic causes of MCPH subtypes 1–12 include mutations in genes encoding microcephalin (MCPH1; MIM*607117), WD-repeat-containing protein 62 (WDR62, MCPH2; MIM*613583), cyclin-dependent kinase 5 regulatory subunit-associated protein 2 (CDK5RAP2, MCPH3; MIM*608201), cancer susceptibility candidate 5 (CASC5, MCPH4; MIM*609173), abnormal spindle-like, microcephaly-associated protein (ASPM, MCPH5; MIM*605481), centromeric protein J (CENPJ, MCPH6; MIM*609279), SCL/TAL1-interrupting locus (STIL, MCPH7; MIM*181590), centrosomal protein 135 kD (CEP135, MCPH8; MIM*611423), centrosomal protein 152 kD (CEP152, MCPH9; MIM*613529), zinc finger protein 335 (ZNF335, MCPH10; MIM*610827), polyhomeotic-like 1 (PHC1, MCPH11; MIM*602978), and cyclin-dependent kinase 6 (CDK6; MCPH12, MIM*603368). A respectable percentage of families with an MCPH phenotype do not carry mutations in the known MCPH genes, indicating further genetic heterogeneity. In 2011, Marchal et al. reported a consanguineous family with a classic MCPH phenotype and demonstrated linkage to chromosome 10q11.23–21.3; thereby they postulated a new and hitherto unrecognized MCPH locus.3 However, within this region, a new candidate gene could not be identified. We reinvestigated this family by a whole-exome sequencing (WES) approach, and identified compound heterozygous mutations in the ASPM gene as the causal gene defect. We studied the index family previously reported by Marchal et al. 20113 with approval of the ethics committee of the Charite (EA1/212/08) and written informed consent of the parents (Fig. 1). The 3 affected children of healthy, consanguineous parents of Turkish descent were born at term without complications and with normal weight and height. Microcephaly was diagnosed through prenatal ultrasound in all patients by the 24th week of gestation, and one further pregnancy (II.4) was terminated through abortion following prenatal microcephaly diagnosis. The relative head circumference decreased further in the youngest and most affected child (from −3 to −7 SD). Motor milestones were delayed, and all 3 children presented with intellectual disability and an expressive speech delay despite normal hearing. First words were spoken between ages 2 and 4.5 years of age, but even by the age of 23 y, only single words were expressed in the eldest patient. In the youngest patient, II.5, a spastic quadriplegia with increased muscle tone and extensor plantar reflex, bilateral atypical transverse palmar creases, and a short left proximal fifth phalanx were apparent. Patient II.3 had fine motor problems and required surgery for strabismus. The 2 patients II.3 and II.5 developed focal and generalized epilepsy at ages 4 and 9 y, respectively, which were successfully treated with carbamazepine and valproic acid. In addition to the microencephaly, cranial magnetic resonance imaging (MRI) revealed a parieto–occipital and left temporo–occipital gyration defect in patient II.3 as well as reduced gyration, parietal polymicrogyria and a myelination defect in patient II.5. The results of abdominal ultrasound, echocardiography, routine blood tests, and metabolic work-up were normal. In all 3 affected children, we identified, by means of WES and Medical Resequencing Analysis Pipeline (MERAP; https://sourceforge.net/projects/merap/), a compound heterozygous mutation in the ASPM gene: (1) the nonsense mutation c.8227C > T (p.R2743X); and (2) the frameshift mutation c.7772_7775delAAAA (p.2591fs) ({type:entrez-nucleotide,attrs:{text:NM_018136,term_id:194248058}}NM_018136). These mutations, which have not been reported so far in the dbSNP138 (http://www.ncbi.nlm.nih.gov/SNP/), the 1000 Genome Project (http://www.1000genomes.org/), the NHLBI GO Exome Sequencing Project (http://evs.gs.washington.edu/EVS/), and our in-house 417 exome data of the Middle East origin, were confirmed by Sanger sequencing, and show perfect cosegregation with biparental inheritance (Fig. 1). Phenotype and clinical presentation are in agreement with the phenotypic spectrum of ASPM mutations.4 However, compound heterozygosity is not to be expected with close consanguinity a priori, though not impossible and not contradictory in principle. Likewise, the occurrence of multiple independent mutations in an isolated inbred population is an unexpected finding. It was first reported on Reunion Island with limb girdle muscular dystrophy, and hence referred to as the “Reunion paradox”.5 Subsequently, several examples have been reported, and until recently, there was no satisfactory explanation.6 Standard population genetic theory has been extended recently, with the result that compound heterozygosity in inbred populations, and, hence, in consanguineous marriages, is severely underestimated (or “misunderestimated” according to George W Bush) by neglecting population sub-structuring and other phenomena.7 There is a lesson to be learned8 when autozygosity mapping is applied as a research strategy for elucidating the genetic causes of rare recessive diseases, and here we report another example where the correct molecular diagnosis was missed previously by applying incomplete and insufficient, though simplistic and standard, population genetic theory. Figure 1. Compound heterozygous ASPM mutation in family with MCPH. Pedigree and results of Sanger sequencing.

Acyl-coenzyme A:cholesterol acyltransferase (ACAT) catalyzes the formation of intracellular cholesterol esters in various tissues. We recently reported the cloning and expression of human macrophage ACAT cDNA. In the current study, we report the production of specific polyclonal antibodies against ACAT by immunizing rabbits with the recombinant fusion protein composed of glutathione S-transferase and the first 131 amino acids of ACAT protein. Immunoblot analysis showed that the antibodies cross-reacted with a 50-kDa protein band from a variety of human cell lines. These antibodies immunodepleted more than 90% of detergent-solubilized ACAT activities from six different human cell types, demonstrating that the 50-kDa protein is the major ACAT catalytic component in these cells. In multiple human tissues examined, the antibodies recognized protein bands with various molecular weights. These antibodies also cross-reacted with the ACAT protein in Chinese hamster ovary cells. Immunoblot analysis showed that the ACAT protein contents in human fibroblast cells, HepG2 cells, or Chinese hamster ovary cells were not affected by sterol in the medium, demonstrating that the main mechanism for sterol-dependent regulation of ACAT activity in these cells is not change in ACAT protein content. As revealed by indirect immunofluorescent microscopy, the ACAT protein in tissue culture cells was located in the endoplasmic reticulum. This finding, along with earlier studies, suggests that cholesterol concentration in the endoplasmic reticulum may be the major determinant for regulating ACAT activity in the intact cells.

Kinesin-8 proteins are microtubule motors that are often involved in regulation of mitotic spindle length and chromosome alignment. They move towards the plus ends of spindle microtubules and regulate the dynamics of these ends due, at least in some species, to their microtubule depolymerization activity. Plasmodium spp. exhibit an atypical endomitotic cell division in which chromosome condensation and spindle dynamics in the different proliferative stages are not well understood. Genome-wide shared orthology analysis of Plasmodium spp. revealed the presence of two kinesin-8 motor proteins, kinesin-8X and kinesin-8B. Here we studied the biochemical properties of kinesin-8X and its role in parasite proliferation. In vitro, kinesin-8X has motility and depolymerization activities like other kinesin-8 motors. To understand the role of Plasmodium kinesin-8X in cell division, we used fluorescence-tagging and live cell imaging to define its location, and gene targeting to analyse its function, during all proliferative stages of the rodent malaria parasite P. berghei life cycle. The results revealed a spatio-temporal involvement of kinesin-8X in spindle dynamics and an association with both mitotic and meiotic spindles and the putative microtubule organising centre (MTOC). Deletion of the kinesin-8X gene revealed a defect in oocyst development, confirmed by ultrastructural studies, suggesting that this protein is required for oocyst development and sporogony. Transcriptome analysis of Δkinesin-8X gametocytes revealed modulated expression of genes involved mainly in microtubule-based processes, chromosome organisation and the regulation of gene expression, supporting a role for kinesin-8X in cell division. Kinesin-8X is thus required for parasite proliferation within the mosquito and for transmission to the vertebrate host.