Mitotic Spindle Poles Research Articles

Abstract 1642: NuMA exclusive post-translational modification in epithelial cancer cells is a novel target for cell eradication

Abstract The post-translational modification of proteins implicated in mitosis in healthy and in cancer epithelial cells was measured by 2-D gels analysis. The post-translational modifications of NuMA in a variety of human epithelial cancer cells was prevented by treatment with a small phenanthridine, PJ34, while not impaired in similarly treated healthy epithelial cells. NuMA in the cancer cells was phosphorylated by pim1 kinase, and was polyADP-ribosylated by Tankyrase1. These post-translational modifications are inhibited by PJ34. Tankyrase1 is not expressed in healthy epithelial cells, and pim1 kinase was hardly expressed in these cells. Preventing the post-translational modification of NuMA in the epithelial cancer cells also suppressed the protein binding capacity of NuMA. NuMA clustering in the mitotic spindle poles, which stabilizes the mitotic spindle, and is required for the alignment of chromosomes in the spindle mid-zone was impaired. In epithelial cancer cells treated with PJ34, the mitotic spindle poles were distorted, extra-centrosomes were not clustered in multi-centrosomal cancer cells, and chromosomes were dispersed. Chromosomes dispersion violated mitosis termination. Mitosis arrested in the anaphase led to mitotic catastrophe cell death. PJ34 treated epithelial cancer cells were eradicated during 72-96 hours, regardless of their mutations, while treated healthy epithelial cells were not affected, and continued to proliferate. Citation Format: Malka Cohen-Armon. NuMA exclusive post-translational modification in epithelial cancer cells is a novel target for cell eradication [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1642.

Abstract 1638: Regulation of spindle pole integrity by the MASTL-ENSA-aurora a pathway during mitosis

Abstract The preservation of spindle pole integrity is crucial for proper spindle assembly and chromosome segregation in mitosis, yet the precise mechanisms governing spindle pole integrity remain elusive. During mitosis, phosphorylated Ensa (p-ENSA) localizes to spindle poles, and the inhibition of ENSA leads to a range of mitotic defects, including misaligned chromosomes, multipolar spindles, asymmetric bipolar spindles, and centrosome aberrations, resulting in a delayed mitotic progression. Remarkably, the mitotic delay induced by ENSA inhibition is alleviated upon depletion of PP2A-B55α, but spindle pole defects persist. Significantly, we have observed an interaction between ENSA and Aurora A during mitosis, and the inhibition of ENSA diminishes Aurora A expression at the mitotic spindle poles. Intriguingly, the injection of MKI-2-sensitized tumors is associated with heightened chromosomal instability and downregulation of the MASTL-ENSA-Aurora A pathway in an orthotopic breast cancer mouse model. These findings offer new insights into the regulation of spindle pole integrity through the MASTL-ENSA-Aurora A pathway during mitosis, emphasizing the pivotal role of ENSA in recruiting Aurora A to the spindle pole, independently of PP2A-B55α. Citation Format: Seul Kim, Kyoungho Jun, Ye-Hyun Kim, Jae Sung Kim. Regulation of spindle pole integrity by the MASTL-ENSA-aurora a pathway during mitosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1638.

Endosulfine alpha maintains spindle pole integrity by recruiting Aurora A during mitosis

BackgroundThe maintenance of spindle pole integrity is essential for spindle assembly and chromosome segregation during mitosis. However, the underlying mechanisms governing spindle pole integrity remain unclear.MethodsENSA was inhibited by siRNA or MKI-2 treatment and its effect on cell cycle progression, chromosome alignment and microtubule alignment was observed by immunohistochemical staining and western blotting. PP2A-B55α knockdown by siRNA was performed to rescue the phenotype caused by ENSA inhibition. The interaction between ENSA and Aurora A was detected by in situ PLA. Furthermore, orthotopic implantation of 4Tl-luc cancer cells was conducted to confirm the consistency between the in vitro and in vivo relationship of the ENSA-Aurora A interaction.ResultsDuring mitosis, p-ENSA is localized at the spindle poles, and the inhibition of ENSA results in mitotic defects, such as misaligned chromosomes, multipolar spindles, asymmetric bipolar spindles, and centrosome defects, with a delay in mitotic progression. Although the mitotic delay caused by ENSA inhibition was rescued by PP2A-B55α depletion, spindle pole defects persisted. Notably, we observed a interaction between ENSA and Aurora A during mitosis, and inhibition of ENSA reduced Aurora A expression at the mitotic spindle poles. Injecting MKI-2-sensitized tumors led to increased chromosomal instability and downregulation of the MASTL-ENSA-Aurora A pathway in an orthotopic breast cancer mouse model.ConclusionsThese findings provide novel insights into the regulation of spindle pole integrity by the MASTL-ENSA-Aurora A pathway during mitosis, highlighting the significance of ENSA in recruiting Aurora A to the spindle pole, independent of PP2A-B55α.

Morphological analysis of the human sperm tail during the early post-fertilization stage.

After fertilization, the human sperm tail lost its fibrous sheath at the pronuclear stage to become coiled and then attached to one of the first mitotic spindle poles. The tip of the sperm tail was branched and its degree did not change from the pronuclear stage to the second mitosis.

Identification of prognostic biomarkers of invasive ductal carcinoma by an integrated bioinformatics approach

Background: Invasive ductal carcinoma (IDC) is the most common type of breast cancer (BC) worldwide. Nowadays, due to its heterogeneity and high capacity for metastasis, it is necessary to discover novel diagnostic and prognostic biomarkers. Therefore, this study aimed to identify novel candidate prognostic genes for IDC using an integrated bioinformatics approach. Methods: Three expression profile data sets were obtained from GEO (GSE29044, GSE3229, and GSE21422), from which differentially expressed genes (DEGs) were extracted for comparative transcriptome analysis of experimental groups (IDC versus control). Next, STRING was utilized to construct a protein interaction network with the shared DEGs, and MCODE and cytoHubba were used to identify the hub genes, which were then characterized using functional enrichment analysis in DAVID and KEGG. Finally, using the Kaplan-Meier tracer database, we determined the correlation between the expression of hub genes and overall survival in BC. Results: We identified seven hub genes (Kinesin-like protein KIF23 [KIF23], abnormal spindle-like microcephaly [ASPM]-associated protein [ASPMAP], Aurora kinase A [AURKA], Rac GTPase-activating protein 1 [RACGAP1], centromere protein F [CENPF], hyaluronan-mediated motility receptor [HMMR], and protein regulator of cytokinesis 1 [PRC1]), which were abundant in microtubule binding and tubulin binding, pathways linked to fundamental cellular structures including the mitotic spindle, spindle, microtubule, and spindle pole. The role of these genes in the pathophysiology of IDC is not yet well characterized; however, they have been associated with other common types of BC, modulating pathways such as Wnt/β-catenin, the epithelial-to-mesenchymal transition (EMT) process, chromosomal instability (CIN), PI3K/AKT/mTOR, and BRCA1 and BRCA2, playing an important role in its progression and being associated with a poor prognosis, thus representing a way to improve our understanding of the process of tumorigenesis and the underlying molecular events of IDC. Conclusions: Genes identified may lead to the discovery of new prognostic targets for IDC.

Microcephaly-associated protein WDR62 shuttles from the Golgi apparatus to the spindle poles in human neural progenitors.

WDR62 is a spindle pole-associated scaffold protein with pleiotropic functions. Recessive mutations in WDR62 cause structural brain abnormalities and account for the second most common cause of autosomal recessive primary microcephaly (MCPH), indicating WDR62 as a critical hub for human brain development. Here, we investigated WDR62 function in corticogenesis through the analysis of a C-terminal truncating mutation (D955AfsX112). Using induced Pluripotent Stem Cells (iPSCs) obtained from a patient and his unaffected parent, as well as isogenic corrected lines, we generated 2D and 3D models of human neurodevelopment, including neuroepithelial stem cells, cerebro-cortical progenitors, terminally differentiated neurons, and cerebral organoids. We report that WDR62 localizes to the Golgi apparatus during interphase in cultured cells and human fetal brain tissue, and translocates to the mitotic spindle poles in a microtubule-dependent manner. Moreover, we demonstrate that WDR62 dysfunction impairs mitotic progression and results in alterations of the neurogenic trajectories of iPSC neuroderivatives. In summary, impairment of WDR62 localization and function results in severe neurodevelopmental abnormalities, thus delineating new mechanisms in the etiology of MCPH.

Microtubule competition and cell growth recenter the nucleus after anaphase in fission yeast.

Cells actively position their nuclei based on their activity. In fission yeast, microtubule-dependent nuclear centering is critical for symmetrical cell division. After spindle disassembly at the end of anaphase, the nucleus recenters over an ∼90-min period, approximately half of the duration of the cell cycle. Live-cell and simulation experiments support the cooperation of two distinct microtubule competition mechanisms in the slow recentering of the nucleus. First, a push-push mechanism acts from spindle disassembly to septation and involves the opposing actions of the mitotic spindle pole body microtubules that push the nucleus away from the ends of the cell, while a postanaphase array of microtubules baskets the nucleus and limits its migration toward the division plane. Second, a slow-and-grow mechanism slowly centers the nucleus in the newborn cell by a combination of microtubule competition and asymmetric cell growth. Our work underlines how intrinsic properties of microtubules differently impact nuclear positioning according to microtubule network organization and cell size.

The kinesin-5 protein Cut7 moves bidirectionally on fission yeast spindles with activity that increases in anaphase.

Kinesin-5 motors are essential to separate mitotic spindle poles and assemble a bipolar spindle in many organisms. These motors crosslink and slide apart antiparallel microtubules via microtubule plus-end-directed motility. However, kinesin-5 localization is enhanced away from antiparallel overlaps. Increasing evidence suggests this localization occurs due to bidirectional motility or trafficking. The purified fission-yeast kinesin-5 protein Cut7 moves bidirectionally, but bidirectionality has not been shown in cells, and the function of the minus-end-directed movement is unknown. Here, we characterized the motility of Cut7 on bipolar and monopolar spindles and observed movement toward both plus- and minus-ends of microtubules. Notably, the activity of the motor increased at anaphase B onset. Perturbations to microtubule dynamics only modestly changed Cut7 movement, whereas Cut7 mutation reduced movement. These results suggest that the directed motility of Cut7 contributes to the movement of the motor. Comparison of the Cut7 mutant and human Eg5 (also known as KIF11) localization suggest a new hypothesis for the function of minus-end-directed motility and spindle-pole localization of kinesin-5s.

NuMA deficiency causes micronuclei via checkpoint-insensitive k-fiber minus-end detachment from mitotic spindle poles

Micronuclei resulting from improper chromosome segregation foster chromosome rearrangements.1,2 Toprevent micronuclei formation in mitosis, the dynamic plus ends of bundled kinetochore microtubules (k-fibers) must establish bipolar attachment with all sister kinetochores on chromosomes,3 whereas k-fiber minus ends must be clustered at the two opposing spindle poles, which are normally connected with centrosomes.4 The establishment of chromosome biorientation via k-fiber plus ends is carefully monitored by the spindle assembly checkpoint (SAC).5 However, how k-fiber minus-end clustering near centrosomes is maintained and monitored remains poorly understood. Here, we show that degradation of NuMA by auxin-inducible degron technologies results in micronuclei formation through k-fiber minus-end detachment from spindle poles during metaphase in HCT116 colon cancer cells. Importantly, k-fiber minus-end detachment from one pole creates misaligned chromosomes that maintain chromosome biorientation and satisfy the SAC, resulting in abnormal chromosome segregation. NuMA depletion also causes minus-end clustering defects in non-transformed Rpe1 cells, but it additionally induces centrosome detachment from partially focused poles, resulting in highly disorganized anaphase. Moreover, we find that NuMA depletion causes centrosome clustering defects in tetraploid-like cells, leading to an increased frequency of multipolar divisions. Together, our data indicate that NuMA is required for faithful chromosome segregation in human mitotic cells, generally by maintaining k-fiber minus-end clustering but also by promoting spindle pole-centrosome or centrosome-centrosome connection in specific cell types or contexts. Similar to erroneous merotelic kinetochore attachments,6 detachment of k-fiber minus ends from spindle poles evades spindle checkpoint surveillance and may therefore be a source of genomic instability in dividing cells.

CPAP insufficiency leads to incomplete centrioles that duplicate but fragment.

Centrioles are structures that assemble centrosomes. CPAP is critical for centrosome assembly, and its mutations are found in patients with diseases such as primary microcephaly. CPAP's centrosomal localization, its dynamics, and the consequences of its insufficiency in human cells are poorly understood. Here we use human cells genetically engineered for fast degradation of CPAP, in combination with superresolution microscopy, to address these uncertainties. We show that three independent centrosomal CPAP populations are dynamically regulated during the cell cycle. We confirm that CPAP is critical for assembly of human centrioles, but not for recruitment of pericentriolar material on already assembled centrioles. Further, we reveal that CPAP insufficiency leads to centrioles with incomplete microtubule triplets that can convert to centrosomes, duplicate, and form mitotic spindle poles, but fragment owing to loss of cohesion between microtubule blades. These findings further our basic understanding of the role of CPAP in centrosome biogenesis and help understand how CPAP aberrations can lead to human diseases.

Exclusive modifications of NuMA in malignant epithelial cells: A potential therapeutic mechanism

NuMA (nuclear mitotic apparatus) protein is indispensable in the mitosis of human proliferating cells, both malignant and benign. The progression of mitosis requires stable spindles, which depend on the bipolar clustering of NuMA within the spindles. The phenanthridine PJ34 kills malignant epithelial cells during mitosis and targets NuMA. PJ34 treated healthy cells are not impaired. PJ34 exclusively blocks the post-translational modification of NuMA in a variety of malignant epithelial cells, but not in benign cells. This blockage of the post-translational modification of NuMA affects its protein-binding capacity, causing construction faults in the mitotic spindle poles of PJ34-treated cancer cells, leading to Mitotic Catastrophe cell death. PJ34 is a potent PARP1 inhibitor. Therefore its PARP independent exclusive cytotoxicity in human malignant cells, challenges the currently accepted notion that inhibition of PARP1 halts cancer by preventing DNA repair. Certain molecules that act as PARP1 inhibitors kill cancer cells by targeting other proteins and vital mechanisms.

Characterization of a recently synthesized microtubule-targeting compound that disrupts mitotic spindle poles in human cells

We reveal the effects of a new microtubule-destabilizing compound in human cells. C75 has a core thienoisoquinoline scaffold with several functional groups amenable to modification. Previously we found that sub micromolar concentrations of C75 caused cytotoxicity. We also found that C75 inhibited microtubule polymerization and competed with colchicine for tubulin-binding in vitro. However, here we found that the two compounds synergized suggesting differences in their mechanism of action. Indeed, live imaging revealed that C75 causes different spindle phenotypes compared to colchicine. Spindles remained bipolar and collapsed after colchicine treatment, while C75 caused bipolar spindles to become multipolar. Importantly, microtubules rapidly disappeared after C75-treatment, but then grew back unevenly and from multiple poles. The C75 spindle phenotype is reminiscent of phenotypes caused by depletion of ch-TOG, a microtubule polymerase, suggesting that C75 blocks microtubule polymerization in metaphase cells. C75 also caused an increase in the number of spindle poles in paclitaxel-treated cells, and combining low amounts of C75 and paclitaxel caused greater regression of multicellular tumour spheroids compared to each compound on their own. These findings warrant further exploration of C75’s anti-cancer potential.

The Tight Junction Protein ZO-1 Is Dispensable for Barrier Function but Critical for Effective Mucosal Repair

Increased permeability is implicated in the pathogenesis of intestinal disease. Invitro and invivo studies have linked down-regulation of the scaffolding protein ZO-1, encoded by the TJP1 gene, to increased tight junction permeability. This has not, however, been tested invivo. Here, we assessed the contributions of ZO-1 to in vivo epithelial barrier function and mucosal homeostasis. Public Gene Expression Omnibus data sets and biopsy specimens from patients with inflammatory bowel disease (IBD) and healthy control individuals were analyzed. Tjp1f/f;vil-CreTg mice with intestinal epithelial-specific ZO-1 knockout (ZO-1KO.IEC) mice and Tjp1f/f mice littermates without Cre expression were studied using chemical and immune-mediated models of disease as well as colonic stem cell cultures. ZO-1 transcript and protein expression were reduced in biopsy specimens from patients with IBD. Despite mildly increased intestinal permeability, ZO-1KO.IEC mice were healthy and did not develop spontaneous disease. ZO-1KO.IEC mice were, however, hypersensitive to mucosal insults and displayed defective repair. Furthermore, ZO-1-deficient colonic epithelia failed to up-regulate proliferation in response to damage invivo or Wnt signaling invitro. ZO-1 was associated with centrioles in interphase cells and mitotic spindle poles during division. In the absence of ZO-1, mitotic spindles failed to correctly orient, resulting in mitotic catastrophe and abortive proliferation. ZO-1 is, therefore, critical for up-regulation of epithelial proliferation and successful completion of mitosis. ZO-1 makes critical, tight junction-independent contributions to Wnt signaling and mitotic spindle orientation. As a result, ZO-1 is essential for mucosal repair. We speculate that ZO-1 down-regulation may be one cause of ineffective mucosal healing in patients with IBD.

P–193 First cleavage division perpendicular to the pronuclear axis adversely affects the clinical outcome in human embryos

Abstract Study question Does the direction of formation for the first cleavage plane relative to the pronuclear axis affect clinical outcome? Summary answer A first cleavage division perpendicular to the pronuclear axis adversely affects the rate of embryo utilization for transfer or cryopreservation and the pregnancy outcome. What is known already It remains unclear how the first cleavage plane is determined in human embryos. By using time-lapse monitoring, our previous study (presented in ESHRE 2019) suggested that both the axis and locations of male and female pronuclei are involved in determining the first embryonic cleavage plane. Furthermore, by using immunofluorescence analysis, it was also revealed that most analyzed zygotes showed two pericentrin signals aligned around the interface between the male and female pronuclei. Our findings suggest that the pronuclear axis strongly influences the positions of the centrosomes, which become mitotic spindle poles and define the first cleavage plane. Study design, size, duration: From January 2015 to December 2017, time-lapse imaging (EmbryoScope®) of 3397 intracytoplasmic sperm injection (ICSI) oocytes was conducted. Of those, the relationship between the pronuclear axis and the first cleavage plane was analyzed in 607 normally fertilized embryos that cleaved to two cells and were obtained in 2015. Furthermore, of 3397 ICSI oocytes, 749 transferred embryos were classified based on the first cleavage patterns relative to the pronuclear axis, and the pregnancy rate was examined. Participants/materials, setting, methods A straight line connecting the centers of the pronuclei was defined as the 2PN axis. Based on the direction of the first cleavage relative to the 2PN axis, embryos were classified into three groups: parallel, perpendicular and intermediate. Fresh embryos were transferred on Day 2/3 (fresh-ET). Frozen and thawed embryos were transferred on Day 2/3 or Day 5 (F/T-ET). Clinical pregnancy was defined as confirmed gestational sac in the uterine cavity. Main results and the role of chance Of 607 analyzed embryos, 506 produced suitable images and were assigned to one of three groups: parallel (84.4%, n = 427), perpendicular (9.7%, n = 49) and intermediate (5.9%, n = 30). Embryos that formed a cleavage furrow parallel to the 2PN axis were significantly more frequent than others (perpendicular, intermediate) (P < 0.001). The embryo utilization rate for transfer or cryopreservation was significantly lower in the perpendicular group than in the parallel group (30.7% vs. 69.3%, P < 0.01). Furthermore, of 749 transferred embryos, 504 assigned to the parallel and perpendicular groups were selected (n = 470 and n = 34, respectively), and the pregnancy outcome was analyzed. The mean maternal age was not significantly different between groups. The pregnancy rate of embryos was 24.2% (n = 45/186) from fresh-ET and 39.4% (n = 112/284) from F/T-ET in the parallel group, and 0% (n = 0/14) from fresh-ET and 15.0% (n = 3/20) from F/T-ET in the perpendicular group. Regardless of the types of embryo transfer (fresh or F/T), the pregnancy rate was significantly lower in the perpendicular group than in the parallel group (P < 0.01). In addition, one of three patients who became pregnant from the transfer of an embryo in the perpendicular group had a miscarriage. Limitations, reasons for caution Since only ICSI embryos were analyzed in this study, the influence of fertilization methods on subsequent development could not be investigated. Further studies including preimplantation genetic testing for aneuploidy may help determine the reasons why pregnancy rates differ between groups. Wider implications of the findings: We suggest that the 2PN axis is essential for determining the first cleavage plane because it seems to be involved in positioning the mitotic spindle poles. The direction of the first cleavage plane relative to the 2PN axis can be an important indicator for predicting embryo development and pregnancy outcome Trial registration number none

Abstract 2042: A cytoskeletal function for PBRM1: reading methylated microtubules to maintain genomic stability

Abstract The chromatin modifier SETD2 often mutated in clear cell renal cell carcinoma (ccRCC), was recently shown to be a dual-function methyltransferase that “writes” methyl marks on both chromatin and microtubules, revealing α-tubulin methylation as a new posttranslational modification of the mitotic spindle. Here, we report that the polybromo protein PBRM1, the 2nd most mutated gene in ccRCC, is a “reader” for this SETD2-dependent methyl mark on α-tubulin. PBRM1 is a component of the PBAF (Polybromo BRG1 associated factor) chromatin remodeler complex. Our western and immunocytochemistry data in multiple kidney-derived cell lines, including HEK293T, HKC and 786-O, revealed that PBRM1 binds to methylated α-tubulin and localizes to the mitotic spindle and spindle pole during cell division. PBRM1 has six bromo domains, two bromo-associated homology (BAH) domains and one HMG domain. While PBRM1 is known to bind acetylated histones via its bromo domains, our GST pull down assays showed that PBRM1 binds methylated α-tubulin via its two BAH domains. Additional western and immunocytochemical experiments following knockout or re-expression of PBRM1 revealed that PBRM1 recruits other PBAF components to the mitotic spindle to maintain genomic stability. Two clinically established ccRCC mutations (P1048R and C1233W) in PBRM1 BAH domains result in loss of microtubule binding, mislocalization of PBAF, and the inability of PBRM1 to maintain genomic stability, as assessed by increased lagging chromosomes, chromosome bridges, multipolar spindles and micronuclei count. A third pathogenic ccRCC mutation (T1202K) in the PBRM1 BAH domain did not affect microtubule binding and consequently was not associated with mitotic spindle defects or genomic instability. Mass spectrometry and RNASeq confirmed BAH domain mutant PBRM1 still assembled a transcriptionally competent PBAF complex, clearly distinguishing the cytoskeletal from the chromatin impact of these mutations. These data reveal a previously unknown function of PBRM1 beyond reading acetylated histones, and expand the repertoire of chromatin remodelers acting on the cytoskeleton to maintain genomic stability. Citation Format: Menuka Karki, Rahul Jangid, Ramakrishnan Anish, Riyad N. Seervai, Jean-Philippe Bertocchio, Takashi Hotta, Pavlos Msaouel, Sung Y. Jung, Sandra L. Grimm, Cristian Coarfa, Bernard E. Weissman, Ryoma Ohi, Kristen J. Verhey, Courtney H. Hodges, Ruhee Dere, In Young Park, B. V. Venkataram Prasad, W. Kimryn Rathmell, Cheryl L. Walker, Durga N. Tripathi. A cytoskeletal function for PBRM1: reading methylated microtubules to maintain genomic stability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2042.

CK1-mediated phosphorylation of FAM110A promotes its interaction with mitotic spindle and controls chromosomal alignment.

Progression through the cell cycle is driven by cyclin-dependent kinases that control gene expression, orchestration of mitotic spindle, and cell division. To identify new regulators of the cell cycle, we performed transcriptomic analysis of human non-transformed cells expressing a fluorescent ubiquitination-based cell cycle indicator and identified 701 transcripts differentially expressed in G1 and G2 cells. Family with sequence similarity 110 member A (FAM110A) protein is highly expressed in G2 cells and localized at mitotic spindle and spindle poles during mitosis. Depletion of FAM110A impairs chromosomal alignment, delays metaphase-to-anaphase transition, and affects spindle positioning. Using mass spectrometry and immunoprecipitation, we identified casein kinase I (CK1) in complex with FAM110A during mitosis. CK1 phosphorylates the C-terminal domain of FAM110A in vitro, and inhibition of CK1 reduces phosphorylation of mitotic FAM110A. Wild-type FAM110A, but not the FAM110A-S252-S255A mutant deficient in CK1 phosphorylation, rescues the chromosomal alignment, duration of mitosis, and orientation of the mitotic spindle after depletion of endogenous FAM110A. We propose that CK1 regulates chromosomal alignment by phosphorylating FAM110A and promoting its interaction with mitotic spindle.

Mud binds the kinesin-14 Ncd in Drosophila

Maintenance of proper mitotic spindle structure is necessary for error-free chromosome segregation and cell division. Spindle assembly is controlled by force-generating kinesin motors that contribute to its geometry and bipolarity, and balancing motor-dependent forces between opposing kinesins is critical to the integrity of this process. Non-claret dysjunctional (Ncd), a Drosophila kinesin-14 member, crosslinks and slides microtubule minus-ends to focus spindle poles and sustain bipolarity. However, mechanisms that regulate Ncd activity during mitosis are underappreciated. Here, we identify Mushroom body defect (Mud), the fly ortholog of human NuMA, as a direct Ncd binding partner. We demonstrate this interaction involves a short coiled-coil domain within Mud (MudCC) binding the N-terminal, non-motor microtubule-binding domain of Ncd (NcdnMBD). We further show that the C-terminal ATPase motor domain of Ncd (NcdCTm) directly interacts with NcdnMBD as well. Mud binding competes against this self-association and also increases NcdnMBD microtubule binding in vitro. Our results describe an interaction between two spindle-associated proteins and suggest a potentially new mode of minus-end motor protein regulation at mitotic spindle poles.

SOGA1 and SOGA2/MTCL1 are CLASP-interacting proteins required for faithful chromosome segregation in human cells.

CLASPs are key modulators of microtubule dynamics throughout the cell cycle. During mitosis, CLASPs independently associate with growing microtubule plus-ends and kinetochores and play essential roles in chromosome segregation. In a proteomic survey for human CLASP1-interacting proteins during mitosis, we have previously identified SOGA1 and SOGA2/MTCL1, whose mitotic roles remained uncharacterized. Here we performed an initial functional characterization of human SOGA1 and SOGA2/MTCL1 during mitosis. Using specific polyclonal antibodies raised against SOGA proteins, we confirmed their expression and reciprocal interaction with CLASP1 and CLASP2 during mitosis. In addition, we found that both SOGA1 and SOGA2/MTCL1 are phospho-regulated during mitosis by CDK1. Immunofluorescence analysis revealed that SOGA2/MTCL1 co-localizes with mitotic spindle microtubules and spindle poles throughout mitosis and both SOGA proteins are enriched at the midbody during mitotic exit/cytokinesis. GFP-tagging of SOGA2/MTCL1 further revealed a microtubule-independent localization at kinetochores. Live-cell imaging after siRNA-mediated knockdown of SOGA1 and SOGA2/MTCL1 showed that they are independently required for distinct aspects of chromosome segregation. Thus, SOGA1 and SOGA2/MTCL1 are bona fide CLASP-interacting proteins during mitosis required for faithful chromosome segregation in human cells.

Roles of developmentally regulated KIF2A alternative isoforms in cortical neuron migration and differentiation.

KIF2A is a kinesin motor protein with essential roles in neural progenitor division and axonal pruning during brain development. However, how different KIF2A alternative isoforms function during development of the cerebral cortex is not known. Here, we focus on three Kif2a isoforms expressed in the developing cortex. We show that Kif2a is essential for dendritic arborization in mice and that the functions of all three isoforms are sufficient for this process. Interestingly, only two of the isoforms can sustain radial migration of cortical neurons; a third isoform, lacking a key N-terminal region, is ineffective. By proximity-based interactome mapping for individual isoforms, we identify previously known KIF2A interactors, proteins localized to the mitotic spindle poles and, unexpectedly, also translation factors, ribonucleoproteins and proteins that are targeted to organelles, prominently to the mitochondria. In addition, we show that a KIF2A mutation, which causes brain malformations in humans, has extensive changes to its proximity-based interactome, with depletion of mitochondrial proteins identified in the wild-type KIF2A interactome. Our data raises new insights about the importance of alternative splice variants during brain development.

The myosin regulatory light chain Myl5 localizes to mitotic spindle poles and is required for proper cell division.

Myosins are ATP-dependent actin-based molecular motors critical for diverse cellular processes like intracellular trafficking, cell motility, and cell invasion. During cell division, myosin MYO10 is important for proper mitotic spindle assembly, the anchoring of the spindle to the cortex, and positioning of the spindle to the cell mid-plane. However, myosins are regulated by myosin regulatory light chains (RLCs), and whether RLCs are important for cell division has remained unexplored. Here, we have determined that the previously uncharacterized myosin RLC Myl5 associates with the mitotic spindle and is required for cell division. We show that Myl5 localizes to the leading edge and filopodia during interphase and to mitotic spindle poles and spindle microtubules during early mitosis. Importantly, depletion of Myl5 led to defects in mitotic spindle assembly, chromosome congression, and chromosome segregation and to a slower transition through mitosis. Furthermore, Myl5 bound to MYO10 in vitro and co-localized with MYO10 at the spindle poles. These results suggest that Myl5 is important for cell division and that it may be performing its function through MYO10.