Mitotic Spindle Structure Research Articles

HN1 expression contributes to mitotic fidelity through Aurora A-PLK1-Eg5 axis.

Hematological and neurological expressed 1 (HN1) is homolog of Jupiter protein from Drosophila melanogaster where it functions as a microtubule-associated protein. However, in mammalian cells, HN1 is associated partially with y-tubulin in centrosomes, Stathmin for stabilizing microtubules, and Cdh1 for regulating Cyclin B1 for cell cycle regulation. Moreover, HN1 overexpression leads to early mitotic exit as well. Other molecular functions and interactions of HN1 are not clear yet. Here, based on our previous analysis where HN1 was shown to cluster supernumerary centrosomes and maintain mitotic spindle assembly, we further investigated the role of HN1 in centrosome maintenance and mitotic fidelity in PC-3 prostate and MDA-MB231 mammary cancer cell lines. The maturation-associated roles of HN1 during cell division by examining the AuroraA-PLK1 axis involving a plus end kinesin, Eg5 as well as pericentriolar matrix protein (PCM1) as components of centrosomes were established. We found that HN1 co-localized to centrioles with Eg5 and Aurora A to suppress aberrant spindle formation to ensure the fidelity of centriole/centrosome duplication when overexpressed. Consistently, depleting the HN1 expression using siRNA or shRNA resulted in an increased number of dysregulated mitotic spindle structures, where Aurora A as well as PLK1 co-localizations with Eg5 and PCM1 were disrupted. Further, the PLK1 and Aurora A kinase's phosphorylations also decreased, confirming the hypothesis that the cells struggle in mitotic progression, display nuclear and cytokinetic abnormalities with supernumerary but immature mononucleated centrosomes. In summary, we described the role of HN1 in centrosome nucleation/maturation in PLK1-Eg5 axis and concomitant mitotic spindle formation in human cells.

Methodology to Create Auxin-Inducible Degron Tagging System to Control Expression of a Target Protein in Mammalian Cell Lines.

The auxin-inducible degron (AID) system is a versatile tool in cell biology and genetics, enabling conditional protein regulation through auxin-induced degradation. Integrating CRISPR/Cas9 with AID expedites tagging and depletion of a required protein in human and mouse cells. The mechanism of AID involves interactions between receptors like TIR1 and the AID tag fused to the target protein. The presence of auxin triggers protein ubiquitination, leading to proteasome-mediated degradation. We have used AID to explore the mitotic functions of the replication licensing protein CDT1. Swift CDT1 degradation via AID upon auxin addition achieves precise mitotic inhibition, revealing defects in mitotic spindle structure and chromosome misalignment. Using live imaging, we found that mitosis-specific degradation of CDT1 delayed progression and chromosome mis-segregation. AID-mediated CDT1 inhibition surpasses siRNA-based methods, offering a robust approach to probe CDT1's mitotic roles. The advantages of AID include targeted degradation and temporal control, facilitating rapid induction and reversal of degradation-contrasting siRNA's delayed RNA degradation and protein turnover. In summary, the AID technique enhances precision, control, and efficiency in studying protein function and regulation across diverse cellular contexts. In this article, we provide a step-by-step methodology for generating an efficient AID-tagging system, keeping in mind the important considerations that need to be adopted to use it for investigating or characterizing protein function in a temporally controlled manner. Key features • The auxin-inducible degron (AID) system serves as a versatile tool, enabling conditional protein regulation through auxin-induced degradation in cell biology and genetics. • Integration of CRISPR/Cas9 knock-in technology with AID expedites the tagging and depletion of essential proteins in mammalian cells. • AID's application extends to exploring the mitotic functions of the replication licensing protein CDT1, achieving precise mitotic inhibition and revealing spindle defects and chromosome misalignment. • The AID system and its diverse applications advance the understanding of protein function and cellular processes, contributing to the study of protein regulation and function.

CTCF is essential for proper mitotic spindle structure and anaphase segregation.

Mitosis is an essential process in which the duplicated genome is segregated equally into two daughter cells. CTCF has been reported to be present in mitosis and has a role in localizing CENP-E, but its importance for mitotic fidelity remains to be determined. To evaluate the importance of CTCF in mitosis, we tracked mitotic behaviors in wild-type and two different CTCF CRISPR-based genetic knockdowns. We find that knockdown of CTCF results in prolonged mitoses and failed anaphase segregation via time-lapse imaging of SiR-DNA. CTCF knockdown did not alter cell cycling or the mitotic checkpoint, which was activated upon nocodazole treatment. Immunofluorescence imaging of the mitotic spindle in CTCF knockdowns revealed disorganization via tri/tetrapolar spindles and chromosomes behind the spindle pole. Imaging of interphase nuclei showed that nuclear size increased drastically, consistent with failure to divide the duplicated genome in anaphase. Long-term inhibition of CNEP-E via GSK923295 recapitulates CTCF knockdown abnormal mitotic spindles with polar chromosomes and increased nuclear sizes. Population measurements of nuclear shape in CTCF knockdowns do not display decreased circularity or increased nuclear blebbing relative to wild-type. However, failed mitoses do display abnormal nuclear morphologies relative to successful mitoses, suggesting that population images do not capture individual behaviors. Thus, CTCF is important for both proper metaphase organization and anaphase segregation which impacts the size and shape of the interphase nucleus likely through its known role in recruiting CENP-E.

OVOL2 impairs RHO GTPase signaling to restrain mitosis and aggressiveness of Anaplastic Thyroid Cancer

BackgroundAnaplastic Thyroid Cancer (ATC) is an undifferentiated and aggressive tumor that often originates from well-Differentiated Thyroid Carcinoma (DTC) through a trans-differentiation process. Epithelial-to-Mesenchymal Transition (EMT) is recognized as one of the major players of this process. OVOL2 is a transcription factor (TF) that promotes epithelial differentiation and restrains EMT during embryonic development. OVOL2 loss in some types of cancers is linked to aggressiveness and poor prognosis. Here, we aim to clarify the unexplored role of OVOL2 in ATC.MethodsGene expression analysis in thyroid cancer patients and cell lines showed that OVOL2 is mainly associated with epithelial features and its expression is deeply impaired in ATC. To assess OVOL2 function, we established an OVOL2-overexpression model in ATC cell lines and evaluated its effects by analyzing gene expression, proliferation, invasion and migration abilities, cell cycle, specific protein localization through immunofluorescence staining. RNA-seq profiling showed that OVOL2 controls a complex network of genes converging on cell cycle and mitosis regulation and Chromatin Immunoprecipitation identified new OVOL2 target genes.ResultsCoherently with its reported function, OVOL2 re-expression restrained EMT and aggressiveness in ATC cells. Unexpectedly, we observed that it caused G2/M block, a consequent reduction in cell proliferation and an increase in cell death. This phenotype was associated to generalized abnormalities in the mitotic spindle structure and cytoskeletal organization. By RNA-seq experiments, we showed that many pathways related to cytoskeleton and migration, cell cycle and mitosis are profoundly affected by OVOL2 expression, in particular the RHO-GTPase pathway resulted as the most interesting. We demonstrated that RHO GTPase pathway is the central hub of OVOL2-mediated program in ATC and that OVOL2 transcriptionally inhibits RhoU and RhoJ. Silencing of RhoU recapitulated the OVOL2-driven phenotype pointing to this protein as a crucial target of OVOL2 in ATC.ConclusionsCollectively, these data describe the role of OVOL2 in ATC and uncover a novel function of this TF in inhibiting the RHO GTPase pathway interlacing its effects on EMT, cytoskeleton dynamics and mitosis.

Novel mechanism for oscillations in catchbonded motor-filament complexes

Generation of mechanical oscillations is ubiquitous to a wide variety of intracellular processes, ranging from activity of muscle fibers to oscillations of the mitotic spindle. The activity of motors plays a vital role in maintaining the integrity of the mitotic spindle structure and generating spontaneous oscillations. Although the structural features and properties of the individual motors are well characterized, their implications on the functional behavior of motor-filament complexes are more involved. We show that force-induced allosteric deformations in dynein, which result in catchbonding behavior, provide a generic mechanism to generate spontaneous oscillations in motor-cytoskeletal filament complexes. The resultant phase diagram of such motor-filament systems—characterized by force-induced allosteric deformations—exhibits bistability and sustained limit-cycle oscillations in biologically relevant regimes, such as for catchbonded dynein. The results reported here elucidate the central role of this mechanism in fashioning a distinctive stability behavior and oscillations in motor-filament complexes such as mitotic spindles.

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.

Black phosphorus quantum dots reverse the malignant potential and enhance chemosensitivity of human renal cell carcinoma cells by targeting histone deacetylase 1 signal pathway

AbstractTargeted anti‐cancer therapy selectively inhibits the growth of malignant cells by interfering with cancer‐specific signaling pathways, and inflicts minimal toxicity to normal tissues. Here, the current study demonstrates black phosphorus quantum dots (BPQDs) can target histone deacetylase 1 (HDAC1), which is overexpressed in multiple tumors and involved in cancer progression. The BPQDs inhibit HDAC1 activity and impair HDAC1‐mediated deacetylation of the mitotic spindle protein Eg5 in human renal cell carcinoma (RCC) cells, thereby disrupting the mitotic spindle structure and stalling cell cycle progression. Since HDAC1 regulates multiple aspects of cancer progression, BPQDs can neutralize the malignant potential of cancer cells by suppressing their proliferation and migration, and inducing apoptosis. Further in vitro assays demonstrate BPQDs significantly increase the sensitivity of RCC cells to the clinical chemotherapeutic agent, paclitaxel (PTX). Moreover, animal experiments also indicate the combined approach with both BPQDs and PTX displays better efficacy than PTX alone. These findings demonstrate that BPQDs have potential applications in targeted anti‐cancer therapy and can be more effectively treated with chemotherapy.

Allosteric uncoupling of microtubule depolymerase activity from motility in human Kinesin‐5

Human Kinesin‐5 (Eg5) has a large number of known allosteric inhibitors that disrupt its mitotic function. Small‐molecule inhibitors of Eg5 are candidate anti‐cancer agents and important probes for understanding the cellular function. Here we show that Eg5 is capable of more than one type of microtubule interaction, and these activities can be controlled by allosteric agents. While both monastrol and S‐trityl‐L‐cysteine (STC) inhibit Eg5 motility, our data reveal an unexpected ability of these loop5 targeting inhibitors to differentially control a novel Eg5 microtubule depolymerizing activity.Remarkably, small molecule loop5 effectors are able to independently modulate discrete functional interactions between the motor and microtubule track. We establish that motility can be uncoupled from the microtubule depolymerase activity and argue that loop5‐targeting inhibitors of Kinesin‐5 should not all be considered functionally synonymous. Also, the depolymerizing activity of the motor does not contribute to the genesis of monopolar spindles during allosteric inhibition of motility, but instead reveals a new function. We propose that, in addition to its canonical role in participating in the construction of the three‐dimensional mitotic spindle structure, Eg5 also plays a distinct role in regulating the dynamics of individual microtubules, and thereby impacts the density of the mitotic spindle.

Small Molecule Uncoupling of Microtubule Depolymerase Activity from Motility in Human Kinesin-5 During Mitotic Spindle Assembly

Human Kinesin-5 (HsEg5) has a large number of known allosteric inhibitors that disrupt its mitotic function. Small-molecule inhibitors of HsEg5 are candidate anti-cancer agents and important probes for understanding the cellular function. Here we show that HsEg5 is capable of more than one type of microtubule interaction, and these activities can be controlled by allosteric agents. While both monastrol and S-trityl-L-cysteine both inhibit HsEg5 motility, our data reveal an unexpected ability of these loop5 targeting inhibitors to differentially control a novel HsEg5 microtubule depolymerizing activity. Remarkably, small molecule loop5 effectors are able to independently modulate discrete functional interactions between the motor and microtubule track. We establish that motility can be uncoupled from the microtubule depolymerase activity and argue that loop5-targeted inhibitors of Kinesin-5 should not all be considered functionally synonymous. Also, the depolymerizing activity of the motor does not contribute to the genesis of monopolar spindles during allosteric inhibition of motility, but instead reveals a new function. We propose that, in addition to its canonical role in participating in the construction of the three-dimensional mitotic spindle structure, HsEg5 also plays a distinct role in regulating the dynamics of individual microtubules, and thereby impacts the density of the mitotic spindle.

Small molecule allosteric uncoupling of microtubule depolymerase activity from motility in human Kinesin-5 during mitotic spindle assembly

Human Kinesin-5 (Eg5) has a large number of known allosteric inhibitors that disrupt its mitotic function. Small-molecule inhibitors of Eg5 are candidate anti-cancer agents and important probes for understanding the cellular function. Here we show that Eg5 is capable of more than one type of microtubule interaction, and these activities can be controlled by allosteric agents. While both monastrol and S-trityl-L-cysteine inhibit Eg5 motility, our data reveal an unexpected ability of these loop5 targeting inhibitors to differentially control a novel Eg5 microtubule depolymerizing activity. Remarkably, small molecule loop5 effectors are able to independently modulate discrete functional interactions between the motor and microtubule track. We establish that motility can be uncoupled from the microtubule depolymerase activity and argue that loop5-targeting inhibitors of Kinesin-5 should not all be considered functionally synonymous. Also, the depolymerizing activity of the motor does not contribute to the genesis of monopolar spindles during allosteric inhibition of motility, but instead reveals a new function. We propose that, in addition to its canonical role in participating in the construction of the three-dimensional mitotic spindle structure, Eg5 also plays a distinct role in regulating the dynamics of individual microtubules, and thereby impacts the density of the mitotic spindle.

Centromere and Pericentromere Transcription: Roles and Regulation … in Sickness and in Health.

The chromosomal loci known as centromeres (CEN) mediate the equal distribution of the duplicated genome between both daughter cells. Specifically, centromeres recruit a protein complex named the kinetochore, that bi-orients the replicated chromosome pairs to the mitotic or meiotic spindle structure. The paired chromosomes are then separated, and the individual chromosomes segregate in opposite direction along the regressing spindle into each daughter cell. Erroneous kinetochore assembly or activity produces aneuploid cells that contain an abnormal number of chromosomes. Aneuploidy may incite cell death, developmental defects (including genetic syndromes), and cancer (>90% of all cancer cells are aneuploid). While kinetochores and their activities have been preserved through evolution, the CEN DNA sequences have not. Hence, to be recognized as sites for kinetochore assembly, CEN display conserved structural themes. In addition, CEN nucleosomes enclose a CEN-exclusive variant of histone H3, named CENP-A, and carry distinct epigenetic labels on CENP-A and the other CEN histone proteins. Through the cell cycle, CEN are transcribed into non-coding RNAs. After subsequent processing, they become key components of the CEN chromatin by marking the CEN locus and by stably anchoring the CEN-binding kinetochore proteins. CEN transcription is tightly regulated, of low intensity, and essential for differentiation and development. Under- or overexpression of CEN transcripts, as documented for myriad cancers, provoke chromosome missegregation and aneuploidy. CEN are genetically stable and fully competent only when they are insulated from the surrounding, pericentromeric chromatin, which must be silenced. We will review CEN transcription and its contribution to faithful kinetochore function. We will further discuss how pericentromeric chromatin is silenced by RNA processing and transcriptionally repressive chromatin marks. We will report on the transcriptional misregulation of (peri)centromeres during stress, natural aging, and disease and reflect on whether their transcripts can serve as future diagnostic tools and anti-cancer targets in the clinic.

Inhibition of the formation of autophagosome but not autolysosome augments ABT-751-induced apoptosis in TP53-deficient Hep-3B cells.

The objective was to investigate the upstream mechanisms of apoptosis which were triggered by a novel antimicrotubule drug, ABT-751, in a tumor protein p53 ( TP53)-deficient hepatocellular carcinoma-derived Hep-3B cells. A series of in vitro assays indicated that ABT-751 caused the disruption of the mitotic spindle structure, collapse of mitochondrial membrane potential, generation of reactive oxygen species, DNA damage, G 2 /M cell cycle arrest, inhibition of anchorage-independent cell growth and apoptosis in Hep-3B cells accompanied by alteration of the expression levels of several DNA damage checkpoint proteins and cell cycle regulators. Subsequently, ABT-751 triggered apoptosis along with markedly upregulated several proapoptotic proteins involving in extrinsic, intrinsic, and caspase-mediated apoptotic pathways. A pan-caspase inhibitor suppressed ABT-751-induced apoptosis. ABT-751 also induced autophagy soon after the occurrence of apoptosis through the suppression of AKT serine/threonine kinase/mechanistic target of rapamycin signaling pathway. Exogenous expression of the TP53 gene significantly incurred both apoptosis and autophagy in Hep-3B cells. Pharmacological inhibition of autophagosome (early autophagy) but not autolysosome (late autophagy) enhanced ABT-751-induced apoptosis in TP53-deficient Hep-3B cells. Our study provided a new strategy to augment ABT-751-induced apoptosis in TP53-deficient cells.

Congenital microcephaly: A diagnostic challenge during Zika epidemics.

The multiple, wide and diverse etiologies of congenital microcephaly are complex and multifactorial. Recent advances in genetic testing have improved understanding of novel genetic causes of congenital microcephaly. The recent Zika virus (ZIKV) epidemic in Latin America has highlighted the need for a better understanding of the underlying pathological mechanisms of microcephaly including both infectious and non-infectious causes. The diagnostic approach to microcephaly needs to include potential infectious and genetic etiologies, as well as environmental in-utero exposures such as alcohol, toxins, and medications. Emerging genetic alterations linked to microcephaly include abnormal mitotic microtubule spindle structure and abnormal function of centrosomes. We discuss the diagnostic challenge of congenital microcephaly in the context of understanding the links with ZIKV emergence as a new etiological factor involved in this birth defect.

PTEN regulates EG5 to control spindle architecture and chromosome congression during mitosis.

Architectural integrity of the mitotic spindle is required for efficient chromosome congression and accurate chromosome segregation to ensure mitotic fidelity. Tumour suppressor PTEN has multiple functions in maintaining genome stability. Here we report an essential role of PTEN in mitosis through regulation of the mitotic kinesin motor EG5 for proper spindle architecture and chromosome congression. PTEN depletion results in chromosome misalignment in metaphase, often leading to catastrophic mitotic failure. In addition, metaphase cells lacking PTEN exhibit defects of spindle geometry, manifested prominently by shorter spindles. PTEN is associated and co-localized with EG5 during mitosis. PTEN deficiency induces aberrant EG5 phosphorylation and abrogates EG5 recruitment to the mitotic spindle apparatus, leading to spindle disorganization. These data demonstrate the functional interplay between PTEN and EG5 in controlling mitotic spindle structure and chromosome behaviour during mitosis. We propose that PTEN functions to equilibrate mitotic phosphorylation for proper spindle formation and faithful genomic transmission.

Myosin-10 independently influences mitotic spindle structure and mitotic progression.

The iconic bipolar structure of the mitotic spindle is of extreme importance to proper spindle function. At best, spindle abnormalities result in a delayed mitosis, while worse outcomes include cell death or disease. Recent work has uncovered an important role for the actin-based motor protein myosin-10 in the regulation of spindle structure and function. Here we examine the contribution of the myosin tail homology 4 (MyTH4) domain of the myosin-10 tail to the protein's spindle functions. The MyTH4 domain is known to mediate binding to microtubules and we verify the suspicion that this domain contributes to myosin-10's close association with the spindle. More surprisingly, our data demonstrate that some but not all of myosin-10's spindle functions require microtubule binding. In particular, myosin-10's contribution to spindle pole integrity requires microtubule binding, whereas its contribution to normal mitotic progression does not. This is demonstrated by the observation that dominant negative expression of the wild-type MyTH4 domain produces multipolar spindles and an increased mitotic index, whereas overexpression of a version of the MyTH4 domain harboring point mutations that abrogate microtubule binding results in only the mitotic index phenotype. Our data suggest that myosin-10 helps to control the metaphase to anaphase transition in cells independent of microtubule binding. © 2016 Wiley Periodicals, Inc.

The conserved apicomplexan Aurora kinase TgArk3 is involved in endodyogeny, duplication rate and parasite virulence.

Aurora kinases are eukaryotic serine/threonine protein kinases that regulate key events associated with chromatin condensation, centrosome and spindle function and cytokinesis. Elucidating the roles of Aurora kinases in apicomplexan parasites is crucial to understand the cell cycle control during Plasmodium schizogony or Toxoplasma endodyogeny. Here, we report on the localization of two previously uncharacterized Toxoplasma Aurora-related kinases (Ark2 and Ark3) in tachyzoites and of the uncharacterized Ark3 orthologue in Plasmodium falciparum erythrocytic stages. In Toxoplasma gondii, we show that TgArk2 and TgArk3 concentrate at specific sub-cellular structures linked to parasite division: the mitotic spindle and intranuclear mitotic structures (TgArk2), and the outer core of the centrosome and the budding daughter cells cytoskeleton (TgArk3). By tagging the endogenous PfArk3 gene with the green fluorescent protein in live parasites, we show that PfArk3 protein expression peaks late in schizogony and localizes at the periphery of budding schizonts. Disruption of the TgArk2 gene reveals no essential function for tachyzoite propagation in vitro, which is surprising giving that the P. falciparum and P. berghei orthologues are essential for erythrocyte schizogony. In contrast, knock-down of TgArk3 protein results in pronounced defects in parasite division and a major growth deficiency. TgArk3-depleted parasites display several defects, such as reduced parasite growth rate, delayed egress and parasite duplication, defect in rosette formation, reduced parasite size and invasion efficiency and lack of virulence in mice. Our study provides new insights into cell cycle control in Toxoplasma and malaria parasites and highlights Aurora kinase 3 as potential drug target.

Quantifying the Effect of Electric Fields in the Frequency Range of 100-500 khz on Mitotic Spindle Structures

It is well established that electric fields can influence cells: Low frequency electric fields (f 1 MHz) cause tissue heating. More recently, it was shown that exposing dividing cells to low intensity (∼1 V/cm) electric fields in the frequency range of 100 - 500 kHz leads to disruption of the mitotic process, and ultimately cell death. This discovery motivated the development of Tumor Treating Fields (TTFields) therapy: a non-invasive treatment that utilizes electric fields in the intermediate frequency range to treat solid tumors. TTFields have been approved by the US Food and Drug Administration for the treatment of recurrent glioblastoma multiforme since 2011.It is thought that TTFields exert their effect on cells by inhibiting tubulin polymerization through interactions of the electric fields with the highly polarized tubulin dimers. However, only few studies have been performed that quantify the intracellular intensity of electric fields, in the frequency range of 100-500 kHz, and how these fields disrupt tubulin polymerization. Here we present the results of computational simulations and in-vitro studies that address these issues. The simulations show that the frequency range of 100-500 kHz marks a transition region in which the magnitude of the electric field inside cells increases significantly, and that the threshold at which this increase occurs depends on the dielectric properties of the cell's membrane and cytoplasm. The experiments show that exposure of cell cultures to TTFields leads to a 10-20% increase in the amount of unpolymerized tubulin dimers within the cells, suggesting that TTFields influence tubulin polymerization dynamics. These studies provide new insight into the biophysical mechanisms that underlie TTFields therapy.

Novel POC1A mutation in primordial dwarfism reveals new insights for centriole biogenesis.

POC1A encodes a WD repeat protein localizing to centrioles and spindle poles and is associated with short stature, onychodysplasia, facial dysmorphism and hypotrichosis (SOFT) syndrome. These main features are related to the defect in cell proliferation of chondrocytes in growth plate. In the current study, we aimed at identifying the molecular basis of two patients with primordial dwarfism (PD) in a single family through utilization of whole-exome sequencing. A novel homozygous p.T120A missense mutation was detected in POC1A in both patients, a known causative gene of SOFT syndrome, and confirmed using Sanger sequencing. To test the pathogenicity of the detected mutation, primary fibroblast cultures obtained from the patients and a control individual were used. For evaluating the global gene expression profile of cells carrying p.T120A mutation in POC1A, we performed the gene expression array and compared their expression profiles to those of control fibroblast cells. The gene expression array analysis showed that 4800 transcript probes were significantly deregulated in cells with p.T120A mutation in comparison to the control. GO term association results showed that deregulated genes are mostly involved in the extracellular matrix and cytoskeleton. Furthermore, the p.T120A missense mutation in POC1A caused the formation of abnormal mitotic spindle structure, including supernumerary centrosomes, and changes in POC1A were accompanied by alterations in another centrosome-associated WD repeat protein p80-katanin. As a result, we identified a novel mutation in POC1A of patients with PD and showed that this mutation causes the formation of multiple numbers of centrioles and multipolar spindles with abnormal chromosome arrangement.

Candida albicans Kinesin Kar3 Depends on a Cik1-Like Regulatory Partner Protein for Its Roles in Mating, Cell Morphogenesis, and Bipolar Spindle Formation.

Candida albicans is a major fungal pathogen whose virulence is associated with its ability to transition from a budding yeast form to invasive hyphal filaments. The kinesin-14 family member CaKar3 is required for transition between these morphological states, as well as for mitotic progression and karyogamy. While kinesin-14 proteins are ubiquitous, CaKar3 homologs in hemiascomycete fungi are unique because they form heterodimers with noncatalytic kinesin-like proteins. Thus, CaKar3-based motors may represent a novel antifungal drug target. We have identified and examined the roles of a kinesin-like regulator of CaKar3. We show that orf19.306 (dubbed CaCIK1) encodes a protein that forms a heterodimer with CaKar3, localizes CaKar3 to spindle pole bodies, and can bind microtubules and influence CaKar3 mechanochemistry despite lacking an ATPase activity of its own. Similar to CaKar3 depletion, loss of CaCik1 results in cell cycle arrest, filamentation defects, and an inability to undergo karyogamy. Furthermore, an examination of the spindle structure in cells lacking either of these proteins shows that a large proportion have a monopolar spindle or two dissociated half-spindles, a phenotype unique to the C. albicans kinesin-14 homolog. These findings provide new insights into mitotic spindle structure and kinesin motor function in C. albicans and identify a potentially vulnerable target for antifungal drug development.

CG10126, A Calcium‐Binding Microtubule‐Associated Protein, Promotes Mitosis during Drosophila Development

The Epidermal Growth Factor Receptor (EGFR) is a receptor tyrosine kinase that regulates signaling pathways critical for development and proliferation in Drosophila. EGFR activity is also highly upregulated in a number of human cancers, making identification of its downstream targets important for understanding both basic cellular processes and disease states. We have identified CG10126, a Drosophila homolog of the human protein Calcyphosine‐like, as a transcriptional target of EGFR signaling. Calcyphosine encodes a calcium‐binding protein regulated in several human cancers, but little is known of its function. Here we examine the role of CG10126 during Drosophila development. We expressed RNA interference constructs at various points in development. Reducing the level of CG10126 in eye imaginal discs produced adults with small eyes, while reducing its level ubiquitously in embryos was generally lethal. To further examine the role of CG10126, we used mass spectrometry to identify the binding partners of CG10126 in the presence or absence of calcium. Specifically in the presence of calcium, CG10126 bound α‐ and β‐tubulin and several spindle assembly proteins, suggesting that it may influence formation of the mitotic spindle. Consistent with this, embryos with reduced CG10126 show a diminished number of mitotic nuclei and abnormal spindle structures. Calcium is indispensable for cell cycle regulation; our results suggest that calcium waves present during mitosis may enable CG10126 to influence microtubule dynamics and spindle assembly. Together, our studies suggest that CG10126, a downstream target of EGFR signaling, is a microtubule‐associated protein that affects cell proliferation by regulating mitotic spindle formation.