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Mitotic kinetochores are initially captured by dynamic microtubules via a ‘search-and-capture’ mechanism. The microtubule motor, dynein, is critical for kinetochore capture as it has been shown to transport microtubule-attached chromosomes towards the spindle pole during early mitosis. In metaphase, the kinetochore localized, microtubule-binding complex, Ndc80, plays a central role in stabilizing kinetochore-microtubule (kMT) attachments. It is not yet clear, however, if Ndc80, which is recruited to kinetochores very early during mitosis contributes to initial kMT capture. Here, by combining CRISPR/Cas9-mediated knockout and RNAi technology with assays specifically targeted to study kMT capture, we show that mitotic cells lacking Ndc80 exhibit severe defects in this function during prometaphase. Rescue experiments show that Ndc80 mutants deficient in microtubule-binding are unable to execute proper kMT capture. While cells inhibited of dynein alone are predominantly able to make initial kMT attachments, cells co-depleted of Ndc80 and dynein show severe defects in kMT capture. Further, we demonstrate a novel physical interaction between Ndc80 and dynein during prometaphase. Thus, our studies, for the first time, identify a distinct event in the formation of initial kMT attachments, which is directly mediated by Ndc80 followed by a coordinated function with dynein, both of which are required for efficient kMT capture and proper chromosome alignment.

Chromosome segregation is mediated by spindle microtubules that attach to the kinetochore via dynamic protein complexes, such as Ndc80, Ska, Cdt1 and ch-TOG during mitotic metaphase. While experimental studies have previously shown that these proteins and protein complexes are all essential for maintaining a stable kinetochore-microtubule (kMT) interface, their exact roles in this mitotic metaphase remains elusive. In this study, we employed experimental and computational methods in order to characterize how these proteins can strengthen kMT attachments in both nonload-bearing and load-bearing conditions, typical of prometaphase and metaphase, respectively. Immunofluorescence staining of HeLa cells showed that the levels of Ska and Cdt1 significantly increased from prometaphase to metaphase, while levels of the Ndc80 complex remained unchanged. Our new computational model showed that by incorporating binding and unbinding of each protein complex coupled with a biased diffusion mechanism, the displacement of a possible complex formed by Ndc80-Ska-Cdt1 is significantly higher than that of Ndc80 alone or Ndc80-Ska. In addition, when we incorporate Ndc80/ch-TOG in the model, rupture force and time of attachment of the kMT interface increases. These results support the hypothesis that Ndc80-associated proteins strengthen kMT attachments, and that the interplay between kMT protein complexes in metaphase ensures stable attachments.

In mitosis, faithful chromosome segregation is orchestrated by the dynamic interactions between the spindle microtubules (MTs) emanating from the opposite poles and the kinetochores of chromosomes. However, the precise mechanism that coordinates the coupling of kinetochore components to dynamic MTs has been a long-standing question. Microtubule (MT) associated proteins (MAPs) regulate MT nucleation, dynamics, MT-mediated transport and MT cross-linking in cells. Especially during mitosis, MAPs play an essential role not only in determining the spindle length, position and orientation but also in facilitating robust kinetochore-microtubule (kMT) attachments by linking the kinetochores to spindle MTs efficiently. MT-stability imparted by the MAPs is critical to ensure accurate chromosome segregation. This review primarily focuses on the specific function of non-motor kinetochore MAPs, their recruitment to kinetochores and their MT-binding properties. We also attempt to synthesize and strengthen our understanding of how these MAPs work in coordination with the kinetochore-bound Ndc80 complex (the key component of the MT-binding interface in metaphase and anaphase) to establish stable kMT attachments and control accurate chromosome segregation during mitosis.

In mitosis, faithful chromosome segregation is orchestrated by the dynamic interactions between the spindle microtubules (MTs) emanating from the opposite poles and the kinetochores of the chromosomes. However, the precise mechanism that coordinates the coupling of the kinetochore components to dynamic MTs has been a long-standing question. Microtubule (MT)-associated proteins (MAPs) regulate MT nucleation and dynamics, MT-mediated transport and MT cross-linking in cells. During mitosis, MAPs play an essential role not only in determining spindle length, position and orientation but also in facilitating robust kinetochore-microtubule (kMT) attachments by linking the kinetochores to spindle MTs efficiently. The stability of MTs imparted by the MAPs is critical to ensure accurate chromosome segregation. This review primarily focuses on the specific function of non-motor kinetochore MAPs, their recruitment to kinetochores and their MT-binding properties. We also attempt to synthesize and strengthen our understanding of how these MAPs work in coordination with the kinetochore-bound Ndc80 complex (the key component at the MT-binding interface in metaphase and anaphase) to establish stable kMT attachments and control accurate chromosome segregation during mitosis.

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Chromosome alignment and segregation during mitosis depends critically on kinetochoremicrotubule (kMT) attachments that are mediated by the function of the molecular motor cytoplasmic dynein, and the kinetochore microtubule (MT) binding complex, Ndc80. The RZZ (Rod-ZW10-Zwilch) complex is central to this coordination as it has an important role in dynein recruitment and has recently been reported to have a key function in the regulation of stable kMT attachment formation in C. elegans. However, the mechanism by which kMT attachments are controlled by the coordinated function of these protein complexes to drive chromosome motility during early mitosis is still unclear. In this manuscript, we provide evidence to show that Ndc80 and dynein directly antagonize each other’s MT-binding. We also find that severe chromosome alignment defects induced by depletion of dynein, or the dynein adapter spindly, are rescued by codepletion of the RZZ component, Rod, in human cells. Interestingly, the rescue of chromosome alignments defects was independent of Rod function in activation of the spindle assembly checkpoint and was accompanied by a remarkable restoration of stable kMT attachments. Furthermore, rescue of chromosome alignment was critically dependent on the plus-end-directed motility of CENP-E, as cells codepleted of CENP-E along with Rod and dynein were unable to establish stable kMT attachments or align their chromosomes properly. Taken together, our findings support the idea that the dynein motor may control the function of the Ndc80 complex in stabilizing kMT attachments either directly by interfering with Ndc80-MT binding, and/or indirectly by modulating the Rod-mediated inhibition of Ndc80.

Study of the various regulatory events of the cell cycle in a phase-dependent manner provides a clear understanding about cell growth and division. The synchronization of cell populations at specific stages of the cell cycle has been found to be very useful in such experimental endeavors. Synchronization of cells by treatment with chemicals that are relatively less toxic can be advantageous over the use of pharmacological inhibitory drugs for the study of consequent cell cycle events and to obtain specific enrichment of selected mitotic stages. Here, we describe the protocol for synchronizing human cells at different stages of the cell cycle, including both in S phase and M phase with a double thymidine block and release procedure for studying the functionality of mitotic proteins in chromosome alignment and segregation. This protocol has been extremely useful for studying the mitotic roles of multifunctional proteins which possess established interphase functions. In our case, the mitotic role of Cdt1, a protein critical for replication origin licensing in G1 phase, can be studied effectively only when G2/M-specific Cdt1 can be depleted. We describe the detailed protocol for depletion of G2/M-specific Cdt1 using double thymidine synchronization. We also explain the protocol of cell fixation, and live cell imaging using high resolution confocal microscopy after thymidine release. The method is also useful for analyzing the function of mitotic proteins under both physiological and perturbed conditions such as for Hec1, a component of the Ndc80 complex, as it enables one to obtain large sample sizes of mitotic cells for fixed and live cell analysis as we show here.

Nucleophosmin (NPM) is an abundantly expressed multifunctional nucleolar phosphoprotein. Here we show that depletion of NPM by RNA interference causes defects in cell division, followed by an arrest of DNA synthesis due to activation of a p53-dependent checkpoint response in HeLa cells. Depletion of NPM leads to mitotic arrest due to spindle checkpoint activation. The mitotic cells arrested by NPM depletion have defects in chromosome congression, proper mitotic spindle and centrosome formation, as well as defects in kinetochore-microtubule attachments. Loss of NPM thus causes severe mitotic defects and delayed mitotic progression. These findings indicate that NPM is essential for mitotic progression and cell proliferation.

The cytoplasmic linker protein (CLIP)-170, an outer kinetochore protein, has a role in kinetochore-microtubule attachment and chromosome alignment during mitosis. However, the mechanism by which CLIP-170 is involved in chromosome alignment is not known. Here, we show that CLIP-170 colocalizes with Polo-like kinase 1 (PLK1) at kinetochores during early mitosis. Depletion of CLIP-170 results in a significant reduction in PLK1 recruitment to kinetochores and causes kinetochore-fiber (K-fiber) instability and defects in chromosome alignment at the metaphase plate. These phenotypes are dependent on the phosphorylation of CLIP-170 at a CDK1-dependent site, T287, as ectopic expression of wild-type CLIP-170, but not the expression of a non-phosphorylatable mutant, CLIP-170-T287A, restores PLK1 localization at kinetochores and rescues K-fiber stability and chromosome alignment in CLIP-170-depleted cells. These data suggest that CLIP-170 acts as a novel recruiter and spatial regulator of PLK1 at kinetochores during early mitosis, promoting K-fiber stability and chromosome alignment for error-free chromosome segregation.

The cytoplasmic linker protein (CLIP)-170, an outer kinetochore protein, has a role in kinetochore-microtubule attachment and chromosome alignment during mitosis. However, the mechanism by which CLIP-170 is involved in chromosome alignment is not known. Here, we show that CLIP-170 colocalizes with Polo-like kinase 1 (PLK1) at kinetochores during early mitosis. Depletion of CLIP-170 results in a significant reduction in PLK1 recruitment to kinetochores and causes kinetochore-fiber (K-fiber) instability and defects in chromosome alignment at the metaphase plate. These phenotypes are dependent on the phosphorylation of CLIP-170 at a CDK1-dependent site, T287, as ectopic expression of wild-type CLIP-170, but not the expression of a non-phosphorylatable mutant, CLIP-170-T287A, restores PLK1 localization at kinetochores and rescues K-fiber stability and chromosome alignment in CLIP-170-depleted cells. These data suggest that CLIP-170 acts as a novel recruiter and spatial regulator of PLK1 at kinetochores during early mitosis, promoting K-fiber stability and chromosome alignment for error-free chromosome segregation.

To explore how ethanolic extract of Coccinia indica affects normal and diabetic rats.The case-controlled animal study was conducted in June 2008 at the Department of Biochemistry and Molecular Biology, University of Dhaka, Bangladesh. Two groups of 10 male rats each - one streptozotocin-induced diabetics and the other normal - were fed orally aqueous suspension of residue extracted from C. indica leaves with 60% ethanol after 18 hours of fasting. After 90 minutes of oral administration, the rats were sacrificed, and blood level of glucose and free fatty acids and hepatic arginase activity were analysed.The blood sugar level had significantly decreased by 23% (p0.01) and 28% (p0.001) in the normal and diabetic rats. Level of blood-free fatty acid was depressed by 15% (p0.01) and 25% (p0.001) in the two groups respectively. Moreover, the activity of hepatic arginase, a key urea cycle enzyme, was significantly depressed by 14% (p0.05) and 22% (p0.02) in the normal and diabetic groups.Results suggested that C. indica extract had anti-hyperglycaemic and anti-ureogenic effects on the diabetic rats as judged by the decreased level of blood glucose and fatty acid and hepatic arginase activity.

Mohammed Abdullahel Amin, Sachihiro Matsunaga, Susumu Uchiyama, Kiichi Fukui; Depletion of nucleophosmin leads to distortion of nucleolar and nuclear structures in HeLa cells. Biochem J 1 November 2008; 415 (3): 345–351. doi: https://doi.org/10.1042/BJ20081411., NPM (nucleophosmin; also known as B23) is an abundantly and ubiquitously expressed multifunctional nucleolar phosphoprotein, which is involved in numerous cellular processes, including ribosome biogenesis, protein chaperoning and centrosome duplication; however, the role of NPM in the cell cycle still remains unknown. In the present study, we show dynamic localization of NPM throughout the cell cycle of HeLa cells. Using a combination of RNAi (RNA interference) and three-dimensional microscopy we show that NPM is localized at the chromosome periphery during mitosis. We also demonstrate that depletion of NPM causes distortion of nucleolar structure as expected and leads to unexpected dramatic changes in nuclear morphology with multiple micronuclei formation. The defect in nuclear shape of NPM-depleted cells, which is clearly observed by live-cell imaging, is due to the distortion of cytoskeletal (α-tubulin and β-actin) structure, resulting from the defects in centrosomal microtubule nucleation. These results indicate that NPM is an essential protein not only for the formation of normal nucleolar structure, but also for the maintenance of regular nuclear shape in HeLa cells.

The cytoplasmic linker protein (CLIP)-170 localizes to kinetochores and is suggested to function in stable attachment of kinetochores to microtubule ends. Here we show that defects in kinetochore–microtubule attachment and chromosome alignment in CLIP-170-depleted cells were rescued by co-depletion of p150glued, a dynactin subunit required for kinetochore localization of CLIP-170. CLIP-170 recruited p150glued to microtubule ends. Kinetochore localization at microtubule ends was perturbed by CLIP-170 depletion, which was rescued by co-depleting p150glued. Our results imply that CLIP-170 tethers kinetochores to microtubule ends against the dynein-mediated poleward force to slide kinetochores along microtubules, facilitating the stable kinetochore attachment to microtubules.