Coordination between the Ndc80 complex and dynein is essential for microtubule plus-end capture by kinetochores during early mitosis.

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 toward the spindle pole during prometaphase. The microtubule-binding nuclear division cycle 80 (Ndc80) complex that is recruited to kinetochores in prophase is known to play a central role in forming kinetochore-microtubule (kMT) attachments in metaphase. It is not yet clear, however, how Ndc80 contributes to initial kMT capture during prometaphase. Here, by combining CRISPR/Cas9-mediated knockout and RNAi technology with assays specific to study kMT capture, we show that mitotic cells lacking Ndc80 exhibit substantial 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 use an in vitro total internal reflection fluorescence microscopy assay to reconstitute microtubule capture events, which suggest that Ndc80 and dynein coordinate with each other for microtubule plus-end capture and that the phosphorylation status of Ndc80 is critical for productive kMT capture. A novel interaction between Ndc80 and dynein that we identify in prometaphase extracts might be critical for efficient plus-end capture. Thus, our studies, for the first time, identify a distinct event in the formation of initial kMT attachments, which is directly mediated by Ndc80 and in coordination with dynein is required for efficient kMT capture and chromosome alignment.

Author Correction: Lateral attachment of kinetochores to microtubules is enriched in prometaphase rosette and facilitates chromosome alignment and bi-orientation establishment.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Lateral attachment of kinetochores to microtubules is enriched in prometaphase rosette and facilitates chromosome alignment and bi-orientation establishment.

Faithful chromosome segregation is ensured by the establishment of bi-orientation; the attachment of sister kinetochores to the end of microtubules extending from opposite spindle poles. In addition, kinetochores can also attach to lateral surfaces of microtubules; called lateral attachment, which plays a role in chromosome capture and transport. However, molecular basis and biological significance of lateral attachment are not fully understood. We have addressed these questions by focusing on the prometaphase rosette, a typical chromosome configuration in early prometaphase. We found that kinetochores form uniform lateral attachments in the prometaphase rosette. Many transient kinetochore components are maximally enriched, in an Aurora B activity-dependent manner, when the prometaphase rosette is formed. We revealed that rosette formation is driven by rapid poleward motion of dynein, but can occur even in its absence, through slow kinetochore movements caused by microtubule depolymerization that is supposedly dependent on kinetochore tethering at microtubule ends by CENP-E. We also found that chromosome connection to microtubules is extensively lost when lateral attachment is perturbed in cells defective in end-on attachment. Our findings demonstrate that lateral attachment is an important intermediate in bi-orientation establishment and chromosome alignment, playing a crucial role in incorporating chromosomes into the nascent spindle.

CLIP-170 tethers kinetochores to microtubule plus ends against poleward force by dynein for stable kinetochore-microtubule attachment.

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.

CLIP-170 recruits PLK1 to kinetochores during early mitosis for chromosome alignment.

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.

A creeper, Coccinia indica, has anti-hyperglycaemic and anti-ureogenic effects in diabetic rats.

OBJECTIVE: To explore how ethanolic extract of Coccinia indica affects normal and diabetic rats. METHODS: 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. RESULTS: The blood sugar level had significantly decreased by 23% (p <0.01) and 28% (p <0.001) in the normal and diabetic rats. Level of blood-free fatty acid was depressed by 15% (p <0.01) and 25% (p <0.001) in the two groups respectively. Moreover, the activity of hepatic arginase, a key urea cycle enzyme, was significantly depressed by 14% (p < 0.05) and 22% (p < 0.02) in the normal and diabetic groups. CONCLUSION: 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.

Nucleophosmin is required for chromosome congression, proper mitotic spindle formation, and kinetochore-microtubule attachment in HeLa cells.

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.

Depletion of nucleophosmin leads to distortion of nucleolar and nuclear structures in HeLa cells.

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 (alpha-tubulin and beta-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.

Fibrillarin, a nucleolar protein, is required for normal nuclear morphology and cellular growth in HeLa cells.

Fibrillarin is a key small nucleolar protein in eukaryotes, which has an important role in pre-rRNA processing during ribosomal biogenesis. Though several functions of fibrillarin are known, its function during the cell cycle is still unknown. In this study, we confirmed the dynamic localization of fibrillarin during the cell cycle of HeLa cells and also performed functional studies by using a combination of immunofluorescence microscopy and RNAi technique. We observed that depletion of fibrillarin has almost no effect on the nucleolar structure. However, fibrillarin-depleted cells showed abnormal nuclear morphology. Moreover, fibrillarin depletion resulted in the reduction of the cellular growth and modest accumulation of cells with 4n DNA content. Our data suggest that fibrillarin would play a critical role in the maintenance of nuclear shape and cellular growth.