Central Spindles Research Articles

O-069 The first mitotic spindle instability impacts nucleation, fragmentation, and development in human embryos

Abstract Study question How multinucleation and fragmentation occur in the first mitosis in human embryos? Summary answer The first mitotic spindles had heterogeneity, and the shapes and movements of the spindles were strongly associated with multinucleation and fragmentation. What is known already Studies have shown that many problems occur during the first mitosis, such as fragmentation and multinucleation. However, the cell biologic mechanisms whereby these errors occur during the first mitosis in human embryos remain unknown. To clarify this aspect, it is necessary to analysis the dynamics of DNA and cytoskeleton using live human embryos. Although microinjections of DNA or mRNA into mammalian embryos were widely used for live imaging during early mitosis, the method into human poses the risk of ethical issues. Therefore, we established a method used for real-time imaging in human embryos using chemical fluorescent labeling. Study design, size, duration Frozen human two-pronuclear stage embryos were donated for research by couples who had completed fertility treatment. We obtained written informed consent from patients who donated human embryos. The mean age of patients at embryo freezing was 32.00 ± 3.03 (SD) years. We imaged thirty-one living human embryos using chemical fluorescent labeling and confocal microscopy to visualize DNA and tubulin. Participants/materials, setting, methods Thirty-one human embryos were cultured in medium containing the live-cell stain probes SPY555-DNA and SPY650-Tubulin. The Embryos were imaged using an LSM980 laser scanning confocal microscope every 10–20 min for a period of eight hours. Three-dimensional (3D) visualizations of human embryos were performed using the Imaris software. The first spindle shapes were quantified by image analysis and calculated the aspect ratio of the spindles using ImageJ. Main results and the role of chance We discovered that over 81% of two-pronuclear zygotes became multinucleated 2-cell stage embryos. We demonstrated that there was variation in the first mitotic spindles. There was a significant difference between the aspect ratio (AR) of the spindles leading to the mononuclear and multinuclear types at the 2-cell stage (p < 0.005). In addition, the low-AR spindles were unstable and often had more defocused poles than the high-AR spindles, which lead to multinucleation at the 2-cell stage (55 % vs. 0%, p < 0.05). More embryo having high-aspect ratio spindles tend to develop blastocysts than those having low aspect-ratio spindles (p < 0.05). Moreover, the movement of the central spindles varied after chromosome segregation. The embryos with more fragments at two cell stage had a significantly longer period for the central spindles to transform into midbodies compared to those with fewer fragments. The central spindles of embryos with more fragments moved a longer distance from the beginning of furrow ingression to the completion of the first division (p < 0.05). These results suggest that the first mitotic spindle instability and heterogeneity cause many errors such as multinucleation, fragmentation, and embryonic development. Limitations, reasons for caution Imaged human embryos were donated from a fertility clinic, and detailed patient information, such as underlying disease and method of ovarian stimulation, was not available. It is possible that the human zygotes used in this study had a bias in patient characteristics. Wider implications of the findings Our study showed that there were instability and heterogeneity in the first mitotic spindle, which was strongly associated with multinucleation, fragmentation, and embryonic development. Revealing the mechanism underlying spindle formation in cleavage-stage embryos will contribute to improving the success rate of assisted reproduction technologies in humans. Trial registration number not applicable

High expression of CCDC69 is correlated with immunotherapy response and protective effects on breast cancer

BackgroundAs a molecule controlling the assembly of central spindles and recruitment of midzone component, coiled-coil domain-containing protein 69 (CCDC69) plays an important role in multiple cancers. Currently, the relationships between CCDC69 and immune infiltration or immunotherapy in breast cancer remain unclear.MethodsThe expression and prognostic significance of CCDC69 in breast cancer were comprehensively analyzed by quantitative real-time PCR, immunohistochemical staining and various databases. The data source of differentially expressed genes, gene set enrichment analysis, and immune cell infiltration analysis came from The Cancer Genome Atlas (TCGA) database. Single-cell analysis based on IMMUcan database was used. The protein-protein interaction network was developed applying STRING, Cytoscape, CytoHubba, and GeneMANIA. TISIDB was employed in analyzing the CCDC69 co-expressed immune related genes. The correlations between CCDC69 and immunotherapy or immune-related scores were analyzed by CAMOIP and TISMO. Ctr-db was also used to conduct drug sensitivity analysis.ResultsThe mRNA of CCDC69 was downregulated in breast cancer tissues compared with normal tissues. Higher CCDC69 expression was associated with a better breast cancer prognosis. Enrichment analysis showed that the co-expression genes of CCDC69 were mainly related to immune-related pathways. The expression of CCDC69 was found to be positively correlated with multiple tumor-suppression immune infiltration cells, especially T cells and dendritic cells. Meanwhile, high CCDC69 expression can predict better immunotherapy responses when compared with low CCDC69 expression. After the interferon-gamma treatment, the CCDC69 expression was elevated in vitro. CCDC69 expression was a reliable predictor for the response status of two therapeutic strategies in breast cancer.ConclusionsOur research revealed the clinical significance of CCDC69 in breast cancer and validated the critical roles of CCDC69 in the tumor immune infiltration and immunotherapy responses.

Sleep spindles in the healthy brain from birth through 18 years.

Sleep spindles are present from birth and reflect cognitive functions across the lifespan, but normative values for this cognitive biomarker across development are lacking. This study aims to establish normative spindle features over development. All available normal 19-channel electroencephalograms from developmentally normal children between February 2002 and June 2021 in the MGH EEG lab were analyzed. Approximately, 20 000 spindles were hand-marked to train and validate an automated spindle detector across ages. Normative values for spindle rate, duration, frequency, refractory period, and interhemispheric lag are provided for each channel and each age. Sleep EEGs from 567 developmentally normal children (range 0 days to 18 years) were included. The detector had excellent performance (F1 = 0.47). Maximal spindle activity is seen over central regions during infancy and adolescence and frontopolar regions during childhood. Spindle rate and duration increase nonlinearly, with the most rapid changes during the first 4 months of life and between ages 3 and 14 years. Peak spindle frequency follows a U-shaped curve and discrete frontal slow and central fast spindles are evident by 18 months. Spindle refractory periods decrease between ages 1 and 14 years while interhemispheric asynchrony decreases over the first 3 months of life and between ages 1 and 14 years. These data provide age- and region-specific normative values for sleep spindles across development, where measures that deviate from these values can be considered pathological. As spindles provide a noninvasive biomarker for cognitive function across the lifespan, these normative measures can accelerate the discovery and diagnosis in neurodevelopmental disorders.

Asymmetry in sleep spindles and motor outcome in infants with unilateral brain injury.

To determine whether interhemispheric difference in sleep spindles in infants with perinatal unilateral brain injury could link to a pathological network reorganization that underpins the development of unilateral cerebral palsy (CP). This was a multicentre retrospective study of 40 infants (19 females, 21 males) with unilateral brain injury. Sleep spindles were detected and quantified with an automated algorithm from electroencephalograph records performed at 2 months to 5 months of age. The clinical outcomes after 18 months were compared to spindle power asymmetry (SPA) between hemispheres in different brain regions. We found a significantly increased SPA in infants who later developed unilateral CP (n=13, with the most robust interhemispheric difference seen in the central spindles. The best individual-level prediction of unilateral CP was seen in the centro-occipital spindles with an overall accuracy of 93%. An empiric cut-off level for SPA at 0.65 gave a positive predictive value of 100% and a negative predictive value of 93% for later development of unilateral CP. Our data suggest that automated analysis of interhemispheric SPA provides a potential biomarker of unilateral CP at a very early age. This holds promise for guiding the early diagnostic process in infants with a perinatally identified brain injury. Unilateral perinatal brain injury may affect the development of electroencephalogram (EEG) sleep spindles. Interhemispheric asymmetry in sleep spindles can be quantified with automated EEG analysis. Spindle power asymmetry can be a potential biomarker of unilateral cerebral palsy.

Microtubule and Actin Cytoskeletal Dynamics in Male Meiotic Cells of Drosophila melanogaster.

Drosophila dividing spermatocytes offer a highly suitable cell system in which to investigate the coordinated reorganization of microtubule and actin cytoskeleton systems during cell division of animal cells. Like male germ cells of mammals, Drosophila spermatogonia and spermatocytes undergo cleavage furrow ingression during cytokinesis, but abscission does not take place. Thus, clusters of primary and secondary spermatocytes undergo meiotic divisions in synchrony, resulting in cysts of 32 secondary spermatocytes and then 64 spermatids connected by specialized structures called ring canals. The meiotic spindles in Drosophila males are substantially larger than the spindles of mammalian somatic cells and exhibit prominent central spindles and contractile rings during cytokinesis. These characteristics make male meiotic cells particularly amenable to immunofluorescence and live imaging analysis of the spindle microtubules and the actomyosin apparatus during meiotic divisions. Moreover, because the spindle assembly checkpoint is not robust in spermatocytes, Drosophila male meiosis allows investigating of whether gene products required for chromosome segregation play additional roles during cytokinesis. Here, we will review how the research studies on Drosophila male meiotic cells have contributed to our knowledge of the conserved molecular pathways that regulate spindle microtubules and cytokinesis with important implications for the comprehension of cancer and other diseases.

Functional midbody assembly in the absence of a central spindle.

Contractile ring constriction during cytokinesis is thought to compact central spindle microtubules to form the midbody, an antiparallel microtubule bundle at the intercellular bridge. In Caenorhabditis elegans, central spindle microtubule assembly requires targeting of the CLASP family protein CLS-2 to the kinetochores in metaphase and spindle midzone in anaphase. CLS-2 targeting is mediated by the CENP-F-like HCP-1/2, but their roles in cytokinesis and midbody assembly are not known. We found that although HCP-1 and HCP-2 mostly function cooperatively, HCP-1 plays a more primary role in promoting CLS-2-dependent central spindle microtubule assembly. HCP-1/2 codisrupted embryos did not form central spindles but completed cytokinesis and formed functional midbodies capable of supporting abscission. These central spindle-independent midbodies appeared to form via contractile ring constriction-driven bundling of astral microtubules at the furrow tip. This work suggests that, in the absence of a central spindle, astral microtubules can support midbody assembly and that midbody assembly is more predictive of successful cytokinesis than central spindle assembly.

Relationship between Cortical Thickness and EEG Alterations during Sleep in the Alzheimer's Disease.

Recent evidence showed that EEG activity alterations that occur during sleep are associated with structural, age-related, changes in healthy aging brains, and predict age-related decline in memory performance. Alzheimer’s disease (AD) patients show specific EEG alterations during sleep associated with cognitive decline, including reduced sleep spindles during NREM sleep and EEG slowing during REM sleep. We investigated the relationship between these EEG sleep alterations and brain structure changes in a study of 23 AD patients who underwent polysomnographic recording of their undisturbed sleep and 1.5T MRI scans. Cortical thickness measures were correlated with EEG power in the sigma band during NREM sleep and with delta- and beta-power during REM sleep. Thinning in the right precuneus correlated with all the EEG indexes considered in this study. Frontal–central NREM sigma power showed an inverse correlation with thinning of the left entorhinal cortex. Increased delta activity at the frontopolar and temporal regions was significantly associated with atrophy in some temporal, parietal, and frontal cortices, and with mean thickness of the right hemisphere. Our findings revealed an association between sleep EEG alterations and the changes to AD patients’ brain structures. Findings also highlight possible compensatory processes involving the sources of frontal–central sleep spindles.

Sleep spindles in children with restless sleep disorder, restless legs syndrome and normal controls

ObjectiveTo analyze and identify differences in sleep spindles in children with restless sleep disorder (RSD), restless legs syndrome (RLS) and normal controls. MethodsPSG (polysomnography) from children with RSD, RLS and normal controls were analyzed. Sleep spindle activity was detected on one frontal and one central electrode, for each epoch of N2 and N3 sleep. Sleep spindle density, duration and intensity (density × duration) were then obtained and used for analysis. ResultsThirty-eight children with RSD, twenty-three children with RLS and twenty-nine controls were included. The duration of frontal spindles in sleep stage N2 was longer in children with RSD than in controls. Frontal spindle density and intensity tended to be increased in RSD children. No significant differences were found for central spindles. ConclusionChildren with RSD had longer frontal spindles. This finding may contribute to explain the occurrence of excessive movement activity during sleep and the presence of daytime symptoms. SignificanceRecent research has demonstrated that children with RSD have increased NREM instability and sympathetic activation during sleep. Analyzing sleep spindles in children with RSD in comparison with children with RLS and controls adds to our understanding of the pathophysiology or RSD and its effects on daytime impairment.

The PP1 regulator PPP1R2 coordinately regulates AURKA and PP1 to control centrosome phosphorylation and maintain central spindle architecture

BackgroundMaintenance of centrosome number in cells is essential for accurate distribution of chromosomes at mitosis and is dependent on both proper centrosome duplication during interphase and their accurate distribution to daughter cells at cytokinesis. Two essential regulators of cell cycle progression are protein phosphatase 1 (PP1) and Aurora A kinase (AURKA), and their activities are each regulated by the PP1 regulatory subunit, protein phosphatase 1 regulatory subunit 2 (PPP1R2). We observed an increase in centrosome number after overexpression of these proteins in cells. Each of these proteins is found on the midbody in telophase and overexpression of PPP1R2 and its mutants increased cell ploidy and disrupted cytokinesis. This suggests that the increase in centrosome number we observed in PPP1R2 overexpressing cells was a consequence of errors in cell division. Furthermore, overexpression of PPP1R2 and its mutants increased midbody length and disrupted midbody architecture. Additionally, we show that overexpression of PPP1R2 alters activity of AURKA and PP1 and their phosphorylation state at the centrosome.ResultsOverexpression of PPP1R2 caused an increase in the frequency of supernumerary centrosomes in cells corresponding to aberrant cytokinesis reflected by increased nuclear content and cellular ploidy. Furthermore, AURKA, PP1, phospho PPP1R2, and PPP1R2 were all localized to the midbody at telophase, and PP1 localization there was dependent on binding of PPP1R2 with PP1 and AURKA as well as its phosphorylation state. Additionally, overexpression of both PPP1R2 and its C-terminal AURKA binding site altered enzymatic activity of AURKA and PP1 at the centrosome and disrupted central spindle structure.ConclusionsResults from our study reveal the involvement of PPP1R2 in coordinating PP1 and AURKA activity during cytokinesis. Overexpression of PPP1R2 or its mutants disrupted the midbody at cytokinesis causing accumulation of centrosomes in cells. PPP1R2 recruited PP1 to the midbody and interference with its targeting resulted in elongated and severely disrupted central spindles supporting an important role for PPP1R2 in cytokinesis.

The protective effect of daytime sleep on planning and risk-related decision-making in emerging adults.

We assessed the effect of a daytime sleep opportunity on planning and risk-related decision-making in emerging adults using multiple neurobehavioral assessments. A total of 136 healthy emerging adults (20.0 ± 1.5 years), 65% female, performed the Risky-Gains Task and the Tower of London test twice. Between these assessments, they were randomized to either have a sleep opportunity monitored by polysomnography (Sleep group, n = 101) or to stay awake (Wake group, n = 35). During Test 2, in comparison to the Sleep group, the Wake group showed increased sleepiness, worse planning ability and more decrease in reaction times when selecting risky choices. Changes in Tower of London test steps used and Risky-Gains Task response time correlated with the number of central and frontal fast sleep spindles, respectively. These results indicate that among emerging adults who commonly have poor sleep patterns, a daytime sleep opportunity was related to better planning ability, better psychomotor vigilance and stable response speeds in risk-related decision-making. Changes in planning and risk-related decision-making correlated with the number of sleep spindles during the nap, supporting a specific role for sleep in modulating planning and potentially other higher-order cognitive functions.

Sleep Spindle Features and Neurobehavioral Performance in Healthy School-Aged Children.

In adults, central fast-frequency sleep spindles are involved in learning and memory functions. The density of local spindles is higher than global spindles, emphasizing the importance of local plastic neural processes. In children, findings on the association of spindles with cognition are more variable. Hence, we aim to study whether the local spindles are also important for neurobehavioral performance in children. We studied the correlations between local (occurring in only one channel: Fp1, Fp2, C3, or C4), bilateral, and diffuse (occurring in all four channels) spindles and neurobehavioral performance in 17 healthy children (median age 9.6 years). Local spindles were not as frequent as bilateral spindles (P-values < 0.05). Central spindle types had significant correlations with sensorimotor and language functions (e.g., the density of bilateral central spindles correlated positively with the Object Assembly in NEPSY, r = 0.490). Interestingly, frontopolar spindles correlated with behavior (e.g., the more bilateral the frontopolar spindles, the less hyperactive the children, r = -0.618). In children, the local spindles, but also more widespread central spindles, seem to be involved in the cognitive processes. Based on our findings, it is important that ageadjusted frequency limits are used in studies evaluating the frequencies of spindles in children.

Sleep spindle characteristics in adolescents

ObjectiveSleep changes substantially during adolescence; however, our understanding of age-related differences in specific electroencephalographic waveforms during this developmental period is limited. MethodSigma power, spindle characteristics and cognitive data were calculated for fast (∼13 Hz) central and slow (∼11 Hz) frontal sleep spindles for a large cross-sectional sample of adolescents (N = 134, aged 12–21 years, from the National Consortium on Alcohol and NeuroDevelopment in Adolescence (NCANDA) study). ResultsOlder age (and advanced pubertal development) was associated with lower absolute sigma power and greater fast spindle density, with spindles having a shorter duration and smaller amplitude and occurring at a faster average frequency than at a younger age. Spindle characteristics were not directly associated with cognition. An indirect relationship (age * density) provided some evidence for an association between better episodic memory performance and greater spindle density only for younger adolescents. ConclusionSpindle characteristics in adolescents differed according to age, possibly reflecting underlying differences in thalamo-cortical connectivity, and may play a role in episodic memory early in adolescence. SignificanceSleep spindles may serve as a marker of adolescent development, likely reflecting brain maturational status. Investigating specific spindle characteristics, in addition to sigma power, is necessary to fully characterize spindles during adolescence.

Shortening of Microtubule Overlap Regions Defines Membrane Delivery Sites during Plant Cytokinesis

Different from animal cells that divide by constriction of the cortex inward, cells of land plants divide by initiating a new cell-wall segment from their center. For this, a disk-shaped, membrane-enclosed precursor termed the cell plate is formed that radially expands toward the parental cell wall [1-3]. The synthesis of the plate starts with the fusion of vesicles into a tubulo-vesicular network [4-6]. Vesicles are putatively delivered to the division plane by transport along microtubules of the bipolar phragmoplast network that guides plate assembly [7-9]. How vesicle immobilization and fusion are then locally triggered is unclear. In general, a framework for how the cytoskeleton spatially defines cell-plate formation is lacking. Here we show that membranous material for cell-plate formation initially accumulates along regions of microtubule overlap in the phragmoplast of the moss Physcomitrella patens. Kinesin-4-mediated shortening of these overlaps at the onset of cytokinesis proved to be required to spatially confine membrane accumulation. Without shortening, the wider cell-plate membrane depositions evolved into cell walls that were thick and irregularly shaped. Phragmoplast assembly thus provides a regular lattice of short overlaps on which a new cell-wall segment can be scaffolded. Since similar patterns of overlaps form in central spindles of animal cells, involving the activity of orthologous proteins [10, 11], we anticipate that our results will help uncover universal features underlying membrane-cytoskeleton coordination during cytokinesis.

CPAP Treatment Partly Normalizes Sleep Spindle Features in Obstructive Sleep Apnea.

Objective. Obstructive sleep apnea (OSA) decreases sleep spindle density and frequency. We evaluated the effects of continuous positive airway pressure (CPAP) treatment on different features of sleep spindles. Methods. Twenty OSA patients underwent two night polysomnographies in a diagnostic phase and one night polysomnography after 6 months of CPAP treatment. The control group comprised 20 healthy controls. Sleep spindles were analyzed by a previously developed automated method. Unilateral and bilateral spindles were identified in central and frontopolar brain locations. Spindle density and frequency were determined for the first and last half of the NREM time. Results. The density of bilateral central spindles, which did not change in the untreated OSA patients, increased towards the morning hours during CPAP treatment and in the controls. Central spindles did not become faster with sleep in OSA patients and the central spindles remained slow in the left hemisphere even with CPAP. Conclusion. CPAP treatment normalized spindle features only partially. The changes may be associated with deficits in thalamocortical spindle generating loops. Significance. This study shows that some sleep spindle changes persist after CPAP treatment in OSA patients. The association of these changes to daytime symptoms in OSA patients needs to be further evaluated.

Aurora B inhibitor barasertib prevents meiotic maturation and subsequent embryo development in pig oocytes

Barasertib, a highly selective Aurora B inhibitor, has been widely used in a variety of cells to investigate the role of Aurora B kinase, which has been implicated in various functions in the mitotic process. However, effects of barasertib on the meiotic maturation process are not fully understood, particularly in porcine oocyte meiotic maturation. In the present study, the effects of barasertib on the meiotic maturation and developmental competence of pig oocytes were investigated, and the possible roles of Aurora B were also evaluated in porcine oocytes undergoing meiosis. Initially, we examined the expression and subcellular localization of Aurora B using Western blot analysis and immunofluorescent staining. Aurora B was found to express and exhibit specific dynamic intracellular localization during porcine oocyte meiotic maturation. Aurora B was observed around the chromosomes after germinal vesicle breakdown. Then it was transferred to the spindle region after metaphase I stage, and was particularly concentrated at the central spindles at telophase I stage. barasertib treatment resulted in the failure of polar body extrusion in pig oocytes, with a larger percentage of barasertib-treated oocytes remaining at the pro–metaphase I stage. Additional results reported that barasertib treatment had no effect on chromosome condensation but resulted in a significantly higher percentage of the treated oocytes with aberrant spindles and misaligned chromosomes during the first meiotic division. In addition, inhibition of Aurora B with lower concentrations of barasertib during pig oocyte meiotic maturation decreased the subsequent embryo developmental competence. Thus, these results illustrate that barasertib has significant effects on porcine oocyte meiotic maturation and subsequent development through Aurora B inhibition, and this regulation is related to its effects on spindle formation and chromosome alignment during the first meiotic division in porcine oocytes.

Versatile kinetochore components control central spindle assembly

Versatile kinetochore components control central spindle assembly

Susceptibility to declarative memory interference is pronounced in primary insomnia.

Sleep has been shown to stabilize memory traces and to protect against competing interference in both the procedural and declarative memory domain. Here, we focused on an interference learning paradigm by testing patients with primary insomnia (N = 27) and healthy control subjects (N = 21). In two separate experimental nights with full polysomnography it was revealed that after morning interference procedural memory performance (using a finger tapping task) was not impaired in insomnia patients while declarative memory (word pair association) was decreased following interference. More specifically, we demonstrate robust associations of central sleep spindles (in N3) with motor memory susceptibility to interference as well as (cortically more widespread) fast spindle associations with declarative memory susceptibility. In general the results suggest that insufficient sleep quality does not necessarily show up in worse overnight consolidation in insomnia but may only become evident (in the declarative memory domain) when interference is imposed.

Germline deletion of FAK-related non-kinase delays post-natal cardiomyocyte mitotic arrest

The cardiomyocyte phenotypic switch from a proliferative to terminally differentiated state impacts normal heart development and pathologic myocardial remodeling, yet the signaling mechanisms that regulate this vital process are incompletely understood. Studies from our lab and others indicate that focal adhesion kinase (FAK) is a critical regulator of cardiac growth and remodeling and we found that expression of the endogenous FAK inhibitor, FAK-related non kinase (FRNK) coincided with postnatal cardiomyocyte arrest. Mis-expression of FRNK in the embryonic heart led to pre-term lethality associated with reduced cardiomyocyte proliferation and led us to speculate that the postnatal FRNK surge might be required to promote quiescence in this growth promoting environment. Herein, we provide strong evidence that endogenous FRNK contributes to post-mitotic arrest. Depletion of FRNK promoted DNA synthesis in post-natal day (P) 10 hearts accompanied by a transient increase in DNA content and multi-nucleation by P14, indicative of DNA replication without cell division. Interestingly, a reduction in tri- and tetra-nucleated cardiomyocytes, concomitant with an increase in bi-nucleated cells by P21, indicated the possibility that FRNK-depleted cardiomyocytes underwent eventual cytokinesis. In support of this conclusion, Aurora B-labeled central spindles (a hallmark of cytokinesis) were observed in tetra-nucleated P20 FRNK(-/-) but not wt cardiomyocytes, while no evidence of apoptosis was observed. Moreover, hearts from FRNK null mice developed ventricular enlargement that persisted until young adulthood which resulted from myocyte expansion rather than myocyte hypertrophy or interstitial growth. These data indicate that endogenous FRNK serves an important role in limiting DNA synthesis and regulating the un-coupling between DNA synthesis and cytokinesis in the post-natal myocardium.

TheDrosophilaRZZ complex: roles in membrane traffic and cytokinesis

The Zw10 protein, in the context of the conserved Rod-Zwilch-Zw10 (RZZ) complex, is a kinetochore component required for proper activity of the spindle assembly checkpoint in both Drosophila and mammals. In mammalian and yeast cells, the Zw10 homologues, together with the conserved RINT1/Tip20p and NAG/Sec39p proteins, form a second complex involved in vesicle transport between Golgi and ER. However, it is currently unknown whether Zw10 and the NAG family member Rod are also involved in Drosophila membrane trafficking. Here we show that Zw10 is enriched at both the Golgi stacks and the ER of Drosophila spermatocytes. Rod is concentrated at the Golgi but not at the ER, whereas Zwilch does not accumulate in any membrane compartment. Mutations in zw10 and RNAi against the Drosophila homologue of RINT1 (rint1) cause strong defects in Golgi morphology and reduce the number of Golgi stacks. Mutations in rod also affect Golgi morphology, whereas zwilch mutants do not exhibit gross Golgi defects. Loss of either Zw10 or Rint1 results in frequent failures of spermatocyte cytokinesis, whereas Rod or Zwilch are not required for this process. Spermatocytes lacking zw10 or rint1 function assemble regular central spindles and acto-myosin rings, but furrow ingression halts prematurely due to defective plasma membrane addition. Collectively, our results suggest that Zw10 and Rint1 cooperate in the ER-Golgi trafficking and in plasma membrane formation during spermatocyte cytokinesis. Our findings further suggest that Rod plays a Golgi-related function that is not required for spermatocyte cytokinesis.

Phosphorylation of Ran-binding Protein-1 by Polo-like Kinase-1 Is Required for Interaction with Ran and Early Mitotic Progression

Polo-like kinase-1 (Plk1) is essential for progression of mitosis and localizes to centrosomes, central spindles, midbody, and kinetochore. Ran, a small GTPase of the Ras superfamily, plays a role in microtubule dynamics and chromosome segregation during mitosis. Although Ran-binding protein-1 (RanBP1) has been reported as a regulator of RanGTPase for its mitotic functions, the action mechanism between Ran and RanBP1 during mitosis is still unknown. Here, we demonstrated in vitro and in vivo phosphorylation of RanBP1 by Plk1 as well as the importance of phosphorylation of RanBP1 in the interaction between Plk1 and Ran during early mitosis. Both phosphorylation-defective and N-terminal deletion mutant constructs of RanBP1 disrupted the interaction with Ran, and depletion of Plk1 also disrupted the formation of a complex between Ran and RanBP1. In addition, the results from both ectopic expression of phosphorylation-defective mutant construct and a functional complementation on RanBP1 deficiency with this mutant indicated that phosphorylation of RanBP1 by Plk1 might be crucial to microtubule nucleation and spindle assembly during mitosis.