Early Mitotic Cells Research Articles

Extract of bulbus of Fritillaria cirrhosa induces spindle multipolarity in human-derived colonic epithelial NCM460 cells through promoting centrosome fragmentation.

Bulbus of Fritillaria cirrhosa D. Don (BFC), an outstanding antitussive and expectorant herbal drug used in China and many other countries, has potential but less understood genotoxicity. Previously, we have reported that aqueous extract of BFC compromised the spindle assembly checkpoint and cytokinesis in NCM460 cells. Here, we found that one remarkable observation in BFC-treated NCM460 cells was multipolar mitosis, a trait classically compromises the fidelity of chromosome segregation. More detailed investigation revealed that BFC-induced spindle multipolarity in metaphases and ana-telophases in a dose- and time-dependent manner, suggesting BFC-induced multipolar spindle conformation was not transient. The frequency of multipolar metaphase correlated well to that of multipolar ana-telophases, indicating that BFC-induced multipolar metaphases often persisted through anaphase. Unexpectedly, BFC blocked the proliferation of binucleated cells, suggesting spindle multipolarity was not downstream of BFC-induced cytokinesis failure. Exposure of BFC to early mitotic cells, rather than S/G2 cells, contributed greatly to spindle multipolarity, indicating BFC might disrupt centrosome integrity rather than induce centrosome overduplication. The immunofluorescence results showed that the centrosomes were severely fragmented by a short-term treatment of BFC and the extent of centrosome fragmentation in early mitotic cells was larger than this in S/G2 cells. Consistently, several genes (e.g. p53, Rb centrin-2, Plk-4, Plk-1 and Aurora-A) involved in regulating centrosome integrity were significantly deregulated by BFC. Together, our results suggest that BFC causes multipolar spindles primarily by inducing centrosome fragmentation. Coupling these results to our previous observations, we recommend the risk/benefit ratio should be considered in the practical use of BFC.

Heterotrimeric G-protein, Gi1, is involved in the regulation of proliferation, neuronal migration, and dendrite morphology during cortical development in vivo.

Heterotrimeric G-proteins are composed of α, β, and γ subunits, and function as signal transducers. Critical roles of the α-subunits of Gi/o family heterotrimeric G-proteins, Gαi2, and Gαo1, have so far been reported in brain development and neurodevelopmental disorders. In this study, we tried to clarify the role of Gαi1, α-subunit of another Gi/o family member Gi1, during corticogenesis, based on the recent identification of its gene abnormalities in neurodevelopmental disorders. In western blot analyses, Gαi1 was found to be expressed in mouse brain in a developmental stage-dependent manner. Morphological analyses revealed that Gαi1 was broadly distributed in cerebral cortex with relatively high expression in the ventricular zone (VZ) at embryonic day (E) 14. Meanwhile, Gαi1 was enriched in membrane area of yet unidentified early mitotic cells in the VZ and the marginal zone at E14. Acute knockdown of Gαi1 with in utero electroporation in cerebral cortex caused cell cycle elongation of the neural progenitor cells and promoted their cell cycle exit. Gαi1-deficient cortical neurons also exhibited delayed radial migration during corticogenesis, with abnormally elongated leading processes and hampered nucleokinesis. In addition, silencing of Gαi1 prevented basal dendrite development. The migration and dendritic phenotypes were at least partially rescued by an RNAi-resistant version of Gαi1. Collectively, these results strongly suggest a crucial role of Gi1 in cortical development, and disturbance of its function may cause deficits in synaptic network formation, leading to neurodevelopmental disorders.

Wwc2 Is a Novel Cell Division Regulator During Preimplantation Mouse Embryo Lineage Formation and Oogenesis.

Formation of the hatching mouse blastocyst marks the end of preimplantation development, whereby previous cell cleavages culminate in the formation of three distinct cell lineages (trophectoderm, primitive endoderm and epiblast). We report that dysregulated expression of Wwc2, a genetic paralog of Kibra/Wwc1 (a known activator of Hippo-signaling, a key pathway during preimplantation development), is specifically associated with cell autonomous deficits in embryo cell number and cell division abnormalities. Division phenotypes are also observed during mouse oocyte meiotic maturation, as Wwc2 dysregulation blocks progression to the stage of meiosis II metaphase (MII) arrest and is associated with spindle defects and failed Aurora-A kinase (AURKA) activation. Oocyte and embryo cell division defects, each occurring in the absence of centrosomes, are fully reversible by expression of recombinant HA-epitope tagged WWC2, restoring activated oocyte AURKA levels. Additionally, clonal embryonic dysregulation implicates Wwc2 in maintaining the pluripotent epiblast lineage. Thus, Wwc2 is a novel regulator of meiotic and early mitotic cell divisions, and mouse blastocyst cell fate.

Investigating nucleo-cytoplasmic shuttling of the human DEAD-box helicase DDX3

The human DEAD-box helicase DDX3 is a multi-functional protein involved in the regulation of gene expression and additional non-conventional roles as signalling adaptor molecule that are independent of its enzymatic RNA remodeling activity. It is a nucleo-cytoplasmic shuttling protein and it has previously been suggested that dysregulation of its subcellular localization could contribute to tumourigenesis. Indeed, both tumour suppressor and oncogenic functions have been attributed to DDX3. In this study, we investigated the regulation of DDX3’s nucleocytoplasmic shuttling. We confirmed that an N-terminal conserved Nuclear Export Signal (NES) is required for export of human DDX3 from the nucleus, and identified three regions within DDX3 that can independently facilitate its nuclear import. We also aimed to identify conditions that alter DDX3’s subcellular localisation. Viral infection, cytokine treatment and DNA damage only induced minor changes in DDX3’s subcellular distribution as determined by High Content Analysis. However, DDX3’s nuclear localization increased in early mitotic cells (during prophase) concomitant with an increase in DDX3 expression levels. Our results are likely to have implications for the proposed use of (nuclear) DDX3 as a prognostic biomarker in cancer.

DNA double-strand breaks and Aurora B mislocalization induced by exposure of early mitotic cells to H2O2 appear to increase chromatin bridges and resultant cytokinesis failure

Aneuploidy, an abnormal number of chromosomes that is a hallmark of cancer cells, can arise from tetraploid/binucleated cells through a failure of cytokinesis. Reactive oxygen species (ROS) have been implicated in various diseases, including cancer. However, the nature and role of ROS in cytokinesis progression and related mechanisms has not been clearly elucidated. Here, using time-lapse analysis of asynchronously growing cells and immunocytochemical analyses of synchronized cells, we found that hydrogen peroxide (H2O2) treatment at early mitosis (primarily prometaphase) significantly induced cytokinesis failure. Cytokinesis failure and the resultant formation of binucleated cells containing nucleoplasmic bridges (NPBs) seemed to be caused by increases in DNA double-strand breaks (DSBs) and subsequent unresolved chromatin bridges. We further found that H2O2 induced mislocalization of Aurora B during mitosis. All of these effects were attenuated by pretreatment with N-acetyl-L-cysteine (NAC) or overexpression of Catalase. Surprisingly, the PARP inhibitor PJ34 also reduced H2O2-induced Aurora B mislocalization and binucleated cell formation. Results of parallel experiments with etoposide, a topoisomerase IIα inhibitor that triggers DNA DSBs, suggested that both DNA DSBs and Aurora B mislocalization contribute to chromatin bridge formation. Aurora B mislocalization also appeared to weaken the “abscission checkpoint”. Finally, we showed that KRAS–induced binucleated cell formation appeared to be also H2O2-dependent. In conclusion, we propose that a ROS, mainly H2O2 increases binucleation through unresolved chromatin bridges caused by DNA damage and mislocalization of Aurora B, the latter of which appears to augment the effect of DNA damage on chromatin bridge formation.

A potential physiological role for bi-directional motility and motor clustering of mitotic kinesin-5 Cin8 in yeast mitosis.

The bipolar kinesin-5 Cin8 switches from minus- to plus-end-directed motility under various conditions in vitro The mechanism and physiological significance of this switch remain unknown. Here, we show that under high ionic strength conditions, Cin8 moves towards and concentrates in clusters at the minus ends of stable and dynamic microtubules. Clustering of Cin8 induces a switch from fast minus- to slow plus-end-directed motility and forms sites that capture antiparallel microtubules (MTs) and induces their sliding apart through plus-end-directed motility. In early mitotic cells with monopolar spindles, Cin8 localizes near the spindle poles at microtubule minus ends. This localization is dependent on the minus-end-directed motility of Cin8. In cells with assembled bipolar spindles, Cin8 is distributed along the spindle microtubules. We propose that minus-end-directed motility is required for Cin8 clustering near the spindle poles before spindle assembly. Cin8 clusters promote the capture of microtubules emanating from the neighboring spindle poles and mediate their antiparallel sliding. This activity is essential to maximize microtubule crosslinking before bipolar spindle assembly and to induce the initial separation of the spindle poles.

Acquisition of Oocyte Polarity.

Acquisition of oocyte polarity involves complex translocation and aggregation of intracellular organelles, RNAs, and proteins, along with strict posttranscriptional regulation. While much is still unknown regarding the formation of the animal-vegetal axis, an early marker of polarity, animal models have contributed to our understanding of these early processes controlling normal oogenesis and embryo development. In recent years, it has become clear that proteins with self-assembling properties are involved in assembling discrete subcellular compartments or domains underlying subcellular asymmetries in the early mitotic and meiotic cells of the female germline. These include asymmetries in duplication of the centrioles and formation of centrosomes and assembly of the organelle and RNA-rich Balbiani body, which plays a critical role in oocyte polarity. Notably, at specific stages of germline development, these transient structures in oocytes are temporally coincident and align with asymmetries in the position and arrangement of nuclear components, such as the nuclear pore and the chromosomal bouquet and the centrioles and cytoskeleton in the cytoplasm. Formation of these critical, transient structures and arrangements involves microtubule pathways, intrinsically disordered proteins (proteins with domains that tend to be fluid or lack a rigid ordered three-dimensional structure ranging from random coils, globular domains, to completely unstructured proteins), and translational repressors and activators. This review aims to examine recent literature and key players in oocyte polarity.

Phosphorylation of Astrin Regulates Its Kinetochore Function

The error-free segregation of chromosomes, which requires the precisely timed search and capture of chromosomes by spindles during early mitotic and meiotic cell division, is responsible for genomic stability and is achieved by the spindle assembly checkpoint in the metaphase-anaphase transition. Mitotic kinases orchestrate M phase events, such as the reorganization of cell architecture and kinetochore (KT) composition with the exquisite phosphorylation of mitotic regulators, to ensure timely and temporal progression. However, the molecular mechanisms underlying the changes of KT composition for stable spindle attachment during mitosis are poorly understood. Here, we show that the sequential action of the kinase Cdk1 and the phosphatase Cdc14A control spindle attachment to KTs. During prophase, the mitotic spindle protein Spag5/Astrin is transported into centrosomes by Kinastrin and phosphorylated at Ser-135 and Ser-249 by Cdk1, which, in prometaphase, is loaded onto the spindle and targeted to KTs. We also demonstrate that Cdc14A dephosphorylates Astrin, and therefore the overexpression of Cdc14A sequesters Astrin in the centrosome and results in aberrant chromosome alignment. Mechanistically, Plk1 acts as an upstream kinase for Astrin phosphorylation by Cdk1 and targeting phospho-Astrin to KTs, leading to the recruitment of outer KT components, such as Cenp-E, and the stable attachment of spindles to KTs. These comprehensive findings reveal a regulatory circuit for protein targeting to KTs that controls the KT composition change of stable spindle attachment and chromosome integrity.

Human embryos commonly form abnormal nuclei during development: a mechanism of DNA damage, embryonic aneuploidy, and developmental arrest.

What is the prevalence and developmental significance of morphologic nuclear abnormalities in human preimplantation embryos? Nuclear abnormalities are commonly found in human IVF embryos and are associated with DNA damage, aneuploidy, and decreased developmental potential. Early human embryonic development is complicated by genomic errors that occur after fertilization. The appearance of extra-nuclear DNA, which has been observed in IVF, may be a result of such errors. However, the mechanism by which abnormal nuclei form and the impact on DNA integrity and embryonic development is not understood. Cryopreserved human cleavage-stage embryos (n = 150) and cryopreserved blastocysts (n = 105) from clinical IVF cycles performed between 1997 and 2008 were donated for research. Fresh embryos (n = 60) of poor quality that were slated for discard were also used. Immunohistochemical, microscopic and cytogenetic analyses at different developmental stages and morphologic grades were performed. Embryos were fixed and stained for DNA, centromeres, mitotic activity and DNA damage and imaged using confocal microscopy. Rates of abnormal nuclear formation were compared between morphologically normal cleavage-stage embryos, morphologically normal blastocysts, and poor quality embryos. To control for clinical and IVF history of oocytes donors, and quality of frozen embryos within our sample, cleavage-stage embryos (n = 52) were thawed and fixed at different stages of development and then analyzed microscopically. Cleavage-stage embryos (n = 9) were thawed and all blastomeres (n = 62) were disaggregated, imaged and analyzed for karyotype. Correlations were made between microscopic and cytogenetic findings of individual blastomeres and whole embryos. The frequency of microscopic nuclear abnormalities was lower in blastocysts (5%; 177/3737 cells) than in cleavage-stage embryos (16%, 103/640 blastomeres, P < 0.05) and highest in arrested embryos (65%; 44/68 blastomeres, P < 0.05). DNA damage was significantly higher in cells with microscopic nuclear abnormalities (γH2AX (phosphorylated (Ser139) histone H2A.X): 87.1%, 74/85; replication protein A: 72.9%, 62/85) relative to cells with normal nuclear morphology (γH2AX: 9.3%, 60/642; RPA: 5.6%, 36/642) (P < 0.05). Blastomeres containing nuclear abnormalities were strongly associated with aneuploidy (Fisher exact test, two-tailed, P < 0.01). The embryos used were de-identified, and the clinical and IVF history was unknown. This study explores a mechanism of abnormal embryonic development post-fertilization. While most of the current data have explored abnormal meiotic chromosome segregation in oocytes as a primary mechanism of reproductive failure, abnormal nuclear formation during early mitotic cell division in IVF embryos also plays a significant role. The detection of abnormal nuclear formation may have clinical application in noninvasive embryo selection during IVF. The study was supported by Columbia University and the New York Stem Cell Foundation. Authors declare no competing interest.

A monoclonal antibody specific for prophase phosphorylation of histone deacetylase 1: a readout for early mitotic cells

Histone deacetylases (HDACs) are modification enzymes that regulate a plethora of biological processes. HDAC1, a crucial epigenetic modifier, is deregulated in cancer and subjected to a variety of post-translational modifications. Here, we describe the generation of a new monoclonal antibody that specifically recognizes a novel highly dynamic prophase phosphorylation of serine 406-HDAC1, providing a powerful tool for detecting early mitotic cells.

MUS81 promotes common fragile site expression.

Fragile sites are chromosomal loci with a propensity to form gaps or breaks during early mitosis, and their instability is implicated as being causative in certain neurological disorders and cancers. Recent work has demonstrated that the so-called common fragile sites (CFSs) often impair the faithful disjunction of sister chromatids in mitosis. However, the mechanisms by which CFSs express their fragility, and the cellular factors required to suppress CFS instability, remain largely undefined. Here, we report that the DNA structure-specific nuclease MUS81-EME1 localizes to CFS loci in early mitotic cells, and promotes the cytological appearance of characteristic gaps or breaks observed at CFSs in metaphase chromosomes. These data indicate that CFS breakage is an active, MUS81-EME1-dependent process, and not a result of inadvertent chromatid rupturing during chromosome condensation. Moreover, CFS cleavage by MUS81-EME1 promotes faithful sister chromatid disjunction. Our findings challenge the prevailing view that CFS breakage is a nonspecific process that is detrimental to cells, and indicate that CFS cleavage actually promotes genome stability.

Temporal Germ Cell Development Strategy during Mixed Spermatogenesis within the Male Mediterranean Gecko, Hemidactylus turcicus (Reptilia: Gekkonidae)

Abstract The testes of Hemidactylus turcicus are composed of seminiferous tubules lined with continuous germinal epithelia in which multiple germ cell morphologies can be found during the active months of sperm development. Spermatogenesis is quiescent during September, with only spermatogonia A and spermatogonia B present in the seminiferous epithelia and minimal mitotic activity is observed. Recrudescence begins in October and the early stages of spermatogenesis progress through November. The onset of spermiation is observed in December and continues through August with the heaviest sperm release occurring in June and July. Multiple generations of late elongated spermatids are found in association with early mitotic and meiotic cells during the months of December–August. This temporal germ cell development strategy is similar to that described in other squamates and anamniotes and is different from the spatial development exhibited by birds and mammals, in which germ cell populations collectively progre...

The histone deacetylase inhibitor LBH589 inhibits expression of mitotic genes causing G2/M arrest and cell death in head and neck squamous cell carcinoma cell lines

Head and neck squamous cell carcinoma represents a complex set of neoplasms arising in diverse anatomical locations. The site and stage of the cancer determine whether patients will be treated with single or multi-modality therapy. The HDAC inhibitor LBH589 is effective in treating some haematological neoplasms and shows promise for certain epithelial neoplasms. As with other human cancer cell lines, LBH589 causes up-regulation of p21, G2/M cell cycle arrest, and cell death of human HNSCC cell lines, as measured using flow cytometry and cDNA microarrays. Global RNA expression studies following treatment of the HNSCC cell line FaDu with LBH589 reveal down-regulation of genes required for chromosome congression and segregation (SMC2L1), sister chromatid cohesion (DDX11), and kinetochore structure (CENP-A, CENP-F, and CENP-M); these LBH589-induced changes in gene expression coupled with the down-regulation of MYC and BIRC5 (survivin) provide a plausible explanation for the early mitotic arrest and cell death observed. When LBH589-induced changes in gene expression were compared with gene expression profiles of 41 primary HNSCC samples, many of the genes that were down-regulated by LBH589 showed increased expression in primary HNSCC, suggesting that some patients with HNSCC may respond to treatment with LBH589.

Ectopic expression of plasma membrane targeted subunits of the Ndc80-complex as a tool to study kinetochore biochemistry

Genomic stability depends on the normal function of the kinetochore, a multi-protein assemblage, which consists of over 80 molecules including both constitutive and transiently binding components. Information regarding the spatial–temporal assembly of kinetochore subcomplexes is often limited by technical difficulties in their isolation. To study kinetochore subcomplex formation, we targeted separately Hec1 and Spc24, two subunits of the Ndc80 kinetochore compilation, to the plasma membrane by fusing them with the amino-terminal palmitoylation and myristoylation (pm) sequence of the receptor tyrosine kinase Fyn. We found that in early mitotic cells, pm-GFP–Hec1 and pm-GFP–Spc24 fusion proteins localised to the plasma membrane and were able to recruit all subunits of the Ndc80 complex (Ndc80/Hec1, Nuf2, Spc24 and Spc25) to these foci. In interphase cells, only Hec1–Nuf2 and Spc24–Spc25 heterodimers accumulated to the plasma membrane foci. The results propose that the assembly of Ndc80 tetramer can take place outside of the kinetochore but requires co-factors that are only present in mitotic cells. These findings provide the first experimental evidence on the successful employment of the plasma membrane targeting technique in the study of kinetochore biochemistry.

Perturbation of Incenp function impedes anaphase chromatid movements and chromosomal passenger protein flux at centromeres

Incenp is an essential mitotic protein that, together with Aurora B, Survivin, and Borealin, forms the core of the chromosomal passenger protein complex (CPC). The CPC regulates various mitotic processes and functions to maintain genomic stability. The proper subcellular localization of the CPC and its full catalytic activity require the presence of each core subunit in the complex. We have investigated the mitotic tasks of the CPC using a function blocking antibody against Incenp microinjected into cells at different mitotic phases. This method allowed temporal analysis of CPC functions without perturbation of complex assembly or activity prior to injection. We have also studied the dynamic properties of Incenp and Aurora B using fusion protein photobleaching. We found that in early mitotic cells, Incenp and Aurora B exhibit dynamic turnover at centromeres, which is prevented by the anti-Incenp antibody. In these cells, the loss of centromeric CPC turnover is accompanied by forced mitotic exit without the execution of cytokinesis. Introduction of anti-Incenp antibody into early anaphase cells causes abnormalities in sister chromatid separation through defects in anaphase spindle functions. In summary, our data uncovers new mitotic roles for the CPC in anaphase and proposes that CPC turnover at centromeres modulates spindle assembly checkpoint signaling.

Depletion of the cap-associated isoform of translation factor eIF4G induces germline apoptosis in C. elegans

During mammalian programmed cell death, cleavage of the translation initiation factor 4G proteins (eIF4GI and eIF4GII) by caspase-3 induces the cap-independent synthesis of pro-apoptotic proteins. Apoptosis occurs naturally in the gonad to remove germ cells that are not selected to grow as oocytes and mature into eggs. Here, we describe two major isoforms of Caenorhabditis elegans eIF4G that are derived from a single gene (ifg-1) and their separate roles in germline homeostasis. Full length IFG-1 protein (170 kDa isoform) differs from the shorter isoform (130 kDa) by the inclusion of the N-terminal domain containing the putative eIF4E-binding site required for mRNA cap recognition. Depletion of the cap-associated p170 isoform induced CED-4 expression in oocytes and markedly increased germline apoptotic events, but did not prevent early mitotic germ cell proliferation. Loss of both p170 and p130 suppressed germ cell proliferation and arrested larval development. Evidence suggests that eIF4G isoforms are differentially utilized during oogenesis to regulate germ cell apoptosis. We propose that an alternative mechanism to eIF4G cleavage may be employed in germ cells by changing the availability of the p170 isoform.

APC/CCdc20 Controls the Ubiquitin-Mediated Degradation of p21 in Prometaphase

During the G1/S transition, p21 proteolysis is mediated by Skp2; however, p21 reaccumulates in G2 and is degraded again in prometaphase. How p21 degradation is controlled in mitosis remains unexplored. We found that Cdc20 (an activator of the ubiquitin ligase APC/C) binds p21 in cultured cells and identified a D box motif in p21 necessary for APC/C(Cdc20)-mediated ubiquitylation of p21. Overexpression of Cdc20 or Skp2 destabilized wild-type p21; however, only Skp2, but not Cdc20, was able to destabilize a p21(D box) mutant. Silencing of Cdc20 induced an accumulation of p21, increased the fraction of p21 bound to Cdk1, and inhibited Cdk1 activity in p21(+/+) prometaphase cells, but not in p21(-/-) cells. Thus, in prometaphase Cdc20 positively regulates Cdk1 by mediating the degradation of p21. We propose that the APC/C(Cdc20)-mediated degradation of p21 contributes to the full activation of Cdk1 necessary for mitotic events and prevents mitotic slippage during spindle checkpoint activation.

Shugoshin 2 Regulates Localization of the Chromosomal Passenger Proteins in Fission Yeast Mitosis

Fission yeast has two members of the Shugoshin family, Sgo1 and Sgo2. Although Sgo1 has clearly been established as a protector of centromere cohesion in meiosis I, the roles of Sgo2 remain elusive. Here we show that Sgo2 is required to ensure proper chromosome biorientation upon recovery from a prolonged spindle checkpoint arrest. Consistent with this, Sgo2 is essential for maintaining the Passenger proteins on centromeres upon checkpoint activation. Interestingly, lack of Sgo2 has a more penetrant effect on the localization of Survivin than on the two other Passenger proteins INCENP and Aurora B, and the Survivin-INCENP complex but not the INCENP-Aurora B complex is destabilized in the absence of Sgo2. Finally we show that the conserved C-terminus of Sgo2 is crucial to maintain Sgo2 and Passenger proteins localization on centromeres upon prolonged checkpoint activation. Taken together, our results demonstrate that Sgo2 is important for chromosome biorientation and that it controls docking of the Passenger proteins on chromosomes in early mitotic cells.

Endocytosis Resumes during Late Mitosis and Is Required for Cytokinesis

Recent work has underscored the importance of membrane trafficking events during cytokinesis. For example, targeted membrane secretion occurs at the cleavage furrow in animal cells, and proteins that regulate endocytosis also influence the process of cytokinesis. Nonetheless, the prevailing dogma is that endosomal membrane trafficking ceases during mitosis and resumes after cell division is complete. In this study, we have characterized endocytic membrane trafficking events that occur during mammalian cell cytokinesis. We have found that, although endocytosis ceases during the early stages of mitosis, it resumes during late mitosis in a temporally and spatially regulated pattern as cells progress from anaphase to cytokinesis. Using fixed and live cell imaging, we have found that, during cleavage furrow ingression, vesicles are internalized from the polar region and subsequently trafficked to the midbody area during later stages of cytokinesis. In addition, we have demonstrated that cytokinesis is inhibited when clathrin-mediated endocytosis is blocked using a series of dominant negative mutants. In contrast to previous thought, we conclude that endocytosis resumes during the later stages of mitosis, before cytokinesis is completed. Furthermore, based on our findings, we propose that the proper regulation of endosomal membrane traffic is necessary for the successful completion of cytokinesis.

Roles of Pdk1p, a Fission Yeast Protein Related to Phosphoinositide-dependent Protein Kinase, in the Regulation of Mitosis and Cytokinesis

Proteins related to the phosphoinositide-dependent protein kinase family have been identified in the majority of eukaryotes. Although much is known about upstream mechanisms that regulate the PDK1-family of kinases in metazoans, how these kinases regulate cell growth and division remains unclear. Here, we characterize a fission yeast protein related to members of this family, which we have termed Pdk1p. Pdk1p localizes to the spindle pole body and the actomyosin ring in early mitotic cells. Cells deleted for pdk1 display multiple defects in mitosis and cytokinesis, all of which are exacerbated when the function of fission yeast polo kinase, Plo1p, is partially compromised. We conclude that Pdk1p functions in concert with Plo1p to regulate multiple processes such as the establishment of a bipolar mitotic spindle, transition to anaphase, placement of the actomyosin ring and proper execution of cytokinesis. We also present evidence that the effects of Pdk1p on cytokinesis are likely mediated via the fission yeast anillin-related protein, Mid1p, and the septation initiation network.