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* Context.--Mitotic count is an important histologic criterion for grading and prognostication in phyllodes tumors (PTs). Counting mitoses is a routine practice for pathologists evaluating neoplasms, but different microscopes, variable field selection, and areas have led to possible misclassification. Objective.--To determine whether 10 high-power fields (HPFs) or whole slide mitotic counts correlated better with PT clinicopathologic parameters using digital pathology (DP). We also aimed to find out whether this study might serve as a basis for an artificial intelligence (Al) protocol to count mitosis. Design.--Representative slides were chosen from 93 cases of PTs diagnosed between 2014 and 2015. The slides were scanned and viewed with DP. Mitotic counting was conducted on the whole slide image, before choosing 10 HPFs and demarcating the tumor area in DP. Values of mitoses per millimeter squared were used to compare results between 10 HPFs and the whole slide. Correlations with clinicopathologic parameters were conducted. Results.--Both whole slide counting of mitoses and 10 HPFs had similar statistically significant correlation coefficients with grade, stromal atypia, and stromal hypercellularity. Neither whole slide mitotic counts nor mitoses per 10 HPFs showed statistically significant correlations with patient age and tumor size. Conclusions.--Accurate mitosis counting in breast PTs is important for grading. Exploring machine learning on digital whole slides may influence approaches to training, testing, and validation of a future Al algorithm. doi: 10.5858/arpa.2019-0435-OA, Phyllodes tumors (PTs) of the breast are biphasic neoplasms composed of proliferation of both stromal and epithelial components. According to the latest 5th edition of the World Health Organization Classification [...]

Little is known about how cells regulate and integrate distinct biosynthetic pathways governing differentiation and cell division. For B lineage cells it is widely accepted that activated cells must complete several rounds of mitosis before yielding antibody-secreting plasma cells. However, we report that marginal zone (MZ) B cells, innate-like naive B cells known to generate plasma cells rapidly in response to blood-borne bacteria, generate functional plasma cells despite cell-cycle arrest. Further, short-term Notch2 blockade in vivo reversed division-independent differentiation potential and decreased transcript abundance for numerous mTORC1- and Myc-regulated genes. Myc loss compromised plasma cell differentiation for MZ B cells, and reciprocally induced ectopic mTORC1 signaling in follicular B cells enabled division-independent differentiation and plasma cell-affiliated gene expression. We conclude that ongoing in situ Notch2/mTORC1 signaling in MZ B cells establishes a unique cellular state that enables rapid division-independent plasma cell differentiation., Introduction Differentiation is often intricately tied to the cell cycle. For instance, precursors for red blood cells must proliferate to yield functional progeny (1), and embryonic stem cells are more [...]

Abstract: Mahogunin RING Finger 1 (MGRN1) is a ubiquitin E3 ligase known to affect spindle tilt in mitotic cells by regulating α-tubulin ubiquitination and polymerization. In cell culture systems we [...]

Asymmetric cell division, the partitioning of cellular components in response to polarizing cues during mitosis, has roles in differentiation and development (1). It is important for the self-renewal of fertilized [...]

Accurate chromosome segregation requires timely dissolution of chromosome cohesion after chromosomes are properly attached to the mitotic spindle. Separase is absolutely essential for cohesion dissolution in organisms from yeast to [...]

At the onset of metazoan cell division the nuclear envelope breaks down to enable capture of chromosomes by the microtubule-containing spindle apparatus (1). During anaphase, when chromosomes have separated, the nuclear envelope is reassembled around the forming daughter nuclei (1,2). How the nuclear envelope is sealed, and how this is coordinated with spindle disassembly, is largely unknown. Here we show that endosomal sorting complex required for transport (ESCRT)-III, previously found to promote membrane constriction and sealing during receptor sorting, virus budding, cytokinesis and plasma membrane repair (3-6), is transiently recruited to the reassembling nuclear envelope during late anaphase. ESCRT-III and its regulatory AAA (ATPase associated with diverse cellular activities) ATPase VPS4 are specifically recruited by the ESCRT-III-like protein CHMP7 to sites where the reforming nuclear envelope engulfs spindle microtubules. Subsequent association of another ESCRT-III-like protein, IST1, directly recruits the AAA ATPase spastin to sever microtubules. Disrupting spastin function impairs spindle disassembly and results in extended localization of ESCRT-III at the nuclear envelope. Interference with ESCRT-III functions in anaphase is accompanied by delayed microtubule disassembly, compromised nuclear integrity and the appearance of DNA damage foci in subsequent interphase. We propose that ESCRT-III, VPS4 and spastin cooperate to coordinate nuclear envelope sealing and spindle disassembly at nuclear envelope-microtubule intersection sites during mitotic exit to ensure nuclear integrity and genome safeguarding, with a striking mechanistic parallel to cytokinetic abscission (7)., Previous studies have shown that ESCRT-III is recruited to the membrane bridge connecting daughter cells during cytokinesis (5,8). Interestingly, cell lines stained with an antibody against the ESCRT-III subunit chromatin [...]

Octoploid triticale were derived from common wheat (Triticum aestivum L. 'Mianyang11') x rye (Secale cereale L. 'Kustro'), and some progeny were obtained by the backcrossing of triticale with 'Mianyang11' followed by self-fertilization. In situ hybridization using rye genomic DNA and repetitive sequences pAs1 and pSc119.2 as probes was used to analyze the mitotic chromosomes of these progeny. Three wheat-rye 1R monosomic addition lines and a wheat line (12FT-1685) containing a 1R and a 1BL.1RS translocation chromosome were identified. Abnormal mitosis was observed in the two lines. During mitosis of a 1R monosomic addition line (3-8-20-1R-2), lagging chromosomes, micronuclei, chromosomal bridges, and the one pole segregation of 1R chromosome were observed. Abnormal mitotic behaviour of chromosomes was also observed in some of the self-progeny plants of lines 12FT-1685 and 3-8-20-1R-2. These progeny contained 1R chromosome or 1R chromosome arm. In addition, 4B chromosomes were absent from one of the progeny of 3-8-20-1R-2. This abnormal mitotic behaviour of chromosomes was not observed in two other 1R monosomic addition lines. These results indicate that a single 1R chromosome added to wheat might cause abnormal mitotic behaviour of both wheat and rye chromosomes and different genetic variations might occurr among the sibling 1R monosomic addition lines. Key words: abnormal mitotic behaviour, monosomic addition line, wheat, rye. Des triticales octoploides ont ete derives du croisement entre le ble tendre (Triticum aestivum L. 'Mianyang11') et le seigle (Secale cereale L. 'Krusto'), tandis que d'autres ont ete obtenus au terme d'un retrocroisement des triticales avec 'Mianyang11' et d'une autofecondation. Des hybridations in situ a l'aide de sondes composees de l'ADN genomique du seigle ou des sequences repetees pAs1 et pSc119.2 ont ete realisees pour analyser le comportement des chromosomes mitotiques chez ces plantes. Les auteurs ont identifie trois lignees d'addition monosomique ble-seigle 1R et une lignee de ble (12FT-1685) contenant le 1R et une translocation 1BL.1RS. Des mitoses anormales ont ete observees chez les deux lignees. Au cours de la mitose chez une lignee d'addition monosomique 1R (3-8-20-1R-2), des chromosomes retardataires, des micronoyaux, des ponts chromosomiques et une segregation unipolaire du chromosome 1R ont ete observes. Un comportement mitotique anormal des chromosomes a egalement ete observe chez certains descendants de l'autofecondation des lignees 12FT-1685 et 3-8-20-1R-2. Ces progenitures contenaient le chromosome 1R ou un bras 1R. De plus, les chromosomes 4B etaient absents chez l'un des descendants de 3-8-20-1R-2. Ce comportement mitotique anormal des chromosomes n'a pas ete observe chez deux autres lignees d'addition monosomique 1R. Ces resultats indiquent que l'addition d'un seul chromosome 1R au genome du ble peut causer un comportement mitotique anormal tant des chromosomes du ble que du seigle et que differents changements genetiques peuvent survenir parmi des lignees d'addition monosomique 1R sceurs. [Traduit par la Redaction] Mots-cles: comportement mitotique anormal, lignee d'addition monosomique, ble, seigle., Introduction Wide hybridization is one of the stresses that might trigger reorganization of parental genomes (McClintock 1978) and has been widely used in cereal breeding programs (Feuillet et al. 2008; [...]

Polo-like kinase 1 (Plk1) is a conserved key regulator of the [G.sub.2]/M transition, but its upstream spatiotemporal regulators remain unknown. With the help of immunofluorescence, co-immunoprecipitation, and glutathione S-transferase (GST) pulldown assay, we found that calmodulin (CaM) is one such regulatory molecule that associates with Plk1 from [G.sub.2] to metaphase. More importantly, this interaction results in considerable stimulation of Plk1 kinase activity leading to hyperphosphorylation of Cdc25C. Our results provide new insight into the role of CaM as an upstream regulator of Plk1 activation during mitotic entry. Key words: colocalization, protein interaction, Plk1 activation, calmodulin, [G.sub.2]/M transition. La kinase Plk1 (Polo-like kinase 1) est un regulateur conserve qui joue un role cle de la transition [G.sub.2]/M, mais la recherche de ses regulateurs spatiotemporels demeure complexe. A l'aide de l'immunofluorescence, de la co-immunoprecipitation et du test de precipitation d'affinite sur GST, nous avons trouve que la calmoduline (CaM) est une de ces proteines regulatrices qui s'associe a Plk1 de [G.sub.2] a la metaphase. Qui plus est, l'interaction resulte en une stimulation considerable de l'activite kinase de Plk1 ce qui mene a l'hyper-phosphorylation de Cdc25C. Nos resultats donnent un nouvel aper^u du role de la CaM en tant que regulateur agissant en amont de l'activation de Plk1 lors de l'entree en mitose. [Traduit par la Redaction] Mots-cles: co-localisation, interaction proteique, activation de Plk1, calmoduline, transition [G.sub.2]/M., Introduction Polo-like kinase 1 (Plk1) is a key regulator of various mitotic events from the beginning to the end of mitosis (Archambault and Glover 2009; Barr et al. 2004; Petronczki [...]

Despite the large size of the Xenopus laevis egg (approximately 1.2 mm diameter), a fertilized egg rapidly proceeds through mitosis in a spatially coordinated fashion. Mitosis is initiated by a [...]

Abstract As part of the life sciences series, this article describes the role of deoxyribonucleic acid and ribonucleic acid, genes and chromosomes. The processes of mitosis and meiosis are discussed [...]

The RNA-binding protein [p54.sup.nrb] is involved in many nuclear processes including transcription, RNA processing, and retention of hyperedited RNAs. In interphase cells, [p54.sup.nrb] localizes to the nucleoplasm and concentrates with protein partners in the paraspeckles via an interaction with the non-coding RNA Neat1. During mitosis, [p54.sup.nrb] becomes multiphosphorylated and the effects of this modification are not known. In the present study, we show that [p54.sup.nrb] phosphorylation does not affect the interactions with its protein partners but rather diminishes its general RNA-binding ability. Biochemical assays indicate that in vitro phosphorylation of a GST-[p54.sup.nrb] construct by CDK1 abolishes the interaction with 5' splice site RNA sequence. Site-directed mutagenesis shows that the threonine 15 residue, located N-terminal to the RRM tandem domains of [p54.sup.nrb], is involved in this inhibition. In vivo analysis reveals that Neat1 ncRNA co-immunoprecipitates with [p54.sup.nrb] in either interphase or mitotic cells, suggesting that [p54.sup.nrb]-Neat1 interaction is not modulated by phosphorylation. Accordingly, in vitro phosphorylated GST-[p54.sup.nrb] still interacts with PIR-1 RNA, a G-rich Neat1 sequence known to interact with [p54.sup.nrb]. In vitro RNA binding assays show that CDK1-phosphorylation of a GST-[p54.sup.nrb] construct abolishes its interaction with homoribopolymers poly(A), poly(C), and poly(U) but not with poly(G). These data suggest that [p54.sup.nrb] interaction with RNA could be selectively modulated by phosphorylation during mitosis. Key words: [p54.sup.nrb], NonO, RNA-binding proteins, mitosis, paraspeckles, Neat1. La proteine de liaison a l'ARN [p54.sup.nrb] est impliquee dans divers processus nucleaires comme la transcription, la maturation des ARNs et la retention des ARNs hyperedites. En interphase, [p54.sup.nrb] est presente dans le nucleoplasme et est aussi concentree avec ses partenaires proteiques dans les paraspeckles via une interaction avec l'ARN non codant Neat1. En mitose, [p54.sup.nrb] devient multiphosphorylee et les effets de cette modification sont inconnus. Dans la presente etude, nous montrons que la phosphorylation de [p54.sup.nrb] n'affecte pas les interactions avec ses partenaires proteiques mais diminue sa capacite de lier l'ARN. Des tests biochimiques indiquent que la phosphorylation in vitro d' une construction GST-[p54.sup.nrb] par CDK1 abolit son interaction avec des sequences d'ARN du site 5' d'epissage. Par mutagenese dirigee, il est montre que cette inhibition depend de la phosphorylation de la threonine 15 situee dans la partie N-terminale en amont des deux domaines RRM de liaison a l'ARN. Des analyses in vivo revelent que l'ARN non codant Neat1 est co-immunoprecipite avec [p54.sup.nrb] aussi bien dans les conditions interphasiques que mitotiques, suggerant que l'interaction entre [p54.sup.nrb] et Neat1 n'est pas affectee par la phosphorylation mitotique. De fait, GST-[p54.sup.nrb] phosphorylee in vitro se lie a l'ARN PIR-1, une sequence riche en G de Neat1 connue pour interagir avec [p54.sup.nrb]. Des analyses de liaison a l'ARN in vitro montre que la phosphorylation de GST-[p54.sup.nrb] par CDK1 affecte son interaction avec des homoribopolymeres poly(A), poly(C) et poly(U) mais pas les poly (G). Ces resultats suggerent que l'interaction de [p54.sup.nrb] avec l'ARN peut etre modulee selectivement par phosphorylation durant la mitose., Introduction The protein [p54.sup.nrb] (NonO in mouse) is a highly conserved nuclear matrix-associated factor involved in numerous nuclear processes (Shav-Tal and Zipori 2002). This multifunctional protein, characterized by the presence [...]

Many protein kinases are activated by a conserved regulatory step involving T-loop phosphorylation. Although there is considerable focus on kinase activator proteins, the importance of specific T-loop phosphatases reversing kinase activation has been underappreciated. We find that the protein phosphatase 6 (PP6) holoenzyme is the major T-loop phosphatase for Aurora A, an essential mitotic kinase. Loss of PP6 function by depletion of catalytic or regulatory subunits interferes with spindle formation and chromosome alignment because of increased Aurora A activity. Aurora A T-loop phosphorylation and the stability of the Aurora A--TPX2 complex are increased in cells depleted of PP6 but not other phosphatases. Furthermore, purified PP6 acts as a T-loop phosphatase for Aurora A--TPX2 complexes in vitro, whereas catalytically inactive mutants cannot dephosphorylate Aurora A or rescue the PPP6C depletion phenotype. These results demonstrate a hitherto unappreciated role for PP6 as the T-loop phosphatase regulating Aurora A activity during spindle formation and suggest the general importance of this form of regulation. doi/ 10.1083/jcb.201008106

Initiation and maintenance of mitosis require the activation of protein kinase cyclin B--Cdc2 and the inhibition of protein phosphatase 2A (PP2A), which, respectively, phosphorylate and dephosphorylate mitotic substrates. The protein kinase Greatwall (Gwl) is required to maintain mitosis through PP2A inhibition. We describe how Gwl activation results in PP2A inhibition. We identified cyclic adenosine monophosphate--regulated phosphoprotein 19 (Arpp19) and [alpha]-Endosulfine as two substrates of Gwl that, when phosphorylated by this kinase, associate with and inhibit PP2A, thus promoting mitotic entry. Conversely, in the absence of Gwl activity, Arpp19 and [alpha]-Endosulfine are dephosphorylated and lose their capacity to bind and inhibit PP2A. Although both proteins can inhibit PP2A, endogenous Arpp19, but not [alpha]-Endosulfine, is responsible for PP2A inhibition at mitotic entry in Xenopus egg extracts. 10.1126/science.1197048

Asymmetric segregation of P granules during the first four divisions of the Coenorhobditis elegans embryo is a classic example of cytoplasmic partitioning of germline determinants. It is thought that asymmetric partitioning of P granule components during mitosis is essential to distinguish germline from soma. We have identified a mutant (pptr-1) in which P granules become unstable during mitosis and P granule proteins and RNAs are distributed equally to somatic and germline blastomeres. Despite symmetric partitioning of P granule components, pptr-1 mutants segregate a germline that uniquely expresses P granules during postembryonic development. pptr-1 mutants are fertile, except at high temperatures. Hence, asymmetric partitioning of maternal P granules is not essential to specify germ cell fate. Instead, it may serve to protect the nascent germline from stress. 10.1126/science.1193697

Entry into mitosis in eukaryotes requires the activity of cyclin-dependent kinase 1 (Cdk1). Cdk1 is opposed by protein phosphatases in two ways: They inhibit activation of Cdk1 by dephosphorylating the protein kinases Wee1 and Myt1 and the protein phosphatase Cdc25 (key regulators of Cdk1), and they also antagonize Cdk1's own phosphorylation of downstream targets. A particular form of protein phosphatase 2A (PP2A) containing a B558 subunit (PP2A-B55[delta]) is the major protein phosphatase that acts on model CDK substrates in Xenopus egg extracts and has antimitotic activity. The activity of PPPA-B55[delta] is high in interphase and low in mitosis, exactly opposite that of Cdk1. We report that inhibition of PP2A-B55[delta] results from a small protein, known as [alpha]-endosulfine (Ensa), that is phosphorylated in mitosis by the protein kinase Greatwall (Gwl). This converts Ensa into a potent and specific inhibitor of PP2A-B55[delta]. This pathway represents a previousLy unknown element in the control of mitosis. 10.1126/science.1195689

We identified the mitotic kinesin-like protein 2 (MKlp2), a kinesin required for chromosome passenger complex (CPC)-mediated cytokinesis, as a target of the mitotic checkpoint protein Mad2. MKlp2 possesses a consensus Mad2-binding motif required for Mad2 binding. Mad2 prevents MKlp2 from loading onto the mitotic spindle, a prerequisite step for its function as a mitotic kinesin. Furthermore, Mad2 inhibits the ability of MKlp2 to relocate the CPC from centromeres, an essential step to promote cytokinesis. An MKlp2 mutant that is refractory to Mad2-mediated inhibition prematurely translocates to the mitotic spindle and mislocalizes the CPC component Aurora B from the midbody of dividing cells. This correlates with an increased incidence of cytokinesis failure. Together, these findings reveal that MKlp2 is a novel mitotic target of Mad2 necessary for proper mitotic progression and cytokinesis. doi/ 10.1083/jcb.201003095

Lte1 is known as a regulator of mitotic progression in budding yeast. Here we demonstrate phosphorylation-dependent inhibition of polarized bud growth during G2/M by Lte1. Cla4 activity first localizes Lte1 to the polarity cap and thus specifically to the bud. This localization is a prerequisite for subsequent Clb--Cdk-dependent phosphorylation of Lte1 and its relocalization to the entire bud cortex. There, Lte1 interferes with activation of the small GTPases, Ras and Bud1. The inhibition of Bud1 prevents untimely polarization until mitosis is completed and Cdc14 phosphatase is released. Inhibition of Bud1 and Ras depends on Lte1's GEF-like domain, which unexpectedly inhibits these small G proteins. Thus, Lte1 has dual functions for regulation of mitotic progression: it both induces mitotic exit and prevents polarized growth during mitotic arrest, thereby coupling cell cycle progression and morphological development. doi/ 10.1083/jcb.201005070

Upon prolonged activation of the spindle assembly checkpoint, cells escape from mitosis through a mechanism called adaptation or mitotic slippage, which is thought to underlie the resistance of cancer cells to antimitotic drugs. We show that, in budding yeast, this mechanism depends on known essential and nonessential regulators of mitotic exit, such as the Cdc14 early anaphase release (FEAR) pathway for the release of the Cdc14 phosphatase from the nucleolus in early anaphase. Moreover, the RSC (remodel the structure of chromatin) chromatin-remodeling complex bound to its accessory subunit Rsc2 is involved in this process as a novel component of the FEAR pathway. We show that Rsc2 interacts physically with the polo kinase Cdc5 and is required for timely phosphorylation of the Cdc14 inhibitor Net1, which is important to free Cdc14 in the active form. Our data suggest that fine-tuning regulators of mitotic exit have important functions during mitotic progression in cells treated with microtubule poisons and might be promising targets for cancer treatment. doi/ 10.1083/jcb.201007025

Correct assembly of nuclear pore complexes (NPCs), which directly and indirectly control nuclear environment and architecture, is vital to genomic regulation. We previously found that nucleoporin 153 (Nup153) is required for timely progression through late mitosis. In this study, we report that disruption of Nup153 function by either small interfering RNA--mediated depletion or expression of a dominant-interfering Nup153 fragment results in dramatic mistargeting of the pore basket components Tpr and Nup50 in midbody-stage cells. We find a concomitant appearance of aberrantly localized active Aurora B and an Aurora B--dependent delay in abscission. Depletion of Nup50 is also sufficient to increase the number of midbody-stage cells and, likewise, triggers distinctive mislocalization of Aurora B. Together, our results suggest that defects in nuclear pore assembly, and specifically the basket structure, at this time of the cell cycle activate an Aurora B--mediated abscission checkpoint, thereby ensuring that daughter cells are generated only when fully formed NPCs are present. doi/ 10.1083/jcb.201007124

progenitor cells must balance self-amplification and production of differentiated progeny during development and homeostasis. In the epidermis, progenitors divide symmetrically to increase surface area and asymmetrically to promote stratification. In this study, we show that individual epidermal cells can undergo both types of division, and therefore, the balance is provided by the sum of individual cells' choices. In addition, we define two control points for determining a cell's mode of division. First is the expression of the mouse Inscuteable gene, which is sufficient to drive asymmetric cell division (ACD). However, there is robust control of division orientation as excessive ACDs are prevented by a change in the localization of NuMA, an effector of spindle orientation. Finally, we show that p63, a transcriptional regulator of stratification, does not control either of these processes. These data have uncovered two important regulatory points controlling ACD in the epidermis and allow a framework for analysis of how external cues control this important choice. doi/ 10.1083/jcb.201008001

The mitotic spindle is a complex and dynamic structure. Although much has been learned about how spindles assemble and mediate chromosome segregation, how spindles rapidly and irreversibly disassemble during telophase is less clear. We used synthetic lethal screens in budding yeast to identify mutants defective in spindle disassembly. Real-time, live cell imaging analysis of spindle disassembly was performed on nine mutants defective in this process. Results of this analysis suggest that spindle disassembly is achieved by mechanistically distinct but functionally overlapping subprocesses: disengagement of the spindle halves, arrest of spindle elongation, and initiation of interpolar microtubule depolymerization. These subprocesses are largely governed by the anaphase-promoting complex, Aurora B kinase, and kinesin-8. Combinatorial inhibition of these subprocesses yielded cells with hyperstable spindle remnants and dramatic defects in cell cycle progression, establishing that rapid spindle disassembly is crucial for cell proliferation. doi/ 10.1083/jcb.201006028

DNA topoisomerase II[alpha] (Topoll[alpha]) is an essential chromosome-associated enzyme with activity implicated in the resolution of tangled DNA at centromeres before anaphase onset. However, the regulatory mechanism of Topoll[alpha] activity is not understood. Here, we show that PIASy-mediated small ubiquitin-like modifier 2/3 (SUMO2/3) modification of Topoll[alpha] strongly inhibits Topoll[alpha] decatenation activity. Using mass spectrometry and biochemical analysis, we demonstrate that Topoll[alpha] is SUMOylated at lysine 660 (Lys660), a residue located in the DNA gate domain, where both DNA cleavage and religation take place. Remarkably, loss of SUMOylation on Lys660 eliminates SUMOylation-dependent inhibition of Topoll[alpha], which indicates that Lys660 SUMOylation is critical for PIASy-mediated inhibition of Topoll[alpha] activity. Together, our findings provide evidence for the regulation of Topoll[alpha] activity on mitotic chromosomes by SUMOylation. Therefore, we propose a novel mechanism for regulation of centromeric DNA catenation during mitosis by PIASy-mediated SUMOylation of Topoll[alpha]. doi/ 10.1083/jcb.201004033

Cytokinesis requires a membrane-remodeling and fission event termed abscission that occurs after chromosome segregation, cleavage furrow formation, and contraction have completed. In this study, we show how abscission factor recruitment is controlled by the Polo-like kinase 1 (Plk1). At the metaphase--anaphase transition, Plk1 initiates cleavage furrow formation and is then progressively degraded during mitotic exit. During this period, Plk1 phosphorylates the abscission factor Cep55 in trans and prevents its untimely recruitment to the anaphase spindle. A Plk1 phosphorylation site mutant of Cep55 is prematurely recruited to the anaphase spindle and fails to support abscission. Endogenous Cep55 behaves similarly after PIk1 inhibition by the drugs BI2536 or GW842862. Only once PIk1 is degraded can Cep55 target to the midbody and promote abscission. Blocking PIk1 degradation leads to elevated levels of PIk1 at the midbody and the failure of Cep55 recruitment. Thus, PIk1 activity negatively regulates Cep55 to ensure orderly abscission factor recruitment and ensures that this occurs only once cell contraction has completed. doi/ 10.1083/jcb.201008108

How dividing cells monitor the effective transmission of genomes during mitosis is poorly understood. Budding yeast use a signaling pathway known as the spindle position checkpoint (SPC) to ensure the arrival of one end of the mitotic spindle in the nascent daughter cell. An important question is how SPC activity is coordinated with mother-daughter polarity. We sought to identify factors at the bud neck, the junction between mother and bud, which contribute to checkpoint signaling. In this paper, we show that the protein kinase Elm1 is an obligate regulator of the SPC, and this function requires localization of Elm1 to the bud neck. Furthermore, we show that Elm1 promotes the activity of the checkpoint kinase Kin4. These findings reveal a novel function for Elm1 in the SPC and suggest how checkpoint activity may be linked to cellular organization. doi/ 10.1083/jcb.201006092

The actin cytoskeleton undergoes rapid changes in its architecture during mitosis. Here, we demonstrate novel actin assembly dynamics in M phase. An amorphous cluster of actin filaments appears during prometaphase, revolves horizontally along the cell cortex at a constant angular speed, and fuses into the contractile ring after three to four revolutions. Cdk1 activity is required for the formation of this mitotic actin cluster and its revolving movement. Rapid turnover of actin in the filaments takes place everywhere in the cluster and is also required for its cluster rotation during mitosis. Knockdown of Arp3, a component of the actin filament--nucleating Arp2/3 complex, inhibits the formation of the mitotic actin cluster without affecting other actin structures. These results identify Arp2/3 complex as a key factor in the generation of the dynamic actin cluster during mitosis. doi/ 10.1083/jcb.201007136

Cdc20 is an activator of the anaphase-promoting complex/cyclosome that initiates anaphase onset by ordering the destruction of cyclin B1 and securin in metaphase. To study the physiological significance of Cdc20 in higher eukaryotes, we generated hypomorphic mice that express small amounts of this essential cell cycle regulator. In this study, we show that these mice are healthy and not prone to cancer despite substantial aneuploidy. Cdc20 hypomorphism causes chromatin bridging and chromosome misalignment, revealing a requirement for Cdc20 in efficient sister chromosome separation and chromosome--microtubule attachment. We find that cyclin B1 is newly synthesized during mitosis via cytoplasmic polyadenylation element--binding protein-dependent translation, causing its rapid accumulation between prometaphase and metaphase of Cdc20 hypomorphic cells. Anaphase onset is significantly delayed in Cdc20 hypomorphic cells but not when translation is inhibited during mitosis. These data reveal that Cdc20 is particularly rate limiting for cyclin B1 destruction because of regulated de novo synthesis of this cyclin after prometaphase onset. doi/ 10.1083/jcb.201003090

Aurora B is a component of the chromosomal passenger complex (CPC) required for correct spindle-kinetochore attachments during chromosome segregation and for cytokinesis. The chromatin factors that recruit the CPC to centromeres are unknown, however. Here we show that phosphorylation of histone H3 threonine 3 (H3T3ph) by Haspin is necessary for CPC accumulation at centromeres and that the CPC subunit Survivin binds directly to H3T3ph. A nonbinding Survivin-D70A/D71A mutant does not support centromeric CPC concentration, and both Haspin depletion and Survivin-D70A/D71A mutation diminish centromere localization of the kinesin MCAK and the mitotic checkpoint response to taxol. Survivin-D70A/D71A mutation and microinjection of H3T3ph-specific antibody both compromise centromeric Aurora B functions but do not prevent cytokinesis. Therefore, H3T3ph generated by Haspin positions the CPC at centromeres to regulate selected targets of Aurora B during mitosis. 10.1126/science.1189435

We have studied Sds22, a conserved regulator of protein phosphatase 1 (PP1) activity, and determined its role in modulating the activity of aurora B kinase and kinetochore--microtubule interactions. Sds22 is required for proper progression through mitosis and Iocalization of PP1 to mitotic kinetochores. Depletion of Sds22 increases aurora B T-loop phosphorylation and the rate of recovery from monastrol arrest. Phospho--aurora B accumulates at kinetochores in Sds22-depleted cells juxtaposed to critical kinetochore substrates. Sds22 modulates sister kinetochore distance and the interaction between Hecl and the microtubule lattice and, thus, the activation of the spindle assembly checkpoint. These results demonstrate that Sds22 specifically defines PP1 function and Iocalization in mitosis. Sds22 regulates PP1 targeting to the kinetochore, accumulation of phospho--aurora B, and force generation at the kinetochore--microtubule interface. doi/ 10.1083/jcb.200912046

In metazoa, new nuclear pore complexes (NPCs) form at two different cell cycle stages: at the end of mitosis concomitant with the reformation of the nuclear envelope and during interphase. However, the mechanisms of these assembly processes may differ. In this study, we apply high resolution live cell microscopy to analyze the dynamics of single NPCs in living mammalian cells during interphase. We show that nuclear growth and NPC assembly are correlated and occur at a constant rate throughout interphase. By analyzing the kinetics of individual NPC assembly events, we demonstrate that they are initiated by slow accumulation of the membrane nucleoporin Pom121 followed by the more rapid association of the soluble NPC subcomplex Nup107-160. This inverse order of recruitment and the overall much slower kinetics compared with postmitotic NPC assembly support the conclusion that the two processes occur by distinct molecular mechanisms. doi/ 10.1083/jcb.201007076

Intestinal smooth muscle cells are normally quiescent, but in the widely studied model of trinitrobenzene sulfonic acid (TNBS)-induced colitis in the rat, the onset of inflammation causes proliferation that leads to increased cell number and an altered phenotype. The factors that drive this are unclear and were studied in primary cultures of circular smooth muscle cells (CSMC) from the rat colon. While platelet-derived growth factor (PDGF)-AA, fibroblast growth factor (FGF), and epidermal growth factor (EGF) were ineffective, PDGF-BB and insulin-like growth factor-1 (IGF-1) caused significant increase in [[sup.3]H]thymidine incorporation, bromodeoxyuridine uptake, and increased CSMC number, with PDGF-BB ([greater than or equal to] 0.2 nM) substantially more effective than IGF-1. Surprisingly, CSMC lacked expression of PDGF receptor-[beta] (PDGF-R[beta]) upon isolation but by 4 days in vitro, CSMC gained expression of PDGF-R[beta] as shown by quantitative PCR, Western blot analysis, and immunocytochemistry; these CSMC responded to PDGF-BB but not IGF-1. PDGF-BB caused PDGF-R[beta] phosphorylation and mobilization from the surface membrane, leading to activation of both Akt and ERK signaling pathways, which were essential for subsequent proliferation. In contrast, PDGF-AA, FGF, EGF, and IGF-1 were ineffective. In vivo, control CSMC lacked expression of PDGF-R[beta]. However, this changed rapidly with TNBS-colitis, and by day 2 when CSMC proliferation in vivo is maximal, freshly isolated CSMC showed on-going PDGF-R[beta] phosphorylation that was further increased by exogenous PDGF-BB. This suggests that the onset of PDGF-R[beta] expression is a key factor in CSMC growth in vitro and in vivo, where inflammation may damage intrinsic inhibitory mechanisms and thus lead to hyperplasia. smooth muscle cell; intestine; platelet-derived growth factor; rat; mitosis; insulin-like growth factor-1; tissue culture doi: 10.1152/ajpcell.00086.2010.

Somatic loss of wild-type alleles can produce disease traits such as neoplasia. Conversely, somatic loss of disease-causing mutations can revert phenotypes; however, these events are infrequently observed. Here we show that ichthyosis with confetti, a severe, sporadic skin disease in humans, is associated with thousands of revertant clones of normal skin that arise from loss of heterozygosity on chromosome 17q via mitotic recombination. This allowed us to map and identify disease-causing mutations in the gene encoding keratin 10 (KRT10); all result in frameshifts into the same alternative reading frame, producing an arginine-rich C-terminal peptide that redirects keratin 10 from the cytokeratin filament network to the nucleolus. The high frequency of somatic reversion in ichthyosis with confetti suggests that revertant stem cell clones are under strong positive selection and/or that the rate of mitotic recombination is elevated in individuals with this disorder. 10.1126/science.1192280

Budding yeast asymmetric cell division relies upon the precise coordination of spindle orientation and cell cycle progression. The spindle position checkpoint (SPOC) is a surveillance mechanism that prevents cells with misoriented spindles from exiting mitosis. The cortical kinase Kin4 acts near the top of this network. How Kin4 kinase activity is regulated and maintained in respect to spindle positional cues remains to be established. Here, we show that the bud neck--associated kinase Elm1 participates in Kin4 activation and SPOC signaling by phosphorylating a conserved residue within the activation loop of Kin4. Blocking Elm1 function abolishes Kin4 kinase activity in vivo and eliminates the SPOC response to spindle misalignment. These findings establish a novel function for Elm1 in the coordination of spindle positioning with cell cycle progression via its control of Kin4. doi/ 10.1083/jcb.201006151

Von Hippel-Lindau (VHL) tumor suppressor gene mutations predispose carriers to kidney cancer. The protein pVHL has been shown to interact with microtubules (MTs), which is critical to cilia maintenance and mitotic spindle orientation. However, the function for pVHL in the regulation of MT dynamics is unknown. We tracked MT growth via the plus end marker EB3 (end-binding protein 3)-GFP and inferred additional parameters of MT dynamics indirectly by spatiotemporal grouping of growth tracks from live cell imaging. Our data establish pVHL as a near-optimal MT-stabilizing protein: it attenuates tubulin turnover, both during MT growth and shrinkage, inhibits catastrophe, and enhances rescue frequencies. These functions are mediated, in part, by inhibition of tubulin guanosine triphosphatase activity in vitro and at MT plus ends and along the MT lattice in vivo. Mutants connected to the VHL cancer syndrome are differentially compromised in these activities. Thus, single cell--level analysis of pVHL MT regulatory function allows new predictions for genotype to phenotype associations that deviate from the coarser clinically defined mutant classifications. doi/ 10.1083/jcb.201006059

The mechanisms that regulate cytoskeletal remodeling during the transition between mitosis and interphase are poorly understood. In fission yeast the MOR pathway promotes actin polarization to cell tips in interphase, whereas the SIN signaling pathway drives actomyosin ring assembly and cytokinesis. We show that the SIN inhibits MOR signaling in mitosis by interfering with Nak1 kinase-mediated activation of the most downstream MOR component, the NDR family kinase Orb6. Inactivation of the MOR may be a key function of the SIN because attenuation of MOR signaling rescued the cytokinetic defects of SIN mutants and allowed weak SIN signaling to trigger ectopic cytokinesis. Furthermore, failure to inhibit the MOR is toxic when the cell division apparatus is compromised. Together, our results reveal a mutually antagonistic relationship between the SIN and MOR pathways, which is important for completion of cytokinesis and coordination of cytoskeletal remodeling at the mitosisto-interphase transition. doi/ 10.1083/jcb.201002055

Ran is an essential GTPase that controls nucleocytoplasmic transport, mitosis, and nuclear envelope formation. These functions are regulated by interaction of Ran with different partners, and by formation of a Ran-GTP gradient emanating from chromatin. Here, we identify a novel level of Ran regulation. We show that Ran is a substrate for p21-activated kinase 4 (PAK4) and that its phosphorylation on serine-135 increases during mitosis. The endogenous phosphorylated Ran and active PAK4 dynamically associate with different components of the microtubule spindle during mitotic progression. A GDP-bound Ran phosphomimetic mutant cannot undergo RCC1-mediated GDP/GTP exchange and cannot induce microtubule asters in mitotic Xenopus egg extracts. Conversely, phosphorylation of GTP-bound Ran facilitates aster nucleation. Finally, phosphorylation of Ran on serine-135 impedes its binding to RCC1 and RanGAP1. Our study suggests that PAK4-mediated phosphorylation of GDP- or GTP-bound Ran regulates the assembly of Ran-dependent complexes on the mitotic spindle. doi/ 10.1083/jcb.200912056

The ubiquitin ligase anaphase-promoting complex/ cyclosome (APC/C) is activated at prometaphase by mitotic phosphorylation and binding of its activator, Cdc20. This initiates cyclin A degradation, whereas cyclin B1 is stabilized by the spindle checkpoint. Upon checkpoint release, the RXXL destruction box (D box) was proposed to direct cyclin B1 to core APC/C or Cdc20. In this study, we report that endogenous cyclin B1-Cdk1 is recruited to checkpoint-inhibited, phosphorylated APC/C in prometaphase independently of Cdc20 or the cyclin B1 D box. Like cyclin A, cyclin B1 binds the APC/C by the Cdk cofactor Cks and the APC3 subunit. Prior binding to [APC/C.sup.Cdc20] makes cyclin B1 a better APC/C substrate in metaphase, driving mitotic exit and cytokinesis. We conclude that in prometaphase, the phosphorylated APC/C can recruit both cyclin A and cyclin B1 in a Cks-dependent manner. This suggests that the spindle checkpoint blocks D box recognition of APC/C-bound cyclin B1, whereas distinctive complexes between the N terminus of cyclin A and Cdc20 evade checkpoint control. doi/ 10.1083/jcb.200912084

The anaphase-promoting complex/cyclosome (APC/C) is the ubiquitin ligase essential to mitosis, which ensures that specific proteins are degraded at specific times to control the order of mitotic events. The APC/C coactivator, Cdc20, is targeted by the spindle assembly checkpoint (SAC) to restrict APC/C activity until metaphase, yet early substrates, such as cyclin A, are degraded in the presence of the active checkpoint. Cdc20 and the cyclin-dependent kinase cofactor, Cks, are required for cyclin A destruction, but how they enable checkpoint-resistant destruction has not been elucidated. In this study, we answer this problem: we show that the N terminus of cyclin A binds directly to Cdc20 and with sufficient affinity that it can out compete the SAC proteins. Subsequently, the Cks protein is necessary and sufficient to promote cyclin A degradation in the presence of an active checkpoint by binding cyclin A-Cdc20 to the APC/C. doi/ 10.1083/jcb.201001083

A cold-sensitive [gamma]-tubulin allele of Aspergillus nidulans, mipAD159, causes defects in mitotic and cell cycle regulation at restrictive temperatures that are apparently independent of microtubule nucleation defects. Time-lapse microscopy of fluorescently tagged mitotic regulatory proteins reveals that cyclin B, cyclin-dependent kinase 1, and the Ancdc14 phosphatase fail to accumulate in a subset of nuclei at restrictive temperatures. These nuclei are permanently removed from the cell cycle, whereas other nuclei, in the same multinucleate cell, cycle normally, accumulating and degrading these proteins. After each mitosis, additional daughter nuclei fail to accumulate these proteins, resulting in an increase in noncycling nuclei over time and consequent inhibition of growth. Extensive analyses reveal that these noncycling nuclei result from a nuclear autonomous, microtubule-independent failure of inactivation of the anaphase-promoting complex/cyclosome. Thus, [gamma]-tubulin functions to regulate this key mitotic and cell cycle regulatory complex. doi/ 10.1083/jcb.201002105

Using chemical genetics to reversibly inhibit Cdk1, we find that cells arrested in late G2 are unable to delay mitotic entry after irradiation. Late G2 cells detect DNA damage lesions and form [gamma]-H2AX foci but fail to activate Chk1. This reflects a lack of DNA double-strand break processing because late G2 cells fail to recruit RPA (replication protein A), ATR (ataxia telangiectasia and Rad3 related), Rad51, or CtlP (C-terminal interacting protein) to sites of radiation-induced damage, events essential for both checkpoint activation and initiation of DNA repair by homologous recombination. Remarkably, inhibition of Akt/PKB (protein kinase B) restores DNA damage processing and Chk1 activation after irradiation in late G2. These data demonstrate a previously unrecognized role for Akt in cell cycle regulation of DNA repair and checkpoint activation. Because Akt/PKB is frequently activated in many tumor types, these findings have important implications for the evolution and therapy of such cancers. doi/ 10.1083/jcb.201003004

Cellular processes such as cell cycle progression, mitosis, apoptosis, and cell migration are characterized by well-defined events that are modulated as a function of time. Measuring these events in the context of time and its perturbation by small molecule compounds and RNAi can provide mechanistic information about cellular pathways being affected. We have used impedance-based time-dependent cell response profiling (TCRP) to measure and characterize cellular responses to antimitotic compounds or siRNAs. Our findings indicate that small molecule perturbation of mitosis leads to unique TCRP. We have further used this unique TCRP signature to screen 119 595 compound library and identified novel antimitotic compounds based on clustering analysis of the TCRPs. Importantly, 113 of the 117 hit compounds in the TCRP antimitotic cluster were confirmed as antimitotic based on independent assays, thus establishing the robust predictive nature of this profiling approach. In addition, potent and novel agents that induce mitotic arrest either by directly interfering with tubulin polymerization or by other mechanisms were identified. The TCRP approach allows for a practical and unbiased phenotypic profiling and screening tool for small molecule and RNAi perturbation of specific cellular pathways and time resolution of the TCRP approach can serve as a complement for other existing multidimensional profiling approaches. 10.1021/ac1007877