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Die mitotische Spindel ist ideal für die Erforschung der Selbstorganisation und Plastizität molekularer Kollektive im Zytoplasma. Die Geometrie der Spindel ist entscheidend für die korrekte Chromosomentrennung, muss sich aber an die Zellgröße anpassen. Es ist unbekannt, ob und wie Zellen während ihrer Differenzierung die Spindelarchitektur anpassen, was insbesondere während der Gehirnentwicklung relevant ist. Wir untersuchten dies mit Maus-Embryonalstammzellen, die in frühe neuronale Vorläuferzellen differenziert wurden. Wir entwickelten ein automatisiertes Mikroskopieprotokoll um einen umfassenden Datensatz von mitotischen Zellen zu generieren. Außerdem entwickelten wir Spindle3D, ein Werkzeug zur dreidimensionalen Analyse von Spindeln. Überraschenderweise waren die Spindelvolumina in differenzierenden Zellen bis zu 24% kleiner als in pluripotenten Zellen. Während die Wachstumsgeschwindigkeit der Mikrotubuli unverändert blieb, verschob sich in sich differenzierenden Zellen die Nukleation von Mikrotubuli zugunsten der astralen Population. Diese Veränderung der Spindelarchitektur basierte auf der differenzierungsbedingten Verdünnung des Zytoplasmas. Dies aktivierte CPAP, ein Protein, das die Zentrosomenreifung reguliert, was zur Superskalierung des perizentriolären Materials und verstärkte Rekrutierung von gamma-Tubulin an den Zentrosomen und somit zur Umlagerung von Mikrotubuli innerhalb der Spindel führte. Diese Veränderungen der mitotischen Architektur konnten durch externe osmotische Einwirkung in undifferenzierten Zellen nachgestellt werden. Insgesamt verbinden unsere Ergebnisse zelltypspezifische zytoplasmatische Materialeigenschaften mit der Spindelarchitektur., The mitotic spindle provides an excellent system in which to study the plasticity of molecular collectives. To segregate chromosomes accurately, the spindle’s geometry must be adaptive to changes in cell size. It is unknown whether and how differentiating cells adjust spindle architecture, specifically during brain development when spindle defects have severe pathological consequences. Using murine embryonic stem cells, we recapitulated the transition from pluripotency to early neural cell identities in vitro. Aiming at a systematic exploration of spindle and cell morphology throughout this process, we developed an automated microscopy protocol and Spindle3D, a morphometric tool for the analysis of spindles in confocal images. Intriguingly, in cells with comparable cell volume, spindle volumes were up to 24% smaller in cells undergoing differentiation. While microtubule growth speed remained equal, we measured increased nucleation of astral microtubules at the expense of the spindle bulk in differentiating cells. The shift in spindle architecture was explained by the differentiation-driven cytoplasmic dilution. This activated the centrosomal regulator CPAP, causing the superscaling of the pericentriolar material and the concomitant increased recruitment of gamma-tubulin to the centrosomes, redistributing microtubule numbers within the spindle. Mimicking the dilution effect by osmotic challenge reproduced the same mitotic architecture in undifferentiated cells. Collectively, our results link cell state-specific cytoplasmic material properties to spindle architecture.

Large-scale cell flow characterizes gastrulation in animal development. In amniote gastrulation, particularly in avian gastrula, a bilateral vortex-like counter-rotating cell flow, called 'polonaise movements', appears along the midline. Here, through experimental manipulations, we addressed relationships between the polonaise movements and morphogenesis of the primitive streak, the earliest midline structure in amniotes. Suppression of the Wnt/planar cell polarity (PCP) signaling pathway maintains the polonaise movements along a deformed primitive streak. Mitotic arrest leads to diminished extension and development of the primitive streak and maintains the early phase of the polonaise movements. Ectopically induced Vg1, an axis-inducing morphogen, generates the polonaise movements, aligned to the induced midline, but disturbs the stereotypical cell flow pattern at the authentic midline. Despite the altered cell flow, induction and extension of the primitive streak are preserved along both authentic and induced midlines. Finally, we show that ectopic axis-inducing morphogen, Vg1, is capable of initiating the polonaise movements without concomitant PS extension under mitotic arrest conditions. These results are consistent with a model wherein primitive streak morphogenesis is required for the maintenance of the polonaise movements, but the polonaise movements are not necessarily responsible for primitive streak morphogenesis. Our data describe a previously undefined relationship between the large-scale cell flow and midline morphogenesis in gastrulation.

Wee1 is a cell cycle regulator that phosphorylates Cdk1/Cdc2 and inhibits G2/M transition. Loss of Wee1 in fission yeast results in an early onset of mitosis. Interestingly, we found that cells lacking Wee1 require the functional spindle checkpoint for their viability. Genetic analysis indicated that the requirement is not attributable to the early onset of mitosis. Live-cell imaging revealed that some kinetochores are not attached or bioriented in the wee1 mutant. Furthermore, Mad2, a component of the spindle checkpoint known to recognize unattached kinetochores, accumulates in the vicinity of the spindle, representing activation of the spindle checkpoint in the mutant. It appears that the wee1 mutant cannot maintain stable kinetochore-microtubule attachment, and relies on the delay imposed by the spindle checkpoint for establishing biorientation of kinetochores. This study revealed a role of Wee1 in ensuring accurate segregation of chromosomes during mitosis, and thus provided a basis for a new principle of cancer treatment with Wee1 inhibitors.

The integration of artificial intelligence (AI) into biomedical research has given rise to new models and research topics in biomedicine. Whole-cell modeling aims to create a holistic understanding of the cell by integrating diverse data. One method of comprehension is the characterization and imitation of a system. Phenomenological cell models imitate cell structure and behavior based on, for example, images. Thus generative AI image models present one approach to developing such phenomenological models of cell systems. Diffusion models are a popular generative model class for image generation. Briefly, diffusion models consist of a forward and reverse diffusion process, where the forward process iteratively adds noise to an image and the reverse process learns to remove it. Image generation is achieved by sampling from noise and applying the learned reverse process. The generation may be conditioned to achieve a specific output. The diffusion process is computationally expensive to evaluate in pixel space. The latent diffusion model presents a solution by moving the diffusion process to the latent space of an autoencoder. A latent diffusion model has been trained to develop a phenomenological model of cells in mitosis. The aim is to identify spatial and temporal patterns in the dataset, consisting of fluorescence microscopy images of cells in mitosis, and condition the output of the latent diffusion model on labels associated with the data. The latent diffusion can generate images unconditionally and conditionally. The unconditionally generated images appear visually similar, but quantitative metrics suggest the potential for improvement. Qualitative analysis of the conditionally generated images indicates opportunities for enhancement. The analysis from the proposed method for objective assessment of conditionally generated images, feature extraction of images followed by dimension reduction using uniform manifold approximation and projection, concurs with the vi, Integrering av artificiell intelligens (AI) i biomedicinsk forskning har gett upphov till nya modeller och forskningsfrågor inom biomedicin. Helcellsmodellering syftar till att skapa ett kvantitativt perspektiv på cellbiologi och skapa holistisk kunskap om cellen. Ett system kan förstås genom karaktärisering och imitation. Generativ AI är ett tillvägagångssätt för att utveckla modeller som kan imitera och karaktärisera celler baserat på bilder. Diffusionsmodeller är en populär klass av generativa modeller för bildgenerering. Diffusionsmodeller består av en framåt- och bakåtdiffusionsprocess, där den framåtriktade processen iterativt lägger till brus i en bild och den bakåtriktade processen lär sig att ta bort det. Nya bilder genereras genom att tillämpa den inlärda bakåtriktade processen på en bild av brus. Generationen kan göras villkorlig för att forma bilden efter givna villkor. Den beräkningsintensiva diffusionsprocessen kan effektiviseras genom att introducera en "autoencoder" som flyttar diffusionsprocessen från pixelrummets stora dimension till det latenta rummet, som har en mindre dimension. Det utgör basen för en latent diffusionsmodell. För att utveckla en fenomenologisk modell av celler i mitos har en latent diffusionsmodell tränats på fluorescensmikroskopibilder på celler som genomgår mitos. Målet är att identifiera spatiala och temporala mönster i bilderna och skapa en modell som kan villkora bildgenerationen baserat på givna spatiala och temporala villkor associerade med bilderna. Latenta diffusionsmodeller kan skapa bilder både villkorligen och helt fritt från den underliggande datadistributionen. Den fria generationen av bilder resulterar i visuellt lika bilder men kvantitativa mått indikerar att modellen kan förbättras. Villkorligt genererade bilder håller inte samma visuella kvalité. Behovet av tekniker för att utvärdera villkorligt genererade bilder har identifierats och en metod har föreslagits. Metoden involverar att extrahera attribut från bild

The integration of artificial intelligence (AI) into biomedical research has given rise to new models and research topics in biomedicine. Whole-cell modeling aims to create a holistic understanding of the cell by integrating diverse data. One method of comprehension is the characterization and imitation of a system. Phenomenological cell models imitate cell structure and behavior based on, for example, images. Thus generative AI image models present one approach to developing such phenomenological models of cell systems. Diffusion models are a popular generative model class for image generation. Briefly, diffusion models consist of a forward and reverse diffusion process, where the forward process iteratively adds noise to an image and the reverse process learns to remove it. Image generation is achieved by sampling from noise and applying the learned reverse process. The generation may be conditioned to achieve a specific output. The diffusion process is computationally expensive to evaluate in pixel space. The latent diffusion model presents a solution by moving the diffusion process to the latent space of an autoencoder. A latent diffusion model has been trained to develop a phenomenological model of cells in mitosis. The aim is to identify spatial and temporal patterns in the dataset, consisting of fluorescence microscopy images of cells in mitosis, and condition the output of the latent diffusion model on labels associated with the data. The latent diffusion can generate images unconditionally and conditionally. The unconditionally generated images appear visually similar, but quantitative metrics suggest the potential for improvement. Qualitative analysis of the conditionally generated images indicates opportunities for enhancement. The analysis from the proposed method for objective assessment of conditionally generated images, feature extraction of images followed by dimension reduction using uniform manifold approximation and projection, concurs with the vi, Integrering av artificiell intelligens (AI) i biomedicinsk forskning har gett upphov till nya modeller och forskningsfrågor inom biomedicin. Helcellsmodellering syftar till att skapa ett kvantitativt perspektiv på cellbiologi och skapa holistisk kunskap om cellen. Ett system kan förstås genom karaktärisering och imitation. Generativ AI är ett tillvägagångssätt för att utveckla modeller som kan imitera och karaktärisera celler baserat på bilder. Diffusionsmodeller är en populär klass av generativa modeller för bildgenerering. Diffusionsmodeller består av en framåt- och bakåtdiffusionsprocess, där den framåtriktade processen iterativt lägger till brus i en bild och den bakåtriktade processen lär sig att ta bort det. Nya bilder genereras genom att tillämpa den inlärda bakåtriktade processen på en bild av brus. Generationen kan göras villkorlig för att forma bilden efter givna villkor. Den beräkningsintensiva diffusionsprocessen kan effektiviseras genom att introducera en "autoencoder" som flyttar diffusionsprocessen från pixelrummets stora dimension till det latenta rummet, som har en mindre dimension. Det utgör basen för en latent diffusionsmodell. För att utveckla en fenomenologisk modell av celler i mitos har en latent diffusionsmodell tränats på fluorescensmikroskopibilder på celler som genomgår mitos. Målet är att identifiera spatiala och temporala mönster i bilderna och skapa en modell som kan villkora bildgenerationen baserat på givna spatiala och temporala villkor associerade med bilderna. Latenta diffusionsmodeller kan skapa bilder både villkorligen och helt fritt från den underliggande datadistributionen. Den fria generationen av bilder resulterar i visuellt lika bilder men kvantitativa mått indikerar att modellen kan förbättras. Villkorligt genererade bilder håller inte samma visuella kvalité. Behovet av tekniker för att utvärdera villkorligt genererade bilder har identifierats och en metod har föreslagits. Metoden involverar att extrahera attribut från bild

Chromosomal instability (CIN) is a hallmark of cancer aggressiveness, providing genetic plasticity and tumor heterogeneity that allows the tumor to evolve and adapt to stress conditions. CIN is considered a cancer therapeutic biomarker because healthy cells do not exhibit CIN. Despite recent efforts to identify therapeutic strategies related to CIN, the results obtained have been very limited. CIN is characterized by a genetic signature where a collection of genes, mostly mitotic regulators, are overexpressed in CIN-positive tumors, providing aggressiveness and poor prognosis. We attempted to identify new therapeutic strategies related to CIN genes by performing a drug screen, using cells that individually express CIN-associated genes in an inducible manner. We find that the overexpression of targeting protein for Xklp2 (TPX2) enhances sensitivity to the proto-oncogene c-Src (SRC) inhibitor dasatinib due to activation of the Yes-associated protein 1 (YAP) pathway. Furthermore, using breast cancer data from The Cancer Genome Atlas (TCGA) and a cohort of cancer-derived patient samples, we find that both TPX2 overexpression and YAP activation are present in a significant percentage of cancer tumor samples and are associated with poor prognosis; therefore, they are putative biomarkers for selection for dasatinib therapy.

The outer corona plays an essential role at the onset of mitosis by expanding to maximize microtubule attachment to kinetochores.1,2 The low-density structure of the corona forms through the expansion of unattached kinetochores. It comprises the RZZ complex, the dynein adaptor Spindly, the plus-end directed microtubule motor centromere protein E (CENP-E), and the Mad1/Mad2 spindle-assembly checkpoint proteins.3,4,5,6,7,8,9,10 CENP-E specifically associates with unattached kinetochores to facilitate chromosome congression,11,12,13,14,15,16 interacting with BubR1 at the kinetochore through its C-terminal region (2091–2358).17,18,19,20,21 We recently showed that CENP-E recruitment to BubR1 at the kinetochores is both rapid and essential for correct chromosome alignment. However, CENP-E is also recruited to the outer corona by a second, slower pathway that is currently undefined.19 Here, we show that BubR1-independent localization of CENP-E is mediated by a conserved loop that is essential for outer-corona targeting. We provide a structural model of the entire CENP-E kinetochore-targeting domain combining X-ray crystallography and Alphafold2. We reveal that maximal recruitment of CENP-E to unattached kinetochores critically depends on BubR1 and the outer corona, including dynein. Ectopic expression of the CENP-E C-terminal domain recruits the RZZ complex, Mad1, and Spindly, and prevents kinetochore biorientation in cells. We propose that BubR1-recruited CENP-E, in addition to its essential role in chromosome alignment to the metaphase plate, contributes to the recruitment of outer corona proteins through interactions with the CENP-E corona-targeting domain to facilitate the rapid capture of microtubules for efficient chromosome alignment and mitotic progression., The outer corona plays an essential role at the onset of mitosis by expanding to maximize microtubule attachment to kinetochores.1,2 The low-density structure of the corona forms through the expansion of unattached kinetochores. It comprises the RZZ complex, the dynein adaptor Spindly, the plus-end directed microtubule motor centromere protein E (CENP-E), and the Mad1/Mad2 spindle-assembly checkpoint proteins.3,4,5,6,7,8,9,10 CENP-E specifically associates with unattached kinetochores to facilitate chromosome congression,11,12,13,14,15,16 interacting with BubR1 at the kinetochore through its C-terminal region (2091–2358).17,18,19,20,21 We recently showed that CENP-E recruitment to BubR1 at the kinetochores is both rapid and essential for correct chromosome alignment. However, CENP-E is also recruited to the outer corona by a second, slower pathway that is currently undefined.19 Here, we show that BubR1-independent localization of CENP-E is mediated by a conserved loop that is essential for outer-corona targeting. We provide a structural model of the entire CENP-E kinetochore-targeting domain combining X-ray crystallography and Alphafold2. We reveal that maximal recruitment of CENP-E to unattached kinetochores critically depends on BubR1 and the outer corona, including dynein. Ectopic expression of the CENP-E C-terminal domain recruits the RZZ complex, Mad1, and Spindly, and prevents kinetochore biorientation in cells. We propose that BubR1-recruited CENP-E, in addition to its essential role in chromosome alignment to the metaphase plate, cont

The outer corona plays an essential role at the onset of mitosis by expanding to maximize microtubule attachment to kinetochores.1,2 The low-density structure of the corona forms through the expansion of unattached kinetochores. It comprises the RZZ complex, the dynein adaptor Spindly, the plus-end directed microtubule motor centromere protein E (CENP-E), and the Mad1/Mad2 spindle-assembly checkpoint proteins.3,4,5,6,7,8,9,10 CENP-E specifically associates with unattached kinetochores to facilitate chromosome congression,11,12,13,14,15,16 interacting with BubR1 at the kinetochore through its C-terminal region (2091–2358).17,18,19,20,21 We recently showed that CENP-E recruitment to BubR1 at the kinetochores is both rapid and essential for correct chromosome alignment. However, CENP-E is also recruited to the outer corona by a second, slower pathway that is currently undefined.19 Here, we show that BubR1-independent localization of CENP-E is mediated by a conserved loop that is essential for outer-corona targeting. We provide a structural model of the entire CENP-E kinetochore-targeting domain combining X-ray crystallography and Alphafold2. We reveal that maximal recruitment of CENP-E to unattached kinetochores critically depends on BubR1 and the outer corona, including dynein. Ectopic expression of the CENP-E C-terminal domain recruits the RZZ complex, Mad1, and Spindly, and prevents kinetochore biorientation in cells. We propose that BubR1-recruited CENP-E, in addition to its essential role in chromosome alignment to the metaphase plate, contributes to the recruitment of outer corona proteins through interactions with the CENP-E corona-targeting domain to facilitate the rapid capture of microtubules for efficient chromosome alignment and mitotic progression., The outer corona plays an essential role at the onset of mitosis by expanding to maximize microtubule attachment to kinetochores.1,2 The low-density structure of the corona forms through the expansion of unattached kinetochores. It comprises the RZZ complex, the dynein adaptor Spindly, the plus-end directed microtubule motor centromere protein E (CENP-E), and the Mad1/Mad2 spindle-assembly checkpoint proteins.3,4,5,6,7,8,9,10 CENP-E specifically associates with unattached kinetochores to facilitate chromosome congression,11,12,13,14,15,16 interacting with BubR1 at the kinetochore through its C-terminal region (2091–2358).17,18,19,20,21 We recently showed that CENP-E recruitment to BubR1 at the kinetochores is both rapid and essential for correct chromosome alignment. However, CENP-E is also recruited to the outer corona by a second, slower pathway that is currently undefined.19 Here, we show that BubR1-independent localization of CENP-E is mediated by a conserved loop that is essential for outer-corona targeting. We provide a structural model of the entire CENP-E kinetochore-targeting domain combining X-ray crystallography and Alphafold2. We reveal that maximal recruitment of CENP-E to unattached kinetochores critically depends on BubR1 and the outer corona, including dynein. Ectopic expression of the CENP-E C-terminal domain recruits the RZZ complex, Mad1, and Spindly, and prevents kinetochore biorientation in cells. We propose that BubR1-recruited CENP-E, in addition to its essential role in chromosome alignment to the metaphase plate, cont

The nuclear envelope delineates the eukaryotic cell nucleus. The membrane system of the nuclear envelope consists of an outer nuclear membrane and an inner nuclear membrane separated by a perinuclear space. It serves as more than just a static barrier, since it regulates the communication between the nucleoplasm and the cytoplasm and provides the anchoring points where chromatin is attached. Fewer nuclear envelope proteins have been identified in plants in comparison with animals and yeasts. Here, we review the current state of knowledge of the nuclear envelope in plants, focusing on its role as a chromatin organizer and regulator of gene expression, as well as on the modifications that it undergoes to be efficiently disassembled and reassembled with each cell division. Advances in knowledge concerning the mitotic role of some nuclear envelope constituents are also presented. In addition, we summarize recent progress on the contribution of the nuclear envelope elements to telomere tethering and chromosome dynamics during the meiotic division in different plant species., Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

Work in the authors’ laboratories is funded by grants from MINECO (SAF2012-36079 to PB and SAF201238215, SAF2014-57791-REDC, and BFU2014-52125-REDT to MM), and Comunidad de Madrid (S2010/BMD2470)., An attractive strategy for cancer therapy is to stop cell proliferation by means of agents that directly arrest the cell cycle. Microtubule poisons such as taxanes block mitosis, eventually leading to cell death in a process frequently known as mitotic catastrophe. However, some cells are able to bypass this mitotic arrest and survive, thus contributing to chemoresistance to those therapies. We have recently observed that mitotic arrest induces an early autophagic flux response that results in autophagy-dependent mitochondrial degradation and a dramatic energetic deficit. The subsequent increase in the AMP/ATP ratio results in the activation of the metabolic sensor AMPK followed by phosphorylation and activation of PFKFB3, an enzyme required for glycolysis. Thus, mitophagy can be considered as a critical effector of the therapeutic effect of mitotic therapies, while both AMPK and PFKFB3 are critical for survival. The manipulation of these molecular routes may therefore have therapeutic benefits in the presence of microtubule poisons., Depto. de Bioquímica y Biología Molecular, Fac. de Ciencias Biológicas, TRUE, pub

Spores are important as dispersal and survival propagules in fungi. In this study we investigated the variation in number, shape, size and germination mode of ascospores in Morchella galilaea, the only species of the genus Morchella known to fruit in the autumn. Based on the observation of five samples, we first discovered significant variation in the shape and size of ascospores in Morchella. One to sixteen ascospores were found in the asci. Ascospore size correlated negatively with ascospore number, but positively with ascus size, and ascus size was positively correlated with ascospore number. We noted that ascospores, both from fresh collections and dried specimens, germinated terminally or laterally either by extended germ tubes, or via the production of conidia that were formed directly from ascospores at one, two or multiple sites. The direct formation of conidia from ascospores takes place within asci or after ascospores are discharged. Using laser confocal microscopy, we recorded the number of nuclei in ascospores and in conidia produced from ascospores. In most ascospores of M. galilaea, several nuclei were observed, as is typical of species of Morchella. However, nuclear number varied from zero to around 20 in this species, and larger ascospores harbored more nuclei. One to six nuclei were present in the conidia. Nuclear migration from ascospores to conidia was observed. Conidia forming directly from ascospores has been observed in few species of Pezizomycetes; this is the first report of the phenomenon in Morchella species. Morphological and molecular data show that conidial formation from ascospores is not found in all the specimens of this species and, hence, is not an informative taxonomic character in M. galilaea. Our data suggest that conidia produced from ascospores and successive mitosis within the ascus may contribute to asci with more than eight spores. The absence of mitosis and/or nuclear degeneration, as well as cytokinesis defect, likely resu

Spores are important as dispersal and survival propagules in fungi. In this study we investigated the variation in number, shape, size and germination mode of ascospores in Morchella galilaea, the only species of the genus Morchella known to fruit in the autumn. Based on the observation of five samples, we first discovered significant variation in the shape and size of ascospores in Morchella. One to sixteen ascospores were found in the asci. Ascospore size correlated negatively with ascospore number, but positively with ascus size, and ascus size was positively correlated with ascospore number. We noted that ascospores, both from fresh collections and dried specimens, germinated terminally or laterally either by extended germ tubes, or via the production of conidia that were formed directly from ascospores at one, two or multiple sites. The direct formation of conidia from ascospores takes place within asci or after ascospores are discharged. Using laser confocal microscopy, we recorded the number of nuclei in ascospores and in conidia produced from ascospores. In most ascospores of M. galilaea, several nuclei were observed, as is typical of species of Morchella. However, nuclear number varied from zero to around 20 in this species, and larger ascospores harbored more nuclei. One to six nuclei were present in the conidia. Nuclear migration from ascospores to conidia was observed. Conidia forming directly from ascospores has been observed in few species of Pezizomycetes; this is the first report of the phenomenon in Morchella species. Morphological and molecular data show that conidial formation from ascospores is not found in all the specimens of this species and, hence, is not an informative taxonomic character in M. galilaea. Our data suggest that conidia produced from ascospores and successive mitosis within the ascus may contribute to asci with more than eight spores. The absence of mitosis and/or nuclear degeneration, as well as cytokinesis defect, likely resu

Spores are important as dispersal and survival propagules in fungi. In this study we investigated the variation in number, shape, size and germination mode of ascospores in Morchella galilaea, the only species of the genus Morchella known to fruit in the autumn. Based on the observation of five samples, we first discovered significant variation in the shape and size of ascospores in Morchella. One to sixteen ascospores were found in the asci. Ascospore size correlated negatively with ascospore number, but positively with ascus size, and ascus size was positively correlated with ascospore number. We noted that ascospores, both from fresh collections and dried specimens, germinated terminally or laterally either by extended germ tubes, or via the production of conidia that were formed directly from ascospores at one, two or multiple sites. The direct formation of conidia from ascospores takes place within asci or after ascospores are discharged. Using laser confocal microscopy, we recorded the number of nuclei in ascospores and in conidia produced from ascospores. In most ascospores of M. galilaea, several nuclei were observed, as is typical of species of Morchella. However, nuclear number varied from zero to around 20 in this species, and larger ascospores harbored more nuclei. One to six nuclei were present in the conidia. Nuclear migration from ascospores to conidia was observed. Conidia forming directly from ascospores has been observed in few species of Pezizomycetes; this is the first report of the phenomenon in Morchella species. Morphological and molecular data show that conidial formation from ascospores is not found in all the specimens of this species and, hence, is not an informative taxonomic character in M. galilaea. Our data suggest that conidia produced from ascospores and successive mitosis within the ascus may contribute to asci with more than eight spores. The absence of mitosis and/or nuclear degeneration, as well as cytokinesis defect, likely resu

Spores are important as dispersal and survival propagules in fungi. In this study we investigated the variation in number, shape, size and germination mode of ascospores in Morchella galilaea, the only species of the genus Morchella known to fruit in the autumn. Based on the observation of five samples, we first discovered significant variation in the shape and size of ascospores in Morchella. One to sixteen ascospores were found in the asci. Ascospore size correlated negatively with ascospore number, but positively with ascus size, and ascus size was positively correlated with ascospore number. We noted that ascospores, both from fresh collections and dried specimens, germinated terminally or laterally either by extended germ tubes, or via the production of conidia that were formed directly from ascospores at one, two or multiple sites. The direct formation of conidia from ascospores takes place within asci or after ascospores are discharged. Using laser confocal microscopy, we recorded the number of nuclei in ascospores and in conidia produced from ascospores. In most ascospores of M. galilaea, several nuclei were observed, as is typical of species of Morchella. However, nuclear number varied from zero to around 20 in this species, and larger ascospores harbored more nuclei. One to six nuclei were present in the conidia. Nuclear migration from ascospores to conidia was observed. Conidia forming directly from ascospores has been observed in few species of Pezizomycetes; this is the first report of the phenomenon in Morchella species. Morphological and molecular data show that conidial formation from ascospores is not found in all the specimens of this species and, hence, is not an informative taxonomic character in M. galilaea. Our data suggest that conidia produced from ascospores and successive mitosis within the ascus may contribute to asci with more than eight spores. The absence of mitosis and/or nuclear degeneration, as well as cytokinesis defect, likely resu

Joint DNA molecules are natural byproducts of DNA replication and repair. Persistent joint molecules give rise to ultrafine DNA bridges (UFBs) in mitosis, compromising sister chromatid separation. The DNA translocase PICH (ERCC6L) has a central role in UFB resolution. A genome-wide loss-of-function screen is performed to identify the genetic context of PICH dependency. In addition to genes involved in DNA condensation, centromere stability, and DNA-damage repair, we identify FIGNL1-interacting regulator of recombination and mitosis (FIRRM), formerly known as C1orf112. We find that FIRRM interacts with and stabilizes the AAA+ ATPase FIGNL1. Inactivation of either FIRRM or FIGNL1 results in UFB formation, prolonged accumulation of RAD51 at nuclear foci, and impaired replication fork dynamics and consequently impairs genome maintenance. Combined, our data suggest that inactivation of FIRRM and FIGNL1 dysregulates RAD51 dynamics at replication forks, resulting in persistent DNA lesions and a dependency on PICH to preserve cell viability.

During mitosis, chromosomes assemble kinetochores to dynamically couple with spindle microtubules.1,2 Kinetochores also function as signaling hubs directing mitotic progression by recruiting and controlling the fate of the anaphase promoting complex/cyclosome (APC/C) activator CDC-20.3,4,5 Kinetochores either incorporate CDC-20 into checkpoint complexes that inhibit the APC/C or dephosphorylate CDC-20, which allows it to interact with and activate the APC/C.4,6 The importance of these two CDC-20 fates likely depends on the biological context. In human somatic cells, the major mechanism controlling mitotic progression is the spindle checkpoint. By contrast, progression through mitosis during the cell cycles of early embryos is largely checkpoint independent.7,8,9,10 Here, we first show that CDC-20 phosphoregulation controls mitotic duration in the C. elegans embryo and defines a checkpoint-independent temporal mitotic optimum for robust embryogenesis. CDC-20 phosphoregulation occurs at kinetochores and in the cytosol. At kinetochores, the flux of CDC-20 for local dephosphorylation requires an ABBA motif on BUB-1 that directly interfaces with the structured WD40 domain of CDC-20.6,11,12,13 We next show that a conserved STP motif in BUB-1 that docks the mitotic kinase PLK-114 is necessary for CDC-20 kinetochore recruitment and timely mitotic progression. The kinase activity of PLK-1 is required for CDC-20 to localize to kinetochores and phosphorylates the CDC-20-binding ABBA motif of BUB-1 to promote BUB-1-CDC-20 interaction and mitotic progression. Thus, the BUB-1-bound pool of PLK-1 ensures timely mitosis during embryonic cell cycles by promoting CDC-20 recruitment to the vicinity of kinetochore-localized phosphatase activity.

DNA compaction is required for the condensation and resolution of chromosomes during mitosis, but the relative contribution of individual chromatin factors to this process is poorly understood. We developed a physiological, cell-free system using high-speed Xenopus egg extracts and optical tweezers to investigate real-time mitotic chromatin fiber formation and force-induced disassembly on single DNA molecules. Compared to interphase extract, which compacted DNA by ~60%, metaphase extract reduced DNA length by over 90%, reflecting differences in whole-chromosome morphology under these two conditions. Depletion of the core histone chaperone ASF1, which inhibits nucleosome assembly, decreased the final degree of metaphase fiber compaction by 29%, while depletion of linker histone H1 had a greater effect, reducing total compaction by 40%. Compared to controls, both depletions reduced the rate of compaction, led to more short periods of decompaction, and increased the speed of force-induced fiber disassembly. In contrast, depletion of condensin from metaphase extract strongly inhibited fiber assembly, resulting in transient compaction events that were rapidly reversed under high force. Altogether, these findings support a speculative model in which condensin plays the predominant role in mitotic DNA compaction, while core and linker histones act to reduce slippage during loop extrusion and modulate the degree of DNA compaction.

Flowering plants contain a large number of cyclin families, each containing multiple members, most of which have not been characterized to date. Here, we analyzed the role of the B1 subclass of mitotic cyclins in cell cycle control during Arabidopsis development. While we reveal CYCB1;5 to be a pseudogene, the remaining four members were found to be expressed in dividing cells. Mutant analyses showed a complex pattern of overlapping, development-specific requirements of B1-type cyclins with CYCB1;2 playing a central role. The double mutant cycb1;1 cycb1;2 is severely compromised in growth, yet viable beyond the seedling stage, hence representing a unique opportunity to study the function of B1-type cyclin activity at the organismic level. Immunolocalization of microtubules in cycb1;1 cycb1;2 and treating mutants with the microtubule drug oryzalin revealed a key role of B1-type cyclins in orchestrating mitotic microtubule networks. Subsequently, we identified the GAMMA-TUBULIN COMPLEX PROTEIN 3-INTERACING PROTEIN 1 (GIP1/MOZART) as an in vitro substrate of B1-type cyclin complexes and further genetic analyses support an important role in the regulation of GIP1 by CYCB1s.

Tetraploidy is a hallmark of cancer cells, and tetraploidy-selective cell growth suppression is a potential strategy for targeted cancer therapy. However, how tetraploid cells differ from normal diploids in their sensitivity to anti-proliferative treatments remains largely unknown. In this study, we found that tetraploid cells are significantly more susceptible to inhibitors of a mitotic kinesin (CENP-E) than are diploids. Treatment with a CENP-E inhibitor preferentially diminished the tetraploid cell population in a diploid-tetraploid co-culture at optimum conditions. Live imaging revealed that a tetraploidy-linked increase in unsolvable chromosome misalignment caused substantially longer mitotic delay in tetraploids than in diploids upon moderate CENP-E inhibition. This time gap of mitotic arrest resulted in cohesion fatigue and subsequent cell death, specifically in tetraploids, leading to tetraploidy-selective cell growth suppression. In contrast, the microtubule-stabilizing compound paclitaxel caused tetraploidy-selective suppression through the aggravation of spindle multipolarization. We also found that treatment with a CENP-E inhibitor had superior generality to paclitaxel in its tetraploidy selectivity across a broader spectrum of cell lines. Our results highlight the unique properties of CENP-E inhibitors in tetraploidy-selective suppression and their potential use in the development of tetraploidy-targeting interventions in cancer.

INTRODUCTION: During proliferative plant cell division, the new cell wall, called the cell plate, is first built in the middle of the cell and then expands outward to complete cytokinesis. This dynamic process requires coordinated movement and arrangement of the cytoskeleton and organelles. METHODS: Here we use live-cell markers to track the dynamic reorganization of microtubules, nuclei, endoplasmic reticulum, and endomembrane compartments during division and the formation of the cell plate in maize leaf epidermal cells. RESULTS: The microtubule plus-end localized protein END BINDING1 (EB1) highlighted increasing microtubule dynamicity during mitosis to support rapid changes in microtubule structures. The localization of the cell-plate specific syntaxin KNOLLE, several RAB-GTPases, as well as two plasma membrane localized proteins was assessed after treatment with the cytokinesis-specific callose-deposition inhibitor Endosidin7 (ES7) and the microtubule-disrupting herbicide chlorpropham (CIPC). While ES7 caused cell plate defects in Arabidopsis thaliana, it did not alter callose accumulation, or disrupt cell plate formation in maize. In contrast, CIPC treatment of maize epidermal cells occasionally produced irregular cell plates that split or fragmented, but did not otherwise disrupt the accumulation of cell-plate localized proteins. DISCUSSION: Together, these markers provide a robust suite of tools to examine subcellular trafficking and organellar organization during mitosis and cell plate formation in maize.

A landmark event in the transition from interphase to mitosis in metazoans is nuclear envelope breakdown (NEBD). Important mitotic events occur prior to NEBD, including condensation of replicated chromosomes and assembly of kinetochores to rapidly engage spindle microtubules. Here, we show that nuclear-enriched protein phosphatase 4 (PP4) ensures robust assembly of the microtubule-coupling outer kinetochore prior to NEBD. In the absence of PP4, chromosomes exhibit extended monopolar orientation after NEBD and subsequently mis-segregate. A secondary consequence of diminished outer kinetochore assembly is defective sister chromatid resolution. After NEBD, a cytoplasmic activity compensates for PP4 loss, leading to outer kinetochore assembly and recovery of chromosomes from monopolar orientation to significant bi-orientation. The Ndc80-Ska microtubule-binding module of the outer kinetochore is required for this recovery. PP4 associates with the inner kinetochore protein CENP-C; however, disrupting the PP4-CENP-C interaction does not perturb chromosome segregation. These results establish that PP4-dependent outer kinetochore assembly prior to NEBD is critical for timely and proper engagement of chromosomes with spindle microtubules.

The genetic material is packaged differentially at different phases of the cell cycle. In interphase, cells package their genome into heterochromatin and euchromatin domains, where genes are repressed or expressed based on cell identity. Heterochromatin, the gene-repressive structure, is regulated by different factors that control methylation and acetylation on histones. During cell division, these histone modifications are copied onto the newly synthesized DNA. The heterochromatin structure is seeded from nucleation DNA sequence that recruits other factors to spread the domain. In our studies, we found that the factors required for spreading heterochromatin in different genomic regions are highly variable. Notably, we proposed a mechanism by which Fkh2, a transcription factor, recruits Clr6 histone deacetylase complex I” to a nucleated heterochromatin domain that initiates the spread of heterochromatin domains. During mitosis, the chromatin is packaged into condensed chromosomes for faithful segregation of the duplicated genome. Progression through mitosis is regulated by the levels of a family of protein kinase complexes named Cyclin-Dependent Kinases (CDKs). The phosphorylation of numerous CDK substrates drives various mitosis events. We identified a novel phosphate-binding pocket on cyclins that aids in the timing of sequential multisite phosphorylation in CDK substrates and mitotic events. Loss of this pocket causes a mitotic delay in vivo as well as loss of multisite phosphorylation in various CDK substrates in vitro.

Cell divisions are accurately positioned to generate cells of the correct size and shape. In plant cells, the new cell wall is built in the middle of the cell by vesicles trafficked along an antiparallel microtubule and a microfilament array called the phragmoplast. The phragmoplast expands toward a specific location at the cell cortex called the division site, but how it accurately reaches the division site is unclear. We observed microtubule arrays that accumulate at the cell cortex during the telophase transition in maize (Zea mays) leaf epidermal cells. Before the phragmoplast reaches the cell cortex, these cortical-telophase microtubules transiently interact with the division site. Increased microtubule plus end capture and pausing occur when microtubules contact the division site-localized protein TANGLED1 or other closely associated proteins. Microtubule capture and pausing align the cortical microtubules perpendicular to the division site during telophase. Once the phragmoplast reaches the cell cortex, cortical-telophase microtubules are incorporated into the phragmoplast primarily by parallel bundling. The addition of microtubules into the phragmoplast promotes fine-tuning of the positioning at the division site. Our hypothesis is that division site-localized proteins such as TANGLED1 organize cortical microtubules during telophase to mediate phragmoplast positioning at the final division plane.

Tetraploidy is a hallmark of cancer cells, and tetraploidy-selective cell growth suppression is a potential strategy for targeted cancer therapy. However, how tetraploid cells differ from normal diploids in their sensitivity to anti-proliferative treatments remains largely unknown. In this study, we found that tetraploid cells are significantly more susceptible to inhibitors of a mitotic kinesin (CENP-E) than are diploids. Treatment with a CENP-E inhibitor preferentially diminished the tetraploid cell population in a diploid-tetraploid co-culture at optimum conditions. Live imaging revealed that a tetraploidy-linked increase in unsolvable chromosome misalignment caused substantially longer mitotic delay in tetraploids than in diploids upon moderate CENP-E inhibition. This time gap of mitotic arrest resulted in cohesion fatigue and subsequent cell death, specifically in tetraploids, leading to tetraploidy-selective cell growth suppression. In contrast, the microtubule-stabilizing compound paclitaxel caused tetraploidy-selective suppression through the aggravation of spindle multipolarization. We also found that treatment with a CENP-E inhibitor had superior generality to paclitaxel in its tetraploidy selectivity across a broader spectrum of cell lines. Our results highlight the unique properties of CENP-E inhibitors in tetraploidy-selective suppression and their potential use in the development of tetraploidy-targeting interventions in cancer.

Protein kinases play essential roles in orchestrating almost every step during mitosis. Aberrant kinase activity often leads to errors in the cell cycle progression which consequently becomes the underlying cause for developmental defects or abnormal cell proliferation leading to cancer. Tank Binding Kinase 1 (TBK1) is overexpressed in certain cancer types and is activated on the centrosomes during mitosis. Loss of TBK1 impairs cell division resulting in growth defects and the accumulation of multinucleated cells. Therefore, proper activation and localization of TBK1 are essential for mitotic progression. Yet, the upstream regulation of TBK1 and the function of activated TBK1 on the centrosomes is unknown. Also, the cause and consequences of overexpression of TBK1 in cancers remain to be explored. Activation of TBK1 depends on its binding to an adaptor protein which induces a conformational change leading to trans autophoshorylation on serine 172 of its kinase domain. We identified that an established innate immune response protein, NAK Associated Protein1 (NAP1/AZI2), is the adaptor required for binding and activating TBK1 during mitosis. Loss of either NAP1 or TBK1 results in the accumulation of binucleated and multinucleated cells, possibly due to several mitotic and cytokinetic defects seen in these knockout (KO) cells. We establish NAP1 as a cell cycle regulated protein which colocalizes with activated TBK1 on the centrosomes during mitosis. Furthermore, by performing an unbiased quantitative phosphoproteomics analysis during mitosis, the substrates discovered reveal that TBK1 also regulates other known cell cycle regulating kinases such as Aurora A and Aurora B. TBK1 is also an established autophagy protein and since the autophagy machinery is often impaired or remodeled to facilitate rapid cell division, we evaluated the underlying connection between TBK1 activation and autophagy. The data shows that cells lacking the essential autophagy proteins FIP200 or AT

Spores are important as dispersal and survival propagules in fungi. In this study we investigated the variation in number, shape, size and germination mode of ascospores in Morchella galilaea, the only species of the genus Morchella known to fruit in the autumn. Based on the observation of five samples, we first discovered significant variation in the shape and size of ascospores in Morchella. One to sixteen ascospores were found in the asci. Ascospore size correlated negatively with ascospore number, but positively with ascus size, and ascus size was positively correlated with ascospore number. We noted that ascospores, both from fresh collections and dried specimens, germinated terminally or laterally either by extended germ tubes, or via the production of conidia that were formed directly from ascospores at one, two or multiple sites. The direct formation of conidia from ascospores takes place within asci or after ascospores are discharged. Using laser confocal microscopy, we recorded the number of nuclei in ascospores and in conidia produced from ascospores. In most ascospores of M. galilaea, several nuclei were observed, as is typical of species of Morchella. However, nuclear number varied from zero to around 20 in this species, and larger ascospores harbored more nuclei. One to six nuclei were present in the conidia. Nuclear migration from ascospores to conidia was observed. Conidia forming directly from ascospores has been observed in few species of Pezizomycetes; this is the first report of the phenomenon in Morchella species. Morphological and molecular data show that conidial formation from ascospores is not found in all the specimens of this species and, hence, is not an informative taxonomic character in M. galilaea. Our data suggest that conidia produced from ascospores and successive mitosis within the ascus may contribute to asci with more than eight spores. The absence of mitosis and/or nuclear degeneration, as well as cytokinesis defect, likely resu

The kinetochore has been viewed as playing a critical role in many aspects of chromosome dynamics as the cell progresses through mitosis. This includes chromosome congression and alignment on the metaphase plate and sister chromosome separation and segregation to the spindle poles. Defects in kinetochore function result in mitotic errors and aneuploidy. Thus, the finding that chromosome fragments lacking a kinetochore (acentrics) are capable of normal congression, sister separation and segregation is fascinating. My thesis takes advantage of a Drosophila system in which acentrics can be efficiently generated and their behavior analyzed through live fluorescence analysis. In Chapter 1, we review how cells transmit chromosomes through mitosis without canonical kinetochore-microtubule interactions. Decades of research and a collection of studies reveal that microtubule-based mechanisms and DNA-based “tethers” connecting the acentric to the main chromosome mass are responsible for proper segregation of acentrics. In Chapter 2, we discuss the mechanisms by which acentric sister chromatids remain paired and eventually separate from one another during anaphase. Taking advantage of Drosophila transgenic for the I-CreI endonuclease, we efficiently generate broken chromosome fragments lacking centromeres, thereby lacking kinetochores. By using a genetic screen and live analysis, we identify proteins responsible for the separation of acentric chromosomes. We conclude DNA catenations are responsible for keeping acentric sisters paired and Topoisomerase II activity and microtubule plus-end pushing forces are needed to resolve these catenations and separate the acentrics. In Chapter 3, we highlight the remarkable ability of acentrics to congress to the metaphase plate despite the absence of kinetochore-microtubule interactions. Through mutational and live cell analysis, we define the forces acting on chromosome arms and the role of the kinetochore in chromosome congression. Utili

Cytoplasmic divisions are thought to rely on nuclear divisions and mitotic signals. We demonstrate in Drosophila embryos that cytoplasm can divide repeatedly without nuclei and mitotic CDK/cyclin complexes. Cdk1 normally slows an otherwise faster cytoplasmic division cycle, coupling it with nuclear divisions, and when uncoupled, cytoplasm starts dividing before mitosis. In developing embryos where CDK/cyclin activity can license mitotic microtubule (MT) organizers like the spindle, cytoplasmic divisions can occur without the centrosome, a principal organizer of interphase MTs. However, centrosomes become essential in the absence of CDK/cyclin activity, implying that the cytoplasm can employ either the centrosome-based interphase or CDK/cyclin-dependent mitotic MTs to facilitate its divisions. Finally, we present evidence that autonomous cytoplasmic divisions occur during unperturbed fly embryogenesis and that they may help extrude mitotically stalled nuclei during blastoderm formation. We postulate that cytoplasmic divisions occur in cycles governed by a yet-to-be-uncovered clock mechanism autonomous from CDK/cyclin complexes.

Tetraploidy is a hallmark of cancer cells, and tetraploidy-selective cell growth suppression is a potential strategy for targeted cancer therapy. However, how tetraploid cells differ from normal diploids in their sensitivity to anti-proliferative treatments remains largely unknown. In this study, we found that tetraploid cells are significantly more susceptible to inhibitors of a mitotic kinesin (CENP-E) than are diploids. Treatment with a CENP-E inhibitor preferentially diminished the tetraploid cell population in a diploid-tetraploid co-culture at optimum conditions. Live imaging revealed that a tetraploidy-linked increase in unsolvable chromosome misalignment caused substantially longer mitotic delay in tetraploids than in diploids upon moderate CENP-E inhibition. This time gap of mitotic arrest resulted in cohesion fatigue and subsequent cell death, specifically in tetraploids, leading to tetraploidy-selective cell growth suppression. In contrast, the microtubule-stabilizing compound paclitaxel caused tetraploidy-selective suppression through the aggravation of spindle multipolarization. We also found that treatment with a CENP-E inhibitor had superior generality to paclitaxel in its tetraploidy selectivity across a broader spectrum of cell lines. Our results highlight the unique properties of CENP-E inhibitors in tetraploidy-selective suppression and their potential use in the development of tetraploidy-targeting interventions in cancer.

Spores are important as dispersal and survival propagules in fungi. In this study we investigated the variation in number, shape, size and germination mode of ascospores in Morchella galilaea, the only species of the genus Morchella known to fruit in the autumn. Based on the observation of five samples, we first discovered significant variation in the shape and size of ascospores in Morchella. One to sixteen ascospores were found in the asci. Ascospore size correlated negatively with ascospore number, but positively with ascus size, and ascus size was positively correlated with ascospore number. We noted that ascospores, both from fresh collections and dried specimens, germinated terminally or laterally either by extended germ tubes, or via the production of conidia that were formed directly from ascospores at one, two or multiple sites. The direct formation of conidia from ascospores takes place within asci or after ascospores are discharged. Using laser confocal microscopy, we recorded the number of nuclei in ascospores and in conidia produced from ascospores. In most ascospores of M. galilaea, several nuclei were observed, as is typical of species of Morchella. However, nuclear number varied from zero to around 20 in this species, and larger ascospores harbored more nuclei. One to six nuclei were present in the conidia. Nuclear migration from ascospores to conidia was observed. Conidia forming directly from ascospores has been observed in few species of Pezizomycetes; this is the first report of the phenomenon in Morchella species. Morphological and molecular data show that conidial formation from ascospores is not found in all the specimens of this species and, hence, is not an informative taxonomic character in M. galilaea. Our data suggest that conidia produced from ascospores and successive mitosis within the ascus may contribute to asci with more than eight spores. The absence of mitosis and/or nuclear degeneration, as well as cytokinesis defect, likely resu

Whole-genome duplication (WGD) is one of the most common genomic abnormalities in cancers. WGD can provide a source of redundant genes to buffer the deleterious effect of somatic alterations and facilitate clonal evolution in cancer cells. The extra DNA and centrosome burden after WGD is associated with an elevation of genome instability. Causes of genome instability are multifaceted and occur throughout the cell cycle. Among these are DNA damage caused by the abortive mitosis that initially triggers tetraploidization, replication stress and DNA damage associated with an enlarged genome, and chromosomal instability during the subsequent mitosis in the presence of extra centrosomes and altered spindle morphology. Here, we chronicle the events after WGD, from tetraploidization instigated by abortive mitosis including mitotic slippage and cytokinesis failure to the replication of the tetraploid genome, and finally, to the mitosis in the presence of supernumerary centrosomes. A recurring theme is the ability of some cancer cells to overcome the obstacles in place for preventing WGD. The underlying mechanisms range from the attenuation of the p53-dependent G(1) checkpoint to enabling pseudobipolar spindle formation via the clustering of supernumerary centrosomes. These survival tactics and the resulting genome instability confer a subset of polyploid cancer cells proliferative advantage over their diploid counterparts and the development of therapeutic resistance.

The Bub1 and BubR1 kinetochore proteins support proper chromosome segregation and mitotic checkpoint activity. Bub1 and BubR1 are paralogs with Bub1 being a kinase, while BubR1 localizes the PP2A-B56 protein phosphatase to kinetochores in humans. Whether this spatial separation of kinase and phosphatase activity is important is unclear as some organisms integrate both activities into one Bub protein. Here, we engineer human Bub1 and BubR1 proteins integrating kinase and phosphatase activities into one protein and show that these do not support normal mitotic progression. A Bub1-PP2A-B56 complex can support chromosome alignment but results in impairment of the checkpoint due to dephosphorylation of the Mad1 binding site in Bub1. Furthermore, a chimeric BubR1 protein containing the Bub1 kinase domain induces delocalized H2ApT120 phosphorylation, resulting in the reduction of centromeric hSgo2 and chromosome segregation errors. Collectively, these results argue that the spatial separation of kinase and phosphatase activities within the Bub complex is required for balancing its functions in the checkpoint and chromosome alignment.

The Bub1 and BubR1 kinetochore proteins support proper chromosome segregation and mitotic checkpoint activity. Bub1 and BubR1 are paralogs with Bub1 being a kinase, while BubR1 localizes the PP2A-B56 protein phosphatase to kinetochores in humans. Whether this spatial separation of kinase and phosphatase activity is important is unclear as some organisms integrate both activities into one Bub protein. Here, we engineer human Bub1 and BubR1 proteins integrating kinase and phosphatase activities into one protein and show that these do not support normal mitotic progression. A Bub1-PP2A-B56 complex can support chromosome alignment but results in impairment of the checkpoint due to dephosphorylation of the Mad1 binding site in Bub1. Furthermore, a chimeric BubR1 protein containing the Bub1 kinase domain induces delocalized H2ApT120 phosphorylation, resulting in the reduction of centromeric hSgo2 and chromosome segregation errors. Collectively, these results argue that the spatial separation of kinase and phosphatase activities within the Bub complex is required for balancing its functions in the checkpoint and chromosome alignment.

Errors in chromosome segregation during mitosis lead to chromosome instability, resulting in an unbalanced number of chromosomes in the daughter cells. Light microscopy has been used extensively to study chromosome missegregation by visualizing errors of the mitotic spindle. However, less attention has been paid to understanding spindle function in the broader context of intracellular structures and organelles during mitosis. Here, we outline a protocol to visualize chromosomes and endomembranes in mitosis, combining light microscopy and 3D volume electron microscopy, serial block-face scanning electron microscopy (SBF-SEM). SBF-SEM provides highresolution imaging of large volumes and subcellular structures, followed by image analysis and 3D reconstruction. This protocol allows scientists to visualize the whole subcellular context of the spindle during mitosis.

In response to improper kinetochore-microtubule attachments in mitosis, the spindle assembly checkpoint (SAC) assembles the mitotic checkpoint complex (MCC) to inhibit the anaphase-promoting complex/cyclosome, thereby delaying entry into anaphase. The MCC comprises Mad2:Cdc20:BubR1:Bub3. Its assembly is catalysed by unattached kinetochores on a Mad1:Mad2 platform. Mad1-bound closed-Mad2 (C-Mad2) recruits open-Mad2 (O-Mad2) through self-dimerization. This interaction, combined with Mps1 kinase-mediated phosphorylation of Bub1 and Mad1, accelerates MCC assembly, in a process that requires O-Mad2 to C-Mad2 conversion and concomitant binding of Cdc20. How Mad1 phosphorylation catalyses MCC assembly is poorly understood. Here, we characterized Mps1 phosphorylation of Mad1 and obtained structural insights into a phosphorylation-specific Mad1:Cdc20 interaction. This interaction, together with the Mps1-phosphorylation dependent association of Bub1 and Mad1, generates a tripartite assembly of Bub1 and Cdc20 onto the C-terminal domain of Mad1 (Mad1 CTD). We additionally identify flexibility of Mad1:Mad2 that suggests how the Cdc20:Mad1 CTD interaction brings the Mad2-interacting motif (MIM) of Cdc20 near O-Mad2. Thus, Mps1-dependent formation of the MCC-assembly scaffold functions to position and orient Cdc20 MIM near O-Mad2, thereby catalysing formation of C-Mad2:Cdc20.

BACKGROUND: During normal zygotic division, two haploid parental genomes replicate, unite and segregate into two biparental diploid blastomeres.RESULTS: Contrary to this fundamental biological tenet, we demonstrate here that parental genomes can segregate to distinct blastomeres during the zygotic division resulting in haploid or uniparental diploid and polyploid cells, a phenomenon coined heterogoneic division. By mapping the genomic landscape of 82 blastomeres from 25 bovine zygotes, we show that multipolar zygotic division is a tell-tale of whole-genome segregation errors. Based on the haplotypes and live-imaging of zygotic divisions, we demonstrate that various combinations of androgenetic, gynogenetic, diploid, and polyploid blastomeres arise via distinct parental genome segregation errors including the formation of additional paternal, private parental, or tripolar spindles, or by extrusion of paternal genomes. Hence, we provide evidence that private parental spindles, if failing to congress before anaphase, can lead to whole-genome segregation errors. In addition, anuclear blastomeres are common, indicating that cytokinesis can be uncoupled from karyokinesis. Dissociation of blastocyst-stage embryos further demonstrates that whole-genome segregation errors might lead to mixoploid or chimeric development in both human and cow. Yet, following multipolar zygotic division, fewer embryos reach the blastocyst stage and diploidization occurs frequently indicating that alternatively, blastomeres with genome-wide errors resulting from whole-genome segregation errors can be selected against or contribute to embryonic arrest.CONCLUSIONS: Heterogoneic zygotic division provides an overarching paradigm for the development of mixoploid and chimeric individuals and moles and can be an important cause of embryonic and fetal arrest following natural conception or IVF.

A rapidly increasing rate of melanoma diagnosis has been noted over the past three decades, and nearly 1 in 4 skin biopsies are diagnosed as melanocytic lesions. The gold standard for diagnosis of melanoma is the histopathological examination by a pathologist to analyze biopsy material at both the cellular and structural levels. A pathologists diagnosis is often subjective and prone to variability, while deep learning image analysis methods may improve and complement current diagnostic and prognostic capabilities. Mitoses are important entities when reviewing skin biopsy cases as their presence carries prognostic information; thus, their precise detection is an important factor for clinical care. In addition, semantic segmentation of clinically important structures in skin biopsies might help the diagnosis pipeline with an accurate classification. We aim to provide prognostic and diagnostic information on skin biopsy images, including the detection of cellular level entities, segmentation of clinically important tissue structures, and other important factors toward the accurate diagnosis of skin biopsy images. This paper is an overview of our work on analysis of digital whole slide skin biopsy images, including mitotic figure (mitosis) detection, semantic segmentation, diagnosis, and analysis of pathologists viewing patterns, and with new work on melanocyte detection. Deep learning has been applied to our methods for all the detection, segmentation, and diagnosis work. In our studies, deep learning is proven superior to prior approaches to skin biopsy analysis. Our work on analysis of pathologists viewing patterns is the only such work in the skin biopsy literature. Our work covers the whole spectrum from low-level entities through diagnosis and understanding what pathologists do in performing their diagnoses.

All eukaryotic cells depend on faithful cell division to give rise to daughter cells that contain the correct number of chromosomes. Chromosome segregation errors during meiosis in germ cells can lead to aneuploid gametes, which can cause infertility, genetic disorders such as Down Syndrome and miscarriages. Additionally, chromosome segregation errors during mitosis can lead to aneuploid somatic cells, which are a hallmark of cancer. Despite the importance of tightly regulating these two types of cell division, the mechanisms that ensure fidelity during these processes remain active areas of investigation. Proteins that have been shown to be important regulators of fidelity during meiosis in Caenorhabditis elegans (C. elegans) include ZHP-3 and PCH-2. ZHP-3 is a putative SUMO or ubiquitin ligase required for crossover recombination, but whether or not ZHP-3 is regulated to promote recombination remains poorly understood. PCH-2 is a conserved AAA+ ATPase required for timely coordination of the meiotic prophase events pairing, synapsis and recombination, but the mechanism for how it accomplishes this coordination is currently unknown. Additionally, PCH-2 has been shown to be required for both silencing and activation of the spindle assembly checkpoint during mitosis, but how it regulates the checkpoint is still an active area of investigation. Here, I use C. elegans as a model system to investigate the molecular mechanisms of these two proteins during meiotic and mitotic chromosome segregation. In Chapter 1, I investigate whether ZHP-3 is phosphorylated by a conserved DNA damage kinase, CHK-1, to determine if this phosphorylation event is required for ZHP-3’s localization or function during meiosis. I find that while ZHP-3 is phosphorylated by CHK-1 kinase in vitro, mutation of these phosphorylation sites in vivo does not affect ZHP-3’s localization to meiotic chromosomes, or its function in promoting crossover recombination. In Chapter 2, I investigate a different re

Cell is a basic unit of life, Structural and functional unit of all living organisms and is also defined as a mass of protoplasm surrounded by a plasma membrane. It is the fundamental building block, which when joined with comparative cells frames a tissue and organs. The cell goes through a progression of occasions that outcome in the duplication of cell alongside the DNA. This is known as the cell cycle. These occasions incorporate duplication of its genome and combination of the cell organelles followed by division of the cytoplasm. Occasions occurring in a cell cycle is hereditarily controlled. From the single cells that make up the most fundamental life forms to the trillions of cells that establish the mindboggling design of the human body, every single living being on Earth is involved cells. In the view of cell division, this paper tries to provide necessary information about the cell cycle.

TANGLED1 (TAN1) is a microtubule binding, division-site-localized protein that plays a role in division plane orientation in plants. Although many potential TAN1 interacting proteins have been identified, how TAN1 interacts with other proteins to ensure correct division positioning is not well understood. Arabidopsis thaliana tan1 single mutants have very weak division plane orientation defects. However mutating a second gene that encodes the unrelated, division-site-localized protein AUXIN-INDUCED-IN-ROOT-CULTURES9 (AIR9), results in strong synthetic phenotypes in the tan1 air9 double mutant including stunted growth, misoriented divisions, and increased cell file rotation. I designed a course-based undergraduate research experience as a high-throughput strategy to screen for TAN1 interactors. Students participated in a sensitized mutagenesis screen by learning how to use CRISPR-Cas9 technology to create gene knockouts of candidate TAN1 interactor genes in air9 single mutant Arabidopsis plants. I also investigated the functional role of TAN1 interaction with the division-site-localized kinesin PHRAGMOPLAST ORIENTING KINESIN1 (POK1). I found that POK1 was mislocalized in the tan1 air9 double mutant, which suggests that AIR9 and TAN1 are involved in POK1 localization to the division site. Using a yeast two-hybrid screen, I identified POK1 interaction sites within the first 132 highly conserved amino acids of TAN1. When mutagenized TAN1 that lost interaction with POK1 in the yeast two-hybrid system was introduced into the tan1 air9 double mutant, it failed to fully rescue phragmoplast positioning defects and disrupted TAN1 and POK1 recruitment to the division site during metaphase and early telophase. This demonstrated that TAN1 interaction with POK1 is important for stabilizing both proteins at the division site to ensure correct phragmoplast guidance during telophase, and that there are likely other unidentified proteins that further stabilize TAN1 and POK1 at the di

A molecular architecture is proposed for a representative mitotic chromosome, human chromosome 10. This architecture is built on an interphase chromosome structure based on cryo-electron microscopy (cryo-EM) cellular tomography [J. Sedat et al., Proc. Natl. Acad. Sci. U.S.A., in press], thus unifying chromosome structure throughout the complete mitotic cycle. The basic organizational principle for mitotic chromosomes is specific coiling of the 11-nm nucleosome fiber into large scale, ∼200-nm interphase structures, a Slinky [https://en.wikipedia.org/wiki/Slinky; motif cited in S. Bowerman et al., eLife 10, e65587 (2021)], then further modified with subsequent additional coiling for the final mitotic chromosome structure. The final mitotic chromosome architecture accounts for the dimensional values as well as the well-known cytological configurations. In addition, proof is experimentally provided by digital PCR technology that G1 T cell nuclei are diploid with one DNA molecule per chromosome. Many nucleosome linker DNA sequences, the promotors and enhancers, are suggestive of optimal exposure on the surfaces of the large-scale coils.

Cell cycle and cell adhesion are two interdependent cellular processes regulating each other, reciprocally, in every cell cycle phase. The cell adhesion to the extracellular matrix (ECM) via integrin receptors triggers signaling pathways required for the cell cycle progression; the passage from the G1 to S phase and the completion of cytokinesis are the best-understood events. Growing evidence, however, suggests more adhesion-dependent regulatory aspects of the cell cycle, particularly during G2 to M transition and early mitosis. Conversely, the cell cycle machinery regulates cell adhesion in manners recently shown driven mainly by cyclin-dependent kinase 1 (CDK1). This review summarizes the recent findings regarding the role of integrin-mediated cell adhesion and its downstream signaling components in regulating the cell cycle, emphasizing the cell cycle progression through the G2 and early M phases. Further investigations are required to raise our knowledge about the molecular mechanisms of crosstalk between cell adhesion and the cell cycle in detail.

Integrin-mediated adhesion to the extracellular matrix is a key regulator of the cell cycle, as demonstrated for the passage of the G1/S checkpoint and the completion of cytokinetic abscission. Here, integrin-dependent regulation of the cell cycle in G2 and early M phases was investigated. The progression through the G2 and M phases was monitored by live-cell imaging and immunofluorescence staining in adherent and non-adherent fibroblast cells. Non-adherent cells, as well as adherent cells lacking FAK activity due to suppressed expression or pharmacological inhibition, exhibited a prolonged G2 phase and severely defect centrosome separation, resulting in delayed progress through the early mitotic stages. The activation of the critical mitotic regulator PLK1 and its indirect target Eg5, a kinesin-family motor protein driving the centrosome separation, were reduced in the cells lacking FAK activity. Furthermore, the absence of integrin adhesion or FAK activity destabilized the structural integrity of centrosomes and often caused detachment of pericentriolar material from the centrioles. These data identify a novel adhesion-dependent mechanism by which integrins via FAK and PLK1 contribute to the regulation of the cell cycle in the G2 and early M phases, and to the maintenance of genome integrity.

Cell cycle and cell adhesion are two interdependent cellular processes regulating each other, reciprocally, in every cell cycle phase. The cell adhesion to the extracellular matrix (ECM) via integrin receptors triggers signaling pathways required for the cell cycle progression; the passage from the G1 to S phase and the completion of cytokinesis are the best-understood events. Growing evidence, however, suggests more adhesion-dependent regulatory aspects of the cell cycle, particularly during G2 to M transition and early mitosis. Conversely, the cell cycle machinery regulates cell adhesion in manners recently shown driven mainly by cyclin-dependent kinase 1 (CDK1). This review summarizes the recent findings regarding the role of integrin-mediated cell adhesion and its downstream signaling components in regulating the cell cycle, emphasizing the cell cycle progression through the G2 and early M phases. Further investigations are required to raise our knowledge about the molecular mechanisms of crosstalk between cell adhesion and the cell cycle in detail.

Integrin-mediated adhesion to the extracellular matrix is a key regulator of the cell cycle, as demonstrated for the passage of the G1/S checkpoint and the completion of cytokinetic abscission. Here, integrin-dependent regulation of the cell cycle in G2 and early M phases was investigated. The progression through the G2 and M phases was monitored by live-cell imaging and immunofluorescence staining in adherent and non-adherent fibroblast cells. Non-adherent cells, as well as adherent cells lacking FAK activity due to suppressed expression or pharmacological inhibition, exhibited a prolonged G2 phase and severely defect centrosome separation, resulting in delayed progress through the early mitotic stages. The activation of the critical mitotic regulator PLK1 and its indirect target Eg5, a kinesin-family motor protein driving the centrosome separation, were reduced in the cells lacking FAK activity. Furthermore, the absence of integrin adhesion or FAK activity destabilized the structural integrity of centrosomes and often caused detachment of pericentriolar material from the centrioles. These data identify a novel adhesion-dependent mechanism by which integrins via FAK and PLK1 contribute to the regulation of the cell cycle in the G2 and early M phases, and to the maintenance of genome integrity.

Integrin-mediated adhesion to the extracellular matrix is a key regulator of the cell cycle, as demonstrated for the passage of the G1/S checkpoint and the completion of cytokinetic abscission. Here, integrin-dependent regulation of the cell cycle in G2 and early M phases was investigated. The progression through the G2 and M phases was monitored by live-cell imaging and immunofluorescence staining in adherent and non-adherent fibroblast cells. Non-adherent cells, as well as adherent cells lacking FAK activity due to suppressed expression or pharmacological inhibition, exhibited a prolonged G2 phase and severely defect centrosome separation, resulting in delayed progress through the early mitotic stages. The activation of the critical mitotic regulator PLK1 and its indirect target Eg5, a kinesin-family motor protein driving the centrosome separation, were reduced in the cells lacking FAK activity. Furthermore, the absence of integrin adhesion or FAK activity destabilized the structural integrity of centrosomes and often caused detachment of pericentriolar material from the centrioles. These data identify a novel adhesion-dependent mechanism by which integrins via FAK and PLK1 contribute to the regulation of the cell cycle in the G2 and early M phases, and to the maintenance of genome integrity.

© The Author(s), 2022. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Molines, A. T., Lemière, J., Gazzola, M., Steinmark, I. E., Edrington, C. H., Hsu, C.-T., Real-Calderon, P., Suhling, K., Goshima, G., Holt, L. J., Thery, M., Brouhard, G. J., & Chang, F. Physical properties of the cytoplasm modulate the rates of microtubule polymerization and depolymerization. Developmental Cell, 57(4), (2022): 466-479.e6, https://doi.org/10.1016/j.devcel.2022.02.001., The cytoplasm is a crowded, visco-elastic environment whose physical properties change according to physiological or developmental states. How the physical properties of the cytoplasm impact cellular functions in vivo remains poorly understood. Here, we probe the effects of cytoplasmic concentration on microtubules by applying osmotic shifts to fission yeast, moss, and mammalian cells. We show that the rates of both microtubule polymerization and depolymerization scale linearly and inversely with cytoplasmic concentration; an increase in cytoplasmic concentration decreases the rates of microtubule polymerization and depolymerization proportionally, whereas a decrease in cytoplasmic concentration leads to the opposite. Numerous lines of evidence indicate that these effects are due to changes in cytoplasmic viscosity rather than cellular stress responses or macromolecular crowding per se. We reconstituted these effects on microtubules in vitro by tuning viscosity. Our findings indicate that, even in normal conditions, the viscosity of the cytoplasm modulates the reactions that underlie microtubule dynamic behaviors., This work was supported by grants to F.C. (NIH GM115185, NIH GM056836, NIH GM146438), to L.J.H. (American Cancer Society RSG-19-073-01-TBE, Pershing Square Sohn Cancer Award, Chan Zuckerberg Initiative, NIH GM132447 and NIH CA240765), to G.G. (JSPS KAKENHI 17H06471 and 18KK0202), to K.S. (UK’s Biotechnology and Biological Sciences Research Council (BBSRC) grant BB/R004803/1) and to M.T. (ERC Consolidator Grant 771599). I.E.S. was supported by King’s College London through a LIDo (London Interdisciplinary Doctoral programme) iCASE studentship.

© The Author(s), 2022. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Molines, A. T., Lemière, J., Gazzola, M., Steinmark, I. E., Edrington, C. H., Hsu, C.-T., Real-Calderon, P., Suhling, K., Goshima, G., Holt, L. J., Thery, M., Brouhard, G. J., & Chang, F. Physical properties of the cytoplasm modulate the rates of microtubule polymerization and depolymerization. Developmental Cell, 57(4), (2022): 466-479.e6, https://doi.org/10.1016/j.devcel.2022.02.001., The cytoplasm is a crowded, visco-elastic environment whose physical properties change according to physiological or developmental states. How the physical properties of the cytoplasm impact cellular functions in vivo remains poorly understood. Here, we probe the effects of cytoplasmic concentration on microtubules by applying osmotic shifts to fission yeast, moss, and mammalian cells. We show that the rates of both microtubule polymerization and depolymerization scale linearly and inversely with cytoplasmic concentration; an increase in cytoplasmic concentration decreases the rates of microtubule polymerization and depolymerization proportionally, whereas a decrease in cytoplasmic concentration leads to the opposite. Numerous lines of evidence indicate that these effects are due to changes in cytoplasmic viscosity rather than cellular stress responses or macromolecular crowding per se. We reconstituted these effects on microtubules in vitro by tuning viscosity. Our findings indicate that, even in normal conditions, the viscosity of the cytoplasm modulates the reactions that underlie microtubule dynamic behaviors., This work was supported by grants to F.C. (NIH GM115185, NIH GM056836, NIH GM146438), to L.J.H. (American Cancer Society RSG-19-073-01-TBE, Pershing Square Sohn Cancer Award, Chan Zuckerberg Initiative, NIH GM132447 and NIH CA240765), to G.G. (JSPS KAKENHI 17H06471 and 18KK0202), to K.S. (UK’s Biotechnology and Biological Sciences Research Council (BBSRC) grant BB/R004803/1) and to M.T. (ERC Consolidator Grant 771599). I.E.S. was supported by King’s College London through a LIDo (London Interdisciplinary Doctoral programme) iCASE studentship.

In 1996, Kim Nasmyth(1) proposed that the eukaryotic cell cycle is an alternating sequence of transitions from G(1) to S-G(2)-M and back again. These two phases correlate to high activity of cyclin-dependent kinases (CDKs) that trigger S-G(2)-M events and CDK antagonists that stabilize G(1) phase. We associated these "alternative phases'' with the coexistence of two stable steady states of the biochemical reactions among CDKs and their antagonists.(2,3) Transitions between these steady states (G(1)-to-S and M-to-G(1)) are driven by "helper'' proteins. The fact that the transitions are irreversible is guaranteed by a "latching'' property of the molecular switches, as we have argued in previous publications.(4,5) Here, we show that if the latch is broken, then the biochemical reactions can swing back-and-forth across the transitions; either G(1)-S-G(1)-S. (periodic DNA replication without mitosis or cell division) or M-(G(1))-M-(G(1)) ... (periodic Cdc14 release, without fully exiting mitosis). Using mathematical modeling of the molecular control circuit in budding yeast, we provide a fresh account of aberrant cell cycles in mutant strains: endoreplication in the clb1-5 Delta strain(6) and periodic release and resequestration of Cdc14 (an "exit'' phosphatase) in the CLB2kd Delta strain.(7,8) In our opinion, these "endocycles'' are not autonomous oscillatory modules that must be entrained by the CDK oscillator(6,7) but rather inadvertent and deleterious oscillations that are normally suppressed by the CDK latching-gate mechanism.(8)

All eukaryotic cells depend on faithful cell division to give rise to daughter cells that contain the correct number of chromosomes. Chromosome segregation errors during meiosis in germ cells can lead to aneuploid gametes, which can cause infertility, genetic disorders such as Down Syndrome and miscarriages. Additionally, chromosome segregation errors during mitosis can lead to aneuploid somatic cells, which are a hallmark of cancer. Despite the importance of tightly regulating these two types of cell division, the mechanisms that ensure fidelity during these processes remain active areas of investigation. Proteins that have been shown to be important regulators of fidelity during meiosis in Caenorhabditis elegans (C. elegans) include ZHP-3 and PCH-2. ZHP-3 is a putative SUMO or ubiquitin ligase required for crossover recombination, but whether or not ZHP-3 is regulated to promote recombination remains poorly understood. PCH-2 is a conserved AAA+ ATPase required for timely coordination of the meiotic prophase events pairing, synapsis and recombination, but the mechanism for how it accomplishes this coordination is currently unknown. Additionally, PCH-2 has been shown to be required for both silencing and activation of the spindle assembly checkpoint during mitosis, but how it regulates the checkpoint is still an active area of investigation. Here, I use C. elegans as a model system to investigate the molecular mechanisms of these two proteins during meiotic and mitotic chromosome segregation. In Chapter 1, I investigate whether ZHP-3 is phosphorylated by a conserved DNA damage kinase, CHK-1, to determine if this phosphorylation event is required for ZHP-3’s localization or function during meiosis. I find that while ZHP-3 is phosphorylated by CHK-1 kinase in vitro, mutation of these phosphorylation sites in vivo does not affect ZHP-3’s localization to meiotic chromosomes, or its function in promoting crossover recombination. In Chapter 2, I investigate a different re