Mitotic Clusters Research Articles

A comprehensive phenotypic CRISPR-Cas9 screen of the ubiquitin pathway uncovers roles of ubiquitin ligases in mitosis

The human ubiquitin proteasome system, composed of over 700 ubiquitin ligases (E3s) and deubiquitinases (DUBs), has been difficult to characterize systematically and phenotypically. We performed chemical-genetic CRISPR-Cas9 screens to identify E3s/DUBs whose loss renders cells sensitive or resistant to 41 compounds targeting a broad range of biological processes, including cell cycle progression, genome stability, metabolism, and vesicular transport. Genes and compounds clustered functionally, with inhibitors of related pathways interacting similarly with E3s/DUBs. Some genes, such as FBXW7, showed interactions with many of the compounds. Others, such as RNF25 and FBXO42, showed interactions primarily with a single compound (methyl methanesulfonate for RNF25) or a set of related compounds (the mitotic cluster for FBXO42). Mutation of several E3s with sensitivity to mitotic inhibitors led to increased aberrant mitoses, suggesting a role for these genes in cell cycle regulation. Our comprehensive CRISPR-Cas9 screen uncovered 466 gene-compound interactions covering 25% of the interrogated E3s/DUBs.

Regulation of dynamic pigment cell states at single-cell resolution.

Cells bearing pigment have diverse roles and are often under strict evolutionary selection. Here, we explore the regulation of pigmented cells in the purple sea urchin Strongylocentrotus purpuratus, an emerging model for diverse pigment function. We took advantage of single cell RNA-seq (scRNAseq) technology and discovered that pigment cells in the embryo segregated into two distinct populations, a mitotic cluster and a post-mitotic cluster. Gcm is essential for expression of several genes important for pigment function, but is only transiently expressed in these cells. We discovered unique genes expressed by pigment cells and test their expression with double fluorescence in situ hybridization. These genes include new members of the fmo family that are expressed selectively in pigment cells of the embryonic and in the coelomic cells of the adult - both cell-types having immune functions. Overall, this study identifies nodes of molecular intersection ripe for change by selective evolutionary pressures.

Deconstructing Retinal Organoids: Single Cell RNA-Seq Reveals the Cellular Components of Human Pluripotent Stem Cell-Derived Retina.

The rapid improvements in single cell sequencing technologies and analyses afford greater scope for dissecting organoid cultures composed of multiple cell types and create an opportunity to interrogate these models to understand tissue biology, cellular behavior and interactions. To this end, retinal organoids generated from human embryonic stem cells (hESCs) were analyzed by single cell RNA‐sequencing (scRNA‐Seq) at three time points of differentiation. Combinatorial data from all time points revealed the presence of nine clusters, five of which corresponded to key retinal cell types: retinal pigment epithelium (RPE), retinal ganglion cells (RGCs), cone and rod photoreceptors, and Müller glia. The remaining four clusters expressed genes typical of mitotic cells, extracellular matrix components and those involved in homeostasis. The cell clustering analysis revealed the decreasing presence of mitotic cells and RGCs, formation of a distinct RPE cluster, the emergence of cone and rod photoreceptors from photoreceptor precursors, and an increasing number of Müller glia cells over time. Pseudo‐time analysis resembled the order of cell birth during retinal development, with the mitotic cluster commencing the trajectory and the large majority of Müller glia completing the time line. Together, these data demonstrate the feasibility and potential of scRNA‐Seq to dissect the inherent complexity of retinal organoids and the orderly birth of key retinal cell types. Stem Cells 2019;37:593–598

Cell Proliferation Patterns in Early Zebrafish Development

Although cell proliferation is an essential cell behavior for animal development, a detailed analysis of spatial and temporal patterns of proliferation in whole embryos are still lacking for most model organisms. Zebrafish embryos are particularly suitable for this type of analysis due to their transparency and size. Therefore, the main objective of the present work was to analyze the spatial and temporal patterns of proliferation during the first day of zebrafish embryo development by indirect immunofluorescence against phosphorylated histone H3, a commonly used mitotic marker. Several interesting findings were established. First, we found that mitosis metasynchrony among blastomeres could begin at the 2- to 4-cell stage embryos. Second, mitosis synchrony was lost before the midblastula transition (MBT). Third, we observed a novel pattern of mitotic clusters that coincided in time with the mitotic pseudo "waves" described to occur before the MBT. Altogether, our findings indicate that early development is less synchronic than anticipated and that synchrony is not a requirement for proper development in zebrafish.

Dispersal of Golgi matrix proteins during mitotic Golgi disassembly.

During mitosis, the mammalian Golgi disassembles into numerous vesicles and larger membrane structures referred to as clusters or remnants. Following mitosis, the vesicles and clusters reassemble to form an intact Golgi in each daughter cell. One model of Golgi biogenesis states that Golgi matrix proteins remain assembled in mitotic clusters and then serve as a template for Golgi reassembly. To test this idea, we performed a 3D-computational analysis of mitotic cells to determine the extent to which these proteins remain in mitotic clusters. As a control we used brefeldin A-induced Golgi disassembly which causes dispersal of Golgi enzymes, but leaves matrix proteins in remnant structures. Unlike brefeldin A-treated cells, in which matrix proteins were clearly sorted from non-matrix proteins, we observed extensive dispersal of matrix proteins in metaphase cells with no evidence of differential sorting of these proteins from other Golgi proteins. The extensive disassembly of matrix proteins argues against their participation in a stable template and supports a self-assembly mode of Golgi biogenesis.

Partitioning of the Golgi apparatus during mitosis in living HeLa cells.

The Golgi apparatus of HeLa cells was fluorescently tagged with a green fluorescent protein (GFP), localized by attachment to the NH2-terminal retention signal of N-acetylglucosaminyltransferase I (NAGT I). The location was confirmed by immunogold and immunofluorescence microscopy using a variety of Golgi markers. The behavior of the fluorescent Golgi marker was observed in fixed and living mitotic cells using confocal microscopy. By metaphase, cells contained a constant number of Golgi fragments dispersed throughout the cytoplasm. Conventional and cryoimmunoelectron microscopy showed that the NAGT I-GFP chimera (NAGFP)-positive fragments were tubulo-vesicular mitotic Golgi clusters. Mitotic conversion of Golgi stacks into mitotic clusters had surprisingly little effect on the polarity of Golgi membrane markers at the level of fluorescence microscopy. In living cells, there was little self-directed movement of the clusters in the period from metaphase to early telophase. In late telophase, the Golgi ribbon began to be reformed by a dynamic process of congregation and tubulation of the newly inherited Golgi fragments. The accuracy of partitioning the NAGFP-tagged Golgi was found to exceed that expected for a stochastic partitioning process. The results provide direct evidence for mitotic clusters as the unit of partitioning and suggest that precise regulation of the number, position, and compartmentation of mitotic membranes is a critical feature for the ordered inheritance of the Golgi apparatus.

Nuclear distribution of PCNA during embryonic development in Xenopus laevis: a reinvestigation of early cell cycles.

The immunocytological distribution of the proliferating cell nuclear antigen (PCNA), a protein involved in DNA replication, has been examined during the early development of Xenopus laevis. The protein is uniformly detected in nuclei during early stages up to the neurula stage. PCNA is detected by its distinctive cyclical pattern during early development, remaining detectable only during the period of S phase of each cell cycle. Immunological detection of PCNA is therefore a useful and specific non-isotopic marker of S-phase cells in the embryo. PCNA associates with typical karyomeric structures, suggesting that DNA replication starts before the nuclear compartment is entirely formed. At the midblastula transition, a new pattern of PCNA staining becomes apparent. First, a new type of PCNA staining is detected at the nuclear periphery. Second, mitotic clusters with different PCNA distributions suggest that the onset of desynchronization of the cell cycle at this stage is not random.

Clustering of Mitoses in Human Cervical Carcinoma

Mitotic nests have been noted both in tissue culture (Cone, 1968) and in human skin (Rowe and Dixon, 1975). The present paper is concerned with the disposition of mitoses in human tumor tissue. Mitotic indices, cell counts, and mitotic clusters were recorded in 20 carcinomas of the cervix uteri. The significance of the results was verified statistically. In 18 of 20 biopsies, the clustering of mitoses was in excess of chance at the 95% confidence level. Assuming a chance distribution, one would expect only one case (5% of 20 cases) to show significant clustering of mitoses. The incidence of cases which show mitotic clustering is higher in cervical carcinoma than in normal skin and uninvolved skin of psoriatic patients. The clustering phenomenon might be due to an initiating factor derived from a mitotic cell which propagates to other cells and activates them, or to a local deficiency in inhibitor factors which reversibly inhibit mitoses under normal conditions.