Quantitative comparison of a human cancer cell surface proteome between interphase and mitosis

The cell surface is the cellular compartment responsible for communication with the environment. The interior of mammalian cells undergoes dramatic reorganization when cells enter mitosis. These changes are triggered by activation of the CDK1 kinase and have been studied extensively. In contrast, very little is known of the cell surface changes during cell division. We undertook a quantitative proteomic comparison of cell surface‐exposed proteins in human cancer cells that were tightly synchronized in mitosis or interphase. Six hundred and twenty‐eight surface and surface‐associated proteins in HeLa cells were identified; of these, 27 were significantly enriched at the cell surface in mitosis and 37 in interphase. Using imaging techniques, we confirmed the mitosis‐selective cell surface localization of protocadherin PCDH7, a member of a family with anti‐adhesive roles in embryos. We show that PCDH7 is required for development of full mitotic rounding pressure at the onset of mitosis. Our analysis provided basic information on how cell cycle progression affects the cell surface. It also provides potential pharmacodynamic biomarkers for anti‐mitotic cancer chemotherapy.     

Nuclear envelope radiating microtubules in plant cells during interphase mitosis transition

Summary The microtubule distribution during the transition from interphase to the mitotic phase was examined at ultrastructural level in large highly vacuolated cells ofNautilocalyx lynchii and in small non-vacuolated cells ofPisum sativum. Both cell types contain, besides preprophase bands and perinuclear microtubules, also microtubules radiating from the nucleus into the transvacuolar cytoplasmic strands and cytoplasm respectively.This microtubule array appears to be nucleated by the cell's nuclear envelope (NE) or NE-surrounding cytoplasm.It is hypothesized that the microtubules radiating from the nucleusinitially play a role in the mobilization of the nucleus whilelater on a stabilized part of this array anchors the nucleus in the plane of cell division, and thus forms a cytoskeletal link between nucleus and division site.Our results are discussed in the light of previous work on cytoplasmic behaviour during interphase-mitosis transition in highly vacuolated plant cells.     

Localization of Nopp140 within mammalian cells during interphase and mitosis

We investigated distribution of the nucleolar phosphoprotein Nopp140 within mammalian cells, using immunofluorescence confocal microscopy and immunoelectron microscopy. During interphase, three-dimensional image reconstructions of confocal sections revealed that nucleolar labelling appeared as several tiny spheres organized in necklaces. Moreover, after an immunogold labelling procedure, gold particles were detected not only over the dense fibrillar component but also over the fibrillar centres of nucleoli in untreated and actinomycin D-treated cells. Labelling was also consistently present in Cajal bodies. After pulse-chase experiments with BrUTP, colocalization was more prominent after a 10- to 15-min chase than after a 5-min chase. During mitosis, confocal analysis indicated that Nopp140 organization was lost. The protein dispersed between and around the chromosomes in prophase. From prometaphase to telophase, it was also detected in numerous cytoplasmic nucleolus-derived foci. During telophase, it reappeared in the reforming nucleoli of daughter nuclei. This strongly suggests that Nopp140 could be a component implicated in the early steps of pre-rRNA processing.     

Histone phosphorylation in late interphase and mitosis

Histone phosphorylation in late interphase has been investigated employing cells synchronized by the isoleucine-deprivation method, followed by resynchronization at the $\text{G}{}_1\text{S}$ boundary using hydroxyurea. Phosphorylation occurred in both f1 and f2a2 as cells synchronously entered S phase following removal of hydroxyurea. The relative rates of phosphorylation of both species of histone increased in G₂-rich and metaphase-rich cultures. A small amount of histone f3 phosphorylation was also observed in M-rich cultures which was not seen in G₁, S, or G₂-rich cultures. It is concluded that f1 phosphorylation is not dependent on continous DNA replication. These experiments suggest consideration of the concept that f1 phosphorylation is initiated as a preparation for impending cell division.     

Microtubule-associated nuclear envelope proteins in interphase and mitosis

The LINC (linker of nucleoskeleton and cytoskeleton) complex forms a transcisternal bridge across the NE (nuclear envelope) that connects the cytoskeleton with the nuclear interior. This enables some proteins of the NE to communicate with the centrosome and the microtubule cytoskeleton. The position of the centrosome relative to the NE is of vital importance for many cell functions, such as cell migration and division, and centrosomal dislocation is a frequent phenotype in laminopathic disorders. Also in mitosis, a small group of transmembrane NE proteins associate with microtubules when they concentrate in a specific membrane domain associated with the mitotic spindle. The present review discusses structural and functional aspects of microtubule association with NE proteins and how this association may be maintained over the cell cycle.     

Dual functions of Nicotiana benthamiana Rae1 in interphase and mitosis

Rae1 performs multiple functions in animal systems, acting in interphase as an mRNA export factor and during mitosis as a mitotic checkpoint and spindle assembly regulator. In this study we characterized multiple functions of Rae1 in plants. Virus-induced gene silencing of Nicotiana benthamiana Rae1 , NbRae1 , which encodes a protein with four WD40 repeats, resulted in growth arrest and abnormal leaf development. NbRae1 was mainly associated with the nuclear envelope during interphase, and NbRae1 deficiency caused accumulation of poly(A) RNA in the nuclei of leaf cells, suggesting defective mRNA export. In the shoot apex, depletion of NbRae1 led to reduced mitotic activities, accompanied by reduced cyclin-dependent kinase (CDK) activity and decreased expression of cyclin B1, CDKB1-1, and histones H3 and H4. The secondary growth of stem vasculature was also inhibited, indicating reduced cambial activities. Differentiated leaf cells of NbRae1 -silenced plants exhibited elevated ploidy levels. Immunolabeling in BY-2 cells showed that NbRae1 protein localized to mitotic microtubules and the cell plate-forming zone during mitosis, and recombinant NbRae1 directly bound to microtubules in vitro . Inhibition of NbRae1 expression in BY-2 cells using a β-estradiol-inducible RNAi system resulted in severe defects in spindle organization and chromosome alignment and segregation, which correlated with delays in cell cycle progression. Together, these results suggest that NbRae1 plays a dual role in mRNA export in interphase and in spindle assembly in mitosis.     

Immunocytochemical localization of casein kinase II during interphase and mitosis

We have developed specific antibodies to synthetic peptide antigens that react with the individual subunits of casein kinase II (CKII). Using these antibodies, we studied the localization of CKII in asynchronous HeLa cells by immunofluorescence and immunoelectron microscopy. Further studies were done on HeLa cells arrested at the G1/S transition by hydroxyurea treatment. Our results indicate that the CKII alpha and beta subunits are localized in the cytoplasm during interphase and are distributed throughout the cell during mitosis. Further electron microscopic investigation revealed that CKII alpha subunit is associated with spindle fibers during metaphase and anaphase. In contrast, the CKII alpha' subunit is localized in the nucleus during G1 and in the cytoplasm during S. Taken together, our results suggest that CKII may play significant roles in cell division control by shifting its localization between the cytoplasm and nucleus.     

Architecture of the Trypanosoma brucei nucleus during interphase and mitosis

The structural basis of mitosis, spindle organisation and chromosome segregation, in the unicellular parasite Trypanosoma brucei is poorly understood. Here, using immunocytochemistry, fluorescent in situ hybridisation and electron microscopy, we provide a detailed analysis of mitosis in this parasite. We describe the organisation of the mitotic spindle during different stages of mitosis, the complex ultrastructure of kinetochores and the identification of a potential spindle-organising centre in the mitotic nucleus. We investigate the dynamics of chromosome segregation using telomeric and chromosome-specific probes. We also discuss the problems involved in chromosome segregation in the light of the fact that the T. brucei karyotype has 22 chromosomes in the apparent presence of only eight ultrastructurally defined kinetochores. Received: 9 August 1999; in revised form: 15 October 1999 / Accepted: 10 November 1999     

Ribonucleic acid transfer from nucleus to cytoplasm during interphase and mitosis in mouse somatic cells cultured in vitro

Summary Kidney cells from primary cultures of 15-day old mouse embryos were incubated for 2, 5 or 10 min with H³-uridine, then either fixed immediately or incubated again for various periods in a chase medium containing an excess of unlabeled uridine and cytidine. The number of grains over the non-nucleolar part of the nucleus (chromatin), the nucleolus and the cytoplasm were counted on the autoradiograms.The grain count showed that both chromatin and nucleolus incorporate very rapidly H³-uridine from the medium, whereas a time lag elapses before any H³-radioactivity above background is detected in the cytoplasm. Incorporation of H³-uridine into the RNA of the nucleus and the nucleolus is not immediately blocked after chase, suggesting that the labeled precursor pool is not completely washed out from the living cell, or diluted by the excess of unlabeled uridine present in the medium. The grain count over the nucleus and the nucleolus rises for a certain time after chase and then gradually declines; H³-radioactivity appears in the cytoplasm 10 min after chase and keeps rising through a 110-min interval. The experiment, then — even though it suggests that the bulk of cellular RNA is synthesized in the chromatin and the nucleolus and then continuously released into the cytoplasm — does not rule out the possibility that some RNA fraction, characterized by a low turnover rate, is synthesized independently in the cytoplasm.Synthesis of RNA is a continuous process throughout the cell cycle, except during metaphase and anaphase. It ceases at prometaphase after the disappearance of the nucleolus and disintegration of the nuclear membrane, and resumes in early telophase. Part of the chromosomal RNA does not remain associated with the chromosomes through division, but is suddenly released into the cytoplasm when the cell enters metaphase.     

Characterization and dynamics of cytoplasmic F-actin in higher plant endosperm cells during interphase, mitosis, and cytokinesis

We have identified an F-actin cytoskeletal network that remains throughout interphase, mitosis, and cytokinesis of higher plant endosperm cells. Fluorescent labeling was obtained using actin monoclonal antibodies and/or rhodamine-phalloidin. Video-enhanced microscopy and ultrastructural observations of immunogold-labeled preparations illustrated microfilament-microtubule co-distribution and interactions. Actin was also identified in cell crude extract with Western blotting. During interphase, microfilament and microtubule arrays formed two distinct networks that intermingled. At the onset of mitosis, when microtubules rearranged into the mitotic spindle, microfilaments were redistributed to the cell cortex, while few microfilaments remained in the spindle. During mitosis, the cortical actin network remained as an elastic cage around the mitotic apparatus and was stretched parallel to the spindle axis during poleward movement of chromosomes. This suggested the presence of dynamic cross-links that rearrange when they are submitted to slow and regular mitotic forces. At the poles, the regular network is maintained. After midanaphase, new, short microfilaments invaded the equator when interzonal vesicles were transported along the phragmoplast microtubules. Colchicine did not affect actin distribution, and cytochalasin B or D did not inhibit chromosome transport. Our data on endosperm cells suggested that plant cytoplasmic actin has an important role in the cell cortex integrity and in the structural dynamics of the poorly understood cytoplasm-mitotic spindle interface. F-actin may contribute to the regulatory mechanisms of microtubule-dependent or guided transport of vesicles during mitosis and cytokinesis in higher plant cells.     

Microinjection of antibodies to centromere protein CENP-A arrests cells in interphase but does not prevent mitosis

Centromere protein CENP-A is a histone H3-like protein associated specifically with the centromere and represents one of the human autoantigens identified by sera taken from patients with the CREST variant of progressive systemic sclerosis. Injection of whole human autoimmune serum to the centromere into interphase cells disrupts some mitotic events. It has been assumed that this effect is due to CENP-E and CENP-C autoantigens, because of the effects of injecting monospecific sera to those proteins into culture cells. Here we have used an antibody raised against an N-terminal peptide of the human autoantigen CENP-A to determine its function in mitosis and during cell cycle progression. Affinity-purified anti-CENP-A antibodies injected into the nucleus during the early replication stages of the cell cycle caused cells to arrest in interphase before mitosis. These cells showed highly condensed small nuclei, a granular cytoplasm and loss of their division capability. On the other hand, microinjection of nocodazole-blocked HeLa cells in mitosis resulted in the typical punctate staining pattern of CENP-A for centromeres during different stages of mitosis and apparently normal cell division. This was corroborated by time-lapse imaging microscopy analysis of mid-interphase-injected cells, revealing that they undergo mitosis and divide properly. However, a significant delay throughout the progression of mitotic stages was observed. These results suggest that CENP-A is involved predominantly in an essential interphase event at the centromere before mitosis. This may include chromatin assembly at the kinetochore coordinate with late replication of satellite DNA to form an active centromere. Received: 3 August 1998 / Accepted: 18 September 1998     

Synchronization of interphase events depends neither on mitosis nor on cdk1

Human HT2-19 cells with a conditional cdk1 mutation stop dividing upon cdk1 inactivation and undergo multiple rounds of endoreplication. We show herein that major cell cycle events remain synchronized in these endoreplicating cells. DNA replication alternates with gap phases and cell cycle-specific cyclin E expression is maintained. Centrosomes duplicate in synchrony with chromosome replication, giving rise to polyploid cells with multiple centrosomes. Centrosome migration, a typical prophase event, also takes place in endoreplicating cells. The timing of these events is unaffected by cdk1 inactivation compared with normally dividing cells. Nuclear lamina breakdown, in contrast, previously shown to be dependent on cdk1, does not take place in endoreplicating HT2-19 cells. Moreover, breakdown of all other major components of the nuclear lamina, like the inner nuclear membrane proteins and nuclear pore complexes, seems also to depend on cdk1. Interestingly, the APC/C ubiquitin ligase is activated in these endoreplicating cells by fzr but not by fzy. The oscillations of interphase events are thus independent of cdk1 and of mitosis but may depend on APC/Cfzr activity.     

Relationship between interphase AgNOR distribution and nucleolar size in cancer cells

Summary We have studied the relationship between interphase nucleolar organizer region (NOR) distribution and nucleolar size in cancer cells at light-microscopical level. Thirteen cases of formalin-fixed bladder cancer and fifteen cases of methacarn-fixed tumours of different origin were used. Nucleoli of the former cases were stained by Phloxine B and of the latter by Toluidine Blue. Selective visualization of interphase NORs was obtained by carrying out the one-step silver staining reaction for AgNOR proteins (Plotonet al., 1986). The area occupied by Phloxine B- or Toluidine Blue-stained nucleoli and interphase silver-stained NORs was measured by means of an automated image analyser. Both in bladder cancers and in the other tumour lesions nucleolar and interphase AgNOR areas were linearly related (r=0.95 and r=0.96, respectively,P<0.001). The close relationship between the area of nucleoli and that of silver-stained nucleolar structures was maintained even if the silver-staining procedure was prolonged beyond the optimal time length for selective interphase NOR staining. In the latter case, however, single interphase AgNORs were no longet visible within the nucleolar body which was, in fact, homogeneously stained. These data indicate that evaluation of the interphase AgNOR area has the same relevance, in tumour pathology, as whole nucleolar size measurement.     

Changes in the distribution of a major fibroblast protein, fibronectin, during mitosis and interphase

The distribution of a major fibroblast protein, fibronectin, was studied by immunofluorescence and immunoscanning electron microscopy in cultures of human and chicken fibroblasts during different phases of the cell cycle. The main findings were: (a) In interphase cells, the intensity of surface-associated fibronectin fluorescence correlated with that of intracellular fibronectin fluorescence. (b) The intensity of the fluorescence of both surface-associated and intracellular fibronectins was not changed in cells that were synthesizing DNA. (c) Mitotic cells had reduced amounts of surface-associated but not of intracellular fibronectin. The surface fibronectin that remained on meta-, ana-, or telophase cells had a distinct punctate distribution and was also localized to strands attaching the cells to the substratum. Fibronectin strands first reappeared on the surface of flattening cytoplasmic parts of telophase cells. (d) Fibronectin was also detected in extracellular fibrillar material on the growth substratum, particularly around dividing cells. Thus, surface-associated fibrillar fibronectin was present during G(1), S, and G(2) but in cells undergoing mitosis the distribution was altered and the amount appeared to be reduced. The observations on the distribution of surface-associated fibronectin suggest that rather than being involved in growth control this fibronectin plays a structural role in interactions of cells with the environment.