Premitotic Nuclei Research Articles

An open-source platform to quantify subnuclear foci and protein colocalization in response to replication stress

Nuclear reorganization, including the localization of proteins into discrete subnuclear foci, is a hallmark of the cellular response to DNA damage and replication stress. These foci are thought to represent transient environments or repair factories, in which the lesion is sequestered with molecules and co-factors that catalyze repair. For example, nuclear foci contain signaling proteins that recruit transducer proteins. One important class of transducers is the structure-selective endonucleases, such as SLX1-SLX4, MUS81-EME1, and XPF-ERCC1, which remove branched DNA structures that form during repair. The relocalization of structure-selective endonucleases into subnuclear foci provides a visual read-out for the presence of direct DNA damage, replication barriers, or DNA entanglements and can be monitored using fluorescence microscopy. By simultaneously probing for two or more fluorescent signals, fluorescence microscopy can also provide insights into the proximal association of proteins within a local environment. Here, we report an open-source and semi-automated method to detect and quantify subnuclear foci, as well as foci colocalization and the accompanying pixel-based colocalization metrics. We use this pipeline to show that pre-mitotic nuclei contain a basal threshold of foci marked by SLX1-SLX4, MUS81, or XPF. Some of these foci colocalize with FANCD2 and have a high degree of correlation and co-occurrence. We also show that pre-mitotic cells experiencing replication stress contain elevated levels of foci containing SLX1-SLX4 or XPF, but not MUS81. These results point towards a role for SLX1-SLX4 and XPF-ERCC1 in the early cellular response to replication stress. Nevertheless, most of the foci that form in response to replication stress contain either FANCD2 or one of the three endonucleases. Altogether, our work highlights the compositional heterogeneity of subnuclear foci that form in response to replication stress. We also describe a user-friendly pipeline that can be used to characterize these dynamic structures.

Dynein-dependent nuclear dynamics affect morphogenesis inCandida albicansby means of the Bub2p spindle checkpoint

Candida albicans, the most prevalent fungal pathogen of humans, grows with multiple morphologies. The dynamics of nuclear movement are similar in wild-type yeast and pseudohyphae: nuclei divide across the bud neck. By contrast, in hyphae, nuclei migrate 10-20 microm into the growing germ tube before dividing. We analyzed the role of the dynein-dynactin complex in hyphal and yeast cells using time-lapse fluorescence microscopy. Cells lacking the heavy chain of cytoplasmic dynein or the p150(Glued) subunit of dynactin were defective in the position and orientation of the spindle. Hyphal cells often failed to deliver a nucleus to the daughter cell, resulting in defects in morphogenesis. Under yeast growth conditions, cultures included a mixture of yeast and pseudohyphal-like cells that exhibited distinctive defects in nuclear dynamics: in yeast, nuclei divided within the mother cell, and the spindle position checkpoint protein Bub2p ensured the delivery of the daughter nucleus to the daughter cell before cytokinesis; in pseudohyphal-like cells, pre-mitotic nuclei migrated into the daughter and no checkpoint ensured return of a nucleus to the mother cell before cytokinesis. Analysis of double mutants indicated that Bub2p also mediated the pre-anaphase arrest and polarization of pseudohyphal-like cells. Thus, Bub2p has two distinct roles in C. albicans cells lacking dynein: it mediates pre-anaphase arrest and it coordinates spindle disassembly with mitotic exit.

The protein kinase kin1 is required for cellular symmetry in fission yeast

The fission yeast Schizosaccharomyces pombe is a highly polarized unicellular eukaryote with two opposite growing poles in which F-actin cytoskeleton is focused. The KIN1/PAR-1/MARK protein family is composed of conserved eukaryotic serine/threonine kinases which are involved in cell polarity, microtubule stability or cell cycle regulation. Here, we investigate the function of the fission yeast KIN1/PAR-1/MARK member, kin1p. Using a deletion allele ( kin1Δ), we show that kin1 mutation promotes a delay in septation. Kin1p regulates the structure of the new cell end after cytokinesis by modulating cell wall remodeling. Abnormal shaped interphase kin1Δ cells misplace F-actin patches and the premitotic nucleus. Thus, mitotic kin1Δ cells misposition the F-actin ring assembly site that is dependent on the position of the interphase nucleus. The resulting asymmetric cell division produces daughter cells with distinct shapes. Overexpressed kin1p accumulates asymmetrically at the cell cortex and affects cell shape, F-actin organization and microtubules. Our results suggest that correct dosage of kin1p at the cortex is required for spatial organization of the fission yeast cell.

Positioning of medial actin rings affected by eccentrically located nuclei in a fission yeast mutant having large vacuoles.

In Schizosaccharomyces pombe wild-type cells, the nucleus positions in the middle of the cell where the cortical actin ring is assembled prior to septum formation. ste12 mutants contain a few large vacuoles. In a considerable fraction of ste12 cells, the nuclei and septa were eccentrically positioned. Both extension of spindle microtubules in the anaphase and post-mitotic migration of sister nuclei to the cell poles were partially disrupted, probably due to enlarged vacuoles. In spite of the eccentric positioning of nuclei, the cortical actin ring overlays the displaced pre-mitotic nucleus. This observation supports the notion that the nucleus dictates the division site in fission yeast.

Positioning of medial actin rings affected by eccentrically located nuclei in a fission yeast mutant having large vacuoles

In Schizosaccharomyces pombe wild-type cells, the nucleus positions in the middle of the cell where the cortical actin ring is assembled prior to septum formation. ste12 mutants contain a few large vacuoles. In a considerable fraction of ste12 cells, the nuclei and septa were eccentrically positioned. Both extension of spindle microtubules in the anaphase and post-mitotic migration of sister nuclei to the cell poles were partially disrupted, probably due to enlarged vacuoles. In spite of the eccentric positioning of nuclei, the cortical actin ring overlays the displaced pre-mitotic nucleus. This observation supports the notion that the nucleus dictates the division site in fission yeast.

Immunofluorescence Study of Microtubule Organization in Some Polarized Cell Types of Selected Brown Algae

AbstractThe microtubule (Mt) organization in apical cells of Sphacelaria rigidula. as well as in branch initials of S. rigidula and Ectocarpus siliculosus, was studied by immunofluorescence. The apical interphase cells of S. rigidula show an impressive cytoskeleton of Mts, converging on the centrosome(s). A number of Mt bundles are perinuclear, but most of them run in axial orientation from the centrosomes to the cell cortex. The anterior Mt system consists of numerous thin Mt bundles, whereas the posterior system contains fewer and thicker bundles. In cells entering prophase, the cytoplasmic Mts gradually disappear. This process is somewhat faster at the posterior than at the anterior pole of the premitotic nucleus. After mitosis, the cytoplasmic Mts of the apical region appear to be re‐organized more rapidly than those of the basal part of the cell. The apical daughter nucleus retains a lobed shape and condensed chromatin for a longer time, and increases considerably in size between telophase and cytokinesis, compared to the basal one. Duplication of the centrosomes proceeds more rapidly in the anterior region of apical cells than in the basal part. During branch formation in S. rigidula and E. siliculosus, a new polarity axis is established, and the Mts extend towards the protrusion into which the nucleus migrates before mitosis. After nuclear division, one of the daughter nuclei is positioned at the tip of the branch, where the apical Mt focussing point is localized.

Laser microsurgery demonstrates that cytoplasmic strands anchoring the nucleus across the vacuole of premitotic plant cells are under tension. Implications for division plane alignment

ABSTRACT In epidermal cells of the plant Nautilocalyx lynchii, induced to divide by explantation, the nucleus undergoes a series of movements, on cytoplasmic strands, leading to construction of a division plane across the vacuole. In the early stage, the nucleus separates from the cortex, occupying an eccentric position in the cell, suspended across the vacuole by few thin strands. In the central stage, the nucleus occupies a central position anchored to the cortex by more numerous, thicker strands. Finally, the phragmosome forms as a coalescence of cytoplasmic strands across the cell, constituting the division plane within which mitosis and cytokinesis take place. The behaviour and alignment of these strands is therefore important since some are precursors of the division plane. In a previous paper (Flanders et al (1990)J. Cell Biol. 110,1111 – 1122), it was pointed out that the alignment of cytoplasmic strands showed features common to a variety of elements under tension. That is, provided they are free to move relative to the cortex, strands radiating from the nucleus should tend to seek short rather than long routes to the cortex. In this way, strands under tension would move away from distant cell corners where two of the cell’s edges make a three-way junction with a neighbouring wall. This provides a basis for the avoidance of four-way junctions and the maintenance of three-way junctions which are a characteristic feature of most plant tissues. In addition, tensile elements such as soap bubble walls contact rigid surfaces perpendicularly. Perpendicular attachment of the cell plate to the side wall is embodied in Sachs’ rule of cell division and the existence of the premitotic strands in a state of tension would provide a basis for this rule. In this study, laser microsurgery has been used to confirm that strands connecting the premitotic nucleus to the cortex are under tension since the severed ends retract immediately upon severance. However,. the response of the nucleus to the breaking of a cytoplasmic strand by laser depends upon the particular stage. The nucleus is most likely to move during the early stage when it is eccentrically placed and has few cytoplasmic strands. In the middle and phragmosomal stages the nucleus is more resistant. Computer-aided image reconstruction of anti-tubulin-labelled confocal sections indicates that the middle stage is characterised by thick microtubule (MT) bundles, which radiate from the nucleus and which seem to account for. the increasing stabilization of the nucleus. Although such strands are more difficult to sever with the laser, their ends still display elastic properties when cut. It is proposed that tension is likely to influence the alignment of strands as well as the position of the nucleus during division plane formation in vacuolated cells.

Post‐mitotic cellular reorganisation in the diatom Cymatopleura solea: The role of microtubules and the microtubule centre

AbstractCell division in Cymatopleura requires precise and major relocations of the nucleus and chioroplast which have been followed by time‐lapse cinematography and with the electron microscope. These movements are (1) the premitotic nucleus migrating to one end of the cell; (2) after cytokinesis, the daughter nuclei moving back to the cell centre, often oscillating several times while establishing their final location; (3) the single chloroplast folding over and sandwiching the central nucleus; and (4) the folded end of the chloroplast stretching back to fill the empty half of the cell. In all cases, straight, actively moving, transient strands of cytoplasm are associated with the movement of the nucleus and chloroplast, and these often appear to be pulling on the surface or the fold of the chloroplast which undergoes transient distortion.These movements are rapid and colchicine‐sensitive. Ultrastructurally, they appear to be mediated by the prominent microtubule centre (MC) and its associated cytoskeleton of microtubules (MTs) although MTs do not attach directly to either nucleus or chloroplast. The MC is located close to the moving nucleus. Later, it moves ahead of the moving chloroplast and its MTs ensheath the tip. Later still, it is seen embedded in the fold of the chloroplast. In all three situations, MTs from it are seen in the strands of cytoplasm radiating from this area across the vacuole. After these events, the MC resumes its usual interphase situation on the nuclear surface.

Cell-population analysis of the initial period of hepatocyte proliferation in the regenerating mouse liver

Morphological autoradiography was used to determine the hepatocyte percentage in the regenerating liver of mice in S-, G-, M-, post-M phases at various times after partial hepatectomy, namely at 24, 30, 36 and 42 hours. The total number of cells in the above mentioned phases turned out to be very little at 24 and 30 hours. At 36 h., the percentage of cells in S phase (labelled nuclei) as well as in G2 phase (unlabelled morphologically obvious premitotic nuclei) appeared to be high. The two alternative explanation of G2 hepatocytes emerging at 36 hours were verified to find out if they are a) that is called "a reserved G2 population", or b) a population characterized by a shorter (down to 2 or 3 h) time of S phase.

Myogenic cells of regenerating adult chicken muscle can fuse into myotubes after a single cell division in vivo

Autoradiographic studies were carried out on regenerating muscles of adult chickens. Three different muscles of hens were injured, and tritiated thymidine (1 μCi/g) was injected at various times after injury to label replicating muscle precursors. Detailed comparisons of grain counts over premitotic nuclei in samples removed one hour after injection of tritiated thymidine, and of postmitotic myotube nuclei in samples removed 10 days after injury (when labeled precursors had fused to form myotubes), revealed how many times some labeled precursors had divided before fusing into myotubes. DNA synthesis in muscle precursors was initiated 30 h after injury. Grain counts of myotube nuclei indicated that many muscle precursors labeled at the onset of myogenic cell proliferation had divided only once, or twice, before fusing into myotubes. The relationship of these in vivo results to the cell lineage model of myogenesis is discussed.

Postnatal DNA synthesis in hearts of mice: autoradiographic and cytofluorometric investigations.

Proliferative activity of cardiomyocytes in ICR mice aged from 1 to 200 days old was studied by 3H-thymidine autoradiography. To determine the outcome of cardiomyocytic DNA synthesis, the DNA content of the cardiomyocytic nuclei in different age groups was also examined by a cytofluorometric method. Flash labelling experiments with 3H-thymidine showed that the cardiomyocytes synthesised DNA most actively in the first 14 days after birth; the label was equally distributed in various parts of the heart and seen in about 8.8% of the cardiomyocytic nuclei on the tenth day. Cumulative labelling experiments for seven and 14 days showed that about 90% of the cardiomyocytic nuclei became labelled, indicating that most cardiomyocytic nuclei of young growing mice developed in this period. Twenty days after birth DNA synthesis of the cardiomyocytes rapidly decreased, the label being evident in less than 0.2%. From 100 to 200 days DNA synthesising cardiomyocytes were seen occasionally in the subendocardial region of the left ventricle (labelling index 0.04%). The cytofluorometric study showed that under normal conditions cardiomyocytes in mice contained mostly diploid nuclei, with a few double diploid nuclei considered to be premitotic nuclei. DNA synthesised cardiomyocytes in adult mice hearts usually became binucleated cells.

A model of myogenesis in vivo, derived from detailed autoradiographic studies of regenerating skeletal muscle, challenges the concept of quantal mitosis.

We have recently shown that myogenesis following severe injury is prolonged compared with minor injury (McGeachie and Grounds 1987). In this previous autoradiographic study 44 mice were injected with tritiated thymidine at various times after muscle injury (0 to 120 h), and samples were taken 9 d after injury to determine the percentage of labelled myotube nuclei. In the present study the same experimental data are analysed in detail to reveal how many times labelled muscle precursors divided before fusing to form myotubes. Additional mice were prepared and samples removed 1 h after injection of tritiated thymidine to determine the maximum grain counts of premitotic nuclei. When a labelled premitotic nucleus divides, each of the two daughter nuclei will contain half of the original label. The grain counts of nuclei resulting from sequential divisions of a maximally labelled premitotic nucleus, forms the basis for our detailed analysis which can reveal how many times a muscle precursor has divided after labelling. Nine days after injury the autoradiographic grain counts of labelled myotube nuclei were analysed in detail. The results describe an in vivo model of myogenesis which we use to evaluate quantitatively observations derived from tissue culture studies. The analysis shows that, at the onset of myogenesis in regenerating muscle (30 h after injury), muscle precursors divide only twice before fusing to form myotubes. This observation challenges the concept of quantal mitosis as defined by the tissue culture studies of Quinn et al. (1984, 1985).

Polarity and growth of caulonema tip cells of the moss Funaria hygrometrica

In the caulonema tip cells of Funaria hygrometrica, chloroplasts, mitochondria, and dictyosomes have differences in structure which are determined by cell polarity. In contrast to the slowly growing chloronema tip cells the apical cell of the caulonema contains a tip body. Colchicine stops tip growth; it causes the formation of subapical cell protrusions, redistribution of the plastids, and a loss of their polar differentiation. Cytochalasin B inhibits growth and affects the position of cell organelles. After treatment with ionophore A23 187, growth is slower and shorter and wider cells are formed. D2O causes a transient reversion of organelle distribution but premitotic nuclei are not dislocated. In some tip cells the reversion of polarity persists; they continue to grow with a new tip at their base. During centrifugation, colchicine has only a slight influence on the stability of organelle anchorage. The former polar organization of most cells is restored within a few hours after centrifugation, and the cells resume normal growth. In premitotic cells the nucleus and other organelles cannot be retransported, they often continue to grow with reversed polarity. Colchicine retards the redistribution of organelles generally and increases the number of cells that form a basal outgrowth. The interrelationship between the peripheral cytoplasm and the nucleus and the role of microtubules in maintaining and reestablishing cell polarity are discussed.

Peculiarities of changes in DNA content of Amoeba proteus nuclei during interphase : A cytofluorometric study

The generation time of the Amoeba proteus strain C was about 48 h. The relative DNA content was measured using the cytofluorometric technique in isolated nuclei over different intervals after synchronous divisions. The DNA content in the nuclei 48 h after division but prior to the next division exceeded by 2.2–2.9-fold the nuclear DNA content 1 h after division. From this it is inferred that (1) some part of the DNA is replicated in interphase more than once, and (2) not all nuclear DNA of the maternal cell is transmitted to the nuclei of daughter cells during mitosis. The DNA content of 48 h nuclei varied for different groups of amoebae taken from a mass culture at different time. This fact suggests that the DNA content in amoeba premitotic nuclei is not constant. Amplification of genes coding for ribosomal RNA (rRNA) during each amoeba cell cycle is considered as a possible interpretation.

Mitosis and cell division in some cereal rust fungi. I. Fine structure of the interphase and premitotic nuclei

The detailed structure of the nucleus and its associated suborganelles in the rust fungi Puccinia graminis tritici, P. graminis avenue, P. recondita, and P. coronata is described. The non-mitotic nuclei in intercellular hyphae of all of the fungi examined were irregularly oval in shape and had prominent nucleoli, and except for P. recondita, heterochromatin was usually uniformly dispersed. In P. recondita, densely staining patches occurred throughout the nucleus, and this was a distinguishing feature of this species. The nuclei in monokaryotic axenic cultures of P. graminis tritici and P. coronata were larger than those in their respective dikaryotic parasitic hyphae or in a dikaryotic axenic culture of P. graminis tritici.The nucleoli varied in size and composition, depending on the physiologic condition or type of cell. In senescing cells the nucleoli occupied about 10% of the nuclear volume, while in young active cells the respective volume occupied was up to 60%. In haustoria the nucleoli were smaller in size and were composed mainly of fibrillar material. In active intercellular hyphae of all of the fungi examined the nucleoli consisted of about equal granular and fibrillar regions. There was a lighter-staining central region and similar light spaces in the fibrillar zones. These light areas were similar to the nucleoplasm in appearance and were interpreted as lacunae possibly continuous with the nucleoplasm. The rust fungal nucleoli and those of some higher plants were compared.A bipolar spindle pole body (SPB) was a regular feature of non-mitotic nuclei. The SPB consisted of two disc-like structures located some distance apart on a layer of amorphous substance. The SPB was located outside the nucleus in a depression of the nuclear envelope, usually toward one side of the nucleus. Subtending the SPB in the nucleus and joined to the SPB via a large pore in the nuclear envelope was a moderately dense region which consisted of an amorphous layer from which loose threads radiated into the nucleus. Occasionally a thread connected this region to the nucleolus.In several cells the nuclei were deformed with concomitant extrusion of the nucleoli. Nucleolar extrusion was seen in all material examined, and the process is described. Nuclear deformation and nucleolar extrusion were considered to be indicators of premitotic nuclei.

Cell proliferation in embryonic mouse neural tube following urethane exposure

A comparative autoradiographic study of the cell cycle in normal 10-day mouse embryos and in embryos exposed to urethane showed an increase of 4.5 hours in the cycle time of the experimental group. Initially urethane interfered with the migration of some unlabeled premitotic nuclei to the germinal zone, resulting in extraventricular mitoses. A transient increase in the mitotic index and proportion of metaphases suggested a lengthened mitotic time but this was of brief duration and would not contribute significantly to the overall increased cycle time. The G 2 period of cells which incorporated thymidine- 3H in the presence of urethane was significantly lengthened as were the S and G 1 periods and some injured cells died in interphase or mitosis. The new lengthened generation time of embryo neural tubes exposed to urethane resembles that of older, 11-day embryos. This suggests the possibility that cells with a short cycle may be preferentially killed by urethane, leaving the determination of mean cycle time to more differentiated cells.

Preprophase microtubules and stomatal differentiation; some effects of centrifugation on symmetrical and asymmetrical cell division

Young wheat seedlings were centrifuged; premitotic and dividing cells of the leaf epidermis were examined. During the normal symmetrical, and also the first asymmetrical division (forming future guard mother cells), preprophase nuclei resisted sedimentation to a varying extent, dependent somewhat on the size of the preprophase band of microtubules. At other stages of these divisions, the spindle invariably sedimented, with accompanying structural disruption. During the second asymmetrical division (forming the subsidiary cell of the stomatal complex), all stages of division were highly polarized, and one pole remained adjacent to the GMC. Evidence is also presented which indicates that preprophase microtubules move intact into the spindle. The preprophase band is not considered functional specifically in orienting or positioning the premitotic nucleus; it appears to represent a gathering of future spindle structures, prior to these being incorporated, many intact, into the mitotic apparatus. Most spindle microtubules appear structurally altered following the centrifugal procedures. Telophase structures resist disruption more than other stages of division. Some evidence suggests that the subsidiary cell nucleus and cytoplasm at telophase are primarily responsible for the formation of the new, highly curved cell wall. Nucleolar material appeared between chromosome arms in the disrupted spindles, even at prophase and metaphase.

Effects of carbon tetrachloride on mitosis.

WE have observed that carbon tetrachloride produces striking effects on Allium cepa, Allium sativum and Zaphranthus sp. root tips. Root tips were treated for five minutes to a half-hour in carbon tetrachloride and later transferred to normal nutrient medium and fixed at various intervals from half an hour to six hours. Among the prominent changes taking place in the resting nucleus is a dissolution of the nuclear membrane and an outflow of its contents. Similar observations have been made in root tips of Allium cepa treated with chloroform1 and potassium cyanide2. It would appear that the lipoid layer of the nuclear membrane is either dissolved or rendered weak by the chemicals, in consequence of which the nuclear contents flow out. After treatment for 15 min. with carbon tetrachloride, resting and premitotic nuclei break up. A number of chemicals are known to produce this effect, for example, urethane3 and nitrogen mustard4. Perhaps the most characteristic changes brought about by carbon tetrachloride are dissolution of chromatin in the resting nucleus and disorganization of the chromosome arms in the premitotic stages, resulting in their progressively negative reaction to the Feulgen reagent.