Detection of intranuclear forces by the use of laser optics during the recovery process of elongated interphase nuclei in centrifuged protenemal cells ofAdiantum capillus-veneris

For the direct investigation of intranuclear dynamics in living cells, extremely deformed nuclei of basipetally centrifuged protonemal cells of the fernAdiantum capillus-veneris were manipulated by the laser trap and the laser scalpel. Whereas the nucleolus was tightly fixed at the central position inside the non-centrifuged nucleus and proved to be immovable by the optical trap, it could easily be trapped and moved towards three directions inside the bubble-like terminal widening of the basal thread-like extension of centrifuged nuclei. Due to the connection of the nucleolus to the chromatin inside the nuclear thread (NT), moving was not possible against the direction of the nuclear apical main body. Nucleoli in recovered nuclei were again immovable, thus indicating the presence of a dynamic nucleolar anchoring system inside the nucleus. When the nucleolus in the bubble was arrested during the thread shortening process by the optical trap, the acropetal movement of the bubble continued. Probably due to dragging forces, some nucleoli became stretched, and a thick strand of a still unknown composition stretched between the nucleolus and the insertion site of the shortening NT. To assess whether the shrinking of the nuclear envelope (NE) and the shortening of the chromatin inside the NT were independent processes, the chromatin above the bubble was cut inside the NT by the laser scalpel. After severance, a gap between the nucleolus and the end of the chromatin strand in the NT indicated the shortening of the chromatin inside the NT. From these findings it was concluded that a shortening force was existing in the chromatin of the NT and that probably no physical link existed between the chromatin and the NE.     

Interaction of rat sertoli cells with a collagen lattice in vitro

Summary Sertoli cells from rats aged 15, 20, and 25 d were subcultured onto collagen-coated, plastic dishes. If the collagen was released from the plastic surface by rimming, the floating rats of collagen showed uniform shrinkage. If the collagen was allowed to remain attached to the plastic, holes appeared in the collagen with cells from rats aged 25 d but not with those of 15 d. Cells from rats aged 20 d caused fewer and smaller holes to appear. The holes were associated with the formation of clumps of spherical cells from which elongated Sertoli cells extended into the surrounding collagen to end near holes. Rhodamine-phalloidin revealed diffusely distributed actin in the spherical cells in contrast to well-developed microfilaments in the peripheral elongated cells. Addition of cytochalasin B (5 μg/ml) to the medium prevented contraction of the floating rats and the development of holes in the attached collagen. In addition, cytochalasin B caused the peripheral cells to become spherical and to separate from the clumps. Moreover, rhodamine-phalloidin revealed that actin in the peripheral cells occurred as clumps without microfilaments when cytochalasin B was present. When Sertoli cells were subcultured onto silicone rubber films, the cells produced wrinkling of the rubber surface within 4 h of plating. These observations were interpreted to mean that Sertoli cells exert local tractional forces on various substrata. These forces require actin, presumably acting by a contractile mechanism. When the collagen is attached to plastic and the cells are organized into clumps with radiating elongated cells (cells from rats aged 25 d), the tractional forces in the elongated cells reorganize the collagen fibers to produce holes. When cells are uniformly distributed (cells from rats aged 15 d), holes are not formed. When the collagen is released from the plastic surface, tractional forces cause the floating rafts to shrink. These interactions of the cells with collagen are likely to be important in determining the shape of the Sertoli cell in vivo, the polarity of the cell, and its biochemical differentiation. This investigation was supported by grants HD 16525, AM 32236, and GM 32705 from the National Institutes of Health, and from the Shriners of North America.     

The Roles of the Cytoskeleton and the Cell Wall in Nuclear Positioning in Tobacco BY-2 Cells

During the course of cell-wall regeneration in protoplasts isolatedfrom tobacco BY-2 cells, the nucleus changed its position fromthe central region to the cell periphery. This nuclear migrationwas inhibited by 2,6-dichlorobenzonitrile (DBN), suggestingthe involvement of cell walls in nuclear migration in tobaccoBY-2 cells. In spherical cells formed by culturing protoplasts in the presenceof DBN or propyzamide, the nucleus was located in the centralregion of the cells and was tethered by transvacuolar cytoplasmicstrands. Nuclei in the spherical cells were displaced by disruptingthe actin filaments in the cytoplasmic strands by treating thecells with cytochalasin B (CB), suggesting that the positionof the nucleus in the spherical cells is maintained by actinfilaments. As the nuclei were located in the central regionof the cells even in the presence of propyzamide, microtubulesseem not to be involved in nuclear positioning in the sphericalcells. Actin filaments, but not microtubules, also seem to play animportant role in nuclear positioning in elongated cells. Inthese cells, CB greatly enhanced the displacement of the nucleusby centrifugation, while propyzamide showed little effect. (Received July 22, 1987; Accepted January 15, 1988)     

Growth rate,labeling index,and radiation survival of cells grown in the matrigel threadin vitro tumor model

Summary Six rodent cell lines (36B10 rat glioma cells, 9L rat gliosarcoma cells, V79 Chinese hamster lung fibroblasts, EMT6/UW and EMT6/Ro mouse mammary sarcoma cells, and RIF-1 mouse fibrosarcoma cells) were tested for growth in cylindrical threads of Matrigel. These cells grew in the threads with doubling times of 17–23 h, reaching maximum cell densities on the order of 10⁸ cells/ml. Histological sections of these threads showed a heterogeneous cell distribution: cells grew to confluence at the thread surface and at somewhat lower cell densities in the thread core. [H-3]thymidine labeling index and radiation sensitivity were measured for 9L and EMT6/UW cells in Matrigel threads. For both cell types, the labeling index in Matrigel was lower than observed in cell monolayers, with higher labeling indexes at the thread periphery than in the thread core. When these threads were grown in stirred medium, lower thread diameters, higher cell yields per thread, and higher labeling indices were obtained. EMT6 cell monolayers coated with Matrigel were less radiosensitive than cells in uncoated monolayers. This protective effect was eliminated by irradiating in the presence of 1 mg/ml misonidazole. EMT6 cells consume nearly three times as much oxygen (mole/cm³-sec) as do 9L cells, which are equally radiosensitive in monolayers with or without a Matrigel coating. The radiation sensitivity of EMT6/UW cells in Matrigel threads was similar to that for monolayers of plateau phase cells, whereas for 9L cells, the response in threads was more similar to exponentially growing cells. We conclude that Matrigel threads provide an alternativein vitro model for studying the radiation response of cells in a three-dimensional geometry.     

Development of the sperm head in the caddis fly Potamophylax rotundipennis (Insecta,Trichoptera)

The restructuring of the sperm head has been examined in a caddis fly, Potamophylax rotundipennis (Limnephilidae), using light and electron microscopy. The roughly spherical nuclei of young spermatids are transformed into needle-shaped elements in advanced spermatids. During this process, the nuclei transiently become sickle-shaped. Prominent structural changes occur within the nucleus during spermiogenesis. The chromatin of spherical and slightly elongated nuclei has an amorphous appearance, then coarse granules become apparent, chromatin threads are visible in fully elongated nuclei and finally lamellar elements appear. During the changes in chromatin texture, a dense layer, the chromatin rim, develops transiently. This feature of the chromatin surface is interpreted as the structural expression of exchanges between nucleus and cytoplasm. A microtubular manchette is formed at the cytoplasmic face of the nuclear envelope. Whereas the manchette covers the full perimeter of the nucleus in early stages of elongation, gaps in the palisade of microtubules appear before the nuclear diameter decreases and needle-shaped nuclei develop. It is possible that the intermittent deployment of manchette microtubules is involved in reducing the nuclear diameter towards the end of nuclear elongation. The delayed detachment of the chromatin from the posterior pole of the nucleus, observed at the onset of nuclear clongation, points to local modifications of the nuclear envelope responsible for the connection of the centriole adjunct and the flagellum with the posterior pole of the nucleus.     

VEGETATIVE NUCLEAR DIVISION IN NEUROSPORA

A re-examination of the mode of vegetative nuclear division in Neurospora crassa was facilitated by the availability of the mutant “clock” which produces definite growth bands. Since the growth rhythm is correlated with nuclear divisions, stained mycelial mats of this mutant prepared at intervals from the beginning of a growth period provided a sequence of stages of division. In a 28-hour period the following broad features of nuclear behavior were observed: In the early part of the period during rapid mycelial growth, dividing elongated nuclei predominated. At the end of the period the mycelium contained mostly rounded resting nuclei. In the middle of a growth period nuclear forms of various degrees of annularity occurred along with elongated and rounded nuclei. Elongated and rounded nuclei completed division cycles without change in form, although the corresponding stages of the two types were similar. Elongated nuclei assumed a spiral form at the beginning of division. As division proceeded, relaxation of the nuclear gyres was accompanied by a visible duplication of the chromatin thread and the appearance of chromomere-like bodies on the daughter threads. One of the chromomere-like bodies became displaced and was interpreted to be a chromosome or a segment of a chromosome that acts as a mitotic center. All the chromosomes were found to be interconnected and to act as a unit throughout the division cycle. Only after the separation of the daughter chromatin threads could seven chromosomes be counted. Electron microscopic studies complemented the observations with the light microscope. On the basis of the evidence it was concluded that the vegetative nuclear division in Neurospora differs from the classical mitotic pattern in the following respects: (1) absence of visible centrioles, (2) the presence of interconnected chromosomes, (3) the comparatively late appearance of countable chromosomes, and (4) the frequent presence of interzonal connections between separating chromatin threads.     

Cell cycle and cell protein content of Chinese hamster cells grown in culture with daily renewal of medium

The effects of cytochalasin B on cytokinesis, karyokinesis and DNA synthesis of various cells transformed by DNA viruses (SV40, polyoma and adeno 12) and of non-transformed cells were studied. Cytokinesis of all cell lines tested was completely inhibited by cytochalasin B at the concentration 0.5–2.0 μg/ml. After treatment by cytochalasin B, non-transformed cells became bi- or trinucleated without the division of cytoplasm. Three of the virally transformed cells also became bi- or trinucleated with a small number of multinucleate cells. On the other hand, in two SV40-transformed mouse cells, the number of nuclei per cell increased significantly and cells with 5–10 nuclei were frequently observed. Upon removal of cytochalasin B, cytoplasmic division recovered rapidly and consequently mono- or binucleate cells were formed. In all transformed cells, DNA synthesis was not inhibited by cytochalasin B, while DNA synthesis was inhibited in non-transformed cells.     

Opposing microtubule- and actin-dependent forces in the development and maintenance of structural polarity in retinal photoreceptors

We have used embryonic cells grown in vitro to study the roles of microtubules and microfilaments in the development and maintenance of the polarized shape of retinal photoreceptors. After several days in culture, isolated cone photoreceptors displayed a highly elongated, compartmentalized morphology similar to that of photoreceptors in vivo. When treated with the microtubule-depolymerizing agent nocodazole, these elongated photoreceptors became progressively shorter, eventually losing their compartmentalized structure and becoming round. Conversely, treatment with the actin-depolymerizing agent cytochalasin D caused the elongated photoreceptors to lengthen even further. Computer-assisted, quantitative analysis showed that responses of individual cells to both nocodazole and Cytochalasin D were concentration-dependent, graded, and reversible. Immunocytochemical studies suggested the presence of longitudinally oriented actin filaments and microtubules in these photoreceptors, prominent in the region that undergoes the most pronounced length changes in response to cytoskeletal inhibitors. Prior to becoming elongated, photoreceptor precursors could be accurately identified in early retinal cultures. These round cells undergo a stereotyped sequence of morphogenetic transformations during in vitro development, including elongation and compartmentalization of the cell body as well as extension of a single neurite. Treatment with either cytochalasin D or nocodazole completely blocked morphogenesis. In addition, cytochalasin D caused the development of an abnormal, elongated cell process, which formed by a microtubule-dependent mechanism. These nocodazole and cytochalasin D effects also were reversible. Taken together, these data indicate that the complex developmental transformations leading to photoreceptor polarization occur in the absence of intercellular contacts, and are predominantly controlled by intracellular cytoskeletal forces. They suggest the existence of continuously active, oppositely directed, microtubule- and actin-dependent forces, the balance of which is a determining factor in the development as well as the maintenance of the elongated, compartmentalized organization of photoreceptor cells.     

Evidence for the nucleus-centriole association in living cells obtained by ultracentrifugation

The association of centrioles with the interphase nuclei of L- and PE-cells has been studied using ultracentrifugation of a cell monolayer in a culture medium at 20 and 37 degrees C. Ultracentrifugation at 10 000 to 40 000 gav for 15 to 60 min did not cause any changes in the cell length or in the size of the nucleus, but entailed delocalization of nuclei. The distance between it and the centrioles hardly ever changes. Any nucleus-delocalizing centrifugation of non-treated cells also resulted in the centrioles being shifted towards the centripetal nucleus pole. After 30 to 60 min, at 40 000 gav, the cells remained viable and capable of mitosis. More intensive centrifugation (15 min at 70 000 gav) proved to be fatal to the cells. The distance between the centrioles and the nucleus became greater in the cells which were centrifuged after incubation with cytochalasin B. The results are interpreted as lending support to the previously demonstrated [28] association between the centrioles and the nucleus in the interphase cells.     

Chemotactic activity of the peritoneal lavage fluid of guinea pigs with inflammatory processes caused by the inoculation of various types of suture thread

Minced non adsorbable or adsorbable suture threads introduced into peritoneal cavity of guinea pigs elicit at inflammation with mononuclear and giant cells surrounding suture thread fragments. We studied the presence in the peritoneal cavity of chemotactic factors for PMN cells and we compared the results in relation to the different type of the suture threads used (Dexon, Mersilene, Gore-Tex). The peritoneal cavity was washed, the fluids collected and used as chemotactic agents. The chemotactic response was assayed by employing multiwell chemotaxis chambers (Neuro Probe) and PMNs from normal, non-treated guinea pigs. Quantification of the migration was calculate by chemotactic index (A/B) (B = random migration, A = chemotaxis). The results demonstrate that a chemotactic activity is present in peritoneal fluids following the inflammatory process. This activity is evident at 7th day after Dexon and Mersilene inoculation; using PTFE however, it decreases at 14th d, when the inflammatory process is already developing into healing tissue. In conclusion the chronic inflammation determines the appearance of chemotactic factors for PMN cells; it is suggested that reactive, mononuclear cells, involved in the process, could be responsible for their production and release.     

红豆草根瘤侵入线的超微结构研究

用透射电镜对红豆草根瘤侵入线的超微结构进行了观察研究.结果表明,(1)红豆草根瘤侵入线由胞间隙和胞间层细胞壁内陷形成,它们的体积较小,多为管状,基质丰富,含菌很少,常有分叉和1个以上的基质区,而且不同基质区的电子密度、细菌数量和侵入线壁厚度都不相同.(2)红豆草根瘤的侵入线十分丰富,它们不仅大量存在于根瘤分生细胞和幼龄侵染细胞中,也经常出现在发育成熟的侵染细胞内.(3)红豆草根瘤中有一种近似圆形的特殊结构,表面由一层膜包围,其内电子密度较低且无固定结构,且只位于侵染细胞的细胞质中,常在侵入线附近,从不出现在侵染细胞的液泡内和非侵染细胞里面.     

Nuclear actin and myosin as control elements in nucleocytoplasmic transport

Fluorescence redistribution after photobleaching (FRAP) was used to examine the role of actin and myosin in the transport of dextrans through the nuclear pore complex. Anti-actin antibodies added to isolated rat liver nuclei significantly reduced the flux rate of fluorescently labeled 64-kD dextrans. The addition of 3 mM ATP to nuclei, which enhances the flux rate in control nuclei by approximately 250%, had no enhancement effect in the presence of either anti-actin or anti-myosin antibody. Phalloidin (10 microM) and cytochalasin D (1 micrograms/ml) individually inhibited the ATP stimulation of transport. Rabbit serum, anti-fibronectin, and anti-lamins A and C antibodies had no effect on transport. These results suggest a model for nuclear transport in which actin/myosin are involved in an ATP-dependent process that alters the effective transport rate across the nuclear pore complex.     

The role of the cytoskeletal elements during the return to the cell center of their nuclei displaced by centrifugation]

Living cells of the monolayer cultures of embryonal pig kidney epithelium (PKE-cells) and of embryonal bovine tracheal cells (FBT-cells) were ultracentrifuged at 20,000g. The centrifugal force was directed parallel to the surface of the culture slides. Just after centrifugation, the cellular nuclei were displaced to the centrifugal parts of cells. Centrifuged slides with cells were returned to the normal culture conditions, and 22 h later the nuclei were seen to restore their central position in the cells. The motion of the nuclei to the cell center was rather chaotic both in direction and speed. The speed of this motion never exceeded several microns per hour. After nocodasole treatment (0.1-10 mkg/ml) of the cells or in a hypotonic medium, the distance of nuclear dislocation during centrifugation was longer, and the nuclei returned to the cell centers faster than in the control ones. After cytochalasin B treatment (2 mkg/ml), the nuclei moved to the cell centers somewhat more slowly than they did in the control cells. Thus, the establishment of the central position of nuclei in the cells takes place in the absence of microtubules or intermediate filaments. Probably, the central position of nuclei depends mainly on the action part of the cytoskeleton.     

Degradation of nuclear proteins: studies on transplanted B82 cell karyoplast proteins

Karyoplasts were prepared from B82 cells (thymidine kinase deficient mouse L cells) by cytochalasin B mediated enucleation. Morphological measurements show that the nucleus constitutes 89% of a karyoplast by volume. Homokaryons were obtained by Sendai virus mediated karyoplast-B82 cell fusion. Transplanted nuclei were not destroyed catastrophically but were maintained intracellularly for at least 140 h. Transplanted nuclear proteins were degraded with an average half-life of 84 +/- 7 h by processes partially sensitive to inhibition by NH4Cl (50%) and leupeptin (30%). The data therefore suggest that some nuclear proteins are translocated to the cytoplasm for lysosomal degradation.     

Cytochalasin induces spindle fusion in the syncytial blastoderm of the early Drosophila embryo.

Microfilament integrity is needed to maintain the regular arrangement of the spindle microtubules and to guarantee the normal progression of the last syncytial mitoses in Drosophila embryo. To investigate when and how microfilaments participate in this process, we incubated permeabilized embryos with the inhibitor of actin polymerization, cytochalasin B, at different times during the nuclear cycle. Our results suggest that the correct microfilament distribution is only required for the appropriate segregation of nuclei during the 11th, 12th and 13th syncytial mitoses rather than during the 10th mitosis when the spindles are too far apart to interact. When cytochalasin B treatment was performed during the last syncytial mitoses many spindles fuse among them and the regular mitotic progression is perturbed.     

Tumor Cells Genetically Labeled with GFP in the Nucleus and RFP in the Cytoplasm for Imaging Cellular Dynamics

Dual-color fluorescent cells with one color fluorescent protein in the nucleus and another color fluorescent protein in the cytoplasm were genetically engineered. The dual-color cancer cells enable real-time nuclear-cytoplasmic dynamics to be visualized in living cells in vivo as well as in vitro. To obtain the dual-color cells, red fluorescent protein (RFP) was expressed in the cytoplasm of a series of human and rodent cancer cells, and green fluorescent protein (GFP) linked to histone H2B was expressed in the nucleus. Nuclear GFP expression enabled visualization of nuclear dynamics, whereas simultaneous cytoplasmic RFP expression enabled visualization of nuclear-cytoplasmic ratios as well as simultaneous cell and nuclear shape changes. Using the Olympus OV100 Whole-Mouse Imaging System, total sub-cellular dynamics can be visualized in the living dual-color cells in real time in the live mouse after cell injection. Highly elongated cancer cells and nuclei in narrow capillaries were visualized where both the nuclei and cytoplasm deform. Both cytoplasm and nuclei were visualized to undergo extreme deformation during extravasation with cytoplasmic processing exiting vessels first and nuclei following along these processes. The dual-color cells described here thus enable the sub-cellular dynamics of cancer cell trafficking to be imaged in the living animal.     

Effects of cytochalasin B on the cortex of the unfertilized sea urchin egg

The peripheral cytoplasm of the unfertilized sea urchin egg contains approximately 18,000 cortical granules. These granules remain monolayered within the normal boundaries of the cortex when the egg is centrifuged at forces sufficient to stratify other intracellular inclusions. Exposure of unfertilized eggs to the microfilament disrupting agent, cytochalasin B (CB) causes the granules to rearrange into several layers and occasionally to undergo exocytosis or break down in situ. When these eggs are centrifuged, the cortical granules are dislodged from the cortex and migrate centrifugally among the densest intracellular components. In addition, cytoplasmic inclusions, which normally are excluded from the cortex, impinge directly upon the egg plasma membrane in CB-treated, centrifuged eggs. These results are consistent with the existence of a microfilamentous network which confines the cortical granules within and excludes other intracellular inclusions from the cortex of the unfertilized egg.     

Role of multipolar mitoses in the proliferation of multinucleate cells induced by cytochalasin B

A prolonged action of cytochalasin B results in the formation of numerous multipolar mitoses (26%) in Chinese hamster cell cultures. The transition to multipolar mitoses in the presence of cytochalasin B is not accompanied by K-mitotic delay. It is shown that a multipolar mitosis without cytoplasmic division is one of the main causes of multinucleation development in cytochalasin B-treated cultures. After stopping the drug action the cytochalasin B-induced multinucleate cells continue to divide by multipolar mitosis. In this case it completes with cytokinesis and, probably, leads to a decrease in the number of nuclei per cell. The origin of multipolar mitotic apparatus after the action of cytochalasin B is discussed in addition to the role of multipolar mitosis in formation and proliferation of multinucleate cells.     

Blue light-induced branching in Vaucheria. Requirement of nuclear accumulation in the irradiated region

When a narrow region of the fresh water coenocytic alga, Vaucheria terrestris sensu G?tz is irradiated with moderately intense blue light, a branch is induced from the center of the irradiated region after 4-5 h. Movement of organelles and microtubule bundles during the photocytomorphogenetic response were investigated. Chloroplasts in the cortical layer immediately started to accumulate in the blue light-irradiated region and their accumulation almost completely finished 30-40 min after the onset of light when the nuclei residing in endoplasm started to accumulate. Accumulation of nuclei was synchronized with disorientation and shortening of microtubule bundles, which originally run parallel to the cell axis. Not only amiprophos-methyl, a potent microtubule-decomposing reagent, but also cytochalasin A strongly inhibited the branch induction. Amiprophos-methyl completely and cytochalasin A mostly destroyed microtubules and completely inhibited nuclear accumulation, but both drugs allowed the accumulation of chloroplasts in the cortical layer of irradiated region. These indicate that the accumulation of nuclei is indispensable for branch induction while the chloroplast accumulation is insufficient by itself for branch induction. The ineffectiveness of cytochalasin A on chloroplast movement brings the conventional view of sliding movement of chloroplast on a long actin cable into question. The morphological and functional relationship between a nucleus and a microtubular bundle are discussed.     

Degradation of short-lived proteins is decreased by centrifugation

We have examined the effects of enucleation and of inhibitors of mRNA synthesis (actinomycin D and cordycepin) on protein turnover of HeLa cells. Enucleation markedly inhibited the rate of protein degradation for short-lived proteins. However, cells centrifuged in the absence of cytochalasin B at the speed required to obtain cytoplasts showed protein degradation rates identical to those of cytoplasts, while inhibitors of mRNA synthesis did not affect the process. Although enucleation may affect degradation of specific proteins, these results suggest that centrifugation is largely responsible for the inhibition of protein degradation in cytoplasts.