Laser microsurgery in fission yeast; role of the mitotic spindle midzone in anaphase B

INTRODUCTION: During anaphase B in mitosis, polymerization and sliding of overlapping spindle microtubules (MTs) contribute to the outward movement the spindle pole bodies (SPBs). To probe the mechanism of spindle elongation, we combine fluorescence microscopy, photobleaching, and laser microsurgery in the fission yeast Schizosaccharomyces pombe. RESULTS: We demonstrate that a green laser cuts intracellular structures in yeast cells with high spatial specificity. By using laser microsurgery, we cut mitotic spindles labeled with GFP-tubulin at various stages of anaphase B. Although cutting generally caused early anaphase spindles to disassemble, midanaphase spindle fragments continued to elongate. In particular, when the spindle was cut near a SPB, the larger spindle fragment continued to elongate in the direction of the cut. Photobleach marks showed that sliding of overlapping midzone MTs was responsible for the elongation of the spindle fragment. Spindle midzone fragments not connected to either of the two spindle poles also elongated. Equatorial microtubule organizing center (eMTOC) activity was not affected in cells with one detached pole but was delayed or absent in cells with two detached poles. CONCLUSIONS: These studies reveal that the spindle midzone is necessary and sufficient for the stabilization of MT ends and for spindle elongation. By contrast, SPBs are not required for elongation, but they contribute to the attachment of the nuclear envelope and chromosomes to the spindle, and to cell cycle progression. Laser microsurgery provides a means by which to dissect the mechanics of the spindle in yeast.     

Ultraviolet microbeam irradiations of mitotic diatoms: investigation of spindle elongation

Our simple instrumentation for generating a UV-microbeam is described UV microbeam irradiations of the central spindle in the pennate diatom Hantzschia amphioxys have been examined through correlated birefringence light microscopy and TEM. A precise correlation between the region of reduced birefringence and the UV-induced lesion in the microtubules (MTs) of the central spindle is demonstrated. The UV beam appears to dissociate MTs, as MT fragments were rarely encountered. The forces associated with metaphase and anaphase spindles have been studied via localized UV-microbeam irradiation of the central spindle. These spindles were found to be subjected to compressional forces, presumably exerted by stretched or contracting chromosomes. Comparisons are made with the results of other writers. These compressional forces caused the poles of a severed anaphase spindle to move toward each other and the center of the cell. As these poles moved centrally, the larger of the two postirradiational central spindle remnants elongated with a concomitant decrease in the length of the overlap. Metaphase spindles, in contrast, did not elongate nor lose their overlap region. Our interpretation is that the force for anaphase spindle elongation in Hantzschia is generated between half-spindles in the region of MT overlap.     

Cytology of lepidoptera. VI. Immunolocalization of microtubules in detergent-extracted apyrene spermatocytes of Ephestia kuehniella Z

Apyrene meiosis was studied in two wild-type strains, L and Sbr, of the Mediterranean mealmoth, Ephestia kuehniella, using anti-tubulin immunofluorescence. The observations were supplemented by phase-contrast light microscopy of living spermatocytes from strain L. The study revealed that nuclear envelope breakdown, centrosome separation, migration of chromatin elements towards the poles, and spindle elongation also occur in apyrene spermatocytes. However, a conventional metaphase plate is never formed, and chromatin segregation is irregular and delayed. Chromosome laggards are frequent. As a rule, apyrene spindles have a low microtubule content. The two strains, L and Sbr, differ regarding the chromatin behavior during meiosis. In strain L, the developing spindles contain numerous small chromatin clumps which segregate asynchronously. The resulting daughter cells possess about the same amount of chromatin. In contrast, large chromatin clumps exist in strain Sbr at the onset of spindle formation. The chromatin blocks transiently occupy an equatorial position and elongate subsequently parallel to the spindle axis. These elongated chromatin bodies often divide highly unequally. As a consequence, secondary spermatocytes in strain Sbr differ greatly in chromatin content. Subjective assessment shows that the size of the microtubular cytoskeleton is positively correlated with the chromatin content of the cell. Hence, it is hypothesized that the chromatin content determines spindle size. This possibly comes about the number of available kinetochores which are exposed and able to stabilize microtubules of centrosomal origin attached with the kinetochores. However, a direct bearing of chromatin on spindle size is similarly conceivable. Other Lepidoptera species examined so far are compatible with a 'type L' or a 'type Sbr' pattern of apyrene meiosis.     

The mechanism of anaphase spindle elongation: uncoupling of tubulin incorporation and microtubule sliding during in vitro spindle reactivation

To study tubulin polymerization and microtubule sliding during spindle elongation in vitro, we developed a method of uncoupling the two processes. When isolated diatom spindles were incubated with biotinylated tubulin (biot-tb) without ATP, biot-tb was incorporated into two regions flanking the zone of microtubule overlap, but the spindles did not elongate. After biot-tb was removed, spindle elongation was initiated by addition of ATP. The incorporated biot-tb was found in the midzone between the original half-spindles. The extent and rate of elongation were increased by preincubation in biot-tb. Serial section reconstruction of spindles elongating in tubulin and ATP showed that the average length of half-spindle microtubules increased due to growth of microtubules from the ends of native microtubules. The characteristic packing pattern between antiparallel microtubules was retained even in the "new" overlap region. Our results suggest that the forces required for spindle elongation are generated by enzymes in the overlap zone that mediate the sliding apart of antiparallel microtubules, and that tubulin polymerization does not contribute to force generation. Changes in the extent of microtubule overlap during spindle elongation were affected by tubulin and ATP concentration in the incubation medium. Spindles continued to elongate even after the overlap zone was composed entirely of newly polymerized microtubules, suggesting that the enzyme responsible for microtubule translocation either is bound to a matrix in the spindle midzone, or else can move on one microtubule toward the spindle midzone and push another microtubule of opposite polarity toward the pole.     

Microtubule organization during maturation of Xenopus oocytes: assembly and rotation of the meiotic spindles.

Assembly of the meiotic spindles during progesterone-induced maturation of Xenopus oocytes was examined by confocal fluorescence microscopy using anti-tubulin antibodies and by time-lapse confocal microscopy of living oocytes microinjected with fluorescent tubulin. Assembly of a transient microtubule array from a disk-shaped MTOC was observed soon after germinal vesicle breakdown. This MTOC-TMA complex rapidly migrated toward the animal pole, in association with the condensing meiotic chromosomes. Four common stages were observed during the assembly of both M1 and M2 spindles: (1) formation of a compact aggregate of microtubules and chromosomes; (2) reorganization of this aggregate resulting in formation of a short bipolar spindle; (3) an anaphase-B-like elongation of the prometaphase spindle, transversely oriented with respect to the oocyte A-V axis; and (4) rotation of the spindle into alignment with the oocyte axis. The rate of spindle elongation observed in M1 (0.7 microns min-1) was slower than that observed in M2 (1.8 microns min-1). Examination of spindles by immunofluorescence with antitubulin revealed numerous interdigitating microtubules, suggesting that prometaphase elongation of meiotic spindles in Xenopus oocytes results from active sliding of antiparallel microtubules. A substantial number of maturing oocytes formed monopolar microtubule asters during M1, nucleated by hollow spherical MTOCs. These monasters were subsequently observed to develop into bipolar M1 spindles and proceed through meiosis. The results presented define a complex pathway for assembly and rotation of the meiotic spindles during maturation of Xenopus oocytes.     

Physiological evidence for involvement of a kinesin-related protein during anaphase spindle elongation in diatom central spindles

We have developed a new model system for studying spindle elongation in vitro using the pennate, marine diatom Cylindrotheca fusiformis. C. fusiformis can be grown in bulk to high densities while in log phase growth and synchronized by a simple light/dark regime. Isolated spindles can be attained in quantities sufficient for biochemical analysis and spindle tubulin is approximately 5% of the total protein present. The spindle isolation procedure results in a 10-fold enrichment of diatom tubulin and a calculated 40-fold increase in spindle protein. Isolated spindles or spindles in permeabilized cells can elongate in vitro by the same mechanism and with the same pharmacological sensitivities as described for other anaphase B models (Cande and McDonald, 1986; Masuda et al., 1990). Using this model, in vitro spindle elongation rate profiles were developed for a battery of nucleotide triphosphates and ATP analogs. The relative rates of spindle elongation produced by various nucleotide triphosphates parallel relative rates seen for kinesin-based motility in microtubule gliding assays. Likewise ATP analogs that allow discrimination between myosin-, dynein-, and kinesin-mediated motility produce relative spindle elongation rates characteristic of kinesin motility. Also, isolated spindle fractions are enriched for a kinesin related protein as identified by a peptide antibody against a conserved region of the kinesin superfamily. These data suggest that kinesin-like motility contributes to spindle elongation during anaphase B of mitosis.     

Analysis of the role of microfilaments and microtubules in acquisition of bipolarity and elongation of fibroblasts in hydrated collagen gels

Fibroblasts in situ reside within a collagenous stroma and are elongate and bipolar in shape. If isolated and grown on glass, they change from elongate to flat shape, lose filopodia, and acquire ruffles. This shape change can be reversed to resemble that in situ by suspending the cells in hydrated collagen gels. In this study of embryonic avian corneal fibroblasts grown in collagen gels, we describe for the first time the steps in the acquisition of the elongate shape and analyze the effect of cytoskeleton-disrupting drugs on filopodial activity, assumption of bipolarity, and cell elongation within extracellular matrix. We have previously shown by immunofluorescence that filopodia contain actin but not myosin and are free of organelles. The cell cortex is rich in actin and the cytosol, in myosin. By using antitubulin, we show in the present study that microtubules are aligned along the long axis of the bipolar cell body. The first step in assumption of the elongate shape is extension of filopodia by the round cells suspended in collagen, and this is not significantly affected by the drugs we used: taxol to stabilize microtubules; nocodazole to disassemble microtubules; and cytochalasin D to disrupt microfilaments. The second step, movement of filopodia to opposite ends of the cell, is disrupted by cytochalasin, but not by taxol or nocodazole. The third step, extension of pseudopodia and acquisition of bipolarity similarly requires intact actin, but not microtubules. If fibroblasts are allowed to become bipolar before drug treatment, moreover, they remain so in the presence of the drugs. To complete the fourth step, extensive elongation of the cell, both intact actin and microtubules are required. Retraction of the already elongated cell occurs on microtubule disruption, but retraction requires an intact actin cytoskeleton. We suggest that the cell interacts with surrounding collagen fibrils via its actin cytoskeleton to become bipolar in shape, and that microtubules interact with the actin cortex to bring about the final elongation of the fibroblast.     

Distribution of phosphorylated spindle-associated proteins in the diatom Stephanopyxis turris

Mitotic spindles isolated from the diatom Stephanopyxis turris become thiophosphorylated in the presence of ATP gamma S at specific locations within the mitotic apparatus, resulting in a stimulation of ATP-dependent spindle elongation in vitro. Here, using indirect immunofluorescence, we compare the staining pattern of an antibody against thiophosphorylated proteins to that of MPM-2, an antibody against mitosis-specific phosphoproteins, in isolated spindles. Both antibodies label spindle poles, kinetochores, and the midzone. Neither antibody exhibits reduced labeling in salt-extracted spindles, although prior salt extraction inhibits thiophosphorylation in ATP gamma S. Furthermore, both antibodies recognize a 205 kd band on immunoblots of spindle extracts. Microtubule-organizing centers and mitotic spindles label brightly with the MPM-2 antibody in intact cells. These results show that functional mitotic spindles isolated from S. turris are phosphorylated both in vivo and in vitro. We discuss the possible role of phosphorylated cytoskeletal proteins in the control of mitotic spindle function.     

Actin-dependent myoid elongation in teleost rod inner/outer segments occurs in the absence of net actin polymerization.

In the retinas of teleost fish, rod photoreceptors elongate in response to light. Light-activated elongation is mediated by the myoid of the rod inner segment and is actin-dependent. Inner segment F-actin filaments form bundles running parallel to the cell's long axis. We examined the mechanism of rod elongation using mechanically-detached rod fragments, consisting of the motile inner segment and sensory outer segment (RIS-ROS). When RIS-ROS are isolated from dark-adapted green sunfish and cultured in the light, they elongate 15 microns at 0.3-0.6 microns/min. Elongation was inhibited 65% by 0.1 microM Cytochalasin D, suggesting a requirement for actin assembly. To determine the extent of assembly during elongation, we used three approaches to measure the F-actin content in RIS-ROS: detection of pelletable actin by SDS-PAGE after detergent-extraction of RIS-ROS; quantification of fluorescein-phalloidin binding by fluorimetry, fluorescence-activated cell sorting and image analysis; estimation of total F-actin filament length by electron microscopy. All three assays indicated that no net assembly of RIS-ROS F-actin accompanied myoid elongation. An increase in F-actin content within the elongated myoid was counterbalanced by a decrease in F-actin content within the 13 microvillus-like calycal processes located at the end of the inner segment opposite to the growing myoid. O'Connor and Burnside (Journal of Cell Biology 89:517-524, 1981) showed that minus-ends of rod F-actin filaments are oriented towards the elongating myoid while plus-ends are oriented towards the shortening calycal processes. Our observations suggest that RIS-ROS elongation entails actin polymerization at the minus-ends of filaments coupled with depolymerization at the filament plus-ends.     

Scanning force microscopy of DNA molecules elongated by convective fluid flow in an evaporating droplet.

Scanning force microscopy (SFM) was used to image intact, nearly fully elongated lambda bacteriophage DNA molecules, fixed onto freshly cleaved mica surfaces. Molecular elongation and fixation were accomplished using a newly characterized fixation technique, termed "fluid fixation." Here convective fluid flows generated within an evaporating droplet of DNA solution efficiently elongate DNA molecules for fixation onto suitably charged surfaces. SFM images of a very large bacteriophage genome, G, showed the presence of double-stranded bubbles. We speculate that these structures may contain putative replication forks. Overall, the experiments presented here demonstrate the viability of using fluid fixation for the preparation of DNA molecules for SFM imaging. The combination of largely automatable optically based techniques with the high-resolution SFM imaging presented here will likely produce a high-throughput system for detailed physical mapping of genomic DNA or clones.     

The role of tubulin polymerization during spindle elongation in vitro

We describe the effect of exogenous tubulin on reactivation of anaphase spindle elongation in isolated diatom spindles. In the absence of tubulin, spindle elongation is limited to the equivalent of the microtubule overlap zone, but in the presence of tubulin spindle elongation is several times the length of the overlap zone. Biotinylated neurotubulin is incorporated into the overlap zone and around the poles. Before spindles have elongated by the equivalent of the overlap zone, there are two regions of incorporated tubulin flanking this zone. After further elongation, there is one broad zone of incorporated tubulin in the spindle midzone. Spindle elongation and the pattern of tubulin incorporation into the midzone, but not the poles, are ATP-dependent and vanadate-sensitive. These results suggest that tubulin adds onto the ends of microtubules in the overlap zone, which then slide through the midzone as the spindle elongates.     

Gametocytogenesis of Leucocytozoon Smithi

ABSTRACT. Development of young gamelocytes of Leucocytozoon smithi into morphologically mature forms was studied using electron microscopy. Gametocytogenesis began on day seven post inoculation when merozoites, released from ruptured hepatic schizonts, developed into gametocytes within mononuclear phagocytes or leukocytes (monocytes or lymphocytes). No gametocytes were observed in any erythrocytes or polymorphonuclear leukocytes. Two gametocyte forms, round and elongate, were observed. Immature round gametocytes occurred on days 7-10 post inoculation in the deep vasculature of liver, lung and spleen. Mature elongate gametocytes were observed beginning on day 12 post inoculation in both the deep tissue vasculature and peripheral circulation of the turkey host. Growth and elongation of the gametocyte resulted in distortion of the host cell and its nucleus. the host cell nucleus initially was elongated and displaced to one side or indented by the growing parasite. Eventually, the nucleus was laterally compressed or split into two or three fragments. the compressed host cell cytoplasm was displaced longitudinally and stretched over the parasite to form hornlike cytoplasmic extensions from each end. the potential role of microtubules in the elongation of the gametocyte and its host cell, and possibly in the indentation and splitting of the host cell nucleus, is discussed.     

Both chloronemal and caulonemal cells expand by tip growth in the moss Physcomitrella patens

Tip growth is a mode of cell expansion in which all growth is restricted to a small area that forms a tip in an elongating cell. In green plants, tip growth has been shown to occur in root hairs, pollen tubes, rhizoids, and caulonema. Each of these cell types has a longitudinally elongated shape, longitudinally oriented microtubules and actin microfilaments, and a characteristic cytoplasmic organization at the growing tip which is required for growth. Chloronema are elongated cylindrical shaped cells that form during the development of the moss protonema. Since there are no published reports on the precise mode of chloronema elongation and conflicting interpretations of its cytology, the mechanism of cell growth has remained unclear. To determine if chloronema elongate by tip or diffuse growth, time-lapse light microscopy was employed to follow the movement of fluorescent microspheres attached to the surface of growing cells. It is shown here that chloronemal cells elongate by a form of tip growth. However, the slower growth of chloronema compared with caulonema is probably the result of differences in cytological organization of the growing tip.     

Organized microtubule arrays in γ-tubulin-depleted Drosophila spermatocytes

To assess the role of γ-tubulin in spindle assembly in vivo, we have followed meiosis progression by immunofluorescence and time-lapse video microscopy in γTub23C^PI mutant spermatocytes. We have found that centrosomes associate with large numbers of astral microtubules even though γ-tubulin is severely depleted; bipolar meiotic spindles are never assembled; and later in meiosis, the microtubules get organized into a conical structure that is never observed in wild-type cells. Several lines of evidence suggest that these cones may be related to wild-type central spindles. First, they are assembled midway through meiosis and elongate during anaphase. Second, they are constricted during late meiosis, giving rise to a pointed end similar to those that form in each half of the wild-type spindle midzone. Third, Klp3A and Polo, two markers of the wild-type central spindle are also found around the pointed end of the mutant cones. Finally, ectopic cytokinesis furrows are often formed at the distal end of the cone. Our results suggest that microtubule polymerization or stabilization from the centrosome may be possible in a γ-tubulin-independent manner in Drosophila spermatocytes. However, γ-tubulin seems to be essential for spindle assembly in these cells. Finally, our results show that at least part of the central spindle and constriction-ring assembly machinery can operate on microtubule bundles that are not organized as bipolar spindles.     

Characterization and solubilization of an acyl chain elongation system in microsomes of leek epidermal cells

Microsomes prepared from leek epidermal tissue readily elongate stearoyl-CoA to very long chain fatty acid with malonyl-CoA as the C2 unit. In the absence of stearoyl-CoA, but in the presence of ATP, microsomes elongate endogenous free fatty acids. Endogenous CoA is the source of CoA. Palmitoyl, stearoyl, and higher saturated acyl-CoAs are readily elongated by the microsomal system but oleoyl-CoA is ineffective; however, the higher monounsaturated acyl-CoAs can be elongated. Since the very long chain fatty acids of the leek epidermis are all saturated, it would appear that the reaction controlling the nature of the final acyl product is the inactivity of oleoyl-CoA as a substrate. There is no evidence that acyl carrier protein participates in the elongation reactions. Evidence is also presented suggesting that (a) there may be two elongation systems, one responsible for the conversion of stearoyl-CoA to arachidonyl-CoA and the second involved in the conversion of arachidonyl-CoA to very long chain fatty acids, and that (b) the elongation activities may be associated with a large polypeptide.     

Cyclic nucleotide regulation of teleost rod photoreceptor inner segment length

Retinal rod photoreceptors of teleost fish elongate in the light and shorten in the dark. Rod cell elongation and shortening are both mediated by actin-dependent mechanisms that occur in the inner segment myoid and ellipsoid. The intracellular signaling pathways by which light and dark regulate the actin cytoskeleton in the inner segment are unknown. To investigate the intracellular signals that regulate teleost rod motility, we have been using mechanically isolated rod inner/outer segments (RIS-ROS) obtained from the retinas of green sunfish, Lepomis cyanellus. In culture, RIS-ROS retain the ability to elongate in response to light; myoids elongate 15-20 microns in length during 45 min of light culture. A pharmacological approach was taken to investigate the role of cyclic nucleotides, cyclic nucleotide-dependent kinases, and protein phosphatases in the regulation of RIS-ROS motility. Millimolar concentrations of cAMP and cGMP analogues were both found to inhibit light-induced myoid elongation and two cyclic nucleotide analogues, SpCAMPS and 8BrcGMP, promoted myoid shortening after RIS-ROS had elongated in response to light. The cyclic nucleotide- dependent kinase inhibitor, H8, mimicked light by promoting myoid elongation in the dark. The effects of H8 were dose dependent, with maximal elongation occurring at concentrations of approximately 100 microM. Similar to the effects of cyclic nucleotide analogues, the phosphatase inhibitor, okadaic acid (0.1-10 microM), inhibited light- induced elongation and promoted shortening. The results presented here suggest that RIS-ROS motility is regulated by protein phosphorylation: phosphorylation in the dark by cyclic nucleotide-dependent protein kinases promotes rod shortening, while dephosphorylation in the light promotes rod elongation.     

Reactivation of isolated mitotic apparatus: metaphase versus anaphase spindles

Mitotic spindles isolated from sea urchin eggs can be reactivated to undergo mitotic processes in vitro. Spindles incubated in reactivation media containing sea urchin tubulin and nucleotides undergo pole-pole elongation similar to that observed in living cells during anaphase-B. The in vitro behavior of spindles isolated during metaphase and anaphase are compared. Both metaphase and anaphase spindles undergo pole-pole elongation with similar rates, but only in the presence of added tubulin. In contrast, metaphase but not anaphase spindles increase chromosome-pole distance in the presence of exogenous tubulin, suggesting that in vitro, tubulin can be incorporated at the kinetochores of metaphase but not anaphase chromosomes. The rate of spindle elongation, ultimate length achieved, and the increase in chromosome-pole distance for isolated metaphase spindles is related to the concentration of available tubulin. Pole-pole elongation and chromosome-pole elongation does not require added adenosine triphosphate (ATP). Guanosine triphosphate (GTP) will support all activities observed. Thus, the force generation mechanism for anaphase-B in isolated sea urchin spindles is independent of added ATP, but dependent on the availability of tubulin. These results support the hypothesis that the mechanism of force generation for anaphase-B is linked to the incorporation of tubulin into the mitotic apparatus. (If, in addition, a microtubule-dependent motor-protein(s) is acting to generate force, it does not appear to be dependent on ATP as the exclusive energy source.     

Absence of microtubule sliding and an analysis of spindle formation and elongation in isolated mitotic spindles from the yeast Saccharomyces cerevisiae

Mitotic spindles were isolated from a cell division cycle mutant of the budding yeast Saccharomyces cerevisiae by the lysis of sphateroplasts on an air:buffer interface and were negatively stained with 1% gold thioglucose. Isolated spindles were incubated under conditions which promoted the sliding disintegration of parallel preparations of Tetrahymena axonemes, namely the addition of ATP to 20 microM. In no experiment was a corresponding change in microtubule organization of the spindle observed even when spindles were first pretreated with either 1-10 microgram/ml trypsin or 0.2-2% Triton X-100. During these experiments a number of spindles were isolated from cells that had passed through the imposed temperature block, and from the images obtained a detailed model of spindle formation and elongation has been constructed. Two sets of microtubules, one from each spindle pole body (SPB), completely interdigitate to form a continuous bundle, and a series of discontinuous microtubules are then nucleated by each SPB. As the spindle elongates, the number of microtubules continuous between the two SPBs decreases until, at a length of 4 micrometer, only one remains. The spindle, composed of only one microtubule, continues to elongate until it reaches the maximal nuclear dimension of 8 micrometer. The data obtained from negatively stained preparations have been verified in thin sections of wild-type cells. We suggest that, as in the later stages of mitosis only one microtubule is involved in the separation of the spindle poles, the microtubular spindle in S. cerevisiae is not a force-generating system but rather acts as a regulatory mechanism controlling the rate of separation.