Distribution of F-actin during cleavage of the Drosophila syncytial blastoderm.

The process of cleavage during the syncytial blastoderm stage of the Drosophila embryo was studied in fixed whole-mounts using a triple-staining technique. Plasmalemma was stained with Concanavalin A conjugated to tetramethylrhodamine isothiocyanate, the underlying cortical F-actin with a fluorescein derivative of phalloidin, and nuclei with 4',-6 diamidine-2-phenylindole dihydrochloride. The surface caps, which overlie the superficial nuclei at this stage, were found to be rich in F-actin as compared with the rest of the cortex. After the caps formed, they extended over the surface and flattened. Whilst this was occurring the F-actin network within the caps became more diffuse. By the end of the expansion process F-actin had become concentrated at both poles of the caps. The caps then split in two. The cleavage was not accompanied by the formation of any apparent contractile ring of microfilaments across the cap, rather the break region was depleted in F-actin. The cortical actin associated with each half of the old cap then became reorganized around a nucleus to form a new daughter cap, and the cycle began again.

An investigation of microtubule organization and functions in living Drosophila embryos by injection of a fluorescently labeled antibody against tyrosinated alpha-tubulin.

Rhodamine-labeled monoclonal antibodies, which react with tyrosinated alpha-tubulin (clone YL 1/2; Kilmartin, J. V., B. Wright, and C. Milstein, 1982, J. Cell Biol., 93:576-582) and label microtubules in vivo (Wehland, J., M. C. Willingham, and I. Sandoval, 1983, J. Cell Biol., 97:1467-1475) were microinjected into syncytial stage Drosophila embryos. At 1 mg/ml antibody concentration, the microtubule arrays of the surface caps became labeled by YL 1/2 but normal development was found to continue. The results are compared with the data from fixed material particularly with regard to interphase microtubules, centrosome separation, and spindle and midbody formation. At 5 mg/ml antibody concentration the microtubules took up larger quantities of antibodies and clumped around the nuclei. Nuclei with clumped microtubules lost their position in the surface layer and moved into the interior. As a result, the F-actin cap meshwork associated with such nuclei either failed to form or subsided. It is concluded that microtubule activity is required to maintain the nuclei in the surface layer and organize the F-actin meshwork of the caps.

Three distinct distributions of F-actin occur during the divisions of polar surface caps to produce pole cells in Drosophila embryos.

The F-actin distribution was studied during pole cell formation in Drosophila embryos using the phalloidin derivative rhodaminyl-lysine-phallotoxin. Nuclei were also stained with 4'-6 diamidine-2-phenylindole dihydrochloride to correlate the pattern seen with the nuclear cycle. The precursors of the pole cells, the polar surface caps, were found to have an F-actin-rich cortex distinct from that of the rest of the embryo surface and an interior cytoplasm that was less intensely stained but brighter than the cytoplasm deeper in the embryo. They were found to divide once without forming true cells and then a second time when cells formed as a result of a meridional and a basal cleavage. Three distinct distributions of the cortical F-actin have been identified during these cleavages. It is concluded that the first division, which cleaves the polar caps but does not separate them from the embryo, involves very different processes from those that lead to the formation of the pole cells. A contractile-ring type of F-actin organization may not be present during the first cleavage but is suggested to occur during the second.

Microinjected profilin affects cytoplasmic streaming in plant cells by rapidly depolymerizing actin microfilaments.

BACKGROUND: Cytoplasmic streaming is a conspicuous feature of plant cell behaviour, in which organelles and vesicles shuttle along cytoplasmic strands that contain actin filaments. The mechanisms that regulate streaming and the formation of actin filament networks are largely unknown, but in all likelihood involve actin-binding proteins. The monomeric actin-binding protein, profilin, is a key regulator of actin-filament dynamics in animal cells and it has recently been identified in plants as a pollen allergen. We set out to determine whether plant profilin can act as a monomeric actin-binding protein and influence actin dynamics in plant cells in vivo. RESULTS: Recombinant birch-pollen profilin was purified by polyproline affinity chromatography and microinjected into Tradescantia blossfeldiana stamen hair cells. After profilin injection, a rapid and irreversible change in cellular organization and streaming was observed: within 1-3 minutes the transvacuolar cytoplasmic strands became thinner and snapped, and cytoplasmic streaming ceased. Fluorescein-labelled-phalloidin staining confirmed that this was due to depolymerization of actin filaments. To confirm that the effects observed were due to sequestration of monomeric actin, another monomeric actin-binding protein, DNase I, was injected and found to produce comparable results. CONCLUSIONS: Profilin can act as a potent regulator of actin organization in living plant cells. Its rapid effect on the integrity of cytoplasmic strands and cytoplasmic streaming supports a model in which organelle movements depend upon microfilaments that exist in dynamic equilibrium with the pool of monomeric actin.

Large vesicle formation within cells induced by treatment with a mixed surfactant.

MDCK cells have been treated with a mixed surfactant at low concentrations to study the induced morphological changes. The most significant change at the light microscope level was the appearance of multiple large vesicles, which increased in size with time, up to approximately 40 microns in diameter. Vesicle formation was shown to be linked with the uptake of the fluid medium, as judged by the presence of FITC-dextran within the vesicles, but was not a result of pinocytosis because cytochalasin D treatment had no effect on their formation. Furthermore, nile red staining demonstrated that the vesicles did not represent fusion of pre-existing lipid droplets. Transmission electron microscopy (TEM) analysis indicated that the vesicles lacked any obvious structure. It is hypothesised that the vesicles are large mixed structures synthesised as a result of interactions between cell membranes and detergent components after saturation with the surfactants. This effect is contrasted with the diffuse uptake of dyes and fluorescently labelled proteins following simple anionic or ionic detergent treatment. The effect of vesicle formation was reversible if the cells were placed in fresh medium lacking detergent. Other effects of mixed detergent included the loss of rounded compact colonies, an increase in mean cell diameter and the almost complete loss of surface microvilli as seen with scanning electron microscopy (SEM). In the TEM the cell ultrastructure was seen to have changed markedly following detergent treatment, with a loss of rough endoplasmic reticulum and an apparent clumping of the cytoplasmic constituents.

The presence of myofibroblasts in the dermis of patients with Dupuytren's contracture. A possible source for recurrence.

Samples of skin and underlying cord obtained at dermofasciectomy for Dupuytren's contracture have been examined for the presence of smooth muscle alpha-actin (SM alpha-actin), a marker for myofibroblasts. 15 of the 20 samples stained positively for SM alpha-actin corresponding with areas of hypercellular Dupuytren's tissue. In 12 of these 15 samples SM alpha-actin-positive hypercellular Dupuytren's tissue extended into the dermis, in three cases reaching the epidermis. In eight samples, diffusely distributed cells positive for SM alpha-actin and resembling fibroblasts were seen in the dermis. These cells appeared to be separate from the Dupuytren's foci. The presence of hypercellular foci and isolated fibroblasts positive for SM alpha-actin within the dermis may explain the high recurrence rate of Dupuytren's disease after fasciectomy.

The cytoskeleton of the early Drosophila embryo.

The organization and roles of the cytoskeleton are described for a complex developing system (the early Drosophila embryo) at a time when the basic embryonic plan is mapped out. This type of embryo shows a separation of mitosis from cytokinesis during the early stages of development. Most cells are only formed when a syncytium of approximately 6000 nuclei are present. The functions of the cytoskeleton are considered for the process of nuclear migration (pre-blastoderm), which distributes the nuclei throughout the embryo and brings most of them close to the surface. They are also described for the subsequent mitoses of the syncytial blastoderm where the cortex and its well-developed cytoskeleton is reorganized into cell-like surface protrusions known as 'caps' or 'buds'. A comparison is made of the very different cytoskeletal organization present during the cleavages that form the two cell types of early development (pole cell and blastoderm cell), together with information from mutations that affect various aspects of these cleavages via factors laid down during oogenesis.

F-actin organization during the cellularization of the Drosophila embryo as revealed with a confocal laser scanning microscope.

The changes in F-actin organization during the cellularization of the Drosophila embryo have been studied with a confocal laser scanning microscope using fluorescein-phalloidin as a specific stain. Particular study has been made of the changes in the organization of the F-actin network associated with the leading edges of the growing membranes. The role of this actin network in the cellularization process is considered. Other actin-containing structures have also been examined, including the cortical actin layer and a conspicuous region of F-actin aggregates, present beneath the level of the forming cell membranes.

Microtubule arrays present during the syncytial and cellular blastoderm stages of the early Drosophila embryo.

The organization of microtubules within the surface caps of Drosophila embryos is described for the mitotic cycles of the syncytial blastoderm stage (particularly cycle 10), and for the subsequent cellularization process. Tubulin was labelled with the well characterized monoclonal antibody YL 1/2 (Kilmartin et al., J cell biol 93 (1982) 576). Each surface cap was found to contain an array of microtubules running around the nucleus. The microtubules originated at prominent centrosomes located close to the apical surface of each cap nucleus. During mitosis the spindle microtubules stained strongly for tubulin. A novel finding was that the spindle microtubules of the interzone region appeared to reduce their connections with the centrosomes at the end of anaphase. The spindle remnant remained in position during telophase but then became smaller in size, disappearing by interphase. At this phase of the cell cycle duplication of the aster centrosomes occurred. The cellular blastoderm stage was marked by a change in the main axis of microtubule orientation. The centrosomes of each cap separated somewhat and formed initiation centres for the development of a well developed basket of microtubules around each nucleus, but now perpendicular to the surface. The microtubule baskets were seen to extend in parallel with nuclear elongation, but not in concert with growth of the cell membranes, which extended some way beneath the bases of the nuclei.

Primary and secondary chick heart fibroblasts: fast and slow-moving cells show no significant difference in microtubule dynamics.

Highly motile chick heart fibroblasts in primary culture (1 degree CHFs) gradually convert into much slower-moving secondary (2 degrees) cells. The polarized movement of the latter, but not the former, cell type has been found to be dependent on an intact microtubule (MT) network [Middleton et al., 1989, J. Cell Sci. 94:25-32]. To investigate the comparative stability of the MT networks of 1 degree s and 2 degrees s, turnover was investigated by microinjection of biotin-labeled brain tubulin to act as a reporter. MTs in both cell types were found to be very dynamic, with the MT networks effectively disassembled by about 30 min in 1 degree CHFs and 60 min in 2 degrees CHFs, with mainly MT fragments remaining beyond these times. All MTs and fragments were found to have turned over by 1 h in 1 degree CHFs and 80 min in 2 degrees s. Because 2 degrees CHFs were found to be on average six times larger than 1 degree s, the difference in MT turnover time was considered largely due to the size difference. For both 1 degree and 2 degrees cells, the more slowly turning over MTs were generally curly and perinuclear in distribution, resembling stable MTs in other systems, but they appeared significantly earlier in CHFs. However, no discrete subpopulations of slower turning over MTs were found to be associated with either the leading edges or the processes of either cell type. In addition, no major differences were identified in the patterns of modified alpha-tubulin along the MTs or of MT cold or drug stability. It is concluded that MTs do not have a direct structural or skeletal function in maintaining a polarized 2 degrees CHF cell shape, but rather play an ancillary role.

F-actin distribution during the cellularization of the Drosophila embryo visualized with FL-phalloidin.

The changing distribution of polymerized actin during the cellularization of the Drosophila blastoderm was investigated in fixed whole embryos using FL-phalloidin as a specific stain. Prior incubation of FL-phalloidin with F-actin from both rabbit and locust muscle blocked the staining action, whereas G-actin at the same concentration had no effect. At the initiation of cellularization bands of F-actin filaments, shaped into rough hexagons, were found around each forming cell close to the surface bulges. These bands interlinked across the whole embryo. Above the level of the hexagons was a fine meshwork of F-actin associated with many folds of the plasmalemma. Below the hexagons was a layer of small irregular actin aggregates. During the process of cellularization the hexagonal actin network was associated with the tips of the extending plasmalemmas until the cells reached their full length. It is suggested that this actin network acts as a contractile ring system which cleaves the embryo into cells. The network was then found to rapidly break down. Microfilament bundles formed rings associated with the bases of the cells. These are presumed to cleave off the fully formed cells from the underlying yolk sac. During the first phase of cell membrane growth the fine F-actin meshwork remained associated with the apical plasmalemmas. However, the mesh rapidly disappeared during the second period of extension. After this, actin aggregates were visible close to the apical surfaces of the cells. F-actin was also observed to be associated with the newly formed plasmalemmas along their length during the whole of the process of cleavage.

Microtubules rich in modified alpha-tubulin characterize the tail processes of motile fibroblasts.

The organisation of microtubules rich in post-translationally modified alpha-tubulin has been investigated in a fibroblast cell line (NIH-3T3-T15) that can be reversibly transformed. An immunofluorescence microscopy study of the static non-transformed cells has revealed a central distribution of wavy microtubules showing post-translational modifications. When transformed there is a marked increase in cell motility and the appearance of long thin cytoplasmic 'tails'. These tails have been found to contain conspicuous bundles of post-translationally modified microtubules that run down the length of the processes and terminate close to the plasmalemma. Both detyrosinated and acetylated alpha-tubulin are present as major species in these modified microtubules. Such a pattern of modified microtubules is only occasionally seen in the untransformed NIH-3T3-T15 cells. We have also found them to be present in other transformed fibroblast lines. The presence of bundles of microtubules rich in modified alpha-tubulin in the cell tails is correlated with a marked reduction in the numbers of F-actin stress fibres. The possible role of these modified stable microtubules in cell motility is discussed.

Scatter factor affects major changes in the cytoskeletal organization of epithelial cells.

The effects of scatter factor on the cytoskeleton of MDCK and PtK2 cells are described. During the first 6 h after the addition of scatter factor, MDCK cells were found to increase their projected areas twofold, as well as the number and size of their F-actin stress fibers. In contrast PtK2 cells showed no change in their projected areas or in their stress fiber content. However, when both MDCK and PtK2 cells began to separate and scatter after approximately 6 h, the size and number of stress fibers was found to decrease considerably. Unscattered PtK2 cells and cells treated with scatter factor which had yet to scatter showed focal contacts present over the whole ventral surface, as judged by staining for both vinculin and talin. After treated cells separated, both vinculin and talin staining were mainly present in focal contacts on the ventral surfaces of the cell bodies and the distal ends of the processes. However, the cell processes showed few focal contacts along their lengths. The distribution of microtubules and vimentin and keratin intermediate filaments also did not change significantly until scattering had occurred. After cell separation, the processes were always packed with microtubules which were often, but not always, rich in detyrosinated alpha-tubulin and often, but not always, packed with intermediate filaments. All these changes in cytoskeletal organization are consistent with the adoption of a much more motile phenotype. The changes found are compared with those brought about by transformation.

The effects of microinjection of rhodamine-phalloidin on mitosis and cytokinesis in early stage Drosophila embryos.

Rhodamine-phalloidin was microinjected into early stage Drosophila embryos, which were then allowed to develop for various times, fixed, and examined by fluorescence microscopy. A gradient of effects was seen. Close to the site of injection an area of diffuse bright fluorescence was found which included lumps and long strands of fluorescent material. Around this region particular cytoplasmic domains showed a denser F-actin distribution. These domains included the nuclear islands of the preblastoderm, the cortical caps of the syncytial blastoderm, and the contractile ring network which forms during cellularization of the blastoderm. It is proposed that these domains are regions of preferential actin polymerization under the appropriate cellular conditions and that the injected phalloidin causes incorporation of additional polymer into existing structures. Further away the pattern of phalloidin staining corresponded to that found with fixed material. In contrast to the domains of apparent additional F-actin polymerization a reduction of actin incorporated into small aggregates was found, both in syncytial blastoderm stages and during cellularization. This occurred in regions where additional actin had been incorporated into adjacent actin-rich structures. A storage role for the aggregates, which are depleted when F-actin is polymerized, is proposed. Both mitosis and cytokinesis were found to be slowed but the inhibition was only transient. However, most embryos died without differentiating. Rarely, differentiated tissues formed and the musculature was strongly stained by rh-phalloidin. When embryos were injected immediately prior to the start of cellularization cytokinesis was inhibited only locally and continued normally elsewhere. This finding argues against the hypothesis that contraction of an actomyosin network over the whole surface is the only force involved in the cellularization of the blastoderm and that local factors, e.g., plasmalemma extension, must be involved.

Stable and slow-turning-over microtubules characterize the processes of motile epithelial cells treated with scatter factor.

The turnover of microtubules was studied in the processes of PtK2 cells, after treatment with the cytokine scatter factor (SF), using micro-injected biotin-tubulin as a reporter of new microtubule growth. Cells treated with SF became dispersed and fibroblast-like in morphology, showing one or more elongated processes. These processes contained bundles of microtubules, a significant proportion of which did not turn over during incubation times of up to an hour. Short broken pieces of microtubule were frequently found in all parts of the cell, particularly after longer incubation times, suggesting that more-stable microtubules were cut into pieces, which were subsequently degraded. From about half an hour after injection small tangles of stable microtubules were found. Some of these were clearly within the cell bodies. Others were usually larger in size and seemingly located outside the injected cells. These were considered to have formed part of small 'feet' presumed to be broken off during the retraction of trailing processes. The microtubules within the processes were resistant to the effects of both microtubule-depolymerizing drugs and cold under conditions where the processes were maintained. When these microtubules disappeared as the result of longer drug treatment the processes were also lost although, rarely, short processes lacking microtubules were found. It is concluded that the stable microtubules have a major role in process maintenance, although one that is indirect rather than a structural relationship.

Changes in the distribution of cortical myosin during the cellularization of the Drosophila embryo.

Changes in the distribution of myosin during the formation of the cellular blastoderm of Drosophila melanogaster were followed by staining sections of embryos with antibodies to myosin. These were visualized with indirect immunofluorescence. Prior to the start of cell membrane extension myosin is distributed between the nuclear caps as a thin sub-plasmalemma layer. There is also myosin present beneath the surface of the caps. When plasmalemma growth occurs, myosin is associated with the furrow canals, the tips of the advancing membranes. The fluorescence is distributed in an approximately hexagonal pattern around the growth points of each cell. The hexagons are joined up forming a network. It is suggested that this myosin is associated with bundles of microfilaments, orientated parallel to the surface, to form many interlocking contractile rings. The simultaneous contraction of these rings causes the cleavage of the blastoderm. During the first phase of membrane growth, myosin is also associated with the apical surfaces of the forming cells. At this stage these surfaces are rich in microvilli. However, by the time the furrow canals have reached the bases of the cells much of this myosin has disappeared. At about this time the apical surface becomes taut with a loss of the microvilli.

Effects of elevated intracellular magnesium on cytoskeletal integrity.

Increasing the intracellular magnesium concentration of PtK2 cells by 1 mM or more resulted in the disassembly of the interphase microtubule array over a period of 5 min after microinjection. This effect was found to be both transient and fully reversible, with the microtubule arrays reforming after further incubation. These effects were studied using immunofluorescence microscopy of fixed cells, and also in living cells using rhodamine-tubulin or rhodamine-conjugated anti-tubulin antibodies and image intensification and enhancement techniques. Simultaneously and accompanying the disassembly of the microtubule arrays the F-actin stress fibres also disappeared, usually leaving the peripheral and perinuclear F-actin microfilaments intact. In contrast, increasing intracellular magnesium appeared to have no effect on the vimentin-containing intermediate filaments of PtK2 cells. These effects on the cytoskeleton were specific to magnesium and could not be mimicked by either microinjection of injection buffer of equivalent ionic strength or sham injection. Raising the intracellular free calcium to the same extent resulted in the disassembly of the microtubule network, but appeared to have no effect on the F-actin stress fibres.