The cytoskeletal involvement in cellularization of theDrosophila melanogaster embryo

Summary The distribution and arrangement of cytoskeletal components in the early embryo ofDrosophila melanogaster were examined by thin-section electron microscopy to elucidate their involvement in the formation of the cellular blastoderm, a process called cellularization. During the final nuclear division in the cortex of the syncytial blastoderm bundles of astral microtubules were closely associated with the surface plasma membrane along the midline where a new gutter was initiated. Thus the new gutter together with the pre-formed ones compartmentalized the embryo surface to reflect underlying individual daughter nuclei. Subsequently such gutters became deeper by further invagination of the plasma membrane between adjacent nuclei to form so-called cleavage furrows. Nuclei simultaneously elongated in the direction perpendicular to the embryo surface and numerous microtubules from the centrosomes ran longitudinally between the nucleus and the cleavage furrow. Microtubules often appeared to be in close association with the nuclear envelope and the cleavage furrow membrane. The plasma membrane at the advancing tip of the furrow was always undercoated with an electron-dense layer, which could be shown to be mainly composed of 5–6 nm microfilaments. These microfilaments were decorated with H-meromyosin to be identified as actin filaments. As cleavage proceeded, each nucleus with its perikaryon became demarcated by the furrow membrane, which then extended laterally to constrict the cytoplasmic connection between each newly forming cell and the central yolk region. The cytoplasmic strand thus formed possessed a prominent circular bundle of microfilaments which were also decorated with H-meromyosin and bidirectionally arranged, similar in structure to the contractile ring in cytokinesis. These observations strongly suggest that both microtubules and actin filaments play a crucial role in cellularization ofDrosophila embryos.     

Organelle movement and fibrillar elements of the cytoskeleton in the angiosperm pollen tube

Summary Continuous observation of organelles and other cytoplasmic inclusions in the older stretches of living pollen tubes of Iris pseudacorus shows that in the more attentuated parts of the protoplast they move along single, mainly longitudinally oriented fibrils, corresponding to those previously isolated from other species and shown to contain bundles of uniformly polarised actin microfilaments. The traffic associated with each fibril is unidirectional, but organelles move along them independently, sometimes with conspicuously different velocities. Larger columns of cytoplasm passing along the tube are associated with several such fibrils, as revealed in occasional discontinuities and also in columns isolated from the tube in suitable medium without fixation. The dimensions of the individual fibrils suggest that the bundles of actin microfilaments are not likely to be enclosed in a unit membrane corresponding to a tonoplast. If so, the nature of the continuous cavities traversed by numerous fibrils in the older parts of the pollen tube requires reappraisal, since these are more likely to be volumes of attentuated cytoplasm comparable with that of the central cavity of the sieve tube than vacuoles of the normal plant-cell type.     

The spermatogenous body cell of the coniferPicea abies (Norway spruce) contains actin microfilaments

Summary In conifer pollen, the generative cell divides into a sterile stalk cell and a body cell, which subsequently divides to produce two sperm. InPicea abies (Norway spruce, Pinaceae) this spermatogenous body cell contains actin microfilaments. Microfilament bundles follow the spherical contour of the body cell within the cell cortex, and also traverse the cytoplasm and enmesh amyloplasts and other organelles. In addition, microfilaments are associated with the surface of the body cell nucleus. The sterile stalk cell also contains microfilament bundles in the cytoplasm, around organelles, and along the nuclear surface. Within the pollen grain, microfilament bundles traverse the vegetative-cell cytoplasm and are enriched in a webbed cage which surrounds the body cell. Microfilaments were identified with rhodamine-phalloidin and with indirect immunofluo-rescence using a monoclonal antibody to actin. The majority of evidence in literature suggests that the spermatogenous generative cell in angiosperms does not contain actin microfilaments, so the presence of microfilaments within the spermatogenous body cell inP. abies appears to be a fundamental difference in sexual reproduction between conifers and angiosperms.     

Actin-endoplasmic reticulum complexes inDrosera

In epidermal cells ofDrosera tentacles that have been preserved for ultrastructural analysis through high pressure freeze fixation and freeze substitution we describe the frequent occurrence of microfilament (MF)-endoplasmic reticulum (ER) complexes. These are found throughout the cytoplasm where they are observed in close association with the plasmalemma (PL), the tonoplast, nuclei, mitochondria, chloroplasts, and microbodies. The MF component of the complexes is identified as actin based on immunogold labelling with actin antibodies. The actin-ER complexes are prominent in the cortical cytoplasm. In this region a network of predominantly tubular ER occupies an intermediary position in which it associates closely with both the PL and the actin MFs. We suggest that the ER, especially those elements adjacent to the PL in the cortical cytoplasm, stabilizes the actin MFs and provides the necessary anchor against which the forces for cytoplasmic streaming are generated.Abbreviations CF chemical fixation - ER endoplasmic reticulum - FS freeze substitution - HPF high pressure freezing - MF microfilaments - MT microtubules - PL plasmalemma     

Tip-localized actin polymerization and remodeling,reflected by the localization of ADF,profilin and villin,are fundamental for gravity-sensing and polar growth in characean rhizoids

Polar organization and gravity-oriented, polarized growth of characean rhizoids are dependent on the actin cytoskeleton. In this report, we demonstrate that the prominent center of the Spitzenkörper serves as the apical actin polymerization site in the extending tip. After cytochalasin D-induced disruption of the actin cytoskeleton, the regeneration of actin microfilaments (MFs) starts with the reappearance of a flat, brightly fluorescing actin array in the outermost tip. The actin array rounds up, produces actin MFs that radiate in all directions and is then relocated into its original central position in the center of the Spitzenkörper. The emerging actin MFs rearrange and cross-link to form the delicate, subapical meshwork, which then controls the statolith positioning, re-establishes the tip-high calcium gradient and mediates the reorganization of the Spitzenkörper with its central ER aggregate and the accumulation of secretory vesicles. Tip growth and gravitropic sensing, which includes control of statolith positioning and gravity-induced sedimentation, are not resumed until the original polar actin organization is completely restored. Immunolocalization of the actin-binding proteins, actin-depolymerizing factor (ADF) and profilin, which both accumulate in the center of the Spitzenkörper, indicates high actin turnover and gives additional support for the actin-polymerizing function of this central, apical area. Association of villin immunofluorescence with two populations of thick undulating actin cables with uniform polarity underlying rotational cytoplasmic streaming in the basal region suggests that villin is the major actin-bundling protein in rhizoids. Our results provide evidence that the precise coordination of apical actin polymerization and dynamic remodeling of actin MFs by actin-binding proteins play a fundamental role in cell polarization, gravity sensing and gravity-oriented polarized growth of characean rhizoids.Abbreviations ADF Actin-depolymerizing factor - CD Cytochalasin D - MF Microfilament     

State of actin during the cycle of cohesiveness of the cytoplasm in parthenogenetically activated sea urchin egg

By the DNase I inhibition assay it is shown that the cytoplasmic matrix isolated 60 min after procaine activation of Paracentrotus lividus eggs contains about 20% of the total egg actin, mostly in polymerized form (85%). Electron microscopy studies on this cytoplasmic structure after treatment with heavy meromyosin (HMM), reveal that the decorated actin filaments are organized in bundles which are distributed radially, with the arrowheads pointing towards the central region. In addition few microtubules and a network of non-decorated microfilaments of about 3 nm diameter are observed. From the cytoplasmic pH determination and the DNase I inhibition assay on homogenates of eggs which were taken at different times of activation, it cannot be inferred that a direct relationship between the increase in the cytoplasmic pH and the increase in the amount of polymerized actin or of cytoplasmic matrix exists. Activation experiments carried out in the presence of colchicine shows that, although the formation of the cytoplasmic matrix is inhibited, polymerization of actin still occurs. Moreover, from the inhibition effects of cytochalasin B (CB) added before the activator it is shown that polymerization of actin is a necessary step for the organization of the cytoplasmic matrix. However, the cycles of cohesiveness of the cytoplasm observed in the course of the activation process do not appear to depend on cycles of polymerization and depolymerization of actin.     

Immunofluorescent visualization of arrays of transverse cortical actin microfilaments in wheat root-tip cells

Summary Actin microfilaments in isolated root-tip cells from wheat (Triticum aestivum L. cv. Kite) were visualized by immunofluorescence microscopy using two different antiactin monoclonal antibodies. Cells in interphase contain predominantly subcortical bundles of microfilaments, as described in many cell types, but in preprophase and prophase cells, immunodetectable actin is organized solely in ordered arrays of cortical microfilaments that cover the entire surface of the cell, transverse on lateral faces, random on end walls. Intermediate stages with random and transverse microfilaments are also seen on lateral faces. The cell cycle stage-dependent transverse cortical microfilaments described here are previously unreported in higher plant cells.Abbreviations Ig immunoglobulin - MF microfilament     

Ultrastructural and immunochemical analyses of the distribution of microfilaments in seedlings and plants ofGlycine max

Summary Filamentous structures were observed when cytoplasmic extracts of various tissues of soybean plants and seedlings were examined by electron microscopy. Three main lines of evidence indicate that these structures represented microfilaments derived from the soybean tissues: a) the diameter of the filaments was estimated to be 6–7 nm; b) the addition of rabbit heavy meromyosin resulted in the decoration of the filaments, yielding characteristic arrow-head patterns; and c) ATP reversed the decoration of the filaments by heavy meromyosin. When the various anatomical parts of soybean plants and seedlings were compared for the presence of microfilaments, the root tips and radicles showed the highest frequency while the petioles and cotyledons yielded no observable filaments. In order to substantiate these findings, a quantitative radioimmunoassay was developed using rabbit antibodies directed against calf thymus actin. These studies demonstrated that the concentration of actin in extracts of the root tip was 15-fold higher than in those of the petiole and leaf. Similar comparisons of various parts of soybean seedlings showed that the radicle was rich in actin. These results suggest that actin filaments are found predominantly in the subterranean parts of plants.     

Cytoplasmic streaming in Amoeba proteus is inhibited by the actinspecific drug phalloidin

Phalloidin, applied by microinjection into Amoeba proteus inhibits specifically, in a concentration dependent manner, the process of cytoplasmic streaming. Preliminary ultrastructural analysis of phalloidin-injected amoebas shows that extensive arrays of microfilaments are formed. These results are discussed in relation to the specific interaction of phalloidin with actin known to occur in vitro, which involve promotion of actin polymerization and stabilization of F-actin structures.     

The involvement of F-actin inUromyces cell differentiation: The effects of cytochalasin E and phalloidin

Summary The role of F-actin in cell differentiation ofUromyces appendiculatus (bean rust fungus) germlings was examined by treating differentiating and nondifferentiating germlings with the actin-binding drugs cytochalasin E (CE) and phalloidin. Prolonged exposure of urediospores to 5×10^–3–5 × 10^–5 M CE induced nuclear division in up to 28–45% of the resulting germlings, whereas the rate of mitosis in established germlings exposed to these concentrations of CE was significantly lower (4–11%). Germlings treated with CE shifted from polarized apical growth to spherical expansion, cytoplasmic microfilaments were depolymerized, and nuclear inclusions became enlarged. Differentiating germlings exposed to a 10 minute pulse of 5×10^–6M CE before the initiation of septum formation prevented the establishment of the F-actin septal ring and growth of the crosswall delimiting the appressorium. Although these CE treatments resulted in morphological and nuclear events similar to those occurring during normal appressorium formation, transient microfilament depolymerization was not sufficient to induce differentiation. Phalloidin stabilized cytoplasmic microfilaments, especially posteriorly-located microfilaments, but did not affect differentiation, nor did it significantly inhibit the effects of CE.Abbrevations CE cytochalasin E - DAPI 4,6-diamidino-2-phenylindole - DMSO dimethyl sulfoxide - F-actin filamentous actin     

Response of actin microfilaments during phytochrome-controlled growth of maize seedlings

Summary In seedlings of maize (Zea mays L. cv. Percival), growth is controlled by the plant photoreceptor phytochrome. Whereas coleoptile growth is promoted by continuous far-red light, a dramatic block of mesocotyl elongation is observed. The response of the coleoptile is based entirely upon light-induced stimulation of cell elongation, whereas the response of the mesocotyl involves light-induced inhibition of cell elongation. The light response of actin microfilaments was followed over time in the epidermis by staining with fluorescence-labelled phalloidin. In contrast to the underlying tissue, epidermal cells are characterized by dense longitudinal bundles of microfilaments. These bundles become loosened during phases of rapid elongation (between 2–3 days in irradiated coleoptiles, between 5–6 days in dark-grown coleoptiles). The condensed bundles re-form when growth gradually ceases. The response of actin to light is fast. If etiolated mesocotyls are transferred to far-red light, condensation of microfilaments can be clearly seen 1 h after the onset of stimulation together with an almost complete block of mesocotyl elongation. The observations are discussed in relation to a possible role of actin microfilaments in the signal-dependent control of cell elongation.     

Changes of the actin filament system in the green algaMicrasterias denticulata induced by different cytoskeleton inhibitors

Summary Two different techniques have been adapted forMicrasterias denticulata to depict the actin cytoskeleton of both untreated and inhibitor-treated developing cells: the quickstaining method, where the cells are fixed in a mixture of glutaraldehyde and formaldehyde followed by staining with phalloidin without embedding, and the methacrylate method, where the cells are also fixed by aldehydes and where the embedding medium is removed prior to incubation with an actin antibody. Both methods produce sufficient preservation and visualization of actin microfilaments (MFs) and confirm earlier observations on the presence of a cortical actin MF network in both the growing and the nongrowing semicell as well as of a basketlike MF arrangement around the migrating nucleus. The results show that a network of actin MFs is essential for the proper development of the young lobes ofM. denticulata. Early developmental stages expanding uniformly at the beginning of growth lack any netlike actin MF arrangement. The actin cytoskeleton in developing cells treated with the actin-targeting agents cytochalasin D and latrunculin B is markedly influenced. Cytochalasin D, which produces the most pronounced effects, causes a breakdown of the network of actin MFs, resulting in bright actin clusters as well as in short and abnormally thick actin fragments particularly in cortical cell regions. In latrunculin B-treated cells remnants of the former actin MF network are still visible, yet most of the actin cytoskeleton appears collapsed and is reduced to short filament pieces. The disturbance of the actin MF system visualized in the present study correlates with the severe morphological and ultrastructural changes occurring in desmid cells as a consequence of both drugs. The dinitroanilin herbicide oryzalin, known to deploymerize cytoplasmic microtubules, causes also an impairment of the actin cytoskeleton inM. denticulata though not sufficient to influence normal cell growth and differentiation.Abbreviations CB cytochalasin B - CD cytochalasin D - DMSO dimethyl sulfoxide - FA formaldehyde - GA glutaraldehyde - LAT-A latrunculin A - LAT-B latrunculin B - MFs microfilaments - MT microtubule Dedicated to Professor Walter Gustav Url on the occasion of his 70th birthday     

A light and electron microscope study ofPicea glauca (White spruce) somatic embryos

Summary Somatic embryos in embryogenic callus cultures derived from Immature zygotic embryos ofPicea glauca (White spruce) were examined by light and electron microscopy. Somatic embryos consist of an embryonic region of small densely cytoplasmic cells subtended by a suspensor consisting of long highly vacuolated cells. Mitotic figures are frequent in the embryonic cells but are not observed in the suspensor. Cell divisions in the embryonic region apparently produce rows of cells which elongate to form the suspensor. The presence of abundant polysomes, coated membranes and dictyosomes in the cytoplasm of embryonic and upper suspensor cells suggests rapid growth of the embryo. In contrast the basipetal suspensor cells appear to be senescing. While only a few scattered microfilaments are present in the meristematic cells, the upper suspensor cells contain numerous bundles of longitudinally oriented microfilaments. These bundles correspond to actin cables observed in light microscope preparations stained with rhodamine labelled phalloidin and are oriented parallel to the direction of active streaming in these cells.     

Actin and Tubulin Arrays in Cultured Xenopus Melanophores Responding to Melatonin

Melatonin induces pigment granule aggregation in amphibian melanophores. In the studies reported here, we have used fluorescence microscopic techniques to test the hypothesis that such melatonin-induced pigment movement is correlated with alterations in either the actin or tubulin cytoskeletal patterns of cultured Xenopus melanophores. In general, the cytoplasmic domains of the cultured melanophores were flat and thin except in the perinuclear region (especially when the pigment was aggregated). The microtubules and microfilaments were usually found in the same focal plane; however, on occasion, microfilaments were closer to the substratum. Microtubules were arranged in arrays radiating from what are presumed to be cytocenters. A small percentage of the melanophores were very large, had actin-rich circular perimeters and did not respond as rapidly to melatonin treatment as did the other melanophores. Melanophores with either aggregated or dispersed melanosomes had low intensity rhodamine-phalloidin staining of actin filaments compared to nonpigmented cells, whereas the FITC anti-tubulin intensities were comparable in magnitude to that seen in nonpigmented cells. When cells were fixed prior to complete melatonin-induced pigment granule aggregation there was no abrupt diminution in either the tubulin or actin staining at the boundary between pigment granule-rich and pigment granule-poor cytoplasmic domains. Nor could the actin and tubulin patterns in cells with partially aggregated melanosomes be reliably distinguished from those in melanophores in which the melanosomes were either completely dispersed or completely aggregated. These data argue against the hypothesis that melatonin causes consistent large-scale rearrangements of tubulin and actin polymers as it induces pigment aggregation in Xenopus melanophores.     

Actin microfilaments and fibroin secretion in the silkgland cells of Bombyx mori : Effects of cytochalasin B

Bombyx mori posterior silkgland cells exhibit an impressive microfilament apparatus located at the cellular apex. It consists of bundles of packed, long microfilaments of 50–70 Å diameter running along circumferences delimiting the lumen of the gland, perpendicularly to the flow of luminal silk. Microfilaments are closely associated with microtubules of the cytoplasmic ‘radial microtubule system’. Immunolabelling with purified antihuman actin antibodies was used to demonstrate their actin-like nature. Apical microfilaments are sensitive to cytochalasin B (CB) which selectively inhibits the secretion of fibroin. Following the removal of the drug, microfilaments recover their normal morphology and secretion resumes. The possible implication of contraction of microfilaments in the process of secretion is discussed.     

Rearrangement of the actin filament and microtubule cytoskeleton during induction of microspore embryogenesis inBrassica napus L. cv. Topas

Summary Changes in the actin filament and microtubule cytoskeleton were examined during heat- and cytochalasin D-induced embryogenesis in microspores ofBrassica napus cv. Topas by rhodamine phalloidin and immunofluorescence labelling respectively. The nucleus was displaced from its peripheral to a more central position in the cell, and perinuclear actin microfilaments and microtubules extended onto the cytoplasm. Heat treatment induced the formation of a preprophase band of microtubules in microspores; preprophase bands are not associated with the first pollen mitosis. Actin filament association with the preprophase band was not observed. The orientation and position of the mitotic spindle were altered, and it was surrounded with randomly oriented microfilaments. The phragmoplast contained microfilaments and microtubules, as in pollen mitosis I, but it assumed a more central position. Cytoskeletal reorganisation also occurred in microspores subjected to a short cytochalasin D treatment, in the absence of a heat treatment. Cytochalasin D treatment of microspores resulted in dislocated mitotic spindles, disrupted phragmoplasts, and symmetric divisions and led to embryogenesis, confirming that a normal actin cytoskeleton has a role in preventing the induction of embryogenesis.Abbreviations CD cytochalasin D - MF actin microfilament - MT microtubule - PPB preprophase band     

Diameters of microtubules change during rotation of the lipotubuloids of Ornithogalum umbellatum stipule epidermis as a result of varying protofilament monomers sizes and distance between them

Microtubules in lipotubuloids of the Ornithogalum umbellatum stipule epidermis cells change their diameters depending on the motion of the cytoplasmic domains rich in microtubules and lipid bodies. Microtubules fixed during rotary and progressive motion of the lipotubuloids composed of the same number of protofilaments fall into two populations – wide (43–58 nm) and narrow (24–39 nm) in size. Following blockage of the motion with 2,4-dinitrophenol (DNP), the range of this diversity is smaller, microtubules become a medium-sized population (34–48 nm). When DNP is removed and the motion reactivated, 2 populations of microtubules reappear. Analysis of the structure of the microtubule wall revealed that changes in the microtubule diameters resulted from varying distances between the adjacent protofilaments, and stretching and compression of tubulin subunits in the protofilaments.A supposition has been put forward that the changes in the sizes of O. umbellatum microtubule diameters: 1) are connected with the interactions between microtubules and actin microfilaments lying along these microtubules; 2) can be the driving force of the rotary motion of lipotubuloids.     

Actin Distribution in Mitotic Apparatus of Somatic Embryo Cells of Norway Spruce

F-actin distribution was studied in mitotic cells of embryogenic suspension culture of Norway spruce [Picea abies (L.) Karst.]. Actin was present in dividing cells of embryo head during whole mitosis. Transient co-localization of actin microfilaments with preprophase band of microtubules was observed. Weak actin staining occurred with non-kinetochor microtubular fibers in metaphase spindle. F-actin was not localized with kinetochore microtubular fibres in metaphase as well as with shortening kinetochore fibres in late anaphase. On the other hand, abundant actin microfilaments array was formed in the area of late anaphase spindle in equatorial level of the cell between separating chromatids. F-actin was also present in phragmoplast area in telophase. This revised version was published online in July 2006 with corrections to the Cover Date.     

Organization of the cytoskeleton in pollen tubes ofPyrus communis: a study employing conventional and freeze-substitution electron microscopy,immunofluorescence, and rhodamine-phalloidin

Summary The structure and organization of the cytoskeleton in the vegetative cell of germinated pollen grains and pollen tubes ofPyrus communis was examined at the ultrastructural level via chemical fixation and freeze substitution, and at the light microscopic level with the aid of immunofluorescence of tubulin and rhodamine-phalloidin.Results indicate that cortical microtubules and microfilaments, together with the plasma membrane, form a structurally integrated cytoskeletal complex. Axially aligned microtubules are present in cortical and cytoplasmic regions of the pollen grain portion of the cell and the distal region of the pollen tube portion. Cytoplasmic bundles of microfilaments are found in association with elements of endoplasmic reticulum and vacuoles. Axially aligned microfilaments are also found in this region, associated with and independent of the microtubules. Microtubules are lacking in the subapical region where short, axially aligned microfilaments are found in the cell cortex. In the apical region, which also lacks microtubules, a 3-dimensional network of short microfilaments occurs. Microfilaments, but not microtubules, appear to be associated with the vegetative nucleus.     

Morphogenesis in the embryo ofDrosophila melanogaster — Germ band extension

Summary Changes at the ultrastructural level during germ band extension in the embryo ofDrosophila melanogaster are described. Cytoplasmic connections between cells and the yolk sac are present during initial cellular movements. At this time, a continuous system of microfilaments is present adjacent to the membranes in the connections and at the periphery of the yolk sac. As germ band extension progresses, this system becomes discontinuous, and microfilaments are apparent only in the immediate vicinity of the connections. Cytoplasmic connections are disassembled at approximately the midpoint of extension; at the same time, extensive membrane associations develop between germ band cells and between these cells and adjacent yolk sac membranes. Positioning and orientation of cytoplasmic connections suggest that the yolk sac, via these connections, is actively involved in the cellular movements of early germ band extension.This paper is dedicated with respect and affection to Donald F. Poulson