TRACHELOMONAS HISPIDA VAR. CORONATA (EUGLENOPHYCEAE). I. ULTRASTRUCTURE OF CYTOSKELETAL AND FLAGELLAR SYSTEMS1,2

Trachelomonas hispida var. coronata Lemm. has a fibrous, mucilaginous, ovoid, mineralized envelope (lorica), the ornamentation and coloration of which are capricious in culture. Cells exhibit a radial distribution of most organelles: (i) A cortical endoplasmic reticulum, (ii) parietal chloroplasts, and (iii) a median vacuolar region surrounded by several Golgi bodies and diverse vesicles. Associated with the emergent flagellum is a “paraflagellar complex” that consists of dense globules, cross-striated ribbon-like structures, a paraflagellar body, and an array of parallel striated filaments. The stigma consists of a single layer of pigmented granules that partially surrounds the canal/reservoir transition zone where microtubular bands intersect. A microtubular cytoskeleton consists of pellicular microtubules, peri-canal microtubules, stigma-associated microtubules and para-reservoir microtubules. The thickenings on the posterior, concave margins of the pellicular strips suggest that this pellicle is of intermediate complexity between those of Euglena spirogyra (Ehrenb. and Trachelomonas volvocina (Ehrenb.).     

The microtubular cytoskeleton of three dinoflagellates: an immunofluorescence study

Summary The sub-thecal microtubular cytoskeleton of the dinoflagellatesAmphidinium rhynchocephalum, Gymnodinium sanguineum, andGymnodinium. sp has been investigated by indirect immunofluorescence microscopy. In these cells, the majority of cytoskeletal microtubules lie in the anterior-posterior plane. These longitudinal microtubules clearly originate from one of two radially arranged microtubular bands that correspond in location with the anterior and posterior edge of the cingolar depression. Despite the morphological variability of these gymnodinioid dinoflagellates, our data indicate that the microtubular cytoskeleton perfectly reflects the spatial patterning of the epicone and hypocone in each cell.Abbreviations ALB Anterior longitudinal microtubular bundles - ATB Anterior transverse microtubular bands - C cingulum - CLB Cingular longitudinal microtubular bundles - E Epicone - H Hypocone - PLB Posterior longitudinal microtubular bundles - PTB Posterior transverse microtubular bands - S Sulcus     

Microtubular configurations during meiosis and megasporogenesis in Gasteria verrucosa and Chamaenerion angustifolium

Summary In Gasteria and Chamaenerion, microtubular configurations were visualized immunocytochemically during meiosis and megasporogenesis in order to study their relationship to cell development, meiotic divisions and selection of the functional megaspore. In Chamaenerion, the intensity of the fluorescence found in megaspores was weaker than that found in Gasteria. Both plants exhibited concentrations of microtubules around the meiocyte nuclei during pachytene-diplotene. Preprophase bands were not observed. In Chamaenerion, cytoplasmic microtubules radiating from meiocyte nuclei were found at late prophase, the dyad stage and in the functional megaspore; in Gasteria, they were observed only at the dyad stage and in the functional megaspore. During the second meiotic division of Gasteria, dividing cells and their nuclei exhibited differences in volumes. Also, the two microtubular spindles of the dyad cells had different widths. Fluorescence indicating the presence of the cytoskeleton diminished during maturation of the large functional megaspores in both plants, whereas in the three degenerating smaller megaspores, fluorescence intensity persisted. Our conclusion is that only an indirect relationship exists between the organization of the microtubular cytoskeleton and selection of the functional megaspore.     

The microtubule cytoskeleton and the rounding of isolated generative cells ofNicotiana tabacum

Summary Upon squashing of the pollen grain, the isolated generative cell ofNicotiana tabacum looses its spindle shape to become spherical; this phenomenon is independent of the sucrose concentration used. The time necessary for this change can vary from 1 min (0% sucrose) to 20 min (30% sucrose). The microtubular cytoskeleton was studied by means of immunofluorescence and electron microscopy. Just after isolation, 5 to 15 clearly visible bundles in microtubules organized in a basket-like structure are present. After 15 min in medium with 15% sucrose, the microtubular cytoskeleton disappears, and a diffusely spread tubulin can be observed. Neither the addition of 10–20 M taxol to the medium, nor the omission of Ca²⁺ to the medium has any effect on the changes in cell shape and loss of microtubular bundles after isolation.Abbreviations GC Generative cell - SC sperm cell - BK Brewbaker and Kwack - CLSM confocal laser scanning fluorescence micros copy     

Cortical microtubular lattices: Absent from mature mesophyll and necessary for cell division?

Using immunofluorescence microscopy, the cortical microtubular net which is regularly present in cells of young, growing tissue is shown to be absent, or largely reduced, in mature mesophyll cells of Nicotiana plumbaginifolia Viv., N. tabacum L., Petunia hybrida Hort. and Brassica napus L. The onset of division in protoplasts isolated from these fully differentiated tissues is preceded by a period of dedifferentiation. One of the early events during dedifferentiation, as shown for N. plumbaginifolia, is the re-establishment of a net of cortical microtubules, prior to spindle formation. These findings indicate that the presence of the cortical microtubular lattice is a prerequisite for protoplast division. Cell-wall regeneration, which also must precede division, occurs simultaneously with the formation of the lattice. However, the cortical microtubules seem to not exert any influence on the orientation of the microfibrils.     

Spatial congruence between exine pattern,microtubules and endomembranes in Vigna pollen

The role of microtubules and endomembranes in pollen wall pattern formation in Vigna vexillata L. was examined using fluorescence laser scanning confocal microscopy. Indirect immunofluorescence using anti--tubulin antibodies revealed that the arrangement of the cortical microtubular cytoskeleton in microspores resembled the reciprocal of the reticulate ektexine ornamentations of mature V. vexillata pollen. Patches of microtubules in cortical cytoplasm corresponded in location with the lumina of the exine reticulum and with apertural sites. Microtubules were absent from cytoplasm under muri (ridges) of the exine reticulum. Labeling of microspores during the mid-tetrad stage with the endomembrane-specific fluorochrome DiOC₆ produced a pattern similar to that of the microtubules; i.e., DiOC₆ staining was localized in cytoplasm underlying lumina and absent from cortical cytoplasm underlying sites of muri. This report represents the first observation of congruence of the pattern of occurrence of any subcellular organelles with exine pattern and, in particular, the congruence of both microtubules and endomembranes in cortical cytoplasm with the lumina of the reticulate exine.     

Dedifferentiation and microtubule reorganization in the apical cell protoplast ofSphacelaria (Phaeophyceae)

Summary The apical cell ofSphacelaria, a tip-growing filamentous brown alga, and its protoplast constitute a model for the investigation of the consequences of cell wall removal on microtubular cytoskeletal organization and cell polarity. In the apical cell, the microtubular cytoskeleton is strongly polarized and, in most cases, extends from two centrosomes to the cortex where it constitutes a fine meshwork. Observations of microtubule dynamics throughout the cell cycle emphasize the coincidence between orientation of the mitotic axis and cell polarity. Just after protoplast isolation, dramatic alterations of initial polarity are observed, whatever the mitotic stage. In particular, the coincidence between cytoplasmic polarity and polarity of the system nucleus-centrosomes is lost in most cases. 12–24 h after protoplast isolation, the cell shows a more symmetrical organization while a dense cortical microtubular network spreads out concomitantly with wall reformation. Our discussion emphasizes the possible relationship between cell polarity and cell totipotency, and the relevance of such a model for higher plant studies.     

Embryo sac development in Arabidopsis thaliana

Summary Aspects of megasporogenesis in Arabidopsis thaliana have been investigated using a variety of histochemical techniques to visualize general cell organization, DNA and callose in whole ovules and sections by bright field, fluorescence, differential interference contrast and scanning electron microscopy. The microtubular cytoskeleton has been studied using immunofluorescence localization of tubulin in sections and whole cells. The observations deviate from reports of preceding studies in that the megasporocyte was found to undergo both meiotic divisions followed by simultaneous cytokinesis (i.e. without an intermediate dyad stage) to give a multiplanar tetrad of megaspores. This represents a variation of monosporic development not previously described. Polarized distribution of organelles prior to meiosis ensures that the functional megaspore receives the largest share. Aberrant wall formation is common between degenerating megaspores. Localized callose deposition in the tetrad separates these cells from the active megaspore. Their pattern of degeneration and displacement is extremely flexible within the embryo sac space. The microtubular cytoskeleton is extensive and largely cytoplasmic, as distinct from cortical, throughout megasporogenesis. In the megasporocyte, megaspores and one-nucleate embryo sac, randomly oriented microtubules throughout the cells may serve to maintain cytoplasmic integrity and position organelles. Numerous microtubules (MTs) associate closely with the nucleus and some radiate from it, perhaps functioning in nuclear positioning. During meiosis MTs are restricted to the spindle configurations and later to the phragmoplasts which form between daughter nuclei. The lack of interphase cortical arrays suggests that the role of internal influences on cell shape is small.     

Microtubular cytoskeletons of the trophic cells of five eumycetozoans

Summary The trophic cells of the protostelids,Ceratiomyxella tahitiensis, Cavostelium apophysatum, Planoprotostelium aurantium, andClastostelium recurvatum, and the reduced myxomycete,Echinostelium bisporum, were examined with indirect immunofluorescence to determine the overall structure of the microtubular cytoskeletons of each type of cell. All five species have a distinct flagellar apparatus in the amoeboflagellate state, but they vary with respect to the presence of body microtubules, those microtubules which do not focus on the flagellar apparatus. The obligate amoebae ofC. tahitiensis, C. apophysatum andC. recurvatum all have extensive microtubular cytoskeletons, but each is unique to its respective species. The obligate amoeba ofC. tahitiensis has scattered microtubules which often describe curved paths. The microtubules never appear to focus on MTOCs. The microtubular cytoskeleton ofC. apophysatum consists of relatively straight, rigid appearing microtubules. Small subpopulations of these microtubules radiate from MTOCs near the nucleus. The obligate amoeba ofC. recurvatum has a cytoskeleton consisting of numerous microtubules which radiate from a perinuclear MTOC and fill the body of the cell. These results correlate well with earlier ultrastructural observations which suggest that the amoeboflagellate state is homologous in these mycetozoans and that the obligate amoebae, when present, are unique to the various lineages in which they appear.     

Cytoskeletal organization and cell organelle transport in basal epithelial cells of the freshwater sponge Spongilla lacustris

Summary Different antibodies against actin, tubulin and cytokeratin were utilized to demonstrate the spatial organization of the cytoskeleton in basal epithelial cells of the freshwater sponge Spongilla lacustris. Accordingly, actin is localized in a cortical layer beneath the plasma membrane and in distinct fibers within the cytoplasmic matrix. Microtubules exhibit a different distributional pattern by radiating from a perinuclear sheath and terminating at, the cell periphery; in contrast, intermediate filaments are lacking. Cytoplasmic streaming activity was studied by in-vivo staining of mitochondria and endoplasmic reticulum by means of fluorescent dyes. Single-frame analysis of such specimens revealed a regular shuttle movement of mitochondria and other small particles between the cell nucleus and the plasma membrane, which can be stopped in a reversible manner with the use of colcemid or colchicine but not with cytochalasin D. The results point to the microtubular system as a candidate for cell organelle transport, whereas the actomyosin system rather serves for changes in cellular shape and motility.     

阔口尖毛虫纤毛器微管的激光共聚焦显微镜观察

应用激光扫描共聚焦显微术显示经荧光紫杉醇标记的阔口尖毛虫(Oxytricha platystoma)口围带、波动膜、额腹横棘毛、左右缘棘毛等纤毛器的微管类细胞骨架.其口围带基部含小膜托架、托架间连接微管和小膜基部微管束,波动膜基部含发达的微管骨架网,口围带和波动膜后端的汇合处含有口底托架及口后微管束,额腹横棘毛和左、右...     

Changes in the density of microtubular networks in mesophyll cells and mesophyll derived protoplasts of Nicotiana and Triticum during leaf development

Changes in the density of microtubular mesh-works were analysed in mesophyll cells and mesophyll derived protoplasts of Nicotiana tabacum L. and Triticum aestivum L. during leaf development. The main purpose of this study was to test whether the low density, if not lack, of microtubular networks recently described in protoplasts that had been isolated from fully differentiated mesophyll cells happened during protoplast isolation or whether the loss of microtubules actually occurred during differentiation of the leaf tissue. Immunofluorescence microscopy showed that the density of the microtubular cytoskeleton in the leaf tissue decreased steadily after cessation of cell growth in both species. Nevertheless, in Triticum microtubule disappearance was swifter and occurred along a gradient from the base to tip of the leaf, a phenomenon reflecting the differences in the ontogeny between the dicotyledonous Nicotiana and the mono-cotyledonous Triticum leaves. Protein extraction from leaf tissues and Western blot analysis indicated that in both species the disappearance of microtubules was the result of a degradation of tubulin and not only due to a depolymerisation into tubulin subunits. When the cell walls were removed from live cells and the protoplasts released, the original patterns of the microtubules became obscured and, particularly in differentiated cells, the integrity and density of the microtubule strands deteriorated. The potential application of the density of the microtubular cytoskeleton as a marker in studies on differentiation and dedifferentiation in mesophyll cells and protoplasts is discussed.We wish to thank Silke Heichel for excellent technical assistance. We also express our thanks to the group of A.M. Lambert at CNRS, Strasbourg, France, for advice during establishment of our Western blot system. The work was supported by a grant of the German Ministry of Science and Technology (BMFT).     

The flagellar apparatus of the glaucophyte Cyanophora cuspidata

Glaucophytes are a kingdom‐scale lineage of unicellular algae with uniquely underived plastids. The genus Cyanophora is of particular interest because it is the only glaucophyte that is a flagellate throughout its life cycle, making its morphology more directly comparable than other glaucophytes to other eukaryote flagellates. The ultrastructure of Cyanophora has already been studied, primarily in the 1960s and 1970s. However, the usefulness of that work has been undermined by its own limitations, subsequent misinterpretations, and a recent taxonomic revision of the genus. For example, Cyanophora's microtubular roots have been widely reported as cruciate, with rotationally symmetrical wide and thin roots, although the first ultrastructural work described it as having three wide and one narrow root. We examine Cyanophora cuspidata using scanning and transmission electron microscopy, and construct a model of its cytoskeleton using serial‐section TEM. We confirm the earlier model, with asymmetric roots. We describe previously unknown and unsuspected features of its microtubular roots, including (i) a rearrangement of individual microtubules within the posterior right root, (ii) a splitting of the posterior left root into two subroots, and (iii) the convergence and termination of the narrow roots against wider ones in both the anterior and posterior subsystems of the flagellar apparatus. We also describe a large complement of nonmicrotubular components of the cytoskeleton, including a substantial connective between the posterior right root and the anterior basal body. Our work should serve as the starting point for a re‐examination of both internal glaucophyte diversity and morphological evolution in eukaryotes.     

Morphogenesis and Cortical Ultrastructure of Brooklynella hostilis,a Dysteriid Ciliate Ectoparasitic on Marine Fishes*

SYNOPSIS. Morphogenesis, and the cortical structures of Brooklynella hostilis, a cyrtophorine gymnostome ciliate ectoparasitic on marine fishes, were studied from protargol silver-impregnated preparations and with the aid of electron microscopy. The pattern of morphogenesis of Brooklynella is close to that found in less differentiated species of the families Chlamydodontidae (e.g., in the genus Trithigmostoma) and Dysteriidae (e.g., in the genus Hartmanula). The full number of kineties in the opisthe is restored after division from a segment of the left one of the 3 kinetics producing the oral rows. The oral rows consist of a double row of kinetosomes arranged in a zig-zag pattern; only the outer row is ciliated, the inner one being barren. However, the positions of the postciliary and transverse fibers indicate that the oral rows are not homologs of an undulating membrane but are akin to a membranelle. In association with the ventral somatic kinetosomes there are 4 postciliary fibers; a rather aberrant, transversally oriented kinetodesma; 2 microtubular, transverse fibers plus a transverse fibrousspur; and one to several ribbons of subkinetal microtubular fibers. Not directly associated with the kinetosomes are fibrous strands running subpellicularly between the kinetosomes and also deep into the cytoplasm. The cortical structures of Brooklynella are compared with those of some other groups of ciliates of about the same phylogenetic level in which the subkinetal microtubules can also be found– rhynchodine, suctorian, and chonotrich ciliates. The nasse consists of 6–8 nematodesmata not closely associated with the microtubular cytopharyngeal tube. The former have a distinctly developed densely fibrous capitulum containing barren kinetosomes which originally produced the nematodesma during stomatogenesis; the capitulum is connected by a fibrous link to the microtubular shaft. Extending from the oral rows to the capitula are fibrous structures strongly reminiscent of filamentous reticulum in hymenostome and peritrich ciliates. The structure of the posterio-ventral glandular organelle is also described and discussed.     

A new type of microtubular cytoskeleton in microsporogenesis of Lavatera thuringiaca L.

Summary. In Lavatera thuringiaca, kariokinesis and simultaneous cytokinesis during the meiotic division of microsporogenesis follow a procedure similar to that which takes place in the majority of members of the class Angiospermae. However, chondriokinesis occurs in a unique way found only in species from the family Malvaceae. Chondriokinesis in such species is well documented, but the relationship between the tubulin cytoskeleton and rearrangement of cell organelles during meiosis in L. thuringiaca has not been precisely defined so far. In this study, the microtubular cytoskeleton was investigated in dividing microsporocytes of L. thuringiaca by immunofluorescence. The meiotic stages and positions of cell organelles were identified by staining with 4′,6-diamidino-2-phenylindole. We observed that, during prophase I and II, changes in microtubular cytoskeleton configurations have unique features, which have not been described for other plant species. At the end of prophase I, organelles (mostly plastids and mitochondria) form a compact envelope around the nucleus, and the subsequent phases of kariokinesis take place within this arrangement. At this point of cell division, microtubules surround the organelle envelope and separate it from the peripheral cytoplasm, which is devoid of plastids and mitochondria. In telophase I, two newly formed nuclei are tightly surrounded by the cell organelle envelopes, and these are separated by the phragmoplast. Later, when the phragmoplast disappears, cell organelles still surround the nuclei but also move a little, starting to occupy the place of the disappearing phragmoplast. After the breakup of tetrads, the radial microtubule system is well developed, and cell organelles can still be observed as a dense envelope around the nuclei. At a very late stage of sporoderm development, the radial microtubule system disappears, and cell organelles become gradually scattered in the cytoplasm of the microspores. Using colchicines, specific inhibitors of microtubule formation, we investigated the relationship between the tubulin cytoskeleton and the distribution of cell organelles. Our analysis demonstrates that impairment of microtubule organization, which constitutes only a single component of the cytoskeleton, is enough to disturb typical chondriokinesis in L. thuringiaca. This indicates that microtubules (independent of microfilaments) are responsible for the reorganization of cell organelles during meiotic division. Correspondence: D. Tchórzewska, Department of Plant Anatomy and Cytology, Maria Curie-Skłodowska University, Akademicka 19, 20-033 Lublin, Poland.     

Embryogenesis in <Emphasis Type="Italic">Sedum acre</Emphasis> L.: structural and immunocytochemical aspects of suspensor development

The changes in the formation of both the actin and the microtubular cytoskeleton during the differentiation of the embryo-suspensor in Sedum acre were studied in comparison with the development of the embryo-proper. The presence and distribution of the cytoskeletal elements were examined ultrastructurally and with the light microscope using immunolabelling and rhodamine-phalloidin staining. At the globular stage of embryo development extensive array of actin filaments is present in the cytoplasm of basal cell, the microfilament bundles generally run parallel to the long axis of basal cell and pass in close to the nucleus. Microtubules form irregular bundles in the cytoplasm of the basal cell. A strongly fluorescent densely packed microtubules are present in the cytoplasmic layer adjacent to the wall separating the basal cell from the first layer of the chalazal suspensor cells. At the heart-stage of embryo development, in the basal cell, extremely dense arrays of actin materials are located near the micropylar and chalazal end of the cell. At this stage of basal cell formation, numerous actin filaments congregate around the nucleus. In the fully differentiated basal cell and micropylar haustorium, the tubulin cytoskeleton forms a dense prominent network composed of numerous cross-linked filaments. In the distal region of the basal cell, a distinct microtubular cytoskeleton with numerous microtubules is observed in the cytoplasmic layer adjacent to the wall, separating the basal cell from the first layer of the chalazal suspensor cells. The role of cytoskeleton during the development of the suspensor in S. acre is discussed.     

A mycorrhizal fungus changes microtubule orientation in tobacco root cells

Summary Cortical cells of mycorrhizal roots undergo drastic morphological changes, such as vacuole fragmentation, nucleus migration, and deposition of cell wall components at the plant-fungus interface. We hypothesized that the cytoskeleton is involved in these mechanisms leading to cell reorganization. We subjected longitudinal, meristem to basal zone, sections of uninfectedNicotiana tabacum roots to immunofluorescence methods to identify the microtubular (MT) structures associated with root cells. Similar sections were obtained from tobacco roots grown in the presence ofGigaspora margarita, an arbuscular mycorrhizal fungus which penetrates the root via the epidermal cells, but mostly develops in the inner cortical cells. While the usual MT structures were found in uninfected roots (e.g., MTs involved in mitosis in the meristem and cortical hoops in differentiated parenchyma cells), an increase in complexity of MT structures was observed in infected tissues. At least three new systems were identified: (i) MTs running along large intracellular hyphae, (ii) MTs linking hyphae, (iii) MTs binding the hyphae to the host nucleus. The experiments show that mycorrhizal infection causes reorganization of root MTs, suggesting their involvement in the drastic morphological changes shown by the cortical cells.     

Cortical microtubules rearrange during differentiation of vascular cambial derivatives, microfilaments do not

The plant cytoskeleton has been implicated in a variety of morphogenetic events in higher plants. Most of this work, however, has concentrated on epidermal cells or primary tissues. We have investigated the cortical microtubular (CMT) and microfilament (MF) components of the cytoskeleton in a secondary tissue  –  active vascular cambium of Aesculus hippocastanum L. (horse-chestnut)  –  and followed the changes in these components during the early stages of differentiation of fusiform cambial derivatives to axial elements of the secondary vascular system. A correlative approach was used employing indirect immunofluorescence microscopy of α-tubulin on 6 μm sections, and transmission electron microscopy of 60 nm sections. The study has demonstrated a rearrangement of the CMT cytoskeleton, from random to helical, as fusiform vascular cambial cells begin to differentiate as secondary phloem vascular tissue. A similar CMT rearrangement is seen as fusiform cambial cells begin to differentiate as secondary xylem fibres. This rearrangement is interpreted as evidence of determination of cambial derivatives towards vascular development. Axially-oriented MF bundles are present in fusiform cambial cells and their axial orientation is retained in the vascular derivatives at early stages of their development even though the CMTs have become rearranged. Received: 5 August 1996 /  Accepted: 23 September 1996     

Pollen wall formation in Lilium: The effect of chaotropic agents,and the organisation of the microtubular cytoskeleton during pattern development

Using a combination of electron-microscopic and immunocytochemical techniques the behaviour of the microtubular cytoskeleton has been followed throughout microsporogenesis in Lilium henryi Thunb. Cells treated with colchicine at specific stages and then permitted to develop to near maturity were used to investigate any participation by microtubules in the regulation of pollen wall patterning. The microtubular cytoskeleton assumes four principal forms during the meiotic process; in pre-meiosis it resembles that characteristic of meristematic somatic cells, during meiotic prophase it becomes associated with a nuclear envelope and, perhaps, with the chromosomes and, as the nuclear and cell divisions commence, it takes the form of a normal spindle apparatus. In the young microspores, microtubules assume a radial organisation extending from sites at the nuclear envelope to the inner face of the plasma membrane. No firm evidence was found linking any one of these forms of cytoskeleton with the generation of patterning on the cell surface. Experiments with colchicine revealed that the drug would readily dislocate the colpus, but did not affect the general reticulate patterning. The radial cytoskeleton was present during the deposition of the early primexine, but evidence from these and other studies (J.M. Sheldon and H.G. Dickinson 1983, J. Cell. Sci. 63, 191–208; H.G. Dickinson and J.M. Sheldon, 1984, Planta 161, 86–90) indicates patterning to be imprinted upon the plasma membrane prior to the appearance of this type of cytoskeleton. These results are discussed in terms of a recent model proposed to explain pattern generation on the surface of Lilium pollen grains, based on the self-assembly of patterning determinants within the plasma membrane.Abbreviation MTOC microtubule-organising centre     

Actin cytoskeleton modulates calcium signaling during maturation of starfish oocytes

Before successful fertilization can occur, oocytes must undergo meiotic maturation. In starfish, this can be achieved in vitro by applying 1-methyladenine (1-MA). The immediate response to 1-MA is the fast Ca²⁺ release in the cell cortex. Here, we show that this Ca²⁺ wave always initiates in the vegetal hemisphere and propagates through the cortex, which is the space immediately under the plasma membrane. We have observed that alteration of the cortical actin cytoskeleton by latrunculin-A and jasplakinolide can potently affect the Ca²⁺ waves triggered by 1-MA. This indicates that the cortical actin cytoskeleton modulates Ca²⁺ release during meiotic maturation. The Ca²⁺ wave was inhibited by the classical antagonists of the InsP₃-linked Ca²⁺ signaling pathway, U73122 and heparin. To our surprise, however, these two inhibitors induced remarkable actin hyper-polymerization in the cell cortex, suggesting that their inhibitory effect on Ca²⁺ release may be attributed to the perturbation of the cortical actin cytoskeleton. In post-meiotic eggs, U73122 and jasplakinolide blocked the elevation of the vitelline layer by uncaged InsP₃, despite the massive release of Ca²⁺, implying that exocytosis of the cortical granules requires not only a Ca²⁺ rise, but also regulation of the cortical actin cytoskeleton. Our results suggest that the cortical actin cytoskeleton of starfish oocytes plays critical roles both in generating Ca²⁺ signals and in regulating cortical granule exocytosis.