上皮细胞分裂过程中中等纤维的变化

Immunofluorescence microscopy was used to follow the rearrangement of keratin filaments and vimentin filaments during mitosis in Vero and HeLa cell lines. The experiment results showed that the three dimensional organization and structure of intermediate filaments changed drastically during mitosis. The behavior of intermediate filaments was different in these two epithelial cell lines. In mitotic Vero cells the keratin filaments and vimentin filaments maintained their filamentous structure and formed a cage around the mitotic apparatus. In mitotic HeLa cells the keratin filaments and vimentin filaments reorganized extensively and formed granular cytoplasmic bodies. The ratio of granular cytoplasmic body formation changed in different mitotic phase. The interphase intermediate filament network was reconstructed after mitosis. It is proposed that the state of intermediate filament network in these cells is cell cycle-dependent and intermediate filaments may have some skeletal role in mitosis.     

上皮细胞分裂过程中中等纤维的变化

应用制备的血清抗体,采用免疫细胞化学方法观察了两株培养上皮细胞的分裂过程中IF的动态变化过程。实验结果显示,在上皮细胞分裂过程中,IF形态结构及空间分布发生了显著变化,不同细胞之间存在差异,分裂的Vero细胞中角蛋白纤维和波形纤维都维持纤维形态,围绕分裂器形成纤维网罩或纤维束环,随着细胞分裂的进行,IF网的空间组织结构和外观发生动态变化;分裂的HeLa细胞中,角蛋白纤维和波形纤维广泛重组形成颗粒状胞质小体,分裂结束后重建IF网。实验结果表明,IF变化具有细胞周期依赖性和一定的细胞特异性。本文对IF在细胞分裂过程中的功能意义作了讨论。     

Cytoskeletal disorders in human keratinocytes--epidermolysis bullosa simplex]

The cytoskeletons possibly related to pathogenesis in skin disease may be limited to keratin intermediate filaments (KIF) in epidermal keratinocytes. Keratins are divided into two subclasses; 11 acidic (type I) keratins and 8 basic (type II) keratins. Combination of equimolar amounts of type I and type II can form KIF. KIFs in human epidermal basal cells consist of a pair of type I and type II keratins specifically synthesized in the basal cells, and those in spinous cells contain two pairs of keratin; a pair of basal cell keratin and another pair of keratin specific for suprabasal cells. In the first section, molecular biology and differentiation of keratins are reviewed. In the second section, epidermolysis bullosa simplex (EBS) was introduced from the view point of abnormal organization of KIFs. In the epidermis of EBS, clefts are induced in the basal cells by minor trauma or frictions consequently to produce bullae. Electron microscopy reveals small spherical aggregations of tonofilaments (KIFs) in the basal cells. In biopsies, these KIF aggregations might be caused by artifacts during procedures for biopsies, so that, in order to avoid these artifacts, we studied the KIF organization in cultured keratinocytes from a patient by immunofluorescence using anti-keratin antibodies and electron microscopy. Anti-keratin antibodies revealed a formation of small droplet-like aggregations of KIFs in many cultured cells adhering to the culture bottles, which were also suggested by electron microscopy. From these observations, it is suggested that the abnormal organization (droplets) of KIFs might be one of intrinsic factors for the pathogenesis of EBS.(ABSTRACT TRUNCATED AT 250 WORDS)     

中等纤维在几种人体上皮细胞有丝分裂过程中的行为

By indirect immunofluorescence microscopy and electron microscopy, we studied the behavior of intermediate filaments during mitosis in three human epithelial cell lines, derived from normal epidermis (PcaSE-1, from a cancer patient), stratified epithelium (CNE, from nasopharyngeal carcinoma) and simple epithelium (SPC-A-1 from lung adenocarcinoma) respectively. CNE cells and SPC-A-1 cells express two different intermediate filament systems; keratin filaments and vimentin filaments, but PcaSE-1 cells only express keratin filaments. The keratin filament system in PcaSE-1 cells remained intact and encircled the developing mitotic spindle as the cells entered mitosis. In contrast, in CNE cells and SPC-A-1 cells, keratin filaments appeared to disassemble into amorphous cytoplasmic bodies during mitosis. However, their vimentin filaments remained morphologically intact throughout mitosis. We propose; (1) The disassembly of keratin filaments in mitotic epithelial cells is more or less associated with the degree of their cell malignancy rather than with the abundance of keratin filaments in interphase. (2) Intermediate filaments may be involved in the positioning and/or centering of the spindle during mitosis. (3) The possible function of vimentin filament system in CNE cells is positioning and orientation of chromosomes.     

中等纤维在几种人体上皮细胞有丝分裂过程中的行为

本文用间接免疫荧光法和电镜术观察了分别来自人表皮(PcaSE-1)、复层上皮(CNE)和单层上皮(SPC-A-1)的3个上皮细胞系的细胞在有丝分裂过程中中等纤维的行为。结果表明,CNE细胞和SPC-A-1细胞表达两种不同类型的中等纤维系统:角蛋白纤维和波形纤维,而PcaSE-1细胞仅表达角蛋白纤维。当细胞进入有丝分裂时,PcaSE-1细胞的角蛋白纤维维持完整的形态且将有丝分裂纺锤体围绕在细胞中央。相反,在CNE细胞和SPC-A-1细胞中,在细胞有丝分裂时,角蛋白纤维解聚成无定形的胞质小体,然而它们的波形纤维始终保持完整的形态。我们认为(1)在分裂上皮细胞中,角蛋白纤维的解聚与细胞的恶性程度有关,而与间期上皮细胞中是否含有丰富的角蛋白纤维无明显关系。(2)在上皮细胞有丝分裂时,中等纤维可能参于纺锤体的定位和趋中。(3)在分裂CNE细胞中,波形纤维的可能功能是染色体的定位和定向。     

Disruption of the keratin filament network during epithelial cell division

The behaviour of keratin filaments during cell division was examined in a wide range of epithelial lines from several species. Almost half of them show keratin disruption as described previously: by immunofluorescence, filaments are replaced during mitosis by a 'speckled' pattern of discrete cytoplasmic dots. In the electron microscope these ' speckles ' are seen as granules around the cell periphery, just below the actin cortical mesh, with no detectable 10 nm filament structure inside them and no keratin filament bundles in the rest of the cytoplasm. A time course of the filament reorganization was constructed from double immunofluorescence data; filaments are disrupted in prophase, and the filament network is intact again by cytokinesis. The phenomenon is restricted to cells rich in keratin filaments, such as keratinocytes; it is unrelated to the co-existence of vimentin in many of these cells, and vimentin is generally maintained as filaments while the keratin is restructured. Some resistance to the effect may be conferred by an extended cycle time. Filament reorganization takes place within minutes, so that a reversible mechanism seems more likely than one involving de novo protein synthesis, at this metabolically quiet stage of the cell cycle.     

Keratin gene mutations in disorders of human skin and its appendages

Keratins, the major structural protein of all epithelia are a diverse group of cytoskeletal scaffolding proteins that form intermediate filament networks, providing structural support to keratinocytes that maintain the integrity of the skin. Expression of keratin genes is usually regulated by differentiation of the epidermal cells within the stratifying squamous epithelium. Amongst the 54 known functional keratin genes in humans, about 22 different genes including, the cornea, hair and hair follicle-specific keratins have been implicated in a wide range of hereditary diseases. The exact phenotype of each disease usually reflects the spatial expression level and the types of mutated keratin genes, the location of the mutations and their consequences at sub-cellular levels as well as other epigenetic and/or environmental factors. The identification of specific pathogenic mutations in keratin disorders formed the basis of our understanding that led to re-classification, improved diagnosis with prognostic implications, prenatal testing and genetic counseling in severe keratin genodermatoses. Molecular defects in cutaneous keratin genes encoding for keratin intermediate filaments (KIFs) causes keratinocytes and tissue-specific fragility, accounting for a large number of genetic disorders in human skin and its appendages. These diseases are characterized by keratinocytes fragility (cytolysis), intra-epidermal blistering, hyperkeratosis, and keratin filament aggregation in severely affected tissues. Examples include epidermolysis bullosa simplex (EBS; K5, K14), keratinopathic ichthyosis (KPI; K1, K2, K10) i.e. epidermolytic ichthyosis (EI; K1, K10) and ichthyosis bullosa of Siemens (IBS; K2), pachyonychia congenita (PC; K6a, K6b, K16, K17), epidermolytic palmo-plantar keratoderma (EPPK; K9, (K1)), monilethrix (K81, K83, K86), ectodermal dysplasia (ED; K85) and steatocystoma multiplex. These keratins also have been identified to have roles in apoptosis, cell proliferation, wound healing, tissue polarity and remodeling. This review summarizes and discusses the clinical, ultrastructural, molecular genetics and biochemical characteristics of a broad spectrum of keratin-related genodermatoses, with special clinical emphasis on EBS, EI and PC. We also highlight current and emerging model tools for prognostic future therapies. Hopefully, disease modeling and in-depth understanding of the molecular pathogenesis of the diseases may lead to the development of novel therapies for several hereditary cutaneous diseases.     

Microinjection of monoclonal antibodies specific for one intermediate filament protein in cells containing multiple keratins allow insight into the composition of particular 10 nm filaments

Monoclonal antibodies specific for vimentin (V9), keratin 7 (CK 7) and keratin 18 (CK5) have been microinjected into three human epithelial cell lines: HeLa, MCF-7 and RT-4. The effect of the injection on other keratin polypeptides and vimentin filaments has been observed by double label immunofluorescence and in some instances by immunoelectron microscopy using gold labels of different sizes. Microinjection of V9 into HeLa cells causes the vimentin to collapse into a perinuclear cap leaving the keratin filaments unaffected. Injection of CK5 does not affect the vimentin filaments but disrupts the keratin filaments revealing keratin aggregates similar to those seen in some epithelial cell lines during mitosis. The keratin aggregates obtained after microinjection in HeLa contain the keratins 8 and 18 and probably also other keratins, as no residual keratin filaments are observed with a keratin polyclonal antibody of broad specificity. Aggregates in mitotic HeLa cells contain at least the keratins 7, 8, and 18. In MCF-7 cells keratins 8, 18, and 19 are observed in the aggregates seen 3 h after microinjection which, however, show a different morphology from those seen in HeLa cells. In MCF-7 cells a new keratin filament is built within 6 h after the injection which is composed mainly of keratin 8 and 19. The antibody-complexed keratin 18 remains in spherical aggregates of different size. The results suggest that in HeLa cells vimentin and keratin form independent networks, and that individual 10 nm filaments in epithelial cell lines can contain more than two keratins.     

Inducible expression of filaggrin increases keratinocyte susceptibility to apoptotic cell death

Filaggrin is an intermediate filament associated protein that aids the packing of keratin filaments during terminal differentiation of keratinocytes. Premature aggregation of keratin filaments is prevented by filaggrin expression as the inactive precursor, profilaggrin, which is localized in keratohyalin granules in vivo. We have previously shown that filaggrin constructs, when transiently transfected into epithelial cells, lead to a collapsed keratin cytoskeletal network and dysmorphic nuclei with features of apoptosis. The apparent transfection rate is low with filaggrin constructs, supporting their disruptive role but hindering further study. To bypass this problem, we generated stable keratinocyte cell lines that express mature human filaggrin using a tetracycline-inducible promoter system. We found that cell lines expressing filaggrin, but not control cell lines, exhibited increased sensitivity to multiple apoptotic stimuli as measured by morphologic and biochemical criteria. None of the cell lines showed an increase in endogenous expression of filaggrin in response to the same stimuli. Filaggrin expression alone was insufficient to induce apoptosis in these keratinocyte cell lines. We conclude that filaggrin, due to its keratin binding ability, primes cells for apoptosis. Because filaggrin is expressed at a level of the epidermis where keratinocytes are in transition between the nucleated granular and the anucleate cornified layers, we hypothesize that filaggrin aids in the terminal differentiation process by facilitating apoptotic machinery.     

Mathematical Modeling of the Impact of Actin and Keratin Filaments on?Keratinocyte Cell Spreading

Keratin intermediate filaments (IFs) form cross-linked arrays to fulfill their structural support function in epithelial cells and tissues subjected to external stress. How the cross-linking of keratin IFs impacts the morphology and differentiation of keratinocytes in the epidermis and related surface epithelia remains an open question. Experimental measurements have established that keratinocyte spreading area is inversely correlated to the extent of keratin IF bundling in two-dimensional culture. In an effort to quantitatively explain this relationship, we developed a mathematical model in which isotropic cell spreading is considered as a first approximation. Relevant physical properties such as actin protrusion, adhesion events, and the corresponding response of lamellum formation at the cell periphery are included in this model. Through optimization with experimental data that relate time-dependent changes in keratinocyte surface area during spreading, our simulation results confirm the notion that the organization and mechanical properties of cross-linked keratin filaments affect cell spreading; in addition, our results provide details of the kinetics of this effect. These in silico findings provide further support for the notion that differentiation-related changes in the density and intracellular organization of keratin IFs affect tissue architecture in epidermis and related stratified epithelia.     

Morphology, behavior, and interaction of cultured epithelial cells after the antibody-induced disruption of keratin filament organization

The organization of intermediate filaments in cultured epithelial cells was rapidly and radically affected by intracellularly injected monoclonal antikeratin filament antibodies. Different antibodies had different effects, ranging from an apparent splaying apart of keratin filament bundles to the complete disruption of the keratin filament network. Antibodies were detectable within cells for more than four days after injection. The antibody-induced disruption of keratin filament organization had no light-microscopically discernible effect on microfilament or microtubule organization, cellular morphology, mitosis, the integrity of epithelial sheets, mitotic rate, or cellular reintegration after mitosis. Cell-to-cell adhesion junctions survived keratin filament disruption. However, antibody injected into a keratinocyte-derived cell line, rich in desmosomes, brought on a superfasciculation of keratin filament bundles, which appeared to pull desmosomal junctions together, suggesting that desmosomes can move in the plane of the plasma membrane and may only be 'fixed' by their anchoring to the cytoplasmic filament network. Our observations suggest that keratin filaments are not involved in the establishment or maintenance of cell shape in cultured cells.     

A role for disulfide bonding in keratin intermediate filament organization and dynamics in skin keratinocytes

We recently reported that a trans-dimer, homotypic disulfide bond involving Cys367 in keratin 14 (K14) occurs in an atomic-resolution structure of the interacting K5/K14 2B domains and in keratinocyte cell lines. Here we show that a sizable fraction of the K14 and K5 protein pools participates in interkeratin disulfide bonding in primary cultures of mouse skin keratinocytes. By comparing the properties of wild-type K14 with a completely cysteine-free variant thereof, we found that K14-dependent disulfide bonding limited filament elongation during polymerization in vitro but was necessary for the genesis of a perinuclear-concentrated network of keratin filaments, normal keratin cycling, and the sessile behavior of the nucleus and whole cell in keratinocytes studied by live imaging. Many of these phenotypes were rescued when analyzing a K14 variant harboring a single Cys residue at position 367. These findings establish disulfide bonding as a novel and important mechanism regulating the assembly, intracellular organization, and dynamics of K14-containing intermediate filaments in skin keratinocytes.     

Population Structure of <Emphasis Type="Italic">in Vitro</Emphasis> Cultured Keratinocytes

Analysis of behavior of isolated epidermal keratinocytes demonstrated two dominant processes within the first two days of cultivation: formation of cell aggregates and their adhesion to the substrate. Keratinocyte spreading increased in the attached aggregates, where densely packed and well spread cells could be recognized in the aggregate center and periphery, respectively. Cell spreading and proliferation was observed in the following days. Randomly distributed p63-positive cells amounted to 35% in the studied 2–3-day keratinocyte cultures. The process of epidermal cell aggregation was reproduced in the population of basal keratinocytes, where Ki-67-expressing cells amounted to about 20%. Single cells expressing keratin 19 were observed during cell aggregation in the secondary culture. We believe that the aggregation of cultured keratinocytes reflects the formation of structural-functional units (SFUs) in vivo. Disaggregated epidermal keratinocytes maintained the information relevant for self-assembly of three-dimensional structures capable of SFU formation after transplantation into the body.     

Onset of re-epithelialization after skin injury correlates with a reorganization of keratin filaments in wound edge keratinocytes: defining a potential role for keratin 16

Injury to stratified epithelia causes a strong induction of keratins 6 (K6) and 16 (K16) in post-mitotic keratinocytes located at the wound edge. We show that induction of K6 and K16 occurs within 6 h after injury to human epidermis. Their subsequent accumulation in keratinocytes correlates with the profound reorganization of keratin filaments from a pan-cytoplasmic distribution to one in which filaments are aggregated in a juxtanuclear location, opposite to the direction of cell migration. This filament reorganization coincides with additional cytoarchitectural changes and the onset of re-epithelialization after 18 h post-injury. By following the assembly of K6 and K16 in vitro and in cultured cells, we find that relative to K5 and K14, a well- characterized keratin pair that is constitutively expressed in epidermis, K6 and K16 polymerize into short 10-nm filaments that accumulate near the nucleus, a property arising from K16. Forced expression of human K16 in skin keratinocytes of transgenic mice causes a retraction of keratin filaments from the cell periphery, often in a polarized fashion. These results imply that K16 may not have a primary structural function akin to epidermal keratins. Rather, they suggest that in the context of epidermal wound healing, the function of K16 could be to promote a reorganization of the cytoplasmic array of keratin filaments, an event that precedes the onset of keratinocyte migration into the wound site.     

The organizational fate of intermediate filament networks in two epithelial cell types during mitosis

Intermediate filaments (IF) appear to be attached to the nuclear envelope in various mammalian cell types. The nucleus of mouse keratinocytes is enveloped by a cagelike network of keratin-containing bundles of IF (IFB). This network appears to be continuous with the cytoplasmic IFB system that extends to the cell surface. Electron microscopy reveals that the IFB appear to terminate at the level of the nuclear envelope, frequently in association with nuclear pore complexes (Jones, J. C .R., A. E. Goldman, P. Steinert, S. Yuspa, and R. D. Goldman, 1982, Cell Motility, 2:197-213). Based on these observations of nuclear-IF associations, it is of interest to determine the fate and organizational states of IF during mitosis, a period in the cell cycle when the nuclear envelope disassembles. Immunofluorescence microscopy using a monoclonal keratin antibody and electron microscopy of thin and thick sections of mitotic mouse keratinocytes revealed that the IFB system remained intact as the cells entered mitosis and surrounded the developing mitotic spindle. IFB were close to chromosomes and often associated with chromosome arms. In contrast, in HeLa, a human epithelial cell, keratin-containing IFB appear to dissemble as cells enter mitosis (Franke, W. W., E. Schmid, C. Grund, and B. Geiger, 1982, Cell, 30:103-113). The keratin IFB in mitotic HeLa cells appeared to form amorphous nonfilamentous bodies as determined by electron microscopy. However, in HeLa, another IF system composed primarily of a 55,000-mol-wt protein (frequently termed vimentin) appears to remain morphologically intact throughout mitosis in close association with the mitotic apparatus (Celis, J.E., P.M. Larsen, S.J. Fey, and A. Celis, 1983, J. Cell Biol., 97:1429-34). We propose that the mitotic apparatus in both mouse epidermal cells and in HeLa cells is supported and centered within the cell by IFB networks.     

Cytoskeleton and morphogenesis in brown algae

BACKGROUND: Morphogenesis on a cellular level includes processes in which cytoskeleton and cell wall expansion are strongly involved. In brown algal zygotes, microtubules (MTs) and actin filaments (AFs) participate in polarity axis fixation, cell division and tip growth. Brown algal vegetative cells lack a cortical MT cytoskeleton, and are characterized by centriole-bearing centrosomes, which function as microtubule organizing centres. SCOPE: Extensive electron microscope and immunofluorescence studies of MT organization in different types of brown algal cells have shown that MTs constitute a major cytoskeletal component, indispensable for cell morphogenesis. Apart from participating in mitosis and cytokinesis, they are also involved in the expression and maintenance of polarity of particular cell types. Disruption of MTs after Nocodazole treatment inhibits cell growth, causing bulging and/or bending of apical cells, thickening of the tip cell wall, and affecting the nuclear positioning. Staining of F-actin using Rhodamine-Phalloidin, revealed a rich network consisting of perinuclear, endoplasmic and cortical AFs. AFs participate in mitosis by the organization of an F-actin spindle and in cytokinesis by an F-actin disc. They are also involved in the maintenance of polarity of apical cells, as well as in lateral branch initiation. The cortical system of AFs was found related to the orientation of cellulose microfibrils (MFs), and therefore to cell wall morphogenesis. This is expressed by the coincidence in the orientation between cortical AFs and the depositing MFs. Treatment with cytochalasin B inhibits mitosis and cytokinesis, as well as tip growth of apical cells, and causes abnormal deposition of MFs. CONCLUSIONS: Both the cytoskeletal elements studied so far, i.e. MTs and AFs are implicated in brown algal cell morphogenesis, expressed in their relationship with cell wall morphogenesis, polarization, spindle organization and cytokinetic mechanism. The novelty is the role of AFs and their possible co-operation with MTs.     

De novo synthesis and specific assembly of keratin filaments in nonepithelial cells after microinjection of mRNA for epidermal keratin

Poly(A)+ RNA isolated from bovine muzzle epidermis was microinjected into nonepithelial cells containing only intermediate-sized filaments of the vimentin type. In recipient cells keratin polypeptides are synthesized and assemble into intermediate-sized filaments at multiple dispersed sites. We describe the time course and the pattern of de novo assembly of keratin filaments within living cells. These filaments were indistinguishable, by immunofluorescence and immunoelectron microscopic criteria, from keratin filament arrays present in true epithelial cells. The presence of extended keratin fibril meshworks in these injected cells is compatible with cell growth and mitosis. Double immunolabeling revealed that newly assembled keratin was not codistributed with microfilament bundles, microtubules or vimentin filaments. We suggest that assembly mechanisms exist which in vivo sort out newly synthesized cytokeratin polypeptides from vimentin.     

An immunofluorescence study of the effects of ultraviolet radiation on the organization of microfilaments, keratin intermediate filaments, and microtubules in human keratinocytes.

Indirect immunofluorescence microscopy has been used to investigate the ultraviolet (UV) radiation induced disruption of the organization of microfilaments, keratin intermediate filaments, and microtubules in cultured human epidermal keratinocytes. Following irradiation, concurrent changes in the organization of the three major cytoskeletal components were observed in cells incubated under low Ca2+ (0.15 mM) conditions. UV irradiation induced a dose-dependent condensation of keratin filaments into the perinuclear region. This collapse of the keratin network was accompanied by the reorganization of microfilaments into rings and a restricted distribution of microtubules, responses normally elicited by exposure to high Ca2+ (1.05 mM) medium. The UV induced alteration of the keratin network appears to disrupt the interactions between keratin and actin, permitting the reorganization of actin filaments in the absence of Ca2+ stimulation. In addition to the perinuclear condensation of keratin filaments, UV irradiation inhibits the Ca2+ induced formation of keratin alignments at the membrane of apposed cells if UV treatment precedes exposure to high Ca2+ medium. Incubation of keratinocytes in high Ca2+ medium for 24 hours prior to irradiation results in the stabilization of membrane associated keratin alignments and a reduced susceptibility of cytoplasmic keratin filaments to UV induced disruption. Unlike results from investigations with isogenic skin fibroblasts, no UV induced disassembly of microtubules was discernible in irradiated human keratinocytes.     

Mutant keratin expression in transgenic mice causes marked abnormalities resembling a human genetic skin disease.

To explore the relationship between keratin gene mutations and genetic disease, we made transgenic mice expressing a mutant keratin in the basal layer of their stratified squamous epithelia. These mice exhibited abnormalities in epidermal architecture and often died prematurely. Blistering occurred easily, and basal cell cytolysis was evidence at the light and electron microscopy levels. Keratin filament formation was markedly altered, with keratin aggregates in basal cells. In contrast, terminally differentiating cells made keratin filaments and formed a stratum corneum. Recovery of outer layer cells was attributed to down-regulation of mutant keratin expression and concomitant induction of differentiation-specific keratins as cells terminally differentiate, and the fact that these cells arose from basal cells developing at a time when keratin expression was relatively low. Collectively, the pathobiology and biochemistry of the transgenic mice and their cultured keratinocytes bore a resemblance to a group of genetic disorders known as epidermolysis bullosa simplex.     

Effect of brefeldin A and the dynamics of the actin plate on cytokinesis of zygotes in the brown alga,Silvetia babingtonii (Fucales,Phaeophyceae)

In the cytokinesis of brown algae, actin filaments appear like a plate at the intersecting region of microtubules (MTs) that emerge from the centrosomes after mitosis. The function of the actin plate itself is still unknown. To elucidate the relationship between the actin plate, MTs and membrane fusion, without inducing cytoskeleton depolymerization, the effect of brefeldin A (BFA), which prevents the production of vesicles from Golgi bodies, was examined in zygotes of Silvetia babingtonii. The beginning of mitosis was slightly delayed in zygotes under BFA compared with the controls. Almost all zygotes were inhibited for the progression of cytokinesis by BFA treatment. Ultrastructural observations showed that Golgi cisternae became fragmented or curled following continuous treatment with BFA, and the inhibitory status of cytokinesis between zygotes. The next cell cycle started before cytokinesis was completed. Although the appearance of the actin plate was not disturbed by BFA treatment, the behaviour of the actin plate during the transition between the first and second cell cycles could be classified into two patterns: it was either invisible upon the initiation of the next cell cycle, or a portion of it remained even though the next cell cycle had begun. In the latter case, a part of the actin plate seemed to associate with the new partially formed cell partition membrane, and MTs from the centrosomes were bound to it. The actin plate completely disappeared in the next mitosis, then re-emerged in the middle area of the four daughter nuclei. The results of the present study indicated that, under BFA treatment, the actin plate persisted until just before the beginning of the next mitotic phase, when the new, incomplete cell partition membrane was present, and MTs sustained the actin plate until next mitosis.