Surface cap modifications in cold-treatedDrosophila melanogaster embryos

Summary When earlyDrosophila embryos were allowed to develop at 0°C, several abnormalities in the surface cap organization were observed. Scanning electron microscopy showed that exposure to cold mainly lead to the deformation of the cortical caps and to their partial fusion with adjacent caps. The process of cellularization was presumably affected and large uncellularized areas were observed. Rhodamine-phalloidin staining showed that cap deformation was closely related to the altered microfilament distribution, which was presumably responsible for the failure of large syncytial areas to cellularize. During the process of cellularization, F-actin localization did not depend on the microtubules forming the baskets around the elongating nuclei, but was related to the subpopulation of mictotubules radiating from the centrosomes toward the plasma membrane. Only these microtubules seemed to be affected by cold treatment.     

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.     

Observations by a novel method of surface changes during the syncytial blastoderm stage of the Drosophila embryo

Up to now the cleavage process during the syncytial blastoderm stage has proved hard to follow in living Drosophila embryos. However, this can be achieved by microinjection of TMRITC-BSA into the fluid between the vitelline membrane and the embryo surface. The superficial bulges are then visible with epifluorescence optics. Once formed the bulges go through cycles of flattening, expansion, division, and rounding up. A tendency was noted for more divisions to occur at angles closer to 90° than at more acute angles relative to the previous cleavage. However, the cleavage angles were frequently determined by the distribution of surface space around the bulges. As caps became more numerous they squeezed and pushed around each other while expanding. As a result of these movements the surface of the blastoderm becomes uniformly covered with nuclei by the time of cellularization.     

Dynamics of the actin cytoskeleton,hyphal tip growth and the movement of the two nuclei in the dikaryon ofCoprinus cinereus

We first examined the changes in distribution of F-actin during conjugate division in the apical cells of the dikaryon ofCoprinus cinereus using indirect immunofluorescence microscopy, then followed hyphal tip growth and the movement of the two nuclei in the apical cells using differential interference contrast microscopy (DIC). In apical cells with interphase nuclei, F-actin occurred solely as peripheral plaques, which were distributed along the whole length of the cell and were more concentrated at the tips, where they formed caps. In the early prophase of conjugate division, F-actin was transiently concentrated, as diffused form and plaques, at hyphal regions where the two nuclei sit, and this was accompanied by transient disappearance of the actin cap at the hyphal tip in the majority of cells. The actin cap was also present at the tips of growing clamp cells from late prophase through metaphase and disintegrated during anaphase. In telophase, actin rings formed at the future septa. DIC revealed that, in early prophase, when the F-actin array occurs around the two nuclei and the actin cap is absent at hyphal tips, hyphae kept growing and the second nucleus accelerated its forward movement to catch up with the leading nucleus, which was still moving forward.     

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.     

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.     

Retarded nuclear migration in Drosophila embryos with aberrant F-actin reorganization caused by maternal mutations and by cytochalasin treatment

Three X-linked mutations of Drosophila melanogaster, gs(1)N26, gs(1)N441 and paralog, had a common maternal-effect phenotype. Mutant embryos show reduced egg contraction that normally occurs at an early cleavage stage in wild-type embryos. In addition, the mutants exhibited retarded nuclear migration while synchronous nuclear divisions were unaffected. The retarded migration causes nuclei to remain in the anterior part of the embryo retaining their spherical distribution even in a late cleavage stage. This consequently results in an extreme delay in nuclear arrival in the posterior periplasm. A mutant phenocopy was induced in wild-type embryos that were treated with cytochalasin B or D at a very early cleavage stage. Remarkable differences were noticed in the organization of cortical F-actin between the mutants and the wild type throughout the cleavage stage: obvious F-actin aggregates were dispersed in the cortex of mutant embryos, in contrast to the wild type where the cortical F-actin layer was smooth and underlying F-actin aggregates were smaller than those in the mutants; the transition of the distribution pattern of F-actin in the yolk mass, from the centralized to the fragmented type, occurred later in the mutants than in wild type. The results suggest that these mutations affect the mechanism underlying establishment and transition of F-actin organization required for normal egg contraction and nuclear migration in the cleavage embryos.     

C-cadherin控制非洲爪蛙早期胚胎中微丝骨架的合成

上皮细胞间形成的Adherensjunctions复合物通过E—cadherin胞质区段,经由catenin家族蛋白介导,与细胞中微丝骨架系统（micrOfilament）相互作用,参与控制细胞极性、迁移,发育中的形态建成运动以及组织稳态维持等重要生命现象。多方面实验证据表明,cadherin复合物与微丝骨架系统的相互作用是高度动态的;作者前期的工作发现,在非洲爪蛙早期胚胎中,经典cadherin（C-cadherin）在细胞膜上的表达量决定细胞中微丝骨架合成总量。该研究进一步提供实验证据,表明随着囊胚期细胞增殖的进行,囊胚中期以后,细胞表面c—cadherin逐步富集,相应地细胞中微丝骨架的合成量也增加。我们还通过细胞解聚,C-cadherin敲降和过量表达,以及c-cadherin与F-actin共定位分析等实验验证在囊胚期外胚层细胞中,细胞膜C—cadherin表达量与细胞微丝骨架的合成量高度正相关。     

Maternal effect mutations of the sponge locus affect actin cytoskeletal rearrangements in Drosophila melanogaster embryos

In the syncytial blastoderm stage of Drosophila embryogenesis, dome-shaped actin "caps" are observed above the interphase nuclei. During mitosis, this actin rearranges to participate in the formation of pseudocleavage furrows, transient membranous invaginations between dividing nuclei. Embryos laid by homozygous sponge mothers lack these characteristic actin structures, but retain other actin associated structures and processes. Our results indicate that the sponge product is specifically required for the formation of actin caps and metaphase furrows. The specificity of the sponge phenotype permits dissection of both the process of actin cap formation and the functions of actin caps and metaphase furrows. Our data demonstrate that the distribution of actin binding protein 13D2 is unaffected in sponge embryos and suggest that 13D2 is upstream of actin in cortical cap assembly. Although actin caps and metaphase furrows have been implicated in maintaining the fidelity of nuclear division and the positions of nuclei within the cortex, our observations indicate that these structures are dispensible during the early syncytial blastoderm cell cycles. A later requirement for actin metaphase furrows in preventing the nucleation of mitotic spindles between inappropriate centrosomes is observed. Furthermore, the formation of actin caps and metaphase furrows is not a prerequisite for the formation of the hexagonal array of actin instrumental in the conversion of the syncytial embryo into a cellular blastoderm.     

Cytoskeletal asymmetry in Zea mays subsidiary cell mother cells: a monopolar prophase microtubule half-spindle anchors the nucleus to its polar position

Double labeling of microtubules and actin filaments revealed that in prophase subsidiary mother cells of Zea mays a monopolar prophase microtubule "half-spindle" is formed, which lines the nuclear hemisphere distal to the inducing guard mother cell. The nuclear hemisphere proximal to the guard mother cell is lined by an F-actin cap, consisting of a cortical F-actin patch and actin filaments originating from it. The microtubules of the "half-spindle" decline from the nuclear surface and terminate to the preprophase microtubule band. After disintegration of the latter, a bipolar metaphase spindle is organized. The polar F-actin cap persists during mitosis and early cytokinesis, extending to the chromosomes and the subsidiary cell daughter nucleus. In oryzalin treated subsidiary mother cells the prophase nuclei move away from the polar site. Cytochalasin B and latrunculin-B block the polar migration of subsidiary mother cell nuclei, but do not affect those already settled to the polar position. The prophase nuclei of latrunculin-B treated subsidiary mother cells are globally surrounded by microtubules, while the division plane of latrunculin-B treated subsidiary mother cells is misaligned. The prophase nuclei of brick 1 mutant Zea mays subsidiary mother cells without F-actin patch are also globally surrounded by microtubules. The presented data show that the prophase microtubule "half-spindle"-preprophase band complex anchors the subsidiary mother cell nucleus to the polar cell site, while the polar F-actin cap stabilizes the one metaphase spindle pole proximal to the inducing guard mother cell.     

Two distinct distributions of F-actin are present in the hyphal apex of the oomycete Achlya bisexualis

We show that two distinct distributions of F-actin are present in the hyphal apex of the oomycete Achlya bisexualis, that have been chemically fixed with a combination of methylglyoxal and formaldehyde and stained with Alexa phalloidin. In approximately one half of the hyphae examined, an F-actin depleted zone within the apical F-actin cap was observed. The remaining hyphae had a continuous apical cap. In live, growing hyphae two types of cytoplasmic organization were observed at the tips, one in which a clear zone was present which may correlate with the F-actin depleted zone, and one where no such clear zone existed which may represent the continuous cap. We suggest that the F-actin depleted zone may be a structural component of the actin network in a subpopulation of oomycete hyphae and may be comparable to similar F-actin depleted zones at the apices of other tip growing cells such as pollen tubes and root hairs. This observation has implications with regard to models of hyphal extension. Hyphae fixed with formaldehyde alone showed continuous apical F-actin caps. Our ability to resolve the F-actin depleted zone likely reflects the cross-linking capabilities of methylglyoxal. The methylglyoxal-formaldehyde combination fixative gave more stained hyphae, brighter staining and more complete staining of F-actin compared to formaldehyde alone.     

The role of microfilaments in the capping of epidermal growth factor receptor in A431 cells

Capping of the EGF receptor (EGF-R) on the surface of suspended and adherent epidermoid carcinoma cells, A431, is studied. It was induced at 20 degrees C after treating cells with monoclonal antibody to the EGF receptor followed by the second antibody conjugated with FITC. Accumulation of cortical actin under the caps was detected by rhodamine-phalloidin. Destruction of the actin stress-fiber-like bundles was observed during incubation of cells with the ligands at 0 degrees C. Two processes appear to take place at 20 degrees C: redistribution of the EGF-R with cortical actin into the caps within 15-30 min and reconstruction of cytoplasmic actin bundles over 45-60 min. Dihydrocytochalasin B prevented cap formation in adherent cells, but small patches of EGF-R colocalized with actin aggregates under plasma membrane were observed. The function of different actin-containing cytoskeleton structures in the process of capping is discussed.     

Cell age-related differences in the interaction of a potential-sensitive fluorescent dye with nuclear envelopes of Acetabularia mediterranea

Abstract. To study whether an electrical potential difference exists across the nuclear envelope or inner nuclear membrane of plant cells, the authors have used an optical probe of membrane potential, the cationic fluorescent dye, DiOC₆(3) (MW = 572.5). This dye was microinjected into the nucleoplasm of isolated Acetabularia nuclei (which are still surrounded by a thin layer of cytoplasm) and its subnuclear localization visualized by fluorescence microscopy. Striking differences, which seemed to be correlated with the developmental stage of the isolated nucleus, were observed. In nuclei isolated from cells at the stage of early cap stage formation, the dye was restricted to the nuclear envelope. In nuclei isolated from cells with intermediate or fully developed caps, there was increased nucleoplasmic staining, and the staining of the envelope was frequently diminished or abolished. In all nuclei, the dye remained within the nucleus after injection. Cytoplasmic staining was only observed when nuclei isolated from cells at the stage of early cap formation were incubated in a hyper- or hypo-tonic medium. Various ionophores, injected before the dye into the nucleoplasm, had no effect on the subsequent nuclear localization of DiOC₆(3), although they did rapidly induce nucleolar condensation in nuclei isolated from cells at the stage of early cap formation. The results suggested that the electrical properties of Acetabularia nuclear envelopes or inner nuclear membranes change during cell maturation. Furthermore, the retention of the dye in the nucleoplasm under isotonic conditions indicated that the nuclear pores were not open channels for molecules of this size.     

The capping of lymphocytes and other cells, studied by an improved method for immunofluorescence staining of frozen sections.

Experiments have been carried out on the capping by lectins and antibodies of surface receptors of mouse splenic T and B lymphocytes and other cells, in which the surface distribution of the lectin or antibody, and the intracellular distribution of myosin or actin, were determined on the same cells by a double fluorescence technique. For this purpose, a general method for intracellular staining was developed which is intended to preserve sensitive antigens and fragile ultrastructural elements. The method involves mild formaldehyde fixation of the cells or tissues, infusion with concentrated sucrose, rapid freezing, and the preparation of frozen sections thinner than 1 micrometer thickness. The immunofluorescent or other appropriate fluorescent reagents are then applied to the thawed section. In the present experiments, intracellular actin was detected using a fluorescent staining method based on the interaction of F-actin with heavy meromyosin, while intracellular myosin was detected by an indirect immunofluorescence procedure. Our findings were that the formation of a cap by each of the lectins or antibody reagents was always accompanied by a concentration of myosin and actin directly under the cap. These and other results suggest that capping is an active process in which actin and myosin participate directly in the formation of all caps. This proposal carries important new implications for the molecular mechanism of capping.     

Contractile ring formation in Xenopus egg and fission yeast

How actin filaments (F-actin) and myosin II (myosin) assemble to form the contractile ring was investigated with fission yeast and Xenopus egg. In fission yeast cells, an aster-like structure composed of F-actin cables is formed at the medial cortex of the cell during prophase to metaphase, and a single F-actin cable(s) extends from this structure, which seems to be a structural basis of the contractile ring. In early mitosis, myosin localizes as dots in the medial cortex independently of F-actin. Then they fuse with each other and are packed into a thin contractile ring. At the growing ends of the cleavage furrow of Xenopus eggs, F-actin at first assembles to form patches. Next they fuse with each other to form short F-actin bundles. The short bundles then form long bundles. Myosin seems to be transported by the cortical movement to the growing end and assembles there as spots earlier than F-actin. Actin polymerization into the patches is likely to occur after accumulation of myosin. The myosin spots and the F-actin patches are simultaneously reorganized to form the contractile ring bundles. The idea that a Ca signal triggers cleavage furrow formation was tested with Xenopus eggs during the first cleavage. We could not detect any Ca signals such as a Ca wave, Ca puffs or even Ca blips at the growing end of the cleavage furrow. Furthermore, cleavages are not affected by Ca-chelators injected into the eggs at concentrations sufficient to suppress the Ca waves. Thus we conclude that formation of the contractile ring is not induced by a Ca signal at the growing end of the cleavage furrow.     

Microfilament distribution in cold-treated Drosophila embryos.

Cold treatment of Drosophila embryos is observed to result in general alteration of microfilament distribution leading to deformation of the surface caps and to perturbation of the process of cleavage furrow extension. After exposure to low temperature the cortical actin caps underwent several morphological changes, despite the arrested nuclear cycle. These observations are discussed in relation to centrosome behavior during the cell cycle.     

High microfilament concentration results in barbed-end ADP caps.

Current theory and experiments describing actin polymerization suggest that site-specific cleavage of bound nucleotide following F-actin filament formation causes the barbed ends of microfilaments to be capped first with ATP subunits, then with ADP bound to inorganic phosphate (ADP.Pi) at steady-state. The barbed ends of depolymerizing filaments consist of ADP subunits. The decrease in stability of the barbed-end cap accompanying the transition from ADP.Pi to ADP allows nucleotide hydrolysis and subsequent loss of Pi to regulate F-actin filament dynamics. We describe a novel computational model of nucleotide capping that simulates both the spatial and temporal properties of actin polymerization. This model has been used to test the effects of high filament concentration on the behavior of the ATP hydrolysis cycle observed during polymerization. The model predicts that under conditions of high microfilament concentration an ADP cap can appear during steady-state at the barbed ends of filaments. We show that the presence of the cap can be accounted for by a kinetic model and predict the relationship between the nucleotide concentration ratio [ATP]/[ADP], the F-actin filament concentration, and the steady-state distribution of barbed-end ADP cap lengths. The possible consequences of this previously unreported phenomenon as a regulator of cytoskeletal behavior are discussed.     

Microfilament distribution in cold-treated Drosophila embryos

Cold treatment of Drosophila embryos is observed to result in general alteration of microfilament distribution leading to deformation of the surface caps and to perturbation of the process of cleavage furrow extension. After exposure to low temperature the cortical actin caps underwent several morphological changes, despite the arrested nuclear cycle. These observations are discussed in relation to centrosome behavior during the cell cycle.     

玉米根冠中类外连丝的结构特征及其共质传输功能(简报)

胞间连丝是植物体内连接两个相邻细胞原生质体的共质运输通道,在胞间物质的转运和通讯联络上发挥重要作用。胞间连丝的功能在生理上主要体现在它对胞间转运物质的通透性（permeability）,通透性的变化和调节影响到许多生理过程的进展与协调。在植物的不同组织及其发育的不同阶段,不同的物化因素,不同的逆境胁迫以及病原物的侵染均可导致胞间连丝的通透性呈现相应的变动。胞间连丝存在形式的多样性以及对其不同程度和不同方式的修饰,均可对胞间连丝的生理功能及其通透性有明显的影响。[第一段]     

Arp2/3-dependent pseudocleavage [correction of psuedocleavage] furrow assembly in syncytial Drosophila embryos

BACKGROUND: In syncytial blastoderm Drosophila embryos, actin caps assemble during telophase. As the cell cycle progresses through interphase, these small caps expand and fuse to form pseudocleavage furrows that are structurally related to the cleavage furrows that assemble during somatic cell division. The molecular mechanism driving cell cycle coordinated actin reorganization from the caps to the furrows is not understood. RESULTS: We show that Drosophila embryos contain a typical Arp2/3 complex and that components of this complex localize to the margins of the expanding caps, to mature pseudocleavage furrows, and to somatic cell cleavage furrows during the postcellularization embryonic divisions. A mutation that disrupts the arpc1 subunit of Arp2/3 leads to spindle fusions that are characteristic of pseudocleavage furrow disruption. By contrast, this mutation does not significantly affect nuclear positioning during interphase, which is dependent on actin cap function. In vivo analysis of actin reorganization demonstrates that the arpc1 mutation does not prevent assembly of small actin caps but blocks cap expansion and furrow assembly as the cell cycle progresses through interphase. The scrambled gene is also required for cap expansion and furrow assembly, and Scrambled is required for Arp2/3 localization to the cap margins. CONCLUSIONS: The Drosophila Arp2/3 complex and Scrambled protein are required for actin cap expansion and pseudocleavage furrow formation during the syncytial blastoderm divisions. We propose that Scrambled-dependent localization of Arp2/3 to the margins of the expanding caps triggers local actin polymerization that drives cap expansion and pseudocleavage furrow assembly.