A specialized complex of apical organelles facilitates Plasmodium merozoite invasion into the erythrocyte. Even though the apical organelles are crucial to the invasion process, relatively little is known about how they function or their biosynthesis during asexual replication. MAEBL is an erythrocyte binding protein located in the rhoptries and on the surface of mature merozoites and is expressed at the beginning of schizogony before the first nuclear division. Therefore, we have characterized MAEBL as a marker for the biosynthetic pathway of the rhoptry apical organelle during the final phase of intraerythrocytic development and as a marker for the nascent rhoptry vesicle in the immature schizont. An extensive proliferation of the endoplasmic reticulum occurred at the onset of schizogony and was seen as a complex but transient tubule array near the parasite surface. Both the rhoptry protein MAEBL and surface protein MSP-1 appeared to be present in this tubular reticular network together with endoplasmic reticulum markers. MAEBL then transits through Golgi bodies positioned near the parasite plasma membrane, directly adjacent to the network. Rhoptry organelle precursors are seen at the three to four nuclei stage of schizont development, remaining near the plasma membrane throughout schizogony. These studies constitute the first direct evidence that proteins of the rhoptry organelles transit through compartments of the 'classical' secretory pathway.

cmu1-1 is a new mutation of Chlamydomonas reinhardtii that causes a change in cell shape due to an alteration of cytoplasmic microtubule organization. cmu1 mutant cells were first identified based on their altered cell shape. Unlike wild-type cells, which are ellipsoid, cmu1 cells tend to be either round or egg-shaped with the flagella extending from the narrow end of the cell. Electron microscopic comparison of mutant and wild-type cells indicated that microtubule distribution was altered in the mutant cells. Immunofluorescence microscopy using anti-beta-tubulin antibodies revealed that, in wild-type cells, microtubules arise from the anterior end of the cell in the region of the basal bodies, pass posteriorly subjacent to the plasma membrane, and terminate near the posterior end of the cell. In mutant cells, the microtubules also arise from the basal body region but then become disarrayed. They frequently curl back anteriorly or wrap around the equator of the cell; some microtubules also extend completely to the posterior end of the cell, then turn back toward the anterior end. No changes in the basal body region were detected by electron microscopy. Some cmu1 cells had multiple nuclei or an aberrant number of flagella, both of which may be due to defects in cell division, a process dependent upon microtubules. Thus, cmu1-1, which was generated by insertional mutagenesis and is tagged, appears to encode a protein that plays an essential role in the spatial organization of cytoplasmic microtubules involved in both interphase and mitotic functions.

Abstract The cytoskeleton is composed of a dynamic network of filament systems that form the scaffolding and machinery responsible for spatially and temporally integrating a variety of cell functions. In addition to maintaining cell shape and polarity, cytoskeletal polymers orchestrate a variety of cell movements, including intracellular transport, chromosome segregation, cell locomotion, and cell division. Such activities result from the direct interaction of polymer systems such as actin filaments and microtubules that combine with a vast array of complementary binding proteins and molecular motors to transduce mechanochemical energy into active force production. The exquisite sensitivity and timing of such events is controlled in turn by a complex network of signalling molecules that co-operatively function to stimulate and regulate productive interactions leading to force generation and mechanical transduction (1).

The mitotic spindle has long been recognized to play an essential role in determining the position of the cleavage furrow during cell division, however little is known about the mechanisms involved in this process, One attractive hypothesis is that signals from the spindle may function to induce reorganization of cortical structures and transport of actin filaments to the equator during cytokinesis. While an important idea, few experiments have directly tested this model. In the present study, we have used a variety of experimental approaches to identify microtubule-dependent effects on key cortical events during normal cell cleavage, including cortical flow, reorientation of actin filaments, and formation of the contractile apparatus. Single-particle tracking experiments showed that the microtubule disrupting drug nocodazole induces an inhibition of the movements of cell surface receptors following anaphase onset, while the microtubule stabilizing drug taxol causes profound changes in the overall pattern of receptor movements. These effects were accompanied by a related set of changes in the organization of the actin cytoskeleton. In nocodazole-treated cells, the three-dimensional organization of cortical actin filaments appeared less ordered than in controls. Measurements with fluorescence-detected linear dichroism indicated a decrease in the alignment of filaments along the spindle axis, In contrast, actin filaments in taxol-treated cells showed an increased alignment along the equator on both the ventral and dorsal cortical surfaces, mirroring the redistribution pattern of surface receptors, Together, these experiments show that spindle microtubules are involved in directing bipolar flow of surface receptors and reorganization of actin filaments during cell division, thus acting as a stimulus for positioning cortical cytoskeletal components and organizing the contractile apparatus of dividing tissue culture cells.

The mechanism of cytokinesis is an old problem in cell biology that has received fresh attention recently with a large variety of powerful approaches and experimental systems. Significant advances have been made on the structure of the cortical cytoskeleton, the identification of proteins and genes involved, and the regulatory mechanism. Many surprises have surfaced within the past two years, leading us toward a major revision in our understanding of this important process.

Eggs and embryos have been used in a vast array of biochemical, biophysical, and cytological studies to ask and answer seminal questions on fertilization, signal transduction, cell cycle regulation, exocytosis, gene expression, cellular induction, and differentiation. Historically, the echinoderm egg and embryo have been particularly fruitful in pushing the envelope of the understanding of complex synergistic interactions among cell signaling events, membrane traffic, and cytoskeletal reorganizations that occur during the course of normal cellular and embryonic differentiation. Nowhere is this better exemplified than in the extraordinary works of Rappaport, Hiramoto, and others who have shown the important relationship that exists between the mitotic spindle and the cortical membrane actin cytoskeleton leading to the induction and formation of the contractile apparatus driving cytokinesis. This chapter focuses on the actin cytoskeleton and its interaction with egg and embryonic membranes during fertilization and morphogenesis. It highlights the known sea urchin egg actin-binding proteins and discusses their structural, functional, and regulatory cytoskeletal-membrane dynamics during development. Fertilization serves as the starting point for reviewing the dynamic interplay between physiological activation and reorganization of the cortical actin-membrane cytoskeleton (filament assembly, cortical granule exocytosis, microvillar elongation, membrane uptake, vesicle sorting) that follows sperm-egg fusion. The chapter provides an appreciation of the sea urchin egdembryo as a cell and developmental model for studying the mechanisms underlying cytoskeletal membrane reorganization and its importance to early embryonic morphogenesis and differentiation.

The current hypothesis of cytokinesis suggests that contractile forces in the cleavage furrow are generated by a circumferential band of actin filaments. However, relatively little is known about the global organization of actin filaments in dividing cells. To approach this problem we have used fluorescence-detected linear dichroism (FDLD) microscopy to measure filament orientation, and digital optical sectioning microscopy to perform three-dimensional reconstructions of dividing NRK cells stained with rhodamine-phalloidin. During metaphase, actin filaments in the equatorial region show a slight orientation along the spindle axis, while those in adjacent regions appear to be randomly distributed. Upon anaphase onset and through cytokinesis, the filaments become oriented along the equator in the furrow region, and along the spindle axis in adjacent regions. The degree of orientation appears to be dependent on cell-cell and cell-substrate adhesions. By performing digital optical sectioning microscopy on a highly spread NRK subclone, we show that actin filaments organize as a largely isotropic cortical meshwork in metaphase cells and convert into an anisotropic network shortly after anaphase onset, becoming more organized as cytokinesis proceeds. The conversion is most dramatic on the adhering ventral surface which shows little or no cleavage activity, and results in the formation of large bundles along the equator. On the dorsal surface, where cleavage occurs actively, actin filaments remain isotropic, showing only subtle alignment late in cytokinesis. In addition, stereo imaging has led to the discovery of a novel set of filaments that are associated with the cortex and traverse through the cytoplasm. Together, these studies provide important insights into the process of actin remodeling during cell division and point to possible additional mechanisms for force generation.

Although the distribution of filamentous actin is well characterized in many cell types, the distribution of nonfilamentous actin remains poorly understood. To determine the relative distribution of filamentous and nonfilamentous actin in cultured NRK cells, we have used a number of labeling agents that differ with respect to their specificities toward the filamentous or nonfilamentous form, including monoclonal and polyclonal anti-actin antibodies, vitamin D-binding protein (DBP), and fluorescent phalloidin. Numerous punctate structures were identified that bind poorly to phalloidin but stain positively with several anti-actin antibodies. These bead structures also stain with DBP, suggesting that they are enriched in non-filamentous actin. Similar punctate structures were observed after the microinjection of fluorescently labeled actin into living cells, allowing us to examine their dynamics in living cells. The actin-containing punctate structures were observed predominantly in the region behind lamellipodia, particularly in spreading cells induced by wounding confluent monolayers. Time-lapse recording of cells injected with fluorescent actin indicated that they form continuously near the leading edge and move centripetally toward the nucleus. Our results suggest that at least part of the unpolymerized actin molecules are localized at discrete sites, possibly as complexes with monomer sequestering proteins. These structures may represent transient storage sites of G-actin within the cell which can be transformed rapidly into actin filaments upon stimulation by specific signals.

We have identified a novel high molecular weight, vesicle-associated protein (VAP-1) in the eggs of the sea urchin Strongylocentrotus purpuratus. Biochemical fractionation and immunofluorescence analysis of unfertilized eggs indicate that VAP-1 is a peripheral membrane protein associated with microsomal membrane fractions. Sequence analysis of partial VAP-1 cDNA clones reveals that the protein contains at least four RNA-binding consensus sequences. The RNA-binding sequences are separated by several glycine rich domains and this organization, RNA-binding domains separated by glycine rich sequences, is common to several RNA-binding proteins including the heterogeneous ribonuclear protein A1 and nucleolin. The characteristics of VAP-1 suggest that the protein may function as a multidomain RNA-binding protein. The possibility that VAP-1 may play a role in nuclear RNA processing is also discussed.

Myosin light chain kinase (MLCK) is thought to regulate the contractile activity in smooth and non-muscle cells, and may play an important role in controlling the reorganization of the actin-myosin cytoskeleton during cell division. To test this hypothesis we have microinjected the 61-kD catalytic fragment of MLCK into mitotic cells, and examined the effects of unregulated MLCK activity on cell division. The microinjection of active 61 kD causes both a significant delay in the transit time from nuclear envelope breakdown to anaphase onset, and an increase in motile surface activity during and after metaphase. Control experiments with intact MLCK or with inactive catalytic fragment suggest that these effects are specifically induced by the unregulated myosin light chain kinase activity. Immunofluorescence analysis suggests that delays in mitosis are coupled to disruptions of spindle structures, while increased surface motility may be related to changes in the organization of actin and myosin at the cell cortex. Most importantly, despite the expression of strong phenotypes, 61 kD-injected cells still form functional cleavage furrows that progress through cytokinesis at rates identical to those of control cells. Together, these results suggest that the activity of MLCK can affect mitosis and cortical activities, however additional control mechanisms are likely involved in the regulation of cytokinesis.

Thyone sperm undergo an explosive acrosome reaction resulting in the extension of a 90 microns long acrosomal process. In unreacted sperm, profilamentous actin is sequestered within the profilactin cup (Tilney: Journal of Cell Biology 69:73-89 1976), which consists of four major polypeptides: actin, profilin, and a 250/235 kDa equimolar doublet (TS 250/235). Dialysis of profilactin preparations into an actin assembly buffer resulted in the formation of acrosomal-like macromolecular aggregates containing actin, TS 250/235, and several other polypeptides as detected by SDS-PAGE. TS 250/235 was purified by subjecting extracts of pH solubilized profilactin cups to DEAE and phosphocellulose ion exchange chromatography. TS 250/235 demonstrated immunocrossreactivity with affinity purified polyclonal antibodies raised against S. purpuratus egg spectrin. As determined by biotinylated-calmodulin overlays, both subunits of TS 250/235 bound calmodulin in a Ca(++)-sensitive manner. Electron microscopy of low angle, rotary shadowed replicas of TS 250/235 revealed an elongate rod-shaped molecule with an average contour length of 203 nm. By indirect immunofluorescence, TS 250/235 was found to be uniformly distributed throughout the profilactin cup of the unreacted sperm. This distribution of TS 250/235 correlated with the location of monomeric actin as determined by localization studies utilizing fluorescent-DNase-1. Upon sperm activation, the cellular distribution of TS 250/235 dramatically changed and was observed both along the length and at the base of the extended acrosomal process.

Annals of the New York Academy of SciencesVolume 582, Issue 1 p. 295-296 Calsequestrin-Containing Endoplasmic Reticulum Concentrates in the Mitotic Spindle during Cell Division in the Sea Urchin JOHN HENSON, JOHN HENSON Department of Anatomy and Cellular Biology Harvard Medical School Boston, Massachusetts 02215Search for more papers by this authorSTEPHEN BEAULIEU, STEPHEN BEAULIEU Department of Anatomy and Cellular Biology Harvard Medical School Boston, Massachusetts 02215Search for more papers by this authorDOUGLAS FISHKIND, DOUGLAS FISHKIND Department of Anatomy and Cellular Biology Harvard Medical School Boston, Massachusetts 02215Search for more papers by this authorEDWARD BONDER, EDWARD BONDER Department of Biological Science Rutgers University Newark, New Jersey 07102Search for more papers by this authorDJAMAL LEBECHE, DJAMAL LEBECHE Department of Physiology Boston University School of Medicine Boston, Massachusetts 02118Search for more papers by this authorBENJAMIN KAMINER, BENJAMIN KAMINER Department of Physiology Boston University School of Medicine Boston, Massachusetts 02118Search for more papers by this authorMARK TERASAKI, MARK TERASAKI Laboratory of Neurobiology NINDS, National Institutes of Health Bethesda, Maryland 20892Search for more papers by this authorDAVID BEGG, DAVID BEGG Department of Anatomy and Cellular Biology Harvard Medical School Boston, Massachusetts 02215Search for more papers by this author JOHN HENSON, JOHN HENSON Department of Anatomy and Cellular Biology Harvard Medical School Boston, Massachusetts 02215Search for more papers by this authorSTEPHEN BEAULIEU, STEPHEN BEAULIEU Department of Anatomy and Cellular Biology Harvard Medical School Boston, Massachusetts 02215Search for more papers by this authorDOUGLAS FISHKIND, DOUGLAS FISHKIND Department of Anatomy and Cellular Biology Harvard Medical School Boston, Massachusetts 02215Search for more papers by this authorEDWARD BONDER, EDWARD BONDER Department of Biological Science Rutgers University Newark, New Jersey 07102Search for more papers by this authorDJAMAL LEBECHE, DJAMAL LEBECHE Department of Physiology Boston University School of Medicine Boston, Massachusetts 02118Search for more papers by this authorBENJAMIN KAMINER, BENJAMIN KAMINER Department of Physiology Boston University School of Medicine Boston, Massachusetts 02118Search for more papers by this authorMARK TERASAKI, MARK TERASAKI Laboratory of Neurobiology NINDS, National Institutes of Health Bethesda, Maryland 20892Search for more papers by this authorDAVID BEGG, DAVID BEGG Department of Anatomy and Cellular Biology Harvard Medical School Boston, Massachusetts 02215Search for more papers by this author First published: April 1990 https://doi.org/10.1111/j.1749-6632.1990.tb21687.xAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume582, Issue1Cytokinesis: Mechanisms of Furrow Formation During Cell DivisionApril 1990Pages 295-296 RelatedInformation

A recent study from our laboratory on the sea urchin egg suggested that spectrin was not solely restricted to the plasma membrane, but instead had a more widespread distribution on the surface of a variety of membranous inclusions. (E. M. Bonder et al., 1989, Dev. Biol. 134, 327-341). In this report we extend our initial findings and provide experimental and ultrastructural evidence for the presence of spectrin on three distinct classes of cytoplasmic vesicles. Immunoblot analysis of membrane fractions prepared from egg homogenates establishes that spectrin coisolates with vesicle-enriched fractions, while indirect immunofluorescence microscopy on cryosections of centrifugally stratified eggs demonstrates that spectrin specifically associates with cortical granules, acidic vesicles, and yolk platelets in vivo. Immunogold ultrastructural localization of spectrin on cortices isolated from eggs and early embryos details the striking distribution of spectrin on the cytoplasmic surface of the plasma membrane and the membranes of cortical granules, acidic vesicles, and yolk platelets, while quantitative studies show that relatively equivalent amounts of spectrin are present on the different membrane surfaces both before and after fertilization. These data, in combination with the localization of numerous spectrin crosslinks between actin filaments in surface microvilli, suggest that spectrin plays a pivotal role in structuring the cortical membrane-cytoskeletal complex of the egg and the embryo.