Of mice, frogs and flies: generation of membrane asymmetries in early development

Embryonic development begins with cleavage of the fertilized egg. Cleavage comprises two major processes: cytokinesis and formation of a polarized epithelial cell layer. The focus of this review is comparison of the generation of membrane polarity during embryonic cleavage in three different developmental model systems. In mammalian embryos, as exemplified by analysis of the mouse, generation of distinct membrane domains is uncoupled from cleavage divisions and is initiated in a specific developmental phase, called compaction. In Xenopus laevis embryos, generation of polarized blastomeres occurs simultaneously with cytokinesis. The origin of specific membrane domains of X. laevis polar blastomeres, however, can be traced back to oogenesis. Finally, in Drosophila melanogaster, generation of polarized cells occurs at cellularization. The relevance of cell adhesion, cell junctions and cytocortical scaffolds will be discussed for each of the model systems. Despite enormous morphologic differences, the three models share many common features; in particular, many important molecular interactions are conserved.     

Simulation of the mechanism of determining the position of the cleavage furrow in cytokinesis of sea urchin eggs

In cytokinesis of sea urchin eggs, the numerical density of astral microtubules extending close to the cell surface has been thought to determine the position of the cleavage furrow. In the present study, a new model was constructed to simulate the relationship between the microtubule density and the furrow formation. In the model, gradients of the microtubule density drive fluid membrane proteins whose accumulation triggers the formation of contractile-ring microfilaments. The model could explain the behavior of the cleavage furrow under various experimental conditions. These simulations revealed two aspects of furrow formation. One is that in some cases, the cleavage furrow appears in a surface region where the microtubule density has neither a minimum nor a maximum. In all furrow regions, however, the second derivative of the microtubule-density function has large positive values. Membrane proteins greatly slow down to accumulate in such a region. The other is that the cleavage furrow is mobile, not fixed in one position, because of the fluidity of membrane proteins. These results strongly suggested that the mitotic apparatus determines the position of the cleavage furrow by redistributing membrane proteins through gradients of the microtubule density at the cell surface.     

Studies on the arrangement of glucocorticoid receptors in the plasma membrane of S-49 lymphoma cells.

The presence of glucocorticoid receptors is required for glucocorticoid-mediated lymphocytolysis to take place. However, the explicit mechanism of involvement of this receptor continues to be debated. We have recently presented evidence that this response is mediated by a specialized form of the glucocorticoid receptor that resides in the plasma membrane (mGR). Using sequential cell separation techniques ("immunopanning," fluorescent cell sorting, and soft agar cloning), a resultant population of membrane receptor-enriched cells have remained stable and provided material for further analysis. The mGR patching and capping phenomenon originally observed with fluoresceinated monoclonal antibody techniques was verified here with electron micrographic analysis using colloidal gold-conjugated antibody. Using 3H-labeled monoclonal antibody, a radioimmunoassay for membrane receptors was developed. Trypsin treatment removed the membrane receptor antigenic site from the surface of cells. Peptide mapping of receptor purified from plasma membranes reveals several trypsin and alpha-chymotrypsin cleavage sites. Larger fragments resulted from cleavage of the membrane receptor of cells enriched for mGR versus those found in cells depleted of the membrane form, although most of the resulting fragments are shared by the two forms. Confirmation of previous studies correlating membrane receptor with the mechanism of glucocorticoid sensitivity is now extended to include elimination of the lymphocytolysis effect in membrane receptor-stripped (trypsinized) S-49 cells.     

Involvement of bristle coat structures in surface membrane formations and membrane interactions during coenocytotomic cleavage in caps of Acetabularia mediterranea

In the multinucleate cap rays of the green alga Acetabularia mediterranea the cell surface increases dramatically within a short time period during the final stages of coenocytotomic cleavage. In early stages of cyst formation the cytoplast is traversed by numerous large and prolate cleavage vesicles which are characterized by typical columellar or spinous coat structures. The cleavage vesicles are closely associated with the surface of plastids and, to a lesser degree, of mitochondria. This intimate association seems to be mediated by regularly spaced, densely stained intermembranous cross-bridge structures and is maintained throughout cleavage. These cleavage vesicles contain a finely fibrillar material structurally similar to the hyaline layer of mucilage that fills the space between the plasma membrane and cell wall. They line up with invaginations of the plasmalemma and vacuole membranes and, together with smaller vesicles interspersed, constitute preformed "perforation lines" for the final separation of the coenoblast portions. Equidistantly spaced plaques of attachment of such vesicles with surface membrane are described. We hypothesize (a) that the cleavage vesicle membrane is the immediate precursor to the new postcoenocytotomic surface membrane, (b) that the cleavage vesicle coat structures are integrated into the subsurface coat of the plasma membrane, (c) that growth of the laterally attached cleavage vesicles by intussusception of small fuzzy-coated vesicles is confined to their "free ends," (d) that the intermembranous cross- bridge elements are related to bristle coat structures and play a role in the establishment of the cleavage lines, and (e) that the coenocytotomic cleavage process is organized so that adjacent plastids are separated in a way that guarantees the inclusion of several plastids in each cyst.     

Early development of the kelp fly,Coelopa frigida (Diptera). II. Morphology of cleavage and blastoderm formation

Cleavage and blastoderm formation in Coelopa frigida are extremely rapid developmental processes. In short (6–7 minutes) successive cell cycles, nuclei multiply and spread out through the egg. The movement seems to be aided by endoplasmic vesicles and cisternae which are in direct contact with the nuclear membrane. The first cells to separate from the egg plasmodium in early superficial cleavage stages are the pole cells. Precursor material from multivesicular bodies forms the pole cell membranes. The primary nuclei from the posterior pole region are removed from the blastoderm by the pole cell segregation. Blastoderm nuclei from the regions adjacent to the posterior pole migrate into the residual periplasm after pole cell segregation has been completed and constitute the blastoderm nuclei in that region of the egg. Nucleoli are not revealed during internal cleavage. They appear in pole cells shortly after their segregation. The generation time of the blastoderm nuclei increases after the twelfth cleavage. Concurrently, nucleoli form in the blastoderm nuclei and permanent cell membranes separate individual blastoderm cells. After blastoderm cells have been separated from each other, they remain in contact with the interior yolk sac by means of cytoplasmic canals. This contact is maintained at least during the early phases of blastokinesis. Observations on nuclear migration and rapid membrane formation are discussed as examples of protein assembly from subunits as an alternative to de novo protein synthesis in early stages of development.     

Cleavage furrow positioning

To complete the cell cycle, the cleavage furrow draws the plasma membrane toward the cell center, pinching the cytoplasm into two lobes that are subsequently separated into two cells. The position of the cleavage furrow is induced by the mitotic spindle during early anaphase. Although the mechanism of cleavage furrow positioning is not understood at a molecular level, recent results suggest that it might be mediated by local relief from the inhibitory effects of microtubules.     

Effect of sodium dodecyl sulfate on polar lobe formation and function in Ilyanassa obsoleta embryos

Polar lobes, anucleate vegetal pole protrusions formed by Ilyanassa obsoleta embryos, serve as a mechanism for shunting morphogenetic determinants to one cell during the first two cleavages. Polar lobe material becomes segregated in the CD cell during first cleavage and in the D cell during second cleavage, resulting in a very unequal four-cell stage. Larval structures including external shell, foot, operculum, statocysts, and eyes develop only when polar lobe material is present. Treatment with the anionic detergent sodium dodecyl sulfate (SDS) before and during the first cleavage inhibited polar lobe formation and equalized cleavage, as the lobe material was distributed to two cells. No polar lobes formed during second clevage in SDS-equalized embryos, and the four-cell stage consisted of four equal cells with reduced cell contacts. SDS inrreversibly inhibited polar lobe formation without affecting cytokinesis. Although 27% of the larvae from SDS-equalized embryos had one or more lobe-dependent structures duplicated, morphogenesis was impaired: more than 40% of such larvae failed to form shell and/or statocysts. When cells were separated after equalized first cleavage and raised as pairs, the pairs of resulting larvae duplicated lobe-dependent structures with the same frequency as whole equalized embryos. Possible explanations for impaired morphogenesis in SDS-treated embryos are discussed.     

On the mechanism of electrical coupling between cells of earlyXenopus embryos

Summary The mechanism of electrical coupling between cells of earlyXenopus embryos has been studied by examination of the nonjunctional membrane resistances and capacitances as a function of cleavage stage, the junctional and nonjunctional membrane resistances as functions of time during the first cleavage, and the electrical properties of the primitive blastocoel. The changes in membrane resitances and capacitances during the first two cleavages may be completely explained by the addition of new membrane, identical in specific resistance and capacitance to the original membrane, at a constant rate to furrows which are electrically connected to the perivitelline space. Microelectrode recording from the primitive blastocoel indicates that there is no electrical difference detectable between it and the perivitelline space. These results are discussed in the context of current theories of the mechanism of intercellular electrotonic coupling.     

Distribution of receptors and functions on cell surfaces: quantitation of ligand-receptor mobility and a new model for the control of plasma membrane topography

The long-range movements of membrane ligand-receptor complexes into surface caps and into the pseudopods of cells performing phagocytosis, the uropods of motile cells and the cleavage furrows of dividing cells appear to be analogous processes. A common mechanism to explain these movements must take into account several recent observations. First, laser photobleaching studies have indicated that Concanavalin A-receptor movement occurs unidirectionally; and analyses of Con A redistribution by quantitative video intensification microscopy (QUAVIM) have shown that movement may exceed the maximum rates measured for protein diffusion in membranes. These are the results predicted for a process of directed migration but not for a process of diffusion with entrapment. In addition it has been found that membrane receptors may segregate out of as well as into cap, pseudopod, uropod and cleavage furrow regions and that topographical heterogeneity on asymmetric cells is not restricted to membrane molecular determinants but extends to a range of endocytic functions and to a macromolecular complex, the coated pit. All dynamic surface events are arrested during mitosis. A new model for the regulation of plasma membrane topography has been developed from these diverse quantitative, functional and morphological data. Its essence is the entrainment of selected membrane determinants on membrane waves directed towards regions such as caps, pseudopods, uropods and cleavage furrows. The waves are initiated by tension due to asymmetric microfilament-membrane interaction.     

Transforming growth factor-alpha and beta-amyloid precursor protein share a secretory mechanism

Cleavage and release of membrane protein ectodomains, a regulated process that affects many cell surface proteins, remains largely uncharacterized. To investigate whether cell surface proteins are cleaved through a shared mechanism or through multiple independent mechanisms, we mutagenized Chinese hamster ovary (CHO) cells and selected clones that were unable to cleave membrane-anchored transforming growth factor alpha (TGF-alpha). The defect in TGF-alpha cleavage in these clones is most apparent upon cell treatment with the protein kinase C (PKC) activator PMA, which stimulates TGF-alpha cleavage in wild-type cells. The mutant clones do not have defects in TFG-alpha expression, transport to the cell surface or turnover. Concomitant with the loss of TGF-alpha cleavage, these clones have lost the ability to cleave many structurally unrelated membrane proteins in response to PMA. These proteins include beta-amyloid precursor protein (beta-APP), whose cleavage into a secreted form avoids conversion into the amyloidogenic peptide A beta, and a group of cell surface proteins whose release into the medium is stimulated by PMA in wild type CHO cells but not in mutants. The mutations prevent cleavage by PKC- dependent as well as PKC-independent mechanisms, and thus affect an essential component that functions downstream of these various signaling mechanisms. We propose that regulated cleavage and secretion of membrane protein ectodomains is mediated by a common system whose components respond to multiple activators and act on susceptible proteins of diverse structure and function.     

Dynamic rearrangement of surface proteins is essential for cytokinesis

Cytokinesis is a complex process that involves dynamic cortical rearrangement. Our recent time-lapse recordings of the mouse egg unexpectedly revealed a high motility of the second polar body (2pb). Experiments to address its underlying mechanism show that neither mechanical compression by the zona pellucida nor the connection via the mid-body is required for the 2pb movement. Time-lapse recordings establish that the 2pb moves together with the cell membrane. These recordings, in which cell surface proteins are labeled with fluorescent latex-microbeads or monovalent antibodies against whole mouse proteins, indicate that the majority of the surface proteins dynamically accumulate in the cleavage furrow at every cell division. Comparable dynamics of the cell surface proteins, and specifically of E-cadherin, are also observed in cultured epithelial cells. The surface protein dynamics are closely correlated with, and dependent on, those of the underlying cortical actin. The cortical actin network may form a scaffold for membrane proteins and thereby transfer them during contractile ring formation toward the cleavage furrow. Immobilization of surface proteins by tetravalent lectin-mediated crosslinking results in the failure of cleavage, demonstrating that the observed protein dynamics are essential for cytokinesis. We propose that dynamic rearrangement of the cell surface proteins is a common feature of cytokinesis, playing a key role in modifying the mechanical properties of the cell membrane during cortical ingression.     

On the Modeling of Endocytosis in Yeast

The cell membrane deforms during endocytosis to surround extracellular material and draw it into the cell. Results of experiments on endocytosis in yeast show general agreement that 1) actin polymerizes into a network of filaments exerting active forces on the membrane to deform it, and 2) the large-scale membrane deformation is tubular in shape. In contrast, there are three competing proposals for precisely how the actin filament network organizes itself to drive the deformation. We use variational approaches and numerical simulations to address this competition by analyzing a meso-scale model of actin-mediated endocytosis in yeast. The meso-scale model breaks up the invagination process into three stages: 1) initiation, where clathrin interacts with the membrane via adaptor proteins; 2) elongation, where the membrane is then further deformed by polymerizing actin filaments; and 3) pinch-off. Our results suggest that the pinch-off mechanism may be assisted by a pearling-like instability. We rule out two of the three competing proposals for the organization of the actin filament network during the elongation stage. These two proposals could be important in the pinch-off stage, however, where additional actin polymerization helps break off the vesicle. Implications and comparisons with earlier modeling of endocytosis in yeast are discussed.     

Molecular dynamics simulations to the bidirectional adhesion signaling pathway of integrin αVβ3

The bidirectional force transmission process of integrin through the cell membrane is still not well understood. Several possible mechanisms have been discussed in literature on the basis of experimental data, and in this study, we investigate these mechanisms by free and steered molecular dynamics simulations. For the first time, constant velocity pulling on the complete integrin molecule inside a dipalmitoyl-phosphatidylcholine membrane is conducted. From the results, the most likely mechanism for inside-out and outside-in signaling is the switchblade model with further separation of the transmembrane helices.     

Numb suppresses the negative complementation at the Notch locus of Drosophila melanogaster, suggesting a putative mechanism for negative complementation

The mutant form of the intracellular asymmetrically localized Numb membrane-bound protein of Drosophila melanogaster suppresses the negative complementation of certain Abruptex (Ax) mutations of the Notch (N) locus encoding a transmembrane receptor protein in which the Ax mutations are mutations in the epidermal growth factor (EGF)-like repeats of the extracellular domain of the receptor. One model for how Ax mutants affect N function is that they are refractory to an antagonistic signal generated by an excess of N ligands. Genetically numb (nb) is an antagonist of N. In the absence of nb, cells follow the same fate as they would in the presence of a gain-of-function N allele, such as Ax. Numb has been shown to interact with the cytoplasmic domain of Notch. It is therefore suggested that numb counteracts the effect of Abruptex on Notch ligand binding, i.e. that Numb is an antagonist to the activation of the Notch signal generated by Notch ligands. Numb might accomplish this by interfering with the proteolytic cleavage of the Notch intracellular domain at the cell membrane. Thus, it seems possible that the mechanism of negative complementation of certain Ax mutants is the failure of this cleavage. Other possible mechanisms for negative complementation are also discussed.     

Cleavage of Mcd1 by caspase-like protease Esp1 promotes apoptosis in budding yeast

Over the last decade, yeast has been used successfully as a model system for studying the molecular mechanism of apoptotic cell death. Here, we report that Mcd1, the yeast homology of human cohesin Rad21, plays an important role in hydrogen peroxide-induced apoptosis in yeast. On induction of cell death, Mcd1 is cleaved and the C-terminal fragment is translocated from nucleus into mitochondria, causing the decrease of mitochondrial membrane potential and the amplification of cell death in a cytochrome c-dependent manner. We further demonstrate that the caspase-like protease Esp1 has dual functions and that it is responsible for the cleavage of Mcd1 during the hydrogen peroxide-induced apoptosis. When apoptosis is induced, Esp1 is released from the anaphase inhibitor Pds1. The activated Esp1 acts as caspase-like protease for the cleavage of Mcd1, which enhances the cell death via its translocation from nucleus to mitochondria.     

Extensional flow of erythrocyte membrane from cell body to elastic tether. I. Analysis.

This is the first of two papers on an analytical and experimental study of the flow of the erythrocyte membrane. In the experiment to be discussed in detail in the second paper, preswollen human erythrocytes are sphered by aspirating a portion of the cell membrane into a small micropipette; and long, thin, membrane filaments or "tethers" are steadily withdrawn from the cell at a point diametrically opposite to the point of aspiration. The aspirated portion of the membrane furnished a "reservoir" of material that replaces the membrane as it flows as a liquid from the nearly spherical cell body to the cylindrical tether. In this paper we show that an application of the principle of conservation of mass permits the tether radius (approximately 200 A or less) to be measured with the light microscope as the tether is formed and extended at a constant rate. A static analysis of the axisymmetric cell deformation and tether formation process reveals that the tether radius is uniquely determined by the isotropic tension in the membrane and the elastic constitutive (material) behavior of the tether itself. A dynamic analysis of the extensional flow process reveals that the tether radius must decrease as the velocity of the tether is increased and that the decrease depends on both the viscosity of the membrane and the elasticity of the tether. The analysis also shows that these two factors (membrane viscosity and tether elasticity) are readily decomposed and determined separately when flow experiments are performed at different isotropic tensions.     

A model of plasma membrane flow and cytosis regulation in growing pollen tubes

A model of cytosis regulation in growing pollen tubes is developed and simulations presented. The authors address the question on the minimal assumptions needed to describe the pattern of exocytosis and endocytosis reported recently by experimental biologists. Biological implications of the model are also treated. Concepts of flow and conservation of membrane material are used to pose an equation system, which describes the movement of plasma membrane in the tip of growing pollen tubes. After obtaining the central equations, relations describing the rates of endocytosis and exocytosis are proposed. Two cytosis receptors (for exocytosis and endocytosis), which have different recycling rates and activation times, suffice to describe a stable growing tube. Simulations show a very good spatial separation between endocytosis and exocytosis, in which separation is shown to depend strongly on exocytic vesicle delivery. In accordance to measurements, most vesicles in the clear zone are predicted to be endocytic. Membrane flow is essential to maintain cell polarity, and bi-directional flow seems to be a natural consequence of the proposed mechanism. For the first time, a model addressing plasma membrane flow and cytosis regulation were posed. Therefore, it represents a missing piece in an integrative model of pollen tube growth, in which cell wall mechanics, hydrodynamic fluxes and regulation mechanisms are combined.     

The patterns of colchicine-induced death of lymphoid cells

It is shown that colchicine injection at doses higher than 1 mg/kg of animal weight induces cell death in thymus, spleen, bone marrow and intestine mucosa. The cell death is accompanied by a regular internucleosomal cleavage of nuclear DNA and by the elimination of the formed fragments from cells. Both the processes begin after a 1.5 hour lag-period and proceed before the outer membrane permeability for supravital dyes increases. DNA degradation is prevented by the inhibitor of protein synthesis cycloheximide. Cytochalasin B does not induce chromatin degradation or cell death and has no effect on radiation death of lymphocytes. A possible role of microtubule destruction as a switch-on mechanism of DNA degradation and cell death is discussed.     

Phosphoinositide function in cytokinesis

In systems as diverse as yeast, slime mold and animal cells, the levels and distribution of phosphatidylinositol phosphates (PIPs) must be strictly regulated for successful cell cleavage. The precise mechanism by which PIPs function in this process remains unknown. Recent experiments are beginning to shed light on the cellular pathways in which PIPs make key contributions during cytokinesis. In particular, PIPs promote proper actin cytoskeletal organization and direct membrane trafficking in dividing cells. Future research will uncover temporal and spatial regulation of the different PIPs, thus elucidating their role in cytoskeletal and membrane events that drive cell cleavage.     

The ultrastructure of the plasma membrane at the division furrow of grasshopper germ cells

Summary Evidence is provided from cinematography and electron microscopy that chemical processes are responsible for both plasma membrane synthesis and cell separation in dividing germ cells of the grasshopper. Polyribosome-like material is deposited as a ring equatorially, which determines the presumptive division plane. The material deposited at the division plane, in synergism with cytoplasmic material and existing plasma membrane, synthesizes the new plasma membrane. The polyribosome-like material polymerizes into helices, and as the coils of the helices tighten less surface becomes available for membrane synthesis. The decreasing rate of plasma membrane synthesis is the mechanism for cell separation. Thus, plasma membrane synthesis and cell separation occur simultaneously, directed by the same dynamic processes.Work supported under the auspices of the U. S. Atomic Energy Commission.