On the mechanisms of cytokinesis in animal cells

We present a model that attempts to explain some aspects of cytokinesis in animal cells. We propose two separate phases of cytokinesis. The first is not dependent on the presence of the mitotic apparatus and involves a general activation of cortical contractile elements resulting in the development of a surface tension. In the second phase the asters of the mitotic apparatus interact and modulate the activities of the tension generating elements in the cortex to produce gradients of surface tension with the highest values being at the equator. Tension generating elements are assumed to be free to move in the plane of the cortex so that they will consequently move up the gradient of tension and accumulate as an equatorial belt of oriented elements i.e. the contractile ring. The model was simulated on a computer and is capable of reproducing some of the wide variety of cleavage configurations that are observed.     

PERIODIC CHANGES IN THE CONTENT OF PROTEIN-BOUND SULFHYDRYL GROUPS AND TENSION AT THE SURFACE OF STARFISH OOCYTES IN CORRELATION WITH THE MEIOTIC DIVISION CYCLE*

The sulfhydryl content of protein and the tension at the surface were measured for starfish oocytes from the first meiotic division to the cleavage stage. A cyclic change in both the protein-SH and the tension at the surface was found to accompany the division cycle, including the first and second meiotic divisions. It is concluded that the unequal meiotic divisions share the same character with the equal divisions of cleavage, with respect to changes both in the protein-SH and the tension at the surface.     

The astral relaxation theory of cytokinesis revisited

Cytokinesis in animal cells is accomplished by the active constriction of the equatorial regions of a cell by an actomyosin-containing contractile ring. The mitotic apparatus specifies the position and orientation of the furrow such that the mitotic spindle is always bisected. Global cortical contractions occur in the cortex of a cell prior to cytokinesis that are independent of the presence of the mitotic apparatus. It was proposed some years ago that the asters of the mitotic apparatus could act to relax the preformed cortical tension in their vicinity. This would produce a differential in tension between the equatorial regions and the adjacent regions of the cortex so that the equatorial regions would contract, forming a cleavage furrow. It can be shown that, as it stands, this theory cannot explain cleavage. However, if cortical contractile elements are assumed to be laterally mobile in the plane of the cortex, then the astral relaxation theory can account for many of the aspects of cleavage, including the formation of the contractile ring. Similar schemes may account for the behaviour of the lamellapodia of motile cells.     

Ultrastructural Basis of the Tension Increase in Sea-Urchin Eggs Prior to Cytokinesis

Cortices of sea-urchin eggs were studied by electron microscopy to identify the structure responsible for the rise in tension at the egg surface prior to cleavage. During anaphase the tension increased and fine filaments of 70–90 Å in diameter appeared in the cell cortex forming a thin mesh-work beneath the cell membrane. The meshwork spread all around the egg cortex without reference to the mitotic axis and the number of filaments seemed to increase up to telophase. Immediately before appearance of the cleavage furrow, the meshwork in the anticipated furrow region became dense. As the furrow appeared the tension began to decrease and the meshwork disappeared. In the progressing furrow region fine filaments of the same size as that of the meshwork-filament were oriented in a bundle to form a contractile ring. Treatment with cytochalasin B suppressed both the tension increase and the formation of the filamentous meshwork. These results suggest that the component filament of the meshwork is an actin microfilament, and that the tension increase at anaphase is due to formation of a meshwork of actin microfilaments from which a contractile ring is subsequently derived at late telophase.     

On fluid-mechanical simulations of cell division and movement

Demonstrations are presented of the possible effects of variable surface tension and dynamical instability in cell cleavage and the formation of a contractile belt. Analysis of a theoretical absorption/desorption model of chemotaxis suggested by these experiments shows that occasional, unidirectional amoeboid movement can occur in an oscillatory field of chemical attractant.     

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.     

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.     

The movement of bound ligands over cell surfaces

The long range movements of membrane-bound ligands into surface caps and into the pseudopods of phagocytizing cells, the uropods of motile cells and the cleavage furrow of dividing cells appear to be analogous processes. A common mechanism to explain these movements must take into account several new and central observations: ligand-receptor complexes can migrate to regions of existing microfilament accumulation; laser photobleaching studies with fluorescent Con A indicate that ligand-receptor movement occurs unidirectionally; video computer analyses of Con A redistribution show that movement may exceed the maximum rates measured for protein diffusion in membranes. These observations are not consistent with models in which ligand-receptor movement occurs by diffusion or by direct interaction with contractile microfilaments. However, they can be satisfied by a new model that proposes the entrainment of selected membrane determinants on membrane waves directed towards regions such as caps, pseudopodia, uropods or cleavage furrow. These oriented waves are initiated by tension due to asymmetric microfilamentmembrane interaction.     

FORCE EXERTED BY THE CLEAVAGE FURROW OF SEA URCHIN EGGS

A drop of ferrofluid injected into the center of a dividing sea urchin egg is deformed into the shape of an hourglass when the cleavage furrow advances. The force applied to the drop is determined from the deformation of the drop and the interfacial tension between the ferrofluid and the protoplasm. The interfacial tension is determined from the deformation of a spherical drop in the protoplasm when a magnetic field is applied, and the force applied to the drop, which is estimated from the deformation by magnetic field of a similar drop in 2 per cent aqueous solution of Triton X-100 and the interfacial tension between the ferrofluid and this solution.
The force applied to the drop in the dividing egg increases during an early stage of cleavage and decreases during a later stage. The force attained a maximum of 9 × 10⁻³ dyne in an egg of Temnopleurus toreumaticus which pinched the drop into two when it divided. Smaller maximum forces, 3.9 × 10⁻³ dyne in the eggs of Temno-pleurus toreumaticus and 2.0 × 10⁻³ dyne in the eggs of Clypeaster japonicus (mean values), were obtained when the furrowing was arrested by the drop. The magnitude of the maximum tension developed in the contractile element located in the furrow cortex is discussed.     

In vivo cleavage of Escherichia coli BIME-2 repeats by DNA gyrase: genetic characterization of the target and identification of the cut site

The Escherichia coli chromosome contains about 300 bacterial interspersed mosaic elements (BIMEs). These elements, located at the 3' end of genes, are composed of three types of alternating repetitive extragenic palindromes (REPs). Based on the type of REP they contain and on their ability to interact with the integration host factor (IHF), BIMEs are subdivided into two families: BIME-1 elements contain an IHF binding site flanked by converging Y and Z1 REPs, whereas BIME-2 elements contain a variable number of alternating Y and Z2 REPs without an IHF site. Although some BIMEs have been implicated in the protection of mRNA against 3' exonucleolytic degradation, the main role of elements belonging to both families remains to be elucidated. In this paper, we used oxolinic acid, a drug that reveals potential sites of DNA gyrase action, to demonstrate that DNA gyrase interacts in vivo with BIME-2 elements. The frequency of cleavage varied from one element to another, and the cleavage pattern observed in elements containing several REPs indicated that DNA gyrase cut DNA every two REPs. A single cleavage site has been identified in the Y REP in six out of seven instances, and the nucleotide sequence of a 44 bp fragment containing the scission point displayed conserved residues at six positions. The lack of one of the conserved residues accounted for the absence of cleavage in most of the Z2 REPs. Our results also showed that cleaved REPs were always associated with another REP, suggesting that a pair of diverging REPs constitutes the target of DNA gyrase. DNA gyrase cleavage at repetitive BIME-2 elements may have consequences for DNA topology and genomic rearrangements.     

Topology of cleavage in the light of Pierre Curie principle

The stages of radial, spiral, and bilateral cleavage, including blastula, are considered as polyhedrons and projections of polyhedrons onto a plane: Wenn, Schlegel, and Euler projections. The blastula spatial organization is characterized by face numerals and Euler characteristics, as well by symmetry groups. The classes of equivalence of polyhedrons have been considered: duality and equal composition. The correspondence between different types of cleavage has been established by shift transformation on Schlegel projections and turn on a spherical noneuclidean surface. Determination of the prospective significance of blastomeres during cleavage was compared with the dichotomous division of the general notion in logic. This in view, the Wenn diagram of four figures has been reflected onto the sphere surface. Blastula faceting is interpreted as a reflection of hereditary information about the prospective significance of blastomeres onto a spherical surface. Topologically, the reflection represents a transformation of the linear orderliness of information contained in the genome into a two-dimensional orderliness of prospective properties on the blastula noneuclidean surface. Therefore, the elements of blastula symmetry can be considered as self in the sense of Pierre Curie principle. Cleavage stages are living colloid crystals.     

Topology of cleavage in light of the Pierre Curie principle

The stages of radial, spiral, and bilateral cleavage, including blastula, are considered as polyhedrons and projections of polyhedrons onto a plane: Wenn, Schlegel, and Euler projections. The blastula spatial organization is characterized by face numerals and Euler characteristics, as well by symmetry groups. The classes of equivalence of polyhedrons have been considered: duality and eqicomposition. The correspondence between different types of cleavage has been established by shift transformation on Schlegel projections and turn on a spherical noneuclidean surface. Determination of the prospective significance of blastomeres during cleavage was compared with the dichotomous division of the general notion in logic. This in view, the Wenn diagram of four figures has been reflected onto the sphere surface. Blastula faceting is interpreted as a reflection of hereditary information about the prospective significance of blastomeres onto a spherical surface. Topologically, the reflection represents a transformation of the linear orderliness of information contained in the genome into a two-dimensional orderliness of prospective properties on the blastula noneuclidean surface. Therefore, the elements of blastula symmetry can be considered as self in the sense of Pierre Curie principle.     

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.     

Distinct RNA structural domains cooperate to maintain a specific cleavage site in the 3'-UTR of IGF-II mRNAs

The insulin-like growth factor II mRNAs are targets for site-specific endonucleolytic cleavage in the 3'-UTR, which results in a very stable 3' cleavage product of 1.8 kb, consisting of 3'-UTR sequences and a poly(A) tail. The 5' cleavage product contains the coding region and is rapidly degraded. Thus, cleavage is thought to provide an additional way to control IGF-II protein synthesis. We had established that cleavage requires two widely separated sequence elements (I and II) in the 3'-UTR that form a stable duplex of 83 nucleotides. The cleavage-site itself is located in an internal loop preceded by two stable stem-loop structures. Furthermore, in a study which was based on RNA folding algorithms, we have shown that there are specific sequence and structural requirements for the cleavage reaction. Here, the functions of the different structural domains in cleavage were assessed by deletion/mutational analyses, and biochemical structure probing assays were performed to characterize better the RNA structures formed and to verify the computer folding predictions. The data suggest that the stem-loop domain contributes to maintain a highly specific c leavage-site by preventing the formation of alternative structures in the cleavage-site domain. Involvement of the nucleotides in the cleavage-site loop itself in non-Watson-Crick interactions may be important for providing a specific recognition surface for an endoribonuclease activity.     

Correlations between the cerebrospinal fluid surface tension value and 1. Concentration of total proteins 2. Number of cell elements

The contribution gives a survey about the problematics of surface tension in biomedical sciences. The paper presents results of a study devoted to the cerebrospinal fluid. The distribution of the surface tension values of this liquid in healthy individuals is presented (n = 33), and further, statistically significant correlations between the cerebrospinal fluid surface tension value and 1. concentration of total proteins expressed by Spearman's coefficient p(s) = (-0.995) and 2. number of cell elements expressed through Spearman's coefficient p(s) = (-0.965) in cerebrospinal fluid are described.     

Instability development in heated human erthrocytes.

Heated human erythrocytes gradually lose their form-maintaining structure as the temperature is increased to 50 degrees C and can behave in some respects as a viscous fluid. We have developed a technique for heating and stressing these cells that is novel, simple and quantitatively precise. We have applied this technique to heated human erythrocytes and have measured instability development in cells. We have employed instability growth theory to calculate a value for an effective surface tension which, in contrast to other methods of membrane surface tension measurement sought to minimize the effects of membrane supporting structural elements. The value obtained for the surface tension of the heated erythrocyte membrane was 0.9 . 10(-6) N/m with a range of variation from 0.4 . 10(-6)N/m to 1.4 . 10(-6) N/m. The methods described may be useful for determining fundamental physical parameters such as internal viscosity and interfacial tension in other systems.     

Membrane protein redistribution during Xenopus first cleavage

A large increase in surface area must accompany formation of the amphibian embryo first cleavage furrow. The additional membrane for this areal expansion has been thought to be provided entirely from cytoplasmic stores during furrowing. We have radioiodinated surface proteins of fertilized, precleavage Xenopus laevis embryos and followed their redistribution during first cleavage by autoradiography. Near the end of first cleavage, membrane of the outer, pigmented surface of the embryo and a short band of membrane at the leading edge of the furrow displayed a high silver grain density, but the remainder of the furrow membrane was lightly labeled. The membrane of the cleavage furrow is thus mosaic in character; the membrane at the leading edge originates in part from the surface of the zygote, but most of the membrane lining the furrow walls is derived from a source inaccessible to surface radioiodination. The furrow membrane adjacent to the outer, pigmented surface consistently showed a very low silver grain density and was underlain by large membranous vesicles, suggesting that new membrane derived from cytoplasmic precursors is inserted primarily in this location, at least during the later phase of cleavage. Radioiodinated membrane proteins and surface-attached carbon particles, which lie in the path of the future furrow, contract toward the animal pole in the initial stages of cleavage while markers in other regions do not. We suggest that the domain of heavily labeled membrane at the leading edge of the definitive furrow contains the labeled elements that are gathered at the animal pole during the initial surface contraction and that they include membrane anchors for the underlying contractile ring of microfilaments.     

Palmitoylation and processing of the lipopeptide surfactant protein C

Pulmonary surfactant, a mixture of lipids and proteins, reduces the surface tension at the air-water interface of the lung alveoli by forming a surface active film. This way, it prevents alveoli from collapsing and facilitates the work of breathing. Surfactant protein C (SP-C) plays an important role in this surfactant function. SP-C is expressed as a proprotein (proSP-C), which becomes posttranslationally modified with palmitate and undergoes several rounds of proteolytical cleavage. This results in the formation of mature SP-C, which is stored in the lamellar bodies (LB) and finally secreted into the alveolar space. Recently, new insights into the sorting, processing and palmitoylation of proSP-C have been obtained by mutagenesis studies. Moreover, reports on the association of development of lung disease with SP-C deficiency have led to new insights into the importance of SP-C for proper surfactant homeostasis. In addition, new information has become available on the role of the palmitoyl chains of SP-C in surface activity. This review summarizes these recent developments in the processing and function of SP-C, with particular emphasis on the signals for and role of palmitoylation of SP-C.     

Alveolar surface forces and lung architecture

The entire alveolar surface is lined by a thin fluid continuum. As a consequence, surface forces at the air-liquid interface are operative, which in part are transmitted to the delicate lung tissue. Morphologic and morphometric analyses of lungs show that the alveolar surface forces exert a moulding effect on alveolar tissue elements. In particular, in lungs at low degrees of inflation, equivalent to the volume range of normal breathing, there is a derecruitment of alveolar surface area with increasing surface tensions which reflects equilibrium configurations of peripheral air spaces where the sum of tissue energy and surface energy is minimum. Thus, changes in surface tension alter the recoil pressure of the lung directly and indirectly by deforming lung tissue and hence changing tissue tensions. However, the interplay between tissue and surface forces is rather complex, and there is a marked volume dependence of the shaping influence of surface forces. With increasing lung volumes the tissue forces transmitted by the fiber scaffold of the lung become the predominant factor of alveolar micromechanics: at lung volumes of 80% total lung capacity or more, the alveolar surface area-volume relation is largely independent of surface tension. Most important, within the range of normal breathing, the surface tension, its variations and the associated variations in surface area are small. The moulding power of surface forces also affects the configuration of capillaries, and hence the microcirculation, of free cellular elements such as the alveolar macrophages beneath the surface lining layer, and of the surfaces of the peripheral airways. Still enigmatic is the coupling mechanism between the fluid continua of alveoli and airways which might also be of importance for alveolar clearance. As to the surface active lining layer of peripheral air spaces, which determines alveolar surface tension, its structure and structure-function relationship are still ill-defined owing to persisting problems of film preservation and fixation. Electron micrographs of alveolar tissue, of lining layers of captive bubbles, and scanning force micrographs of surfactant films transferred on mica plates reveal a complex structural pattern which precludes so far the formulation of an unequivocal hypothesis.