Contractile Network Research Articles

Ca2+ ‐binding proteins and contractility of the infraciliary lattice in Paramecium

Summary— The infraciliary lattice (ICL) is the innermost cortical cytoskeletal network of Paramecium. Its meshes which run around the proximal end of basal bodies form a continuous contractile network beneath the cell surface. We had previously shown that the network, which could be recovered in a contracted form and selectively solubilized by EGTA from an ICL‐enriched cell fraction, was principally composed of 23–24 kDa polypeptides cross‐reacting with antibodies raised against the 22 kDa Ca2+ ‐binding proteins of the ecto‐endoplasmic boundary (EEB), a contractile cytoskeletal network of another ciliate Isotricha prostoma. We show here 1) that the ICL also comprises a 220 kDa polypeptide; 2) that the 23–24 kDa polypeptides are resolved in 2D gels into 11 spots of acidic pI, 7 of which are both Ca2+ ‐binding and cross‐reacting with the anti EEB polypeptides; 3) that the network displays a high Ca2+ ‐affinity as the treshold for solubilization/co‐precipitation of both high and low MW polypeptides is around 10−8 M free Ca2+; 4) that in vivo contraction of the network occurs upon physiological increase of internal calcium concentration. The likely phylogenetic relationships of the 23–24 kDa ICL polypeptides with the calmodulin related family of Ca2+ ‐modulated polypeptides and the functions of the ICL in cell contractility and Ca2+ homeostasis are discussed.

Dynamics of the contractile system in the pseudopodial tips of normally locomoting amoebae, demonstrated in vivo by video-enhancement

Behaviour of the membrane and contractile system was directly recorded in the advancing and retracting frontal zones of spontaneously locomoting or stimulated amoebae. The advancing pseudopodial tips alternately slow down and accelerate. In the slowing phase the frontal hyaline caps are flat and compressed by countercontraction of the cortical actin network beneath the leading edge. At this stage the membrane-cytoskeleton complex splits: the detached contractile layer is retracted inwards, and the membrane lifted outwards. The fluid endoplasm fraction is filtered forward through the detached actin network. This results in a local hydrostatic pressure drop, immediately restores the forward flow of endoplasm and initiates the acceleration phase of the leading edge progression. The frontal membrane, temporarily disconnected from the cytoskeletal layer, is free to slide and extend forward, but the new submembrane contractile network is soon repolymerized. In this way, after making one step forward, the frontal zone recovers its former state, and the cycle is then repeated. The cortex disassembly-reassembly cycles at the leading edge are produced every 2 s, on average. Retraction of the frontal contractile layers is part of the general centripetal cortex flow observed during motor functions of amoebae and many other cells, and is therefore associated with various other backward movements observed within and on the surface of advancing frontal zones of amoebae. The backward movement of the contractile cortex is also responsible for the withdrawal of previously advancing pseudopodia, if the detachment of successive contractile sheets from the frontal membrane ceases. It was demonstrated that the action of attractants and repellents is based on the activation or inhibition, respectively, of rhythmic disassembly of the membrane-cytoskeleton complex at the leading edge.

Myogenesis and histogenesis of skeletal muscle on flexible membranes in vitro

Primary muscle cell cultures consisting of single myocytes and fibroblasts are grown on flexible, optically clear biomembranes. Muscle cell growth, fusion and terminal differentiation are normal. A most effective membrane for these cultures is commercially available Saran Wrap. Muscle cultures on Saran will, once differentiated, contract vigorously and will deform the Saran which is pinned to a Sylgard base. At first, the muscle forms a two-dimensional network which ultimately detaches from the Saran membrane allowing an undergrowth of fibroblasts so that these connective tissue cells completely surround groups of muscle fibers. A three-dimensional network is thus formed, held in place through durable adhesions to stainless steel pins. This three-dimensional, highly contractile network is seen to consist of all three connective tissue compartments seen in vivo, the endomysium, perimysium and epimysium. Finally, this muscle shows advanced levels of maturation in that neonatal and adult isoforms of myosin heavy chain are detected together with high levels of myosin fast light chain 3.

Cytological identification of cell types in the testis of Esox lucius and E. niger

Testes of Esox lucius and Esox niger were investigated histologically, cytochemically, and ultrastructurally in reproductive fish. Intralobular Sertoli cells possessed numerous lipid droplets in Esox lucius, but not in Esox niger. In both species, interlobular cell types included myoid cells and lipid-negative Leydig cells within the extravascular space. Evidence is presented for a contractile network of myoid cells within the testes of these teleosts. The presence of Leydig cells and myoid boundary cells in the testis of Esox lucius refutes the reported homology between lobule boundary cells and Leydig cells in this species.

Mechanics and control of the cytoskeleton in Amoeba proteus

Many models of the cytoskeletal motility of Amoeba proteus can be formulated in terms of the theory of reactive interpenetrating flow (Dembo and Harlow, 1986). We have devised numerical methodology for testing such models against the phenomenon of steady axisymmetric fountain flow. The simplest workable scheme revealed by such tests (the minimal model) is the main preoccupation of this study. All parameters of the minimal model are determined from available data. Using these parameters the model quantitatively accounts for the self assembly of the cytoskeleton of A. proteus: for the formation and detailed morphology of the endoplasmic channel, the ectoplasmic tube, the uropod, the plasma gel sheet, and the hyaline cap. The model accounts for the kinematics of the cytoskeleton: the detailed velocity field of the forward flow of the endoplasm, the contraction of the ectoplasmic tube, and the inversion of the flow in the fountain zone. The model also gives a satisfactory account of measurements of pressure gradients, measurements of heat dissipation, and measurements of the output of useful work by amoeba. Finally, the model suggests a very promising (but still hypothetical) continuum formulation of the free boundary problem of amoeboid motion. by balancing normal forces on the plasma membrane as closely as possible, the minimal model is able to predict the turgor pressure and surface tension of A. proteus. Several dynamical factors are crucial to the success of the minimal model and are likely to be general features of cytoskeletal mechanics and control in amoeboid cells. These are: a constitutive law for the viscosity of the contractile network that includes an automatic process of gelation as the network density gets large; a very vigorous cycle of network polymerization and depolymerization (in the case of A. proteus, the time constant for this reaction is approximately 12 s); control of network contractility by a diffusible factor (probably calcium ion); and control of the adhesive interaction between the cytoskeleton and the inner surface of the plasma membrane.

Initial Characterization of small Proteoglycans Synthesized by Embryonic Chick Leg Muscle-Associated Connective Tissues

Muscle development in the embryonic chick involves the interfacing of two different cell types: myogenic cells forming the contractile network and connective tissue cells which wrap this network into discrete morphologies. Previous reports from this laboratory detailed the characterization of large extracellular matrix proteoglycans synthesized by skeletal muscle. This report describes the initial characterization of small muscle-associated connective tissue proteoglycans synthesized in embryonic chick leg in ovo and by embryonic leg muscle-associated secondary fibroblasts in vitro by CsCl equilibrium density gradient centrifugation. Analysis of the D1 gradient fractions from the in ovo and in vitro material revealed that the proteoglycans were of relatively small size. These molecules eluted from Sepharose CL-2B through a Kav range of 0.6-0.8. Analysis of alkaline borohydride-released glycosaminoglycan chains by Sepharose CL-6B, Sephadex G-25, and thin layer chromatography demonstrated a mixture of three proteoglycan families composed of chondroitin sulfate, dermatan sulfate, and heparan sulfate glycosaminoglycans.

Cell motion, contractile networks, and the physics of interpenetrating reactive flow

In this paper we propose a physical model of contractile biological polymer networks based on the notion of reactive interpenetrating flow. We show how our model leads to a mathematical formulation of the dynamical laws governing the behavior of contractile networks. We also develop estimates of the various parameters that appear in our equations, and we discuss some elementary predictions of the model concerning the general scaling principles that pertain to the motions of contractile networks.

Numerical studies of unreactive contractile networks

We present a finite difference algorithm for integrating the reactive flow model of contractile biological polymer networks on a fixed Eulerian mesh. We discuss the accuracy and limits of the algorithm. To illustrate the application of the algorithm, we carry out a family of computations involving an unreactive contractile network contained in a two-dimensional square reaction vessel. By numerical experiments using different values of the physical parameters of the model, we find that for this simple sort of system two major dynamical modes of contraction are predicted to occur. There is the squeezing type contraction in which the network contracts to a single small clump with gradual expulsion of solution material, and the rending type contraction in which the network tears itself into a number of separate pieces. We find that to a good approximation the transition between the squeezing mode and the rending mode is controlled by a single nondimensional number (the rending number). We discuss the relevance of these results for the analysis of various experimental observations.

Myofibrillogenesis in living cells microinjected with fluorescently labeled alpha-actinin.

Fluorescently labeled alpha-actinin, isolated from chicken gizzards, breast muscle, or calf brains, was microinjected into cultured embryonic myotubes and cardiac myocytes where it was incorporated into the Z-bands of myofibrils. The localization in injected, living cells was confirmed by reacting permeabilized myotubes and cardiac myocytes with fluorescent alpha-actinin. Both living and permeabilized cells incorporated the alpha-actinin regardless of whether the alpha-actinin was isolated from nonmuscle, skeletal, or smooth muscle, or whether it was labeled with different fluorescent dyes. The living muscle cells could beat up to 5 d after injection. Rest-length sarcomeres in beating myotubes and cardiac myocytes were approximately 1.9-2.4 microns long, as measured by the separation of fluorescent bands of alpha-actinin. There were areas in nearly all beating cells, however, where narrow bands of alpha-actinin, spaced 0.3-1.5 micron apart, were arranged in linear arrays giving the appearance of minisarcomeres. In myotubes, alpha-actinin was found exclusively in these closely spaced arrays for the first 2-3 d in culture. When the myotubes became contraction-competent, at approximately day 4 to day 5 in culture, alpha-actinin was localized in Z-bands of fully formed sarcomeres, as well as in minisarcomeres. Video recordings of injected, spontaneously beating myotubes showed contracting myofibrils with 2.3 microns sarcomeres adjacent to noncontracting fibers with finely spaced periodicities of alpha-actinin. Time sequences of the same living myotube over a 24-h period revealed that the spacings between the minisarcomeres increased from 0.9-1.3 to 1.6-2.3 microns. Embryonic cardiac myocytes usually contained contractile networks of fully formed sarcomeres together with noncontractile minisarcomeres in peripheral areas of the cytoplasm. In some cells, individual myofibrils with 1.9-2.3 microns sarcomeres were connected in series with minisarcomeres. Double labeling of cardiac myocytes and myotubes with alpha-actinin and a monoclonal antibody directed against adult chicken skeletal myosin showed that all fibers that contained alpha-actinin also contained skeletal muscle myosin. This was true whether alpha-actinin was present in Z-bands of fully formed sarcomeres or present in the closely spaced beads of minisarcomeres. We propose that the closely spaced beads containing alpha-actinin are nascent Z-bands that grow apart and associate laterally with neighboring arrays containing alpha-actinin to form sarcomeres during myofibrillogenesis.

Distribution of actin filaments in fertilized egg of the ascidian Ciona intestinalis

Microfilaments in the contracting cortex during the bipolar ooplasmic segregation of Ciona intestinalis eggs were examined by two methods, staining with fluorescent phalloidin and decoration with myosin subfragment 1 (S1). Fluorescent (Fl-)phalloidin revealed prominent fluorescence in the contracting cortex between the surface constriction and the vegetal pole of fertilized eggs. The animal pole did not stain. After extraction in Triton X-100, the cortex appeared as a thin layer that easily separated from cytoplasmic mass, especially at the contracting stage after fertilization. This layer also stained strongly with Fl-phalloidin. S1-decoration confirmed that actin filaments were abundant in the thin layer of Triton-extracted cortex. The actin filaments are considered to compose a contractile network covering the vegetal side of the constriction.

The effects of cytochalasin B on the cytoplasmic contractile network revealed by whole-cell transmission electron microscopy

The ultrastructure of the contractile response to cytochalasin B (CB) has been studied using whole-cell electron microscopy. The actin-containing contractile network rapidly condenses into numerous stellate microfilament foci (SMF). These SMF punctuate the cytoplasm, and are frequently associated with an extensive persistent cytoskeleton containing microtubules and intermediate filaments. This association of SMF and persistent cytoskeleton appears to mediate the arborized morphology induced by CB. Eventually SMF aggregate and migrate towards the nucleus. Concomitantly the cell surface is differentiated into clusters of miniblebs which migrate to the nucleus. SMF loss from the periphery resulted in respreading to a flattened angular morphology within which the nucleus was frequently displaced. The role of the actin network, and the mechanism of these CB-induced contractile alterations are discussed.

Palate morphogenesis. I. Immunological and ultrastructural analyses of mouse palate.

Midpalate was analyzed for the presence of nonmuscle contractile systems. The results indicate that increased amounts of actin and myosin are present in cells of regions 2 and 3. A localization of the contractile proteins in cellular projections (filopodia) and in the peripheral cytoplasm of the cell body was confirmed by indirect immunofluorescence studies, using antibodies directed against smooth muscle myosin and against skeletal muscle actin. Specificity of the immunofluorescence reactions was ascertained by immunoabsorption studies using purified myosin and actin. Electron microscopic observations of the mesenchymal cells in region 2 revealed 70A microfilaments along the cell periphery and packed in fliopodia-like projections which course between the cells. These cells, which surround a small ossification center, show no orientation, but extend up to the cranial base perichondrium and down into the shelf between the tongue side epithelium and the ossification center. The cells and projections are attached to each other by adherens and tight-like junctions, forming a putative cohesive contractile network. Putative contractile cells in region 3 are strikingly aligned perpendicular to the oral epithelium and extend one-third of the distance into the shelf. Projections from region 3 cells are contiguous with basement membrane material of the oral epithelium. Axonal bundles and single axons were commonly observed coursing through regions 2 and 3, often seen in close association with the mesenchymal cells. Both clear and dense-core vesicles were found in the axons and cells of these regions. The possible role of these putative nonmuscle contractile cells in palate morphogenesis is discussed.

A synchronized multicellular movement initiated by light and mediated by microfilaments

Our ultrastructural studies of the sperm duct epithelium in the ascidian Ciona intestinalis document the existence of a light-sensitive assembly of microfilaments. This process coincides with the contraction of the sperm duct epithelium following exposure to blue light. Isolated regions are capable of organizing a microfilament system, thus, the receptoral mechanism regulating this process must be distributed along the length of the epithelium. Cells of the isolated dark-adapted duct contain an amorphous material, but few microfilaments. In contrast, when ducts are actively spawning in response to illumination or when isolated segments of the excised duct are exposed to light, the basal plasmalemma becomes highly convoluted, and the adjacent cytoplasm is packed with a microfilamentous complex. Treatment of dark-adapted segments with A23187 will mimic the effects of light, suggesting that ion fluxes may be involved in generating the contractile network. SDS gel electrophoresis indicates that these cells contain a major band with an electrophoretic mobility indistinguishable from that of actin. The light-induced contraction of cells composing the sperm duct epithelium is implicated in effecting sperm release from individuals and, hence, in controlling synchronous spawning within a population.

Microfilaments in cellular and developmental processes.

In our opinion, all of the phenomena that are inhibited by cytochalasin can be thought of as resulting from contractile activity of cellular organelles. Smooth muscle contraction, clot retraction, beat of heart cells, and shortening of the tadpole tail are all cases in which no argument of substance for alternative causes can be offered. The morphogenetic processes in epithelia, contractile ring function during cytokinesis, migration of cells on a substratum, and streaming in plant cells can be explained most simply on the basis of contractility being the causal event in each process. The many similarities between the latter cases and the former ones in which contraction is certain argue for that conclusion. For instance, platelets probably contract, possess a microfilament network, and behave like undulating membrane organelles. Migrating cells possess undulating membranes and contain a similar network. It is very likely, therefore, that their network is also contractile.