Cytoskeletal organization of a cloned hemopoietic stromal cell line during attachment and spreading

The cell membrane of a cloned murine bone marrow stromal cell line D2XRII was extracted in situ using Triton X-100 detergent and the cytoskeletal structure studied during the process of adherence and spreading. During this process, three zones can be identified in the cytoplasm: the perinuclear zone, which was the fixed part of the cell; the peripheral mixed filamentous zone, which formed the core of long cytoplasmic projections; and an outer zone, which formed the boundary of cytoplasmic projections and contained only intermediate filaments. The process of spreading appeared to originate from very long strips of microfilaments emanating from the second zone, crossing the width of the outer zone, and extending beyond for a long distance. The second and third zones then appeared to "stream out" around the axis of this strip, and in this fashion the cytoplasm spreads over the substratum.     

Mobility of GPIIb-IIIa receptors within membranes of surface- and suspension-activated platelets does not depend on assembly and contraction of cytoplasmic actin

Investigations using fibrinogen coupled to colloidal gold (Fgn/Au) have shown that glycoprotein IIb-IIIa (GPIIb-IIIa) receptors are mobile and undergo centripetal reorganization on spread platelets following surface activation. The assembly of cytoplasmic actin and its constriction into an inner filamentous zone have been proposed as the mechanism driving the Fgn/Au receptor complexes across the plasma membrane. The present study has used cytochalasin B (CB), an agent known to inhibit actin assembly and cause breakdown of newly formed actin filaments to test that hypothesis. At a concentration which inhibited pseudopod formation and spreading, CB did not block movement of Fgn/Au receptor complexes toward platelet centers or into the open canalicular system (OCS). Channels of the OCS filled with Fgn/Au receptor complexes were evident in over 90% of the CB-treated, surface-activated platelets. The findings do not support the concept that assembly of cytoplasmic actin and its contraction move receptor-ligand complexes on the platelet plasma membrane.     

Tomographic reconstruction of treponemal cytoplasmic filaments reveals novel bridging and anchoring components

An understanding of the involvement of bacterial cytoplasmic filaments in cell division requires the elucidation of the structural organization of those filamentous structures. Treponemal cytoplasmic filaments are composed of one protein, CfpA, and have been demonstrated to be involved in cell division. In this study, we used electron tomography to show that the filaments are part of a complex with a novel molecular organization that includes at least two distinct features decorating the filaments. One set of components appears to anchor the filaments to the cytoplasmic membrane. The other set of components appears to bridge the cytoplasmic filaments on the cytoplasmic side, and to be involved in the interfilament spacing within the cell. The filaments occupy between 3 and 18% of the inner surface of the cytoplasmic membrane. These results reveal a novel filamentous molecular organization of independent filaments linked by bridges and continuously anchored to the membrane.     

Flow-cytometric detection of surface membrane alterations and concomitant changes in the cytoskeletal actin status of activated platelets.

Occlusive vascular diseases are promoted by a "prethrombotic state" with increased platelet activity. Polymerization of cytoskeletal proteins and exposure of subcellular structures or rebinding of secreted proteins have been characterized as early reactions after platelet activation preceding adhesion and aggregation. Here, we demonstrate the kinetic increase in specific binding of monoclonal antibodies to thrombospondin (P10) and to platelet membrane activation markers CD63 (GP53, a 53 kD lysosomal protein) and CD62 (GMP140, a 140 kD alpha granule protein) by using a flow-cytometric bio-assay and the related change in the actin status by using the DNase-I inhibition assay after stimulation of normal human platelets with 0.2 U/ml thrombin. F-actin was raised from 41% to 51% of total platelet actin content 30 s after stimulation and remained thereafter constant (50% at 60 s). Simultaneously, the percentage of P10, CD63, and CD62 positive platelets was elevated from 5.4%, 24.4%, and 9.1% to 67.4%, 80.2%, and 82.3% respectively. The mean number of P10, CD63, and CD62 antibody binding sites increased from 3,300, 1,715, and 2,146 to 6,400, 6,800, and 9,016 per platelet. Conclusively, changes in the organization of the cytoskeletal protein "actin" and exposure of subcellular structures indicating platelet secretion can be regarded as markers of early platelet activation. Thus, the parallel response in both analytical systems provides further support for the diagnostic concept of flow-cytometric detection of preactivated platelets in the peripheral blood by using fluochrome staining procedures detecting activation dependent structural alterations directly at the cellular level.     

Utrastructure of the cement gland of Xenopus laevis.

To establish a morphological baseline for experimental studies of differentiation using the cement gland as a model, the following observations are added to those on record. The elongated cells of Xenopus laevis cement glands have an internal organization displaying five distinct zones differing in structure and specialized function. The apical zone contains packed secretion vesicles apparently belonging to two different types. The transit zone appears to be devoid of major biosynthetic activity and contains secretion vesicles migrating toward the surface. The zone of biosynthesis is typically organized in concentric regions. The very elongated nucleus lies in the next zone. Finally, the storage zone is characterized by lipid droplets and yolk platelets. Only quantitative differences are observed between cells of young and mature cement glands. Though all cells have the same general organization they may probably be divided into two subtypes according to the structure of their cytoplasm. The epithelial cells surrounding the gland differ according to their position along lateral or basal borders.     

Microfilament reorganization in normal and cytochalasin B treated adherent thrombocytes

Thrombocyte adhesion following activation by a Formvar surface involves a morphologic transition resulting in a fully spread cell. Correlative SEM and whole mount TEM were used to study the cytoskeletal alterations that accompany changes in surface morphology during adhesion. Following initial adhesion, thrombocytes extend slender pseudopods containing longitudinally oriented bundles of filaments that are 13–22 nm in diameter. Concomitant with pseudopod extension, a cytoplasmic hyalomere, consisting of a dense filamentous network, extends between the pseudopods and ultimately results in a fully spread cell. Treatment of thromboyctes with cytochalasin B (10^?5 M) caused clumping of the hyalomere filament network and retraction of the hyalomere. Examination of partially retracted cells revealed that pseudopod filament bundles were continuous with the contracting filamentous network. It is concluded that pseudopod filament bundles and cytoplasmic hyalomere filaments are interconvertible and that their organizational relationship changes in accordance with gross morphologic changes.     

Endothelial cell protrusion and migration in three-dimensional collagen matrices

Many cells display dramatically different morphologies when migrating in 3D matrices vs. on planar substrata. How these differences arise and the implications they have on cell migration are not well understood. To address these issues, we examined the locomotive structure and behavior of bovine aortic endothelial cells (ECs) either inside 3D collagen gels or on 2D surfaces. Using time-lapse imaging, immunofluorescence, and confocal microscopy, we identified key morphological differences between ECs in 3D collagen gels vs. on 2D substrata, and also demonstrated important functional similarities. In 3D matrices, ECs formed cylindrical branching pseudopodia, while on 2D substrata they formed wide flat lamellae. Three distinct cytoplasmic zones were identified in both conditions: (i) a small, F-actin-rich, rapidly moving peripheral zone, (ii) a larger, more stable, intermediate zone characterized by abundant microtubules and small organelles, and (iii) a locomotively inert central zone rich in microtubules, and containing the larger organelles. There were few differences between 2D and 3D cells in the content and behavior of their peripheral and central zones, whereas major differences were seen in the shape and types of movements displayed by the intermediate zone, which appeared critical in distributing cell-matrix adhesions and directing cytoplasmic flow. This morphological and functional delineation of cytoplasmic zones provides a conceptual framework for understanding differences in the behavior of cells in 3D and 2D environments, and indicates that cytoskeletal structure and dynamics in the relatively uncharacterized intermediate zone may be particularly important in cell motility in general.     

Redistribution of the fibrinogen receptor of human platelets after surface activation

We investigated the whole cell distribution of the platelet membrane receptor for fibrinogen in surface-activated human platelets. Fibrinogen-labeled colloidal gold was used in conjunction with platelet whole mount preparations to visualize directly the fibrinogen receptor. Unstimulated platelets fail to bind fibrinogen, and binding was minimal in the stages of activation immediately following adhesion. The amount of fibrinogen bound per platelet increased rapidly during the shape changes associated with surface activation until 7,600 +/- 500 labels were present at saturation. Maximal binding of fibrinogen was followed by receptor redistribution. During the early stages of spreading, fibrinogen labels were uniformly distributed over the entire platelet surface, including pseudopodia, but the labels become progressively centralized as the spreading process continued. In well spread platelets, labels were found over the central regions, whereas peripheral areas were cleared of receptors. Receptor redistribution during spreading was accompanied by cytoskeletal reorganization such that a direct correlation was seen between the development of specific ultrastructural zones and the distribution of surface receptor sites suggesting a link between the surface receptors and the cytoskeleton. The association of fibrinogen receptors with contractile elements of the cytoskeleton, which permits coordinated receptor centralization, is important to the understanding of the role of fibrinogen in normal platelet aggregation and clot retraction.     

Kindlins: essential regulators of integrin signalling and cell-matrix adhesion

Integrin-mediated cell-ECM (extracellular matrix) adhesion is a fundamental process that controls cell behaviour. For correct cell-ECM adhesion, both the ligand-binding affinity and the spatial organization of integrins must be precisely controlled; how integrins are regulated, however, is not completely understood. Kindlins constitute a family of evolutionarily conserved cytoplasmic components of cell-ECM adhesions that bind to beta-integrin cytoplasmic tails directly and cooperate with talin in integrin activation. In addition, kindlins interact with many components of cell-ECM adhesions--such as migfilin and integrin-linked kinase--to promote cytoskeletal reorganization. Loss of kindlins causes severe defects in integrin signalling, cell-ECM adhesion and cytoskeletal organization, resulting in early embryonic lethality (kindlin-2), postnatal lethality (kindlin-3) and Kindler syndrome (kindlin-1). It is therefore clear that kindlins, together with several other integrin-proximal proteins, are essential for integrin signalling and cell-ECM adhesion regulation.     

Studies on the function of cell surface glycoproteins. II. Possible role of surface glycoproteins in the control of cytoskeletal organization and surface morphology

Immunoglobulin from goat antiserum directed against purified surface membranes from transformed BHK21/C13 cells (anti-M) has been shown to cause both control and transformed hamster cells to round and detach from the substrate (see accompanying paper). This paper documents the effects of the antiserum on the cytoskeletal organization and cell surface morphology of control BHK21/C13 cells examined by scanning and transmission electron microscopy. As a result of antiserum-induced rounding, the normally smooth cell surface becomes covered with filopodia and blebs, and the organization of all three components of the filamentous cytoskeleton is altered. In terms of cell surface morphology and cytoskeletal organization, the cells resemble rounded, postmitotic or trypsinized BHK cells rather than cells treated with either anticytoskeletal drugs or lectins. Immunocytochemical and radioimmune assay experiments support the suggestion that the rounding reaction induced by anti-M serum results from the specific interaction of antibodies with molecules on the cell surface. It is suggested that anti-M serum induces alterations in cytoskeletal organization via a transmembrane signal and that cytoskeletal reorganization is a fundamental part of the rounding and detachment process.     

The cytoskeletal organization of epithelial cells in culture

Cytoskeleton organization of cultured normal epithelial cells (epithelium of newborn mouse kidney, mouse and rat hepatocytes) was studied using electron microscopy of platinum replicas. These cells in culture were firmly connected with each other and formed multicellular islands. Pseudopodial activity was observed only at the free edges of marginal cells of the islands. Cytoskeleton in the vicinity of such active edges included several structurally different zones. The most peripheral zone contained dense actin meshwork. More inner "sparse" zone contained loose actin filament network. Next zone in the same direction was the lamella proper. It contained individual microfilaments and their bundles or meshwork patches. Microtubules and intermediate filaments were also present in the lamella proper. The characteristic feature of the central (endoplasmic) region of the marginal cells of the islands was the presence of the submembranous microfilament sheath. Microfilaments in the sheath were densely packed. Individual fibers were visible along a significant distance. The inner cells in the epithelial islands had no zonal organization of the cytoskeleton. The endoplasmic microfilament sheath occupied the whole dorsal cell surface in these cells. Different epithelia studied here had some variations in the relative width of cytoskeletal zones. The organization of cytoskeleton in the epithelial cells has many features in common with that in fibroblasts. Possible mechanisms of establishment of the zonal cytoskeletal organization in both the cell types are discussed.     

Activation induces a rapid reorganization of spectrin in lymphocytes

Lymphocyte activation results in a rapid reorganization of the cytoskeletal protein spectrin. Immediately following an activation signal, there is fragmentation of a spectrin-rich cytoplasmic structure and subsequent translocation of the fragments to defined areas of the plasma membrane in both antigen-specific T cell hybridomas and lymph node T cells. These dramatic changes have been documented by light and electron microscopic immunolocalization and by immunoblot analysis of plasma membrane-enriched preparations. A T cell hybridoma variant lacking the spectrin-rich cytoplasmic structure of the parental line does not redistribute spectrin and produces little or no IL-2 in response to antigen-dependent activation. This suggests a functional link between spectrin distribution and activation potential. We propose that the cytoplasmic structure functions as an organizing center or reservoir for spectrin that is sensitive to signaling at the cell surface.     

Fibrinogen distribution on surfaces and in organelles of ADP stimulated human blood platelets

The fibrinogen distribution in platelet organelles after ADP-stimulation was investigated with anti-human fibrinogen using protein A-gold applied to serial sections. Fibrinogen was detected in the so-called alpha-granules of platelets and also in granule protrusions which were observed after ADP-stimulation. The ends of these protrusions were formed as coated membranes and the tips were often in apposition to the surface connected membranes or the plasmalemma. At such places fusion events and hence signs of an exocytosis could be demonstrated by means of cryofixation and cryosubstitution. Examination of serial sections revealed fibrinogen on all these granule profiles. Surface connected membranes, free surfaces and the characteristic structure of the contact zones of aggregated platelets were also labelled by gold particles but less than anticipated. On the platelet surfaces and surface connected membranes fibrinogen was rarely demonstrable with ferritin-labelled anti-human fibrinogen on washed or thrombin-stimulated, almost fibrinogen free platelets. After addition of human fibrinogen to the thrombin stimulated and disaggregated platelets a part of the platelets aggregated spontaneously and formed characteristic contact zones. Anti-human fibrinogen was observed on the free surfaces, in filamentous bridges between the contact spaces and in a tubular surface connected membrane system with involvement of coated membranes at the central ends of these structures. The results indicate the following: all alpha-granules contain fibrinogen; after ADP-stimulation secretion takes place with involvement of coated membranes; during aggregation fibrinogen binds to platelet surfaces and forms contact spaces; fibrinogen is taken up by the surface connected system with involvement of coated membranes.     

FINE STRUCTURE OF THE MYOEPITHELIUM OF THE ECCRINE SWEAT GLANDS OF MAN

The secretory coils of glutaraldehyde-osmium tetroxide-fixed and Epon-Araldite-embedded eccrine sweat glands from the palms of young men were studied with the electron microscope. The myoepithelial cells lie on the epithelial side of the basement membrane and abut other epithelial elements directly. The irregularly serrated base of the cell has dense thickenings along the plasma membrane which alternate with zones bearing pits; the smooth apical surface lacks dense thickenings, is studded with pits, and conjoined to secretory cells by occasional desmosomes. Masses of myofilaments, 50 A in diameter, fill most of the cell and are associated with irregular dense zones. In cross-section the arrangement of the myofilaments seems identical with that of the I band of striated muscle, and the dense zone has typical Z band structure. A few microtubules and cytoplasmic cores bearing profiles of the endoplasmic reticulum, filamentous mitochondria, and glycogen granules penetrate the fibrillar masses and run parallel to the oriented myofilaments. In the perinuclear zone, Golgi membranes, rough- and smooth-surfaced elements of the endoplasmic reticulum, mitochondria, glycogen, microtubules, lipid, pigment, and dense granules are variable components in the cytoplasm. The interrelationships of the myoepithelial cells with the secretory cells suggest that the former may act as regulators, controlling the flow of metabolites to the secretory epithelium.     

The chondrocyte cytoskeleton in mature articular cartilage: structure and distribution of actin, tubulin, and vimentin filaments.

We investigated the structure of the chondrocyte cytoskeleton in intact tissue sections of mature bovine articular cartilage using confocal fluorescence microscopy complemented by protein extraction and immunoblotting analysis. Actin microfilaments were present inside the cell membrane as a predominantly cortical structure. Vimentin and tubulin spanned the cytoplasm from cell to nuclear membrane, the vimentin network appearing finer compared to tubulin. These cytoskeletal structures were present in chondrocytes from all depth zones of the articular cartilage. However, staining intensity varied from zone to zone, usually showing more intense staining for the filament systems at the articular surface compared to the deeper zones. These results obtained on fluorescently labeled sections were also corroborated by protein contents extracted and observed by immunoblotting. The observed cytoskeletal structures are compatible with some of the proposed cellular functions of these systems and support possible microenvironmental regulation of the cytoskeleton, including that due to physical forces from load-bearing, which are known to vary through the depth layers of articular cartilage.     

The membrane skeleton

One important element that defines cell shape is the membrane skeleton. This filamentous network is closely apposed to the cytoplasmic face of the plasma membrane where it gives mechanical support to the membrane, provides specific attachment sites for cytoskeletal components and helps to organize some integral membrane proteins into domains. The membrane skeleton of erythrocytes has been studied extensively by biochemical and ultrastructural methods, but similar structures in other cell types are just beginning to be defined. In this review, David Pumplin and Robert Bloch draw attention to these nonerythroid skeletons and compare and contrast them with the erythrocyte model.     

Ultrastructure and cytochemistry study of the yolk syncytial layer in the alevin of trout (Salmo fario trutta L.) after hatching

After hatching, the yolk syncytial layer of Salmo fario trutta may be subdivided into two zones, namely, the vitellolysis zone (containing numerous yolk platelets), and the cytoplasmic zone (where yolk platelets are rare). In the vitellolysis zone, two stages in the utilization of the yolk are observed: 1) The first stage, comprises the formation of yolk platelets from coalescent yolk by spherical cutting out and basal scission. This process seems to be achieved by the invagination of fibrillar elements into the coalescent yolk to form individual yolk platelets surrounded by a limiting membrane. 2) The second stage essentially consists of the extrusion or budding of yolk matter from a yolk platelet. Again, where the yolk matter leaves a platelet, fibrillar elements are evident and show an alkaline phosphatase activity. The platelets of the vitellolysis zone have a homogeneous content and variable diameter; they never acquire a heterogeneous and polymorphic aspect which could be interpreted as an intermediate stage in their degradation.     

Cytoskeletal changes in mouse embryonic fibroblasts exposed to colcemid. Electron microscopic research on platinum replicas

Cytoskeletons of colcemid-treated mouse embryo fibroblasts were studied using platinum replica technique. In the control cells, cytoskeletal components were oriented along direction of cell polarization. Structure of the control cytoskeleton changed regularly from the cell active edge to its centre forming several zones. Distribution of microtubules by colcemid led to significant changes in the organization of actin cytoskeleton. Both orientation and zonal differentiation of cytoskeleton disappeared in colcemid-treated fibroblasts. Changes in the fine structure of microfilament sheath were most prominent. Control sheath was composed of stretched tightly packed microfilaments. Colcemid treatment transformed it into fine microfilament meshwork, normally characteristic only for ruffle zone. Alterations of the fine structure of focal contacts and ruffles were also observed in treated cells. The role of microtubules in the organization of intracellular tensions and in the distribution of sites of actin polymerization is discussed.     

Ultrastructural organization of vegetative hyphae of Tuber Albidum Pico

The ultrastructural organization of vegetative hyphae ofTuber albidum, an ectomycorrhizal fungus is described. Three zones are recognized: 1) an apical zone with cytoplasmic vesicles; 2) a subapical zone with protoplasmic organelles; 3) a vacuolated zone. This organization corresponds in its main features to the pattern just described for rapidly growing fungi.     

Patterning of the membrane cytoskeleton by the extracellular matrix

The extracellular matrices of different tissues contain components which affect the migration, morphology and differentiation of many types of cells. These forms of cell behavior often involve dramatic changes in cytoskeletal organization. Extracellular matrix components are recognized by specific cell surface receptors which span the membrane and interact with the actin cytoskeleton. In cultured cells, the matrix receptors are concentrated in sites of cell attachment called focal adhesions. Information that is conveyed from the extracellular matrix to the cytoskeleton may involve matrix components, cell surface receptors, as well as the proteins at the cytoplasmic face of the focal adhesion which link the receptors to the actin cytoskeleton.