Plasmalemmal Undercoat Research Articles

The mechanical properties of a cell, as determined by its actin cytoskeleton, are important for nanoneedle insertion into a living cell

Previously, we reported that a nanoneedle of 200 nm diameter manipulated by an atomic force microscope apparatus could be inserted into a living cell. The insertion probabilities varied according to cell type. However, the nanoneedle was never successfully inserted into artificial liposomes. In the current study, we found that the stress fibers and actin filaments comprising the plasmalemmal undercoat are important, determining factors as to whether a nanoneedle can be successfully inserted into a cell. Depolymerization of microtubules increased both the number of stress fibers and insertion efficiency in NRK cells. These results indicate that the insertion efficiency of a nanoneedle (200 nm in diameter) into a cell with a smaller actin meshwork in its plasmalemmal undercoat is enhanced and the formation of stress fibers obviously contributes to this incremental enhancement. These facts are not only important as technical information to improve the efficiency of cell manipulation but also as observations of the mechanical properties of the native cell cortex.

Ultrastructural observations of astrocyte end-feet in the rat central nervous system.

Astrocytic end-feet in the rat CNS were studied by thin section electron microscopy. Astrocyte processes that enclose neuronal elements extended to blood vessels and the pia mater, where the processes expanded to form end-feet or glial limiting membranes. At the end-feet, cell junctions such as gap junctions and desmosome-like junctions were formed between the astrocyte processes. The end-foot plasma membrane facing the basal lamina was undercoated with electron-dense, layered materials, with an internal substructure of filamentous networks, with which bundles of glial filaments (GFs) appeared to be closely associated via fine filamentous structures, often showing a hemi-desmosome-like appearance. In specimens treated with Triton X-100, the internal substructure of the undercoat was better visualized and the association with GFs was well preserved. At the end-feet, some unique tubular structures were found in spatial relationship to the plasmalemmal undercoat. Plectin visualized by immunofluorescence was localized to astrocytes and their processes, especially at the end-feet facing the pia mater. Immunoelectron microscopy located plectin on fine filamentous structures lying between GFs and the plasmalemmal undercoat. These observations suggest that plasmalemmal undercoats at the astrocyte end feet may serve as attachment sites of GFs to the plasma membrane and that plectin may be involved in such attachment.

Spectrin Expression in Thymus of Embryonic and Young Chickens.

Specrtrin, as a plasmalemmal undercoat protein in cytoskeleton, was observed immunohistochemically in thymus of embryonic and young chickens. Spectrin was negligible in thymic lymphocytes until middle stage of embryo and was then detected later stage of embryonic development. However, thymic lymphocytes expressed the spectirn weakly and the cells staining heavily with ant-spectrin could not find to be dominant before hatching. In young chickens, an apparent increase was not seen in the number of cells stained heavily with anti-spectrin. Ultrastructurally, cytoplasmic spectrins, together with the spectrin localization coinciding with plasma membrane, were detected in aggregated or diffusive form in embryonic lymphocytes. New type of spectrin localization was not seen in young chickens. These results may reflect the spectrin heterogeneity in thymic lymphocytes. In these spectrin-positive-cells (SPC), changes of spectrin characteristics, such as staining intensity or ultrastructural localization could not seem to parallel with acquisition of maturiy of thymic lymphocytes.

表皮ケラチノサイトの細胞接着分子と膜裏打ち蛋白 （１） 細胞接着構造と構成蛋白

表皮ケラチノサイトの細胞接着分子と膜裏打ち蛋白 （１） 細胞接着構造と構成蛋白

Integrins and adhesion molecules: Tumour necrosis factor-α-mediated dyscohesion of epithelial cells is associated with disordered expression of cadherin/β-catenin and disassembly of actin filaments

Tumour necrosis factor (TNF)-alpha induced, in a time- and dose-dependent fashion, dyscohesion (cell-cell dissociation) of the endometrial epithelial cells. TNF-alpha impaired the ability of cells to aggregate and to attain compaction. The cell-cell adherent junction is a specialized region of the plasma membrane where cadherin molecules act as adhesion molecules and actin filaments are densely associated with the plasma membrane through a well-developed plasmalemmal undercoat. Dyscohesion induced by TNF-alpha was associated with the disordered expression of cadherin/beta-catenin at the sites of cell-cell contact. In addition, within the time-frame that dyscohesion was induced, TNF-alpha down-regulated the expression of actin mRNA only at 100 ng/ml without modulating the overall amount of actin protein, its beta-isoform or the amount of ribosylated actin. However, TNF-alpha-mediated dyscohesion of epithelial cells was associated with loss of plasmalemmal undercoat as well as intracytoplasmic aggregates of F-actin and a simultaneous increase in G-actin. The effect of cytochalasin-B, which disrupts actin filaments on cell-cell binding, was less pronounced than the effect of TNF-alpha, suggesting that the effect of this cytokine on dyscohesion is not solely dependent on the disassembly of actin filaments. These findings show that the induction of disordered expression of adhesion molecules, as well as disassembly of actin filaments, are implicated in the dyscohesion induced by TNF-alpha.

Molecular Organization and Functional Regulation of Cell to Cell Junctions in the Endothelium

Intercellular junctions are important structural determinants of endothelial permeability. These organelles are formed by a complex network of transmembrane proteins linked to a well developed plasmalemmal undercoat. One of the typical characteristics of endothelial junctions is their dynamic organization. Endothelial cells are able to rapidly change the architecture of the junctions to allow the passage of plasma constituents and circulating cells. This effect, in most of the cases, is quickly reversible and the endothelium is able to disorganize/reorganize the intercellular junctions within minutes. The mechanisms that regulate the opening and the closure of endothelial junctions are still obscure. It is conceivable that inflammatory agents increase permeability by binding to specific receptors generating intracellular signals which in turn cause cytoskeletal reorganization and opening of interendothelial gaps. Endothelial junctions also control leukocyte extravasation. Once leukocytes have adhered to the endothelium, a coordinated opening of interendothelial clefts occurs. The mechanism by which this takes place is unknown, but it might present characteristics similar to that triggered by soluble mediators.

Some ultrastructural observations on the mode of association of microtubules with the plasmalemma in epidermal tendon cells of the river crab

Summary— The ultrastructural aspects of the association of microtubules (MTs) with the plasmalemma in epidermal tendon cells of the river crab, Polamon dehaani, were studied by thin‐section electron microscopy combined with detergent treatment. In the tendon cell, MTs were linked laterally by anchoring filaments to the plasmalemma via a submembranous electron‐dense layer called the plasmalemmal undercoat. To further clarify how such anchoring filaments are spatially related to the plasmalemma through the undercoat, we carefully examined and compared thin‐section images obtained from various specimen preparations using saponin and Triton X‐100. When the tissues were treated with saponin or Triton, electron‐dense materials in the undercoat were extracted in varying degrees to expose internal substructures. The undercoat appeared to show a two‐layer organization, the inner and outer layers. In more extracted samples, filamentous networks became prominent in the outer layer. Anchoring filaments were seen to attach to such filamentous networks, which in turn were linked to the plasmalemma proper. Thus, it may be reasonable to consider that the filamentous network constitutes the core structure of the plasmalemmal undercoat which is structurally reinforced by extractable electron‐dense materials.

Molecular linkage between cadherins and actin filaments in cell—cell adherens junctions

The cell—cell adherens junction is a site for cadherin-mediated cell adhesion where actin filaments are densely associated with the plasma membrane through its well-developed plasmalemmal undercoat. Recent research has focused on the molecular linkage between cadherins and actin filaments in the undercoat of adherens junctions in order to understand the functions of these undercoat-constitutive proteins in the regulation and signal transduction of cadherin-based cell adhesion.

The association of microtubules with the plasmalemma in epidermal tendon cells of the river crab

The mode of association of microtubules (MTs) with the plasmalemma in epidermal tendon cells of the river crab, Potamon dehaani was studied by thin-section electron microscopy. In the leg muscle, the tendon cells connect striated muscle cells with the cuticle, forming specialized junctions at both ends. At the muscle-tendon cell junction, the apposed plasmalemmas are interdigitated in a zig-zag pattern separated by a uniform space of about 50 nm, where the basal lamina is shared by two cells. At the tendon cell-cuticle junction, the plasmalemma of the tendon cell forms many conical invaginations, into which dense fibrous material extends from the cuticle. Inside the tendon cell, numerous microtubules run parallel to the direction of tension transmission and are arranged into parallel bundles of various sizes. Within such bundles, fine filamentous structures cross-link adjacent MTs. MTs span the entire length of the cell and attach at their both ends to the junctional domains of the plasmalemma. The junctional plasmalemma is characterized by formation of an electron-dense undercoat, through which MTs are connected with the plasmalemma proper. The ultrastructural features of MT association with the plasmalemma are basically the same at both junctions. At the junctions, MTs usually terminate with free ends and are linked laterally to the plasmalemmal undercoat with fine filamentous structures. These observations emphasize the role of the plasmalemmal undercoat as a device of the attachment of MTs to the plasmalemma.

Co-distribution of annexin VI and actin in secretory ameloblasts and odontoblasts of rat incisor

Annexin VI and actin were detected by immunoblot analysis in the enamel- and dentin-related portions of dental tissues. Annexin VI was found mainly in the particulate fraction whereas actin was detected in both the soluble and particulate fractions. By immunoelectron microscopy, annexin VI antibodies conjugated with colloidal gold were seen to label the mitochondria, the cytosol and the nucleus of secretory ameloblasts and odontoblasts of rat incisor. In the processes of these cells, the plasmalemmal undercoat was labeled. Anti-actin antibodies labeled the desmosome-like junctions, the cytosol, and the mitochondria of the cell bodies. Extensive labeling was seen at the periphery of the Tomes' processes and odontoblast processes. These results suggest that annexin VI may play a role in Ca2(+)-regulation in the cell bodies, especially as a calcium receptor protein in the mitochondria. Moreover, annexin VI and actin seem to be co-distributed in secretory processes. Thus, these proteins might be both involved in exocytotic and endocytotic events.

Cytology and organization of reactive astroglia in human cerebellar cortex with severe loss of granule cells: A study on the ataxic form of creutzfeldt-jakob disease

In order to investigate the cellular basis of human astrogliosis, we have selected the cerebellar cortex because it provides a relatively simple and geometrical organization of both neuronal and glial populations. A pathological system with severe and progressive loss of granule cells was studied: the ataxic form of Creutzfeldt-Jakob disease, where the tissue geometry is minimally disturbed. The quantitative study revealed a drastic reduction in the numerical density of granule cells in the Creutzfeldt-Jakob disease cerebellum, and a significant increase in the numerical density of astrocytes. Karyometric analysis showed that the nuclear area was significantly greater in reactive astroglial cells than in normal astroglia. Glial fibrillary acidic protein immunocytochemistry revealed astroglial hypertrophy, but the geometry and spatial domains of astroglial subtypes were strictly preserved. Vimentin expression was detected in Bergmann glia and in certain astrocytes of the granular layer. Ultrastructural analysis showed that reactive astroglia had large nuclei, with expanded interchromatinic regions which contained clusters of interchromatin granules and nuclear bodies, and prominent reticulate nucleoli. In the cytoplasm, hypertrophied bundles of intermediate filaments were observed, some of them associated with the nuclear envelope. Numerous adhering and gap junctions were also found among reactive astroglial cells. Perivascular glial processes showed a terminal web of intermediate filaments and a conspicuous plasmalemmal undercoat. Interendothelial tight junctions were preserved. Our results suggest that the severe loss of granule cells induces a highly ordered astroglial response which tends to preserve the geometry of the astroglial scaffold, the domains of each astroglial subtype, the neuronal microenvironmental conditions and the efficiency of the blood-brain barrier, in order to promote neuron survival.

Cytoskeletal regulation of normal rat glioblasts differentiated by glia maturation factor

The morphological differentiation of normal rat glioblasts induced by glia maturation factor (GMF) was investigated with regard to the reorganization of the cytoskeletal structures by the use of indirect immunofluorescence staining methods. The microfilaments were reorganized from a stress fiber form to a plasmalemmal undercoat structure by GMF, and to a network-like structure by dBcAMP. The GMF- or dBcAMP-induced morphological differentiation was enhanced by cytochalasin D (over 2 ?M) and conversely inhibited by colchicine (over 1 ?M). Phalloidin, an F-actin stabilizer, inhibited more strongly the morphological differentiation induced by dBcAMP than that induced by GMF. Both glia filaments and microtubules existing as a network in control glioblasts assembled after GMF or dBcAMP stimulation to form thick filamentous structures, not only in the cell bodies but also in the processes. The bundle formation of glia filaments was inhibited by colchicine. These findings suggest that glial differentiation depends on both stress fiber disintegration and microtubule assembly, which are closely related to the bundle formation of glia filaments. The GMF- or dBcAMP-induced differentiation was promoted by 25 ?M W-7 and inhibited by 1 mM CaCl(2) plus 4 ?M A23187. At a higher concentration (50?M) W-7 promoted the differentiation even in the presence of CaCl(2) and A23187 at the same concentrations. These findings suggest that the suppression of the calmodulin function is important for morphological differentiation of glioblasts.

Growth inhibition by sialosyl cholesterol of rat glioma cells

Synthesized sialosyl cholesterol (?-sialosyl cholesterol) induced growth inhibition of rat astrocytoma cells (GA-1) or schwannoma cells (354-A) which was detected as a decline of cell growth curves and a suppression of DNA syntheses. The incubation time to initiate growth inhibition of GA-1 was as short as 10 min and that to complete the action was 2 h after the initiation of drug-stimulation, indicating an immediate incorporation of sialosyl cholesterol into glioma cells. The morphological alteration of 354-A, but not GA-1, from an epithelioid cell shape to a spindle-like shape was also detected by the stimulation and ascertained by fluorescence visualization of cytoskeletons: the reorganization of actin filaments distributing as a plasmalemmal undercoat to form stress actin fibers and no detectable change in distribution of tubulins. The results provide a possibility that sialosyl cholesterol incorporated into the plasma membrane may cause growth inhibition of both glioma cells and morphological differentiation of schwannoma cells mostly through alterations of membrane characteristics and/or cytoskeletal anchorage, and also suggest the therapeutic potential of sialosyl cholesterol for brain tumors.

Cytoskeleton in the apical region of mouse taste bud cells.

The cytoskeletal structures in the apical region of mouse taste bud cells were examined by immunocytochemistry and electron microscopy. Immunostaining for actin showed positive reactions in the apical portions of the taste buds. These regions contained bundles of longitudinally oriented filaments (5-7 nm in diameter) extending from the tip of microvilli to the apical cytoplasm of type I, II, and III cells. After incubation with heavy meromyosin, arrowhead formation was observed along these filaments, thus indicating these filaments to be composed of actin. The plasmalemmal undercoat, which was composed of vertical and horizontal layers, was observed on the zonula occludens. It is supposed that this undercoat gives the structural support for the lateral membrane of the apical region in the taste bud cells.

Plasmalemmal undercoat: The cytoskeleton supporting the plasmalemma.

The plasmalemmal undercoat can be defined as the electron-dense material of layered organization closely applied to the cytoplasmic surface of the plasma membrane (plasmalemma) as revealed by thin-section electron microscopy. Though the structures which fulfill this criterion occur widely, most of them have not received the attention they may deserve. The undercoat is a special form of the cytoskeleton-membrane interaction, though it constitutes a part of the cytoskeleton. The significance of the plasmalemmal undercoat may be primarily to provide a structural support for the membrane. With this support, cells can perform many important functions on their limited or whole surfaces. The undercoat may provide a mechanical support to the plasmalemma so that the membrane may acquire rigidity, strength and elasticity. Through association with the membrane, the undercoat may regulate the distribution of integral membrane proteins to form and maintain various functional domains on the plasmalemma. The undercoat may further provide attachment sites for cytoskeletal fibrous components such as actin filaments, intermediate filaments and microtubules.

マウス頭蓋骨内側表面の細胞の細胞内微細構造の走査型電子顕微鏡による観察の試み

The cytoskeleton and plasmalemmal undercoat of bone-surface cells, especially of osteoclasts, have been studied in detail mainly by fluorescence microscopy and transmission electron microscopy, but its three-dimensional organization and its changes under various hormonal or pathological conditions have not fully been understood. To observe the three-dimensional organization of intracellular structures of the bone-surface cells by high resolution scanning electron microscopy (SEM), we used a new method of tissue-preparation for SEM. Parietal bones of the neonatal mice were removed and the inner-surface cells of those were mechanically raptured. Then, the free surface plasmalemma of the cells was detached by adhering it to the polylysine-coated glass. These bones were fixed, dehydrated, and dried by the critical point drying method. In the cells which had morphological characteristics of osteoblasts, filamentous networks of cytoskeleton were observed. These filaments were about 10nm or less in diameter and, thus, were thought to be actin filaments and intermediate filaments. Three-dimensional organization of filamentous networks was more obvious in stereopair micrographs. Membranous organelles to which the filaments attached were also observed.

Microfilament-undercoated plasmalemmal infoldings in a human clear-cell sarcoma

Unusual structures often found in the cytoplasm of tumor cells in a clear-cell sarcoma appeared as multilayered, concentric, oval, spiral, parallel arrays of cisternae in various planes of section. It was demonstrated that the cisternal membrane and cavity were continuous with plasmalemmas of tumor cells and the extracellular space, respectively, suggesting that the structures were formed by the intracytoplasmic infoldings of plasmalemmas. Another characteristic found in the structures was orderly microfilaments with an average diameter of 6.5 nm which were placed between the confronting plasmalemmas in the infoldings. The filaments which underlay the infolded plasmalemmas ran parallel to each other along the cytoplasmic surfaces of plasmalemmas approximately 15 nm apart. The regularly arranged filaments were found in the infolded plasmalemmas, but not beneath any other area of plasmalemmas. The short ends of long filaments appeared to bend toward the inner surfaces of plasmalemma and to be directly connected with the surface proper. These results show that the filaments may be closely associated with the plasmalemmal infoldings and included as the same category of plasmalemmal undercoat. Additionally, the biological significance of the structures is discussed.

The extrarhabdomeral cytoskeleton in photoreceptors of Diptera. II. Plasmalemmal undercoats

The plasmalemmal undercoats of those regions of the photoreceptors of the blowfly Lucilia that flank the central extracellular space of each ommatidium are described from en face and transverse thin sections. Labile structures were stabilized before fixation for electron microscopy by using an inhibitor of thiol proteases, Ep-475, as described in the previous paper (Blest et al., Proc. R. Soc. Lond . B 220, 339-352, 1984). Membranes of R 1-6 are underlain by a closely associated, randomly organized filamentous meshwork. That of the basal region of R 7 is highly organized, and consists of very long, about 8 nm filaments running parallel to each other and to the longitudinal ommatidial axis; these ‘backbone’ filaments are tightly adherent to the plasma membrane, and are spaced some 190-200 nm apart. They are linked by abundant transverse filaments that form a reticulum between them. The degree of ordering of the reticulum in life is not clear, but some well-preserved profiles suggest that it may be high. Replicas obtained by the freeze-fracture technique show that extrarhabdomeral membranes have dense populations of intramembrane particles, just as they do in Drosophila where a genetic analysis has shown them to consist largely of rhodopsin. It is proposed as a working hypothesis that these planar membranes can be regarded as flat equivalents of the microvillar membranes, that some fraction of the integral membrane proteins may be immobilized by bonding to the plasmalemmal undercoat, and that the latter may help to constrain both the translational and rotational movements of rhodopsin molecules.