Attachment Of Filaments Research Articles

Electron-microscopic study of the apical region of the toad bladder epithelial cell.

Antidiuretic hormone (ADH) promotes fusion of cytoplasmic tubules with the luminal membrane and delivery of particles from the tubules to the membrane. The particles are believed to be the water-conducting elements in the membrane. We have employed several scanning (SEM) and transmission electron-microscopic (TEM) techniques to study the relationship of the cytoplasmic tubules to the luminal membrane and to the apical cytoskeleton of the toad bladder epithelial cell. This paper reports the results of freeze-crack SEM and tannic acid-fixed TEM studies, as well as studies with a resinless method of embedding. Freeze-cracked epithelial cells reveal that the tubules are anchored in a matrix of cytoskeleton and granules just below the luminal membrane, and many, if not all, retain their anchorage to the matrix after ADH-induced fusion. Tannic acid-fixed specimens show that the tubules in unstimulated cells lie horizontally. Fusion appears to involve an angulation of the tubules, and this may be the major mode of ADH-induced tubule movement. There are suggestions in the tannic acid sections of filamentous attachments of tubules to the surrounding cytoskeleton. In addition there are prominent microfilament bundles running down the microvilli and a dense concentration of filaments just below the luminal membrane. The presence of these filaments is confirmed in the resinless sections, and their possible role in ADH action is discussed.

The microvillus 110K cytoskeletal protein is an integral membrane protein

A protein of 110,000 MW connects actin filaments to the plasma membrane in microvilli of intestinal epithelial cells. In the present study four independent lines of evidence suggest that the 110K protein is directly bound to the lipid bilayer. The solubilization of the 110K protein requires detergents and removal of detergent after solubilization results in aggregation. The 110K protein partitions into the detergent phase in Triton X-114 solutions. It is selectively incorporated into liposomes. It is specifically labeled with the hydrophobic probe 14C-phenylisothiocyanate. In addition we present a purification scheme for the 110K protein in milligram amounts. This represents the simplest system of membrane to filament attachment, in which an integral membrane protein is also a cytoskeletal protein.

Attachment of vimentin filaments to desmosomal plaques in human meningiomal cells and arachnoidal tissue.

Desmosomal proteins are co-expressed with intermediate-sized filaments (IF) of the cytokeratin type in epithelial cells, and these IF are firmly attached to the desmosomal plaque. In meningiomal and certain arachnoidal cells, however, vimentin IF are attached to desmosomal plaques. Meningiomas obtained after surgery, arachnoid "membranes", and arachnoid granulations at autopsy, as well as meningiomal cells grown in short-term culture have been examined by single and double immunofluorescence and immunoelectron microscopy using antibodies to desmoplakins, vimentin, cytokeratins, glial filament protein, neurofilament protein, and procollagen. In addition, two-dimensional gel electrophoresis of the cytoskeletal proteins has been performed. Using all of these techniques, vimentin was the only IF protein that was detected in significant amounts. The junctions morphologically resembling desmosomes of epithelial cells have been identified as true desmosomes by antibodies specific for desmoplakins and they provided the membrane attachment sites for the vimentin IF. These findings show that anchorage of IF to the cell surface at desmosomal plaques is not restricted to cytokeratin IF as in epithelial cells and desmin IF as in cardiac myocytes, suggesting that binding to desmosomes and hemidesmosomes is a more common feature of IF organization. The co-expression of desmosomal proteins and IF of the vimentin type only defines a new class of cell ("desmofibrocyte") and may also provide an important histodiagnostic criterion.

Specific attachment of desmin filaments to desmosomal plaques in cardiac myocytes.

Intercellular junctions which are similar in ultrastructure and protein composition to typical desmosomes have so far only been found in epithelial cells and in heart tissue, specifically in the intercalated disks of cardiac myocytes and at cell boundaries between Purkinje fiber cells. In epithelial cells the cytoplasmic side of desmosomes, the 'desmosomal plaque', represents a specific attachment structure for the anchorage of intermediate filaments (IF) of the cytokeratin type. Cardiac myocytes do not contain cytokeratin filaments. In primary cultures of rat cardiac myocytes, we have examined by immunofluorescence and electron microscopy, using single and double label techniques, whether other types of IF are attached to the desmosomal plaques of the heart. Antibodies to desmoplakin, the major protein of the desmosomal plaque, have been used to label specifically the desmosomal plaques. It is shown that the desmoplakin-containing structures are often associated with IF stained by antibodies to desmin, i.e., the characteristic type of IF present in these cells. Like cytokeratin filaments in epithelial cells, desmin filaments attach laterally to the desmosomal plaque. They also remain attached to these plaques after endocytotic internalization of desmosomal domains by treatment of the cells with EGTA. These desmin filaments do not appear to attach to junctions of the fascia adherens type and to nexuses (gap junctions). These observations show that anchorage at desmosomal plaques is not restricted to IF of the cytokeratin type and that IF composed of either cytokeratin or desmin, specifically attach, in a lateral fashion, to desmoplakin-containing regions of the plasma membrane. We conclude that special domains exist in these two IF proteins that are involved in binding to the desmosomal plaque.

Proteins involved in the attachment of actin to the plasma membrane.

Proteins that may be involved in two types of actin-membrane association are discussed. The first set includes alpha-actinin, vinculin, fimbrin and a new cytoskeletal protein that are all concentrated in adhesion plaques, those regions of cultured fibroblasts where bundles of actin microfilaments terminate and where the plasma membrane comes close to the underlying substrate. The properties of non-muscle alpha-actinin suggest that it functions to cross-link actin filaments and thereby stabilize microfilament bundles rather than functioning in their attachment to the membrane. Fimbrin also appears to be involved in bundling of filaments rather than in attachment. In contrast, vinculin binds to the ends of actin filaments in vitro and is probably the best candidate for a role in the attachment of actin to membranes at the adhesion plaque. The discovery of a new protein, 215k, of unknown function, in the adhesion plaque suggests that many more proteins remain to be identified in this region. Attachment of actin filaments to other regions of the plasma membrane is also considered and a protein is described that seems to be a spectrin in brain and other tissues. The brain protein resembles erythrocyte spectrin in its physical properties, in binding actin, in being associated with cell membranes and in cross-reacting immunologically. We suggest that the brain protein and erythrocyte spectrin both belong to a family of related proteins (the spectrins) which function in the attachment of actin to membranes in many different cell types.

An ultrastructural study of calcium induced degenerative changes in dissociated heart cells

Dissociated myocytes were prepared by enzymatic perfusion of adult rabbit hearts, and fixed following exposure to various concentrations of Ca 2+. Only slight ultrastructural changes were noted in myocytes which were maintained in relaxation [(Ca 2+) < 0.1 μ m] or in cyclic contractile activity [(Ca 2+) ≅ 0.4 μ m]. In these cells, slight swelling permitted clear demonstration of interesting ultrastructural details such as tight connections between Z bands and sarcolemma, attachment of filaments to these structures, and relationships of sarcoplasmic reticulum and T tubules. When the Ca 2+ concentration was raised above 1.0 μ m, sarcomeres exhibited extreme and irreversible shortening, followed by myofilament disorganization, disappearance of Z lines, aggregation of most thick and some thin filaments forming a central mass, and outward displacement of organelles. Under these conditions numerous filaments were attached randomly to the sarcolemma and to intracellular membranes, demonstrating their inherent capacity to bind to membranous structures. The ultrastructural alterations observed in our experiments are similar to those described for the “calcium paradox”, which is likely to be a common pathogenic mechanism of myocardial cell damage.

Membrane structure of nonactivated and activated human blood platelets as revealed by freeze-fracture: evidence for particle redistribution during platelet contraction.

The distribution of intramembrane particles of nonactivated and activated human blood platelets was studied by freeze-fracture under various experimental conditions to see whether morphological evidence for a structural coupling between the platelet actomyosin system and the fibrin network in a retracting clot could be established. Membrane particles were evenly distributed in nonactivated platelets; the total number (E + P faces) was approximately 1,500/micrometers 2 of membrane, and there were two to three times more particles present on the E face than on the P face. Transformation of discoid platelets to "spiny spheres" by cooling did not change the particle distribution. Platelet activation and aggregation by serum or ADP caused no change in membrane particle density or distribution. Particle distribution was not changed in Ca2+-activated platelets fixed immediately before fibrin formation, but after fibrin formation and during clot retraction, particles were sometimes most frequent on the P face and tended to form distinct clusters, and aggregates of E face pits were observed. Blood platelets contain contractile proteins that are distinct as filaments in platelets in retracting clots. We suggest that the redistribution of particles seen in activated platelets during clot retraction reflects the esablishment of mechanical transmembrane links between the platelet actomyosin system and the fibrin net. The P-face particle clusters may represent sites of force transmission between actin filaments bonded to the inside of the membrane and the fibrin network at the outside. Thus, whereas membrane particles may not be directly involved in the attachment of actin filaments to membranes, the transmission of the force of the contractile system to an exterior substrate apparently involves the intramembrane particles.

Microinjection of Fluorescently Labeled 130K Protein into Living Fibroblasts: Localization at the Ends of Actin Microfilament Bundles and in Close Relation to Fibronectin

Understanding the interactions between cell surface components and the underlying cytoskeleton continues to be a major goal in cell biology. In particular, little is known about the molecules involved in linking actin filaments to the plasma membrane in cells such as fibroblasts. A protein with a molecular weight of 130,000 (the 130K protein) was recently purified from smooth muscle by Geiger and localized to the ends of microfilament bundles in a variety of cell types. Independently we had purified this protein and were studying its possible interactions with other structural proteins when we learned of its interesting location, one that would be consistent with some role in the attachment of actin filaments to the plasma membrane. We were prompted to investigate this interesting protein further by microinjection of the f1uorescent1y labeled protein into living cells, a technique which had recently been successfully applied to the study of α-actinin.

The preparation of a cardiac plasma-membrane fraction containing intercalated discs [proceedings

Conference Article| June 01 1980 The preparation of a cardiac plasma-membrane fraction containing intercalated discs CAMILO A. L. S. COLACO; CAMILO A. L. S. COLACO 1National Institute for Medical Research, Mill Hill, London NW7 1AA, U.K. Search for other works by this author on: This Site PubMed Google Scholar W. HOWARD EVANS W. HOWARD EVANS 1National Institute for Medical Research, Mill Hill, London NW7 1AA, U.K. Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1980) 8 (3): 328. https://doi.org/10.1042/bst0080328 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Twitter LinkedIn Cite Icon Cite Get Permissions Citation CAMILO A. L. S. COLACO, W. HOWARD EVANS; The preparation of a cardiac plasma-membrane fraction containing intercalated discs. Biochem Soc Trans 1 June 1980; 8 (3): 328. doi: https://doi.org/10.1042/bst0080328 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search nav search search input Search input auto suggest search filter All ContentAll JournalsBiochemical Society Transactions Search Advanced Search This content is only available as a PDF. © 1980 Biochemical Society1980 Article PDF first page preview Close Modal You do not currently have access to this content.

Subaxolemmal filamentous network in the giant nerve fiber of the squid (Loligo pealei L.) and its possible role in excitability.

A new technique utilizing the squid giant nerve fiber has been developed which permits direct examination of the inner face of the axolemma by scanning electron microscopy. The axoplasm was removed sequentially in a 15-mm long segment of the fiber by intracellular perfusion with a solution of KF, KCl, Ca++-containing seawater, or with pronase. The action potential of the fibers was monitored during these treatments. After brief prefixation in 1% paraformaldehyde and 1% glutaraldehyde, the perfused segment was opened by a lne could be related to information on the detailed morphology of the cytoplasmic face of the axolemma and the ectoplasm. The results obtained by scanning electron microscopy were further substantiated by transmission electron microscopy of thin sections. In addition, living axons were studied with polarized light during axoplasm removal, and the identification of actin by heavy meromyosin labeling and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis was accomplished. These observations demonstrate that a three-dimensional network of interwoven filaments, consisting partly of an actinlike protein, is firmly attached to the axolemma. The axoplasmic face of fibers in which the filaments have been removed partially after perfusion with pronase displays smooth membranous blebs and large profiles which sppose the axolemma. In fibers where the excitability has been suppressed by pronase perfusion, approximately one-third of the inner face of the axolemma in the perfusion zone is free of filaments. It is hypothesized that the attachment of axoplasm filaments to the axolemma may have a role in the maintenance of the normal morphology of the axolemma, and, thus, in some aspect of excitability.

Actin filament-membrane attachment: are membrane particles involved?

The association of actin filaments with membranes is an important feature in the motility of nonmuscle cells. We investigated the role of membrane particles in the attachment of actin filaments to membranes in those systems in which the attachment site can be identified. Freeze fractures through the end-on attachment site of the acrosomal filament bundles in Mytilus (mussel) and Limulus (horseshoe crab) sperm and the attachment site of the microvillar filament bundles in the brush border of intestinal epithelial cells were examined. There are no particles on the P face of the membrane at these sites in the sperm systems and generally none at these sites in microvilli. In microvilli, the actin filaments are also attached along their lengths to the membrane by bridges. When the isolated brush border is incubated in high concentrations of Mg++ (15 mM), the actin filaments form paracrystals and, as a result, the bridges are in register (330 A period). Under these conditions, alignment of the particles on the P face of the membrane into circumferential bands also occurs. However, these bands are generally separated by 800-900 A, indicating that all the bridges cannot be directly attached to membrane particles. Thus membrane particles are not directly involved in the attachment of actin filaments to membranes.

Organization of an actin filament-membrane complex. Filament polarity and membrane attachment in the microvilli of intestinal epithelial cells.

The association of actin filaments with membranes is now recognized as an important parameter in the motility of nonmuscle cells. We have investigated the organization of one of the most extensive and highly ordered actin filament-membrane complexes in nature, the brush border of intestinal epithelial cells. Through the analysis of isolated, demembranated brush borders decorated with the myosin subfragment, S1, we have determined that all the microvillar actin filaments have the same polarity. The S1 arrowhead complexes point away from the site of attachment of actin filaments at the apical tip of the microvillar membrane. In addition to the end-on attachment of actin filaments at the tip of the microvillus, these filaments are also connected to the plasma membrane all along their lengths by periodic (33 nm) cross bridges. These bridges were best observed in isolated brush borders incubated in high concentrations of Mg++. Their visibility is attributed to the induction of actin paracrystals in the filament bundles of the microvilli. Finally, we present evidence for the presence of myosinlike filaments in the terminal web region of the brush border. A model for the functional organization of actin and myosin in the brush border is presented.

Alpha-actinin: immunofluorescent localization of a muscle structural protein in nonmuscle cells.

Antibodies specific for the skeletal muscle structural protein alpha-actinin are used to localize this protein by indirect immunofluorescence in nonmuscle cells. In cultured nonmuscle cells, alpha-actinin is localized along or between actin filament bundles producing an almost regular periodicity. The protein is also detected in the form of fluorescent plaques at some ends of actin filament bundles, as well as in a filamentous form in some overlap areas of cells. In spreading rat embryo cells, alpha-actinin assumes a focal distribution which corresponds to the vertices of a highly regular actin filament network. The results suggest that alpha-actinin may be involved in the organization of actin filament bundles, in the attachment of actin filaments to the plasma membrane, and in the assembly of actin filaments in areas of cell to cell contact.

C-banding and non-homologous associations in Gyrllus argentinus.

A comparative study of C-banded mitotic and meiotic chromosomes of Gryllus argentinus (Gryllidae) is reported. Improved cytological procedures were followed to establish karyotypes and to detect C-segments. Twenty-eight autosomes plus a sexual system XX, XO were found. Terminal C-heterochromatin in both arms and paracentric segments were detected in most of the chromosomes of the complement. Microdensitometric tracings confirmed the distribution of C-banded segments. Manifold connections through condensed terminal chromomeres were observed at pachytene occurring between two or more bivalents during meiosis. These heterologous associations involved the whole karyotype. End-to-end pachytene associations reacted intensely to the C-procedure. C-segments detected at diakinesis allowed the measurement of the centromere indices of bivalents which resulted in a more precise identification of given chromosome pairs. The relationship of the presence of terminal heterochromatin in mitotic chromosomes, the end-to-end associations through C-segments, the attachment of pachytene filaments to the nuclear membrane and their molecular implications is discussed.

Cytoplasmic Organization in the Receptor Cells of the Crista Ampullaris

Receptor cells of the cristae in the vestibular labyrinth of the bullfrog, Rana catesbiana, show a high degree of morphological organization. Four specialized regions may be distinguished: the apical region, the supranuclear region, the paranuclear region, and the basilar region.The apical region includes a single kinocilium, approximately 40 stereocilia, and many small microvilli all projecting from the apical cell surface into the lumen of the ampulla. A cuticular plate, located at the base of the stereocilia, contains filamentous attachments of the stereocilia, and has the general appearance of a homogeneous aggregation of fine particles (Fig. 1). An accumulation of mitochondria is located within the cytoplasm basal to the cuticular plate.

Fine structure of the mastigont system in Tritrichomonas foetus (Riedmüller).

SYNOPSIS. Tritrichomonas foetus shares many fine‐structural features with the previously described genera of the subfamily Trichomonadinae. These include the arrangement and structure of the kinetosomes, of most rootlet filaments, including the sigmoid filaments of kinetosome #2, as well as those of the parabasal apparatus and of the pelta‐axostyle complex. On the other hand, this species, and presumably all other Tritrichomonas augusta‐type flagellates, differ from Trichomonadinae in certain important details. Among the features which T. foetus does not share with Trichomonadinae are the fine structure of the costa and of the undulating membrane, as well as several organelles not found in the latter subfamily. The costal base of Trichomonadinae is replaced in T. foetus and other Tritrichomonadinae by a comb‐like structure, extending between the costa and the infrakinetosomal body. The suprakinetosomal body, connected to kinetosome #2 in the region of attachment of the sigmoid filaments, and the infrakinetosomal body, which appears to contribute to the proximal marginal lamella, are organelles evidently restricted to Tritrichomonadinae. The undulating membrane consists of 2 parts. The proximal part is a fold‐like differentiation of the dorsal body surface, the dorsal part of which contains the proximal marginal lamella. The distal part of the undulating membrane, with no obvious physical connection to the fold, encloses the distal marginal lamella in its ventral, and the microtubules of the recurrent flagellum in its dorsal area. The organelle of T. foetus which by its size, certain structural characteristics, and relationship with the undulating membrane and some organelles, including the paracostal granules, is analogous to the costa of Trichomonadinae and of Trichomitopsis termopsidis (subfamily Tritrichomonadinae), conforms in the structure of its periodic cross‐striations to that of the parabasal filaments of the latter organisms; its origin corresponds closely to that of parabasal filament 2 of Trichomonadinae.

Ultrastructure of Treponema pallidum Nichols following lysis by physical and chemical methods. I. Envelope, wall, membrane and fibrils.

1. Normal cells of the Nichols non-pathogenic strain of Treponema pallidum were disrupted by one or a combination of physical and chemical procedures. Cells and cellular fragments were then observed in the electron microscope by means of three techniques: negative staining, thin sectioning, and freeze-etching. Changes in typical morphology were noted and individual organelles were isolated for detailed structural analysis. 2. The cell envelope, a triple-layered structure, was sensitive to trypsin, sodium deoxycholate, sodium lauryl sulfate, diethyl ether, hyaluronidase and a combination of lysozyme and rapid freeze-thawing. The envelope contained polygonal subunits that consisted of at least three components. 3. The cover of the protoplasmic cylinder was composed of two unit membranes interpreted to be the cell wall and cell membrane. The cell wall, which retained its spiral shape after removal of the cell envelope, was sensitive to lysozyme. The cell membrane was the site of attachment of the axial filaments. 4. A band of parallel fibrils, attached to the innermost layer of the cell membrane, wrapped around the protoplasmic cylinder in a spiral fashion. The fibrils, sensitive to trypsin, were released from the cell membrane by sodium lauryl sulfate

Attachment of muscle filaments to the outer membrane of the nuclear envelope.

Myofilaments of striated muscles can be recognized in the electron microscope to be in structural continuity with the outer membrane of the nuclear envelope. The very site of insertion of these myofilaments at the membrane surface frequently appears characterized by a dense basal knob of 85–135 A. It is hypothesized that this attachment of myofilaments to the nuclear membrane plays a role in mechanically transmitting the contraction of the fiber to the nucleus, thus bringing about the harmonica-like “folded” appearance of the nucleus which is known for the contracted states of striated, smooth and cardiac muscles.

The ultrastructure of chloroplast of a brown alga Sphacelaria sp. : II. Association between the chloroplast DNA and the photosynthetic lamellae

Further to the observation of the spatial relationship between the ring genophore and the internal membranes of Sphacelaria chloroplasts, the attachment of the DNA filaments and the photosynthetic lamellae can be demonstrated by both thin-sectioning and isolation techniques. DNA filaments released by osmotic shock are found attached to the membrane: The configurations of the filaments are either linear, single loop, double loop, or a combination of a loop and linear. The last two are interpreted as the replicated forms. It is concluded that chloroplast DNA in Sphacelaria exists as a number of replicons of either linear or loop forms intermingled within the genophore. It is logical to assume that the mechanism for the replication and segregation of these replicons is similar to that of bacterial DNA.

Contractility and the fibre systems of Stentor Coeruleus

ABSTRACT Observations of the microtubular ‘km’ system and filamentous ‘M’ systems in living specimens of Stentor coeruleus show that on contraction of the body both systems shorten, thicken and remain straight, but that on initial relaxation they behave differently: the km fibres begin to lengthen later than the M fibres, the latter being thrown into sinuous folds. Electron microscopy of specimens cooled before fixation appears to confirm this difference in behaviour in relaxing specimens. The M fibres of the stalk region are discrete and prominent bundles, but in the adoral half of the body they are extensively linked together by side branches. There are diffuse filamentous attachments between the M fibres and the kinetosomes. The vertical microtubular stacks which make up the km fibres are each linked with a pair of kinetosomes. Each stack contains about 21 microtubules grouped in a 2 + 19 pattern. Crossbridges are present between the microtubules of adjacent stacks. The number of stacks in each km fibre cross-section is smaller in extended specimens than in contracted ones, indicating that the stacks slide upon one another as the body changes its length.