Accumulation Of Microfilaments Research Articles

Polarized thyroid cells in monolayers cultured on collagen gel: their cytoskeleton organization, iodine uptake, and resting membrane potentials.

Porcine thyroid cells were cultured on collagen gel-coated cover glasses. They were reorganized into polarized monolayer cells; the basal cell membranes were in contact with the collagen gel, and the apical ones faced the culture medium. We studied cytoskeleton organization, resting membrane potentials, and iodine uptake of these cells. The quick-freezing and deep-etching replica method provided three-dimensional images of the cytoskeleton organization. Networks of microfilaments were observed under the apical cell membrane. In the deep cytoplasm and near the basal cell membranes, intermediate filaments predominated and were interlinked with the microfilaments. When the cells were cultured in the presence of TSH, TSH induced the formation of microvilli at the apical cell membranes and the accumulation of microfilaments under these membranes; in the deep cytoplasm, the intermediate filaments were more closely interlinked with the microfilaments. The microfilaments were immunostained with antiactin antibody. Thus, collagen is a factor in determining the cell polarity, and TSH further augments polarization through reorganizing the cytoskeletons. Electrophysiological study revealed that the resting membrane potential of cells cultured in the absence of TSH was -46 mV, and that of cells cultured in the presence of TSH was -58 mV. TSH hyperpolarized resting membrane potentials. These cells took up iodine. TSH in the medium augmented this uptake. TSH augments thyroid cell polarization through reorganizing the cytoskeletons and hyperpolarizing the resting membrane potentials and enhances iodine uptake by the cells.

Participation of myofibroblasts in feline mammary carcinoma.

Myofibroblasts were observed at sites of connective tissue proliferation in 17 cases of feline mammary carcinoma (15 adenocarcinomas and 2 solid carcinomas). The cytoplasm of these cells appeared bright red with Mallory-azan stain and dark blue with levanol fast cyanin 5RN, indicating the presence of contractile protein. The appearance of these cells by electron microscopy agreed with other reports on myofibroblasts: indented nuclei, remarkable accumulation of microfilaments in the cytoplasm, organization of desmosomes and hemidesmosomes on the cell surface and the formation of a basement membrane-like structure. Such myofibroblasts were found in both the interstitial connective tissue and fibrous capsule, and in some cases they showed widespread proliferation in the stroma having a myomatous appearance. On the other hand, they could not be detected at the sites of invasion in some cases. These morphological features suggest that the organization and participation of myofibroblasts may be of particular significance, i.e., reaction to tumor cells or transformation together with tumor cells, in feline mammary carcinoma.

Effect of selective enzymatic digestions on skin biopsies from pseudoxanthoma elasticum: an ultrastructural study.

Skin biopsies from patients with pseudoxanthoma elasticum (PXE) were studied by electron microscopy either before or after selective digestions with collagenase, elastase, trypsin, hyaluronidase, chondroitinase AC and ABC, with the aim of identifying an eventual organic component associated with mineralization within the elastin fibers and the chemical nature of the enormous aggregates of filaments very often associated with, but distinct from mineralized elastin fibers. The results obtained, on both embedded thin sections and fresh tissue fragments, showed that elastin fibers, whether mineralized or not, were sensitive only to elastase, and they did not contain significant amounts of materials different from elastin that could be accounted for by ion precipitation; the aggregates of microfilaments in strict connection with altered elastin fibers were mostly sensitive to elastase and hyaluronidase, were partially removed by trypsin and chondroitinase, and were not modified by collagenase, which seems to indicate that the microfilaments consist mainly of abnormally aggregated elastin molecules together with low sulfated proteoglycans. It may be concluded that PXE is a complex genetic disorder of the connective tissue, and that mineralization of elastin is only one of the alterations of the extracellular matrix.

CARCINOSARCOMA OF THE ESOPHAGUS: An Immunohistochemical and Electron Microscopic Study

Immunohistochemical and electron microscopic examinations were made of a carcinosarcoma of the esophagus in an 80-year-old man. An immunohistochemical examination showed that sarcomatous spindle cells were vimentin-positive, whereas squamous carcinoma cells were keratin-positive. No coexistence of vimentin and keratin in a single tumor cell was found. Electron microscopically, the sarcomatous spindle cells were characterized by well-developed rough endoplasmic reticulum, abundant intermediate filaments, and the occasional presence of peripheral aggregates of microfilaments. No definite desmosomes were identified among these cells. These results appear to indicate that most of the spindle-shaped tumor cells assume fibroblastic cellular features and synthesize the intermediate filament protein usually expressed in mesenchymal cells, even though such tumor cells could be epithelial in origin.

37] Ligand-induced patching and capping of surface immunoglobulins

This chapter discusses the patching and capping phenomena that has been described for a variety of membrane components in a number of different cell types. The basic model remains, however, the patching and capping of membrane immunoglobulin (mIg) la of B lymphocytes. With other membrane components, the conditions for obtaining such redistribution phenomena may be significantly different. The chapter states that a detailed model for the mechanisms of patching and capping, and related membrane phenomena such as pinosome formation, microvilli formation, endocytosis, and shedding, has also been developed. The chapter reviews the general characteristics of the capping phenomenon, particularly the energy requirements, temperature dependence, inhibition, reversion by microfilament-directed drugs, and the accumulation of microfilaments under the cap that have led to the suggestion that capping is probably a cellular contractile phenomenon. The role of microtubules in the capping phenomenon is more complex and they do not appear to enhance microfilament mobility but rather to inhibit it. Capping of any membrane component probably occurs as a consequence of microprecipitation resulting in entrapment within the lattice formed in the plasma membrane, of a microfilament-associated membrane component: the “membrane capping component” or “integral protein X.” The hypothesis has been formulated, however, that not only clustered components but also whole domains of membrane might be brought into the cap.

Macrovacuolation induced by cytochalasin: Its relation to the cytoskeleton; morphological and cytochemical observations

At higher doses of cytochalasin (e.g. 3 μg/ml for 3–20 hr), cells of the rat fibroblastoid line, Hmf, undergo extreme retraction, arborization, and subsequent rounding, and develop big cystic vacuoles. These vacuoles are always closely invested by microfilamentous masses, the CD-induced derivatives of the actin-based cytoskeleton, which aggregate in the endoplasm. Vacuolation is progressive (e.g. 12% cells at 6 hr; > 80% at 18 hr), related to total dose (concentration × time); and to congener (CD > CB). Vacuole membranes have the same dimension (85 Å), surface marker 5′-nucleotidase, and junctional specializations as those found at the cell surface; they lack the membrane markers associated with endomembrane systems (e.g. AcPase, TPPase, IDPase) and are not lysosomal. Vacuoles represent internalized plasma membrane; they apparently result from retention in the endoplasm, and fusion, of pinocytotic vesicles originating at the cell surface. Vacuole membrane is always in intimate relation to the actin-based microfilament aggregates that surround the vacuoles, and actin-membrane linker proteins fodrin and vinculin are localized at the vacuole boundaries. Vacuoles and their enveloping actin-filament aggregates are surrounded by arrays of vimentin-based intermediate filaments. A new membranous compartment with characteristics of plasma membrane is thus formed within the cell under the influence of CD. Rounding brought about by other means causes no vacuolization. Macrovacuolation, like the other changes caused by CD, is completely reversible on restoration of cells to normal medium.

Palmar fibromatosis (Dupuytren's contracture). Ultrastructural and enzyme histochemical studies of 43 cases.

Forty three cases of palmar fibromatosis were studied by light and electron microscopy, enzyme histochemistry, and ultrastructural immunohistochemistry. By electron microscopy most of the cells composing the nodules in both the proliferative and the involutional stages were identical to myofibroblasts. The myofibroblasts in the involutional nodules often possessed microfilament aggregates probably representing contraction of micro(actin)filaments in the cytoplasm. The proliferative nodules revealed small perivascular haemorrhages and haemosiderin deposits accompanied by accumulation of macrophages and some lymphocytes; these inflammatory cells possibly secrete a certain growth factor inducing proliferation of genetically abnormal fibroblasts and myofibroblasts. Diaminopeptidase IV was detected in myofibroblasts and fibroblasts by enzyme histochemistry and ultrastructural immunohistochemistry; the enzyme may play a role in the metabolism of intercellular substances. Some perivascular mesenchymal cells, interpreted as variants of myofibroblasts, had moderate activity of alkaline phosphatase.

Membrane cycling and macrovacuolation under the influence of cytochalasin: Kinetic and morphometric studies

Fibroblasts exposed to higher doses of cytochalasin accumulate very big discrete endoplasmic vacuoles, the membrane of which is derived by internalization of plasmalemma. Morphometry confirms that the amount of surface interiorized is equal to the difference between the original cell surface area (before CD) and the reduced surface area measurable after CD-induced rounding. Correspondingly, there is a nearly two-fold increase in the activity of the ectoenzyme 5′-nucleotidase (a marker for plasma membrane) internally within the cytoplasm, after treatment with CD. Macrovacuolation increases cell volume by ~30%. Surface membrane is internalized as micropinocytotic vesicles at a rate measurable by the accumulation of HRP, a marker of fluid-phase pinocytosis. Uptake of HRP is shown to be enhanced at all times during exposure to CD, and is balanced by accelerated exocytic recycling of membrane except during a phase (~4–8 hr) in which pinocytic uptake exceeds exocytosis. Vesicular membrane accumulated intracellularly in this period is retained in the endoplasm, and by successive fusions forms vacuoles in close approximation to microfilament aggregates. Once established, this new macrovacuolar membrane compartment is in dynamic equilibrium with the cell surface, and its membrane is cycled like the plasma membrane, in a mutual exchange of pinosomes between the several vacuoles and the cell surface. In drug-free medium vacuole membrane apparently reverts to the surface by pinocytotic recycling, and the cells recover normal characteristics 4–6 hr after withdrawal of cytochalasin.

An ultrastructural study on periductal elastosis in human breast tumors.

An ultrastructural study on elastosis of human breast tumors was made with special attention to the periductal elastosis and the cell responsible for elastic fiber formation. The elastosis was found prominently in scirrhous type of duct carcinoma. In the area of mild periductal elastosis, the elastic fibers with many microfibrils and a tiny central elastin were seen around the periductal fibroblasts which were characterized by attenuated cytoplasms with aggregates of microfilaments and slightly developed rough endoplasmic reticulum. With the thickening of the periductal wall, such an area was replaced by abundant mature elastic fibers with peripheral microfibrils and a few intervening ordinary fibroblasts. Therefore, it was suggested that the periductal fibroblasts which transformed into ordinary fibroblasts during the development of elastosis were primarily concerned with the elastic fiber formation. In the interlobular tissue in which both fibroblasts and myofibroblasts were present, the elastic fibers were larger than those of the periductal area and had less microfibrils in their periphery. The relationship between microfibrils and elastin during the early elastosis, maturation process of the elastic fibers, and cell modulation of the fibroblasts in the breast elastosis were discussed.

Morphogenesis of coremia and rhizomorphs in the AscomyceteSphaerostilbe repens

The differentiating stages of coremia and rhizomorphs inSphaerostilbe repens were studied by transmission electron microscopy. Vegetative mycelium is characterized by highly cytoplasmic cells rich in ribosomes and mitochondria and with few vacuoles as well as endoplasmic reticulum. Cell walls are thin attaining a maximum thickness of 0.10 μm. During the aggregating phase a prosenchymatous mass of randomly oriented cells is produced by localized elongation and branching of the filaments. The hyphae in this region have the appearance of actively metabolising cells. In the course of the differentiating phase, numerous hyphae of the median zone of the aggregate grow upward and downward to give rise to coremium and rhizomorph primordia respectively. The individual hyphal tips lay parallel to each other and cells of the growing apices retain their meristematic characteristics. At the periphery of the aggregate and to a lesser extent in the subapical rhizomorphic zone, cells reduce their cytoplasmic density as a consequence of a decrease in the number of ribosomes. These cells also increase in size and become isodiametric and vacuolated. During cellular differentiation walls increase steadily in thickness and at the elongating phase they reach 0.30 μm in the rhizomorphic cortex. Mucilaginous material is progressively deposited around hyphae and in the most differentiated zones, coalesce to fill interhyphal spaces. This extracellular matrix seems to play a role in maintaining cohesiveness of the aggregated organs. The tissue in the process of differentiation is scattered with cells highly enriched in mitochondria and with cells virtually undifferentiated. Accumulation of microfilaments takes place in the differentiating zone localized behind the immersed meristematic apex. These structures might be involved in wall synthesis. Glycogen rosettes accumulate in the vegetative mycelium surrounding the aggregating centers, suggesting the possibility of supplying energy during the differentiating processes. The vacuolar system, represented by autophagic vacuoles which are present until the differentiation phase, presumably may also participate in the biochemical changes that occur during aggregation. Coremial cells are characterized by an increase in wall thickness, a highly sinuous plasma-membrane as well as large amounts of mucilaginous compounds accumulated between hyphae, but in all other respects they resemble the cells of actively growing vegetative hyphae.

The detection of effaced podocytes by high resolution light microscopy.

Fifteen cases of renal minimal change disease and five normal glomeruli were studied by high resolution light microscopy (HRLM) and transmission electron microscopy (TEM). Effacement of podocytes usually is accompanied by aggregation of microfilaments in the cytoplasm of the epithelial cells parallel to the glomerular basement membrane (GBM). The aggregated microfilaments were detected as a dense blue line by high resolution light microscopy. The presence of this line appears to be a reliable sign for the detection of diffusely effaced podocytes by light microscopy. However, when effacement of podocytes was not accompanied by the condensation of microfilaments, the detection of this change by HRLM was more difficult.

Acute renal failure and renal tubular squamous metaplasia following treatment with streptozotocin

Nephrotoxicity, in the form of transient proteinuria, azotemia, abnormalities of tubular function, and acute renal failure, is the major toxic condition following administration of streptozotocin. The renal morphologic and ultrastructural abnormalities associated with streptozotocin remain poorly defined. We describe a patient with metastatic islet cell tumor of the pancreas who was treated with 16 weekly courses of 1 g/m2 of streptozotocin without marked change in renal function. Following a six-week hiatus without change in renal function, a single course of 1 g/m2 of streptozotocin was administered and resulted in acute renal failure. Light microscopic examination of the kidneys showed irregularly dilated renal tubules lined by low cuboid epithelium. The cells were pleomorphic and showed some mitoses. Nuclei were irregular and variably hyperchromatic. Electron microscopic examination disclosed large aggregates of fine microfilaments in the proximal convoluted tubules and collecting ducts. Microfilament aggregates were both free in the cytoplasm and membrane bound. Microfilaments were proved to be tonofilaments by the demonstration of keratin within the epithelium, using the immunoperoxidase method. These data suggest that squamous metaplasia may be an important part of streptozotocin renal toxicity, and the suggestion is made that they may be an antecedent of neoplastic change.

Microfilament accumulation and the transport of amino acids and glucose in adult rat hepatocytes cultured on collagen gel/nylon mesh

Transport of α-aminoisobutyric acid in cultured hepatocytes is temperature- and energy-dependent, whereas transport of 2-deoxy-D-glucose is not energy-dependent. In early cultures of hepatocytes (day 2) on a collagen gel/nylon mesh, the cells contain few microfilaments and the transport of amino acids and glucose is 5–7 times more than in late cultures of hepatocytes (day 6) which contain an apical, extensive accumulation of microfilaments. Cytochalasin D has little effect on the transport of amino acids and glucose in day 2 cultures of hepatocytes, but enhances transport of both compounds in day 6 cultures. These findings suggest that the microfilament accumulation in cultured hepatocytes inhibits transport of amino acids and glucose.

Coelomocyte cytoskeletons: Interaction with cytochalasin B

Cytochalasin B (CB) quickly distorts the gross morphology of sea urchin petaloid coelomocytes at low (0.1 μg/ml) concentrations and inhibits the cell's ability to morphologically transform to the filopodial form. Neither the ultrastructure nor the biochemical components of these cells are altered during these initial effects. At 10 μg/ml of CB, however, there is evidence of microfilament aggregation into foci after prolonged (30 min) exposure to CB but this is not correlated with a change in cytoskeletal composition or cell morphology.

Ultrastructure of the parathyroid gland of the japanese lizard in the spring and summer season.

The ultrastructure of the parathyroid glands of adult Japanese lizards (Takydromus tachydromoides) in the spring and summer season was examined. The parenchyma of the gland consists of chief cells arranged in cords or solid masses. Many chief cells contain numerous free ribosomes and mitochondria, well-developed Golgi complexes, a few lysosome-like bodies, some multivesicular bodies and relatively numerous lipid droplets. The endoplasmic reticulum is mainly smooth-surfaced. Cisternae of the rough endoplasmic reticulum are distributed randomly in the cytoplasm. Small coated vesicles of 700-800 Å in diameter are found occasionally in the cytoplasm, especially in the Golgi region. The chief cells contain occasional secretory granules of 150-300 nm in diameter that are distributed randomly in the cytoplasm and lie close to the plasma membrane. Electron dense material similar to the contents of the secretory granules is observed in the enlarged intercellular space. These findings suggest that the secretory granules may be discharged into the intercellular space by an eruptocrine type of secretion. Coated vesicles (invaginations) connected to the plasma membrane and smooth vesicles arranged in a row near the plasma membrane are observed. It is suggested that such coated vesicles may take up extracellular proteins. The accumulation of microfilaments is sometimes recognized. Morphological evidence of synthetic and secretory activities in the chief cells suggests active parathyroid function in the Japanese lizard during the spring and summer season.

Actin microfilaments, cell shape, and secretory processes in isolated rat hepatocytes. Effect of phalloidin and cytochalasin D.

The effects of phalloidin and cytochalasin D, drugs which, respectively, stabilize and destabilize actin microfilaments, have been tested on isolated rat hepatocytes. Both drugs produced a modification of cell shape, characterized by protrusions bulging from the cytoplasm. In phalloidin-treated hepatocytes, an accumulation of actin microfilamentous network was detectable at the base of each protrusion by electron microscopy, immunofluorescence, and HMM decoration. This accumulation of microfilaments was absent in cytochalasin D-treated cells. The release of triglycerides, an index of very low density lipoprotein secretion, was inhibited by phalloidin or cytochalasin D, and accompanied by an increase in cellular triglycerides. At the electron microscope examination, triglyceride accumulation was represented by fat droplets and vesicle-enclosed, very low density lipoprotein-like particles. Total protein and albumin secretion was only very slightly modified by either one of these drugs. With the use of various phalloidin analogs, a correlation was observed between their respective ability to stabilize F-actin in vitro, and their effects on cell shape and triglyceride secretion. In conclusion, phalloidin, and cytochalasin D: (a) modify the shape of isolated hepatocytes; (b) inhibit lipoprotein secretion. These effects possibly result from a modification of actin microfilament function.

Spermatogenesis in males of the free-living nematode, Caenorhabditis elegans

We have studied the morphology of spermatogenesis in the free-living nematode Caenorhabditis elegans with the light and electron microscopes. The gonad of the adult male is a single, reflexed cylindrical structure containing all stages of spermatogenesis arranged in a single wave of development. The primary spermatocytes are at the end of the gonad most distal from its opening into the cloaca. In this region, the cells are syncytial, and there is a central core containing cytoplasm common to all the neighboring cells, an organization reminiscent of the ovary in the hermaphrodite. Moving toward the cloaca one encounters the stages of meiotic prophase. At diplotene and diakinesis the cells contain many Golgi complexes. Some of these Golgi complexes are associated with an urn-shaped vesicle with a dark amorphous collar about its neck. Other Golgi complexes are seen next to aggregates of microfilaments. These “fibrous bodies” become enveloped with a flattened vesicle that forms a boundary two membranes thick. After these two structures have grown in size, their membranes fuse to form a composite structure. The membranes at the site of fusion then develop dark-staining thickenings as they fold into convoluted sacs and tubes. At about this time, the cells go through the two meiotic divisions. At telophase II the composite structures and fibrous bodies cluster at the spindle poles and cleavage furrows not only separate the daughter cells, but they also slough off a substantial volume of cytoplasm. In the resulting spermatid the nucleus condenses to form the small mass of dark-staining chromatin characteristic of sperm. The microfilaments of the fibrous bodies now disappear while the membranes of the composite structures continue to fold. The texture of the cytoplasm becomes more dense and now contains numerous slender, wavy tubular elements. A part of each composite structure now fuses with the plasma membrane of the sperm to make an almost spherical invagination of extracellular space partially filled by the tortuous bits of membrane-bound cytoplasm formed by the foldings of the membranes of the composite structure.

The ultrastructure of immature Sertoli cells. Maturation-like changes during culture and the maintenance of mitotic potentiality.

Cell aggregates dissociated from 10-day-old rat testes form epithelial colonies in culture. These epithelial cells have been identified as supporting cells (immature Sertoli cells) by the close similarity between the nuclear and cytoplasmic structures of the cultured cells and those of supporting cells in vivo. The cultured supporting cells undergo maturation-like changes in serum-enriched media containing follicle stimulating hormone (FSH). These changes include a change in the pattern of chromatin condensation, the development of large nucleoli and nuclear infoldings, a progressive predominance of the smooth over the rough endoplasmic reticulum and the appearance of luminal (apical) differentiations. The differentiations of the luminal surface consist of finger-like cytoplasmic extensions, a regional accumulation of microfilaments and microtubules and the junctional complexes between neighboring cells near the lumen. In the presence of FSH there is a low but sustained mitotic activity in the population of cultured supporting cells. This FSH-dependent mitotic activity was observed from Day 4-15 in culture. It is suggested that while several structural differentiations of the supporting cells develop under these conditions in Vitro, the luminal region of these cells does not differentiate in the same way as the apical region of Sertoli cells in vivo.

Fine structural changes in the liver of methotrexate-treated psoriatics.

Ultrastructure of 55 liver biopsies obtained from 52 patients, all but 1 with psoriasis and all but 5 treated with methotrexate (MTX), have been studied. In MTX-treated psoriatics, liver cell nuclei showed reduced electron opacity with frequent cytoplasmic invaginations and glycogen inclusions. In the mitochondria nonspecific changes, such as pleomorphism, gigantism, paracrystalline inclusions, compartmentalization, signs of division and sequestration via autophagosomy were noted. The pericanalicular ectoplasm exhibited widening with accumulation of microfilaments. Detachment of desmosomal plaques between hepatocytes was common. A striking finding was the hyperplasia of fat-storing perisinusoidal (Ito) cells. No definite correlation was found between morphology, duration of disease, treatment as well as cumulative dose of MTX.

The mechanism of concanavalin A cap formation in leukocytes.

ABSTRACT The process of Concanavalin A (Con A) cap formation on human blood polymorphonuclear leukocytes, monocytes and lymphocytes and rabbit alveolar macrophages has been studied by correlative use of light, fluorescence and electron microscopy. The most important precondition for Con A capping on these cells is the disassembly of cytoplasmic microtubules by colchicine or the glutathione-oxidizing agent, ‘diamide’. Incubation of microtubule-depleted leukocytes with fluorescein-conjugated Con A (F-Con A) leads to the aggregation of lectin into a cap which usually occupies a protuberance at one pole of the cell. F-Con A can also be concentrated at a constriction in the cell body. The protuberance is shown to consist of highly plicated membrane subtended by a network of densely packed microfilaments. Additional microfilaments originate from this network and course into individual plications of the protuberance. However, the formation of the protuberance with its organized structure follows the disassembly of microtubules alone and does not require Con A. Thus when cells are treated with colchicine or diamide, then fixed and labelled with F-Con A the typical changes in cell shape that are associated with capping are observed but lectin is distributed homogeneously over the cell surface. Similarly if cells are first capped with low concentrations of unlabelled lectin, then fixed and incubated with F-Con A, fluorescence is again uniformly distributed over the whole membrane. This indicates that membrane Con A receptors have not been concentrated over the protuberance despite the prior aggregation of microfilaments. By contrast, when precapped cells are labelled with F-Con A before fixation, fluorescence is concentrated through the previously established cap. Thus extensive organization of microfilaments and unlabelled lectin does not inhibit the movement of F-Con A-receptor complexes on unfixed cells. Further, the Con A cap is sufficiently fluid to permit mixing of sequentially formed Con A-receptor complexes. Although the aggregation of microfilaments into a protuberance and the concentration of Con A into the membrane of the protuberance are clearly separable events, microfilaments and Con A-receptor complexes are ultimately found in close association in the cap. This association appears to stabilize the localization of both the surface-bound lectin and the submembranous network of microfilaments. Such stabilization could result from physical interactions between microfilaments and Con A within the protuberance. However, we favour an alternative mechanism in which a region of low membrane fluidity that limits further diffusion is established following microtubule disassembly and is preserved by microfilament-membrane interactions.