Distribution of actin-filament bundles in myoid cells,Sertoli cells,and tunica albuginea of rat and mouse testes

Summary Frozen sections of the rat and mouse testes were stained with either FITC-phalloidin or NBD-phallacidin and viewed with conventional fluorescence and confocal laser microscopes in order to demonstrate the arrangment of actin-filament bundles in myoid cells, Sertoli cells and tunica albuginea. Myoid cells are rich in actin-filament bundles crossing at right angles. These bundles running in different directions can also be visualized by means of electron microscopy. Nerve fibers occur in the vicinity of myoid cells, suggesting a neural control of the cell. At Sertoli cell junctions actin filaments occur at the circumference of the cell, where they show a honeycomb pattern. The ratio of the number of Sertoli cells per myoid cell can be calculated by means of confocal microscopy; this technique may provide a new parameter for determining spermatogenic activity. In the tunica albuginea of the juvenile mouse testis, actin filaments are arranged in an alternate fashion.     

The ultrastructural organization of the basement membrane of Bowman's capsule in the rat renal corpuscle

Summary The basement membrane of Bowman's capsule (BCBM) of the rat was studied by means of a modified tissue-preservation technique for transmission electron microscopy, which avoids the usual thorough fixation in OsO₄ and applies tannic acid and uranyl acetate for staining (Sakai et al. 1986). At most sites the BCBM is multilayered, consisting of one to seven dense layers separated by electron-lucent layers. The latter, which can be termed laminae rarae, contain fine filaments which connect the dense layers to each other and the innermost dense layer to the basal cell membrane of the parietal epithelium. The laminae densae are basically composed of fine filaments arranged in an anastomosing pattern. Individual filaments ranging from 5 to 15 nm in diameter, combine to form filament bundles up to 100 nm in thickness and 1 to 2 m in length. Within a dense layer, filaments and filamentous bundles are oriented mainly in the same direction. Often the inner dense layers do not form a continuous sheet, and the filamentous bundles are arranged in anastomosing or spiral patterns to form a ribbon-like structure that we call a microligament. These microligaments are often embedded in basal furrows of the parietal epithelium and are best developed around the vascular pole. Intracellular actin bundles of the parietal cells are regularly associated with these extracellular ribbon-like structures of the basement membrane. In conclusion, the BCBM has an unusual structure: the laminae densae are characterized by their filamentous nature and are arranged in different patterns, i.e. as a multilayered mat and as microligaments.Fellow of the Deutscher Akademischer Austauschdienst     

The contractile nature of the tubular-like cells of the parietal layer of the Bowman's capsule in male mice. An immunochemical and ultrastructural study.

The tubule-like cells (TLC) of the parietal layer of the Bowman's capsule of the renal corpuscle in normal male mice are provided with bundles of thin microfilaments (50-70 A). Moreover, the ultrastructural observations demonstrated that similar thin filaments (50-70 A) are located in the flattened cells of the parietal layer of the Bowman's capsule and in the tubular cells. All the above cells bind antimyosin-like antibodies. Considering the correspondence between the immunochemical and ultrastructural findings, it is suggested that the microfilaments located in TLC contain a 'myosin-like' protein.     

Peritubular myoid cells from rat seminiferous tubules contain actin and myosin filaments distributed in two independent layers

In the mammalian testis, peritubular myoid cells (PM cells) surround the seminiferous tubules (STs), express cytoskeletal markers of true smooth muscle cells, and participate in the contraction of the ST. It has been claimed that PM cells contain bundles of actin filaments distributed orthogonally in an intermingled mesh. Our hypothesis is that these actin filaments are not forming a random intermingled mesh, but are actually arranged in contractile filaments in independent layers. The aim of this study is to describe the organization of the actin cytoskeleton in PM cells from adult rat testes and its changes during endothelin-1-induced ST contraction. For this purpose, we isolated segments of ST corresponding to the stages IX-X of the spermatogenic cycle (ST segments), and analyzed the actin and myosin filament distribution by confocal and transmission electron microscopy. We found that PM cells have actin and myosin filaments interconnected in thick bundles (AF-MyF bundles). These AF-MyF bundles are distributed in two independent layers: an inner layer toward the seminiferous epithelium, and an outer layer toward the interstitium, with the bundles oriented perpendicularly and in parallel to the main ST axis, respectively. In endothelin-1 contracted ST segments, PM cells increased their thickness and reduced their length in both directions, parallel and perpendicular to the main ST axis. The AF-MyF bundles maintained the same organization in two layers, although both layers appeared significantly thicker. We believe that this is the first time this arrangement of AF-MyF bundles in two independent layers has been shown in smooth muscle cells, and that this organization would allow the cell to generate contractile force in two directions.     

Actin in living and fixed characean internodal cells: identification of a cortical array of fine actin strands and chloroplast actin rings

Summary We report on the novel features of the actin cytoskeleton and its development in characean internodal cells. Images obtained by confocal laser scanning microscopy after microinjection of living cells with fluorescent derivatives of F-actin-specific phallotoxins, and by modified immunofluorescence methods using fixed cells, were mutually confirmatory at all stages of internodal cell growth. The microinjection method allowed capture of 3-dimensional images of high quality even though photobleaching and apparent loss of the probes through degradation and uptake into the vacuole made it difficult to record phallotoxin-labelled actin over long periods of time. When injected at appropriate concentrations, phallotoxins affected neither the rate of cytoplasmic streaming nor the long-term viability of cells. Recently formed internodal cells have relatively disorganized actin bundles that become oriented in the subcortical cytoplasm approximately parallel to the newly established long axis and traverse the cell through transvacuolar strands. In older cells with central vacuoles not traversed by cytoplasmic strands, subcortical bundles are organized in parallel groups that associate closely with stationary chloroplasts, now in files. The parallel arrangement and continuity of actin bundles is maintained where they pass round nodal regions of the cell, even in the absence of chloroplast files. This study reports on two novel structural features of the characean internodal actin cytoskeleton: a distinct array of actin strands near the plasma membrane that is oriented transversely during cell growth and rings of actin around the chloroplasts bordering the neutral line, the zone that separates opposing flows of endoplasm.     

Myosin II filament assemblies in the active lamella of fibroblasts: their morphogenesis and role in the formation of actin filament bundles

The morphogenesis of myosin II structures in active lamella undergoing net protrusion was analyzed by correlative fluorescence and electron microscopy. In rat embryo fibroblasts (REF 52) microinjected with tetramethylrhodamine-myosin II, nascent myosin spots formed close to the active edge during periods of retraction and then elongated into wavy ribbons of uniform width. The spots and ribbons initially behaved as distinct structural entities but subsequently aligned with each other in a sarcomeric-like pattern. Electron microscopy established that the spots and ribbons consisted of bipolar minifilaments associated with each other at their head-containing ends and arranged in a single row in an "open" zig-zag conformation or as a "closed" parallel stack. Ribbons also contacted each other in a nonsarcomeric, network-like arrangement as described previously (Verkhovsky and Borisy, 1993. J. Cell Biol. 123:637-652). Myosin ribbons were particularly pronounced in REF 52 cells, but small ribbons and networks were found also in a range of other mammalian cells. At the edge of the cell, individual spots and open ribbons were associated with relatively disordered actin filaments. Further from the edge, myosin filament alignment increased in parallel with the development of actin bundles. In actin bundles, the actin cross-linking protein, alpha-actinin, was excluded from sites of myosin localization but concentrated in paired sites flanking each myosin ribbon, suggesting that myosin filament association may initiate a pathway for the formation of actin filament bundles. We propose that zig-zag assemblies of myosin II filaments induce the formation of actin bundles by pulling on an actin filament network and that co-alignment of actin and myosin filaments proceeds via folding of myosin II filament assemblies in an accordion-like fashion.     

泰和鸡肾小球旁器的观察

本文用光镜和透射电镜对泰和鸡(乌骨鸡)的肾小球旁器进行了观察。结果表明,泰和鸡的肾小球旁器由球旁细胞、过渡型致密斑、球外间膜细胞和极周细胞所组成。极周细胞在鸟类还属首次报道,它位于肾小囊脏层与壁层上皮移行处,环绕着肾小体的血管极,其结构与哺乳动物的相似。本文还就泰和鸡肾小球旁器的结构与功能的关系作了讨论。     

Stress fibers in situ in proximal tubules of the rat kidney

Actin bundles in proximal tubules of the rat kidney were examined by immunofluorescence and confocal laser microscopy with special reference to their three-dimensional distribution and identification as stress fibers. Renal tubular segments were prepared from the fresh renal cortex by simple homogenization and centrifugation, and fixed in formaldehyde for staining with fluorescent dye-labeled phalloidin. Segments of the proximal tubules could be identified easily on the bases of their diameter, the height of epithelial cells and prominent brush borders. Confocal laser microscopy clearly demonstrated the overall distribution of actin bundles in the whole-mount proximal tubular segments. Actin bundles in the basal cytoplasm of epithelial cells were observed to run parallel to each other and at a right angle to the tubular axis. In the stereo views reconstructed from serial optical sections, the basal actin bundles appeared as straight rods with both ends tapered. They varied in length and width and extended rather short distances of not more than 10 microns. Often, two or more actin bundles were longitudinally aligned in tandem. Some bundles showed irregular bandings along their length. Each bundle was composed of tightly packed actin filaments which could be decorated with heavy meromyosin subfragment-1 to display a bi-directional arrangement within the bundle. Immunostaining of cryostat sections showed that actin bundles contained myosin and vinculin. Enzymatically isolated proximal tubules contracted upon addition of Mg-ATP. These observations collectively suggest that the actin bundles at the base of renal proximal tubule epithelial cells can be listed among the examples of stress fibers in situ.     

Reorganization of actin cytoskeleton of nuclear erythrocytes and leukocytes in fish,frogs, and birds during migration

Using the method of confocal laser scanning microscopy, changes in the spatial organization of actin filaments of nuclear erythrocytes and leukocytes in fish, frogs, and birds during migration were studied. It has been shown that, during movement, in erythrocytes, like in leukocytes, reorganization of cytoskeleton microfilaments occurs. In the course of migration, in amphibians and birds, red blood cells form pseudopodia filled with bundles of actin filaments arranged in parallel, whereas no pseudopodia are formed in fish erythrocytes. The change in the structure of the actin cytoskeleton of nuclear erythrocytes, like in leukocytes, is responsible for the capability of red blood cells to undergo reactions of migration and phagocytosis.     

Confocal Microscopy Observations on Actin Cytoskeleton in the Pollen and Pollen Protoplast of Narcissus

Actin cytoskeleton was localized in the pollen and pollen protoplast of Narcissus cyclamineus using fluorescence labelled phalloidin andconfocal microscopy. In the hydrated pollen (before germination) actin filamem bundles were arranged in a parallel array and at right angles to the long axis of the pollen grain in the cortex. But at the germination pore region(or fur row) the actin filament bundles formed a reticulate network. In the centre of the grain there was also an actin filament network which was more open and had less bundles associated with it than the network underneath the furrow. When the pollen grain started to produce pollen tube, most(if not all) of the actin filament bundles in the pollen grain rearranged into a parallel array pointing towards the tube. The bundles in the array later elongated and extended into the pollen tube. In the pollen protoplast a very tightly-packed actin bundle network was present. Numerous branches and jonts of actin filament bundles could be seen in the network. If the protoplasts were fixed before staining, the bundles aggregated and the branches and joints became less obvious indicating that fixation had affected the nature and arrangement of the actin filament bundles. If the pollen protoplasts were bursted (using the osmotic shock technique) or extracted (using Triton X-100), fragments of actin filament bundles could still be found associated with the membrane ghost indicating that some of the actin filament bundles in the cortex were tightly attached to the membrane. Using a double staining technique, actin filaments and microtubules were co-localized in the pollen protoplast. The co-alignment of some of the actin filament bundles with the microtubule bundles suggested that the actin cytoskeleton and the microtubule cytoskeleton were not distributed at random but in a well organized and orchestrated manner [possibly under the control of a yet undiscovered structure(s). The actin filament cytoskeleton in the generative cells failed to stain either in pollen or pollen tube, but they became stained in the pollen protoplast. The actin cytoskeleton in the generative cell appeared as a loosely organized network made up of short and long actin filament bundles.     

Actin bundles in human hair follicles as revealed by confocal laser microscopy

A confocal laser microscope was used to examine the distribution pattern of actin bundles in whole-mounts of human hair follicles stained with fluorescently labeled phalloidin. Actin bundles were found exclusively in the epithelial outer root sheath of the lower and middle portions of the follicle. In the growth stage, the lower follicle was characterized by well-developed actin bundles arranged circumferentially in the innermost and outermost cell layers of the outer root sheath. Actin bundles in the innermost cells were aligned end-to-end so that they formed complete circular bands surrounding the inner root sheath. In the outermost cells, actin bundles ran underneath the basal plasma membrane to which they attached at both ends. In contrast, in the quiescent stage, actin bundles in the lower follicle were disposed radially toward the follicle surface where they terminated perpendicular to the basal plasma membrane. In the middle follicle, circumferential actin bundles were found only in the intermediate layer of the outer root sheath throughout the hair cycle. Immunofluorescent anti-myosin and anti-α-actinin staining showed a striated pattern along actin bundles. Vinculin was localized at both ends of actin bundles, corresponding to the cell-to-cell or cell-to-substrate adherens junctions. Glycerinated follicles changed in shape on the addition of MgATP, suggesting a contraction of actin bundles. From these observations, we conclude that actin bundles in the hair follicle are comparable to stress fibers and that they serve as a tensile scaffold for the growth and integrity of the follicle. Received: 6 May 1995 / Accepted: 25 October 1995     

THE COLUMNAR CELLS OCCURRING IN THE PARIETAL LAYER OF BOWMAN''S CAPSULE : Cellular Fine Structure and Protein Transport

The renal corpuscles of adult, C3H Swiss, male mice contain testosterone-sensitive, columnar cells in the parietal layer of Bowman's capsule. A study of the normal fine structure of these cells reveals several distinctive characteristics: a microvillous brush border; apical tubular invaginations and apical tubules; an elaborate infolding of the basal surface membrane forming cellular compartments, which contain numerous mitochondria; and a complex group of membrane-limited cytoplasmic inclusions. This appearance is remarkably similar to the fine structure of cells in the proximal convoluted tubule. 1 hr after an in vivo injection of horseradish peroxidase, numerous protein-absorption droplets occur in the columnar cell cytoplasm. The speed and cytomorphology of protein transport by these capsular cells closely resemble the handling of peroxidase by the proximal convoluted tubule. Origins for these testosterone-sensitive cells are discussed briefly. Morphological evidence is presented for the differentiation of squamous cells in Bowman's parietal capsule into columnar cells, which appear structurally and functionally identical with proximal convoluted tubular epithelium.     

Metaplasia of the parietal layer of Bowman's capsule: a histopathological survey of the human kidney

Human kidney sections taken at autopsy were examined to determine the incidence of metaplasia of the Bowman's parietal epithelium. Autopsy records were consulted to determine if there was any correlation between clinical disease, histopathological changes in organ systems and metaplasia of Bowman's capsule. The sections represented both sexes in 9 age groups from 2 to 87 years. The sections were fixed in neutral formalin, embedded in paraffin, sectioned at 6 microns, and stained with hematoxylin and eosin. A total of 129 kidney sections, representing 129 individuals, were evaluated. One hundred renal corpuscles were counted per section and the parietal layer of Bowman's capsule was classified as normal (squamous) or metaplastic (cuboidal). Of the 129 kidneys examined, 69 (53%) had metaplasia of Bowman's capsule. Of the 87 male kidneys, 51 (59%) exhibited metaplasia of Bowman's capsule. Of the 42 female kidneys examined, 18 (43%) of the kidneys had metaplasia of Bowman's capsule. On average, in kidneys with metaplasia, 4% of the renal corpuscles had metaplasia of Bowman's parietal layer. The lesion was present in both sexes in all age groups. The autopsy records revealed that there was no common clinical condition associated with the metaplastic lesion, but metaplasia of Bowman's parietal epithelium was consistently present with hepatic congestion and/or fatty changes.     

Cyclooxygenase and cAMP-dependent protein kinase reorganize the actin cytoskeleton for motility in HeLa cells

The adhesion of a cell to its surrounding matrix is a key determinant in many aspects of cell behavior. Adhesion consists of distinct stages : attachment, cell spreading, motility, and/or immobilization. Interrelated signaling pathways regulate these stages, and many adhesion-related signals control the architecture of the cytoskeleton. The various cytoskeletal organizations then give rise to the specific stages of adhesion. It has been shown that arachidonic acid acts at a signaling branch point during cell attachment. Arachidonic acid is metabolized via lipoxygenase to activate actin polymerization and cell spreading. It is also metabolized by cyclooxygenase to generate small actin bundles. We have used confocal microscopy and indirect immunofluorescence to investigate the structure of these cyclooxygenase dependent actin bundles in HeLa cells. We have also employed cell migration assays and pharmacological modulation of cyclooxygenase and downstream signals. The results indicate that cyclooxygenase and PKA stimulate the formation of actin bundles that contain myosin II and associate with small focal adhesions. In addition, we demonstrate that this cytoskeletal organization correlates with increased cell motility.     

Actin filament turnover regulated by cross-linking accounts for the size, shape, location, and number of actin bundles in Drosophila bristles

Drosophila bristle cells are shaped during growth by longitudinal bundles of cross-linked actin filaments attached to the plasma membrane. We used confocal and electron microscopy to examine actin bundle structure and found that during bristle elongation, snarls of uncross-linked actin filaments and small internal bundles also form in the shaft cytoplasm only to disappear within 4 min. Thus, formation and later removal of actin filaments are prominent features of growing bristles. These transient snarls and internal bundles can be stabilized by culturing elongating bristles with jasplakinolide, a membrane-permeant inhibitor of actin filament depolymerization, resulting in enormous numbers of internal bundles and uncross-linked filaments. Examination of bundle disassembly in mutant bristles shows that plasma membrane association and cross-bridging adjacent actin filaments together inhibits depolymerization. Thus, highly cross-bridged and membrane-bound actin filaments turn over slowly and persist, whereas poorly cross-linked filaments turnover more rapidly. We argue that the selection of stable bundles relative to poorly cross-bridged filaments can account for the size, shape, number, and location of the longitudinal actin bundles in bristles. As a result, filament turnover plays an important role in regulating cytoskeleton assembly and consequently cell shape.     

Desmin-positive epithelial cells outgrowing from rat encapsulated glomeruli

Decapsulated glomeruli, encapsulated glomeruli and tubular fragments were each selected and cultured in order to identify the origin of polygonal epithelial cells in glomerular outgrowths and to characterize them by double-label immunofluorescence microscopy using antibodies against cytoskeletal proteins. Polygonal cells outgrew from less than 1% of decapsulated glomeruli, 34.8 to 65.1% of encapsulated glomeruli and 62.4 to 79.7% of tubular fragments in culture. These data support the idea that polygonal cells in glomerular culture are derived mainly from parietal epithelial cells of Bowman's capsule but not visceral cells, and indicate that polygonal cells of tubular epithelial origin are also present. Polygonal cells from encapsulated glomeruli consisted of intensely vimentin-positive (IV) cells and weakly vimentin-positive (WV) ones. Most of the IV cells showed various degrees of staining with anti-desmin antibody, and some of them also expressed cytokeratins and alpha-smooth muscle actin. By contrast, all of the WV cells were stained with anti-cytokeratin antibody but not with anti-desmin antibody. Polygonal cells in cultures of tubular fragments were negative for desmin. These findings suggest that parietal cells express desmin in culture, even though they show no desmin staining in kidney sections.     

A cytochalasin-sensitive actin filament meshwork is a prerequisite for local wound wall deposition inNitella internodal cells

Summary Reorganization of the actin cytoskeleton following cell wall puncturing of characean internodal cells was studied by immunofluorescence and confocal laser scanning microscopy. Injury locally destroyed the parallel subcortical actin filament bundles and cortical actin strands that are characteristic of unwounded regions. At wounds, a delicate three-dimensional interlaced structure of actin strands, with meshes up to 5 m wide, formed by de novo assembly of isolated filaments and by the elongation of residual subcortical actin bundles and cortical actin strands. The actin meshwork persisted for up to 2 h, corresponding to the duration of intense wound wall secretion. Actin filament bundles continuous with the subcortical bundles outside the wound then regenerated, their parallel alignment probably assisted by endoplasmic flow. Cytochalasin D concentrations that arrested cytoplasmic streaming completely inhibited the formation of the actin meshwork, wound wall deposition and recovery of actin bundles. Concentrations that only reduced streaming velocity delayed meshwork formation and wound walls were thinner than in controls. The actual amount of F-actin within the meshwork, however, was clearly greater in the presence of low cytochalasin concentrations. In late stages of recovery, the actin bundles became very thick and intervening spaces became wider thereby forming a conspicuous, three-dimensional lattice that was continuous with interwebbing subcortical bundles and cortical actin around the periphery of the wound. Our experiments suggest that actin meshwork formation is a prerequisite for plasma membrane-directed transport of vesicles involved in wounding-induced exocytosis in characean internodes. Stabilization of the meshwork by subinhibitory concentrations of cytochalasin D is probably caused by actinbinding properties of the drug that either induce bundling or impede function of associated proteins.Abbreviations AFW artificial fresh water - BSA bovine serum albumin - CLSM confocal laser scanning microscope (microscopy) - DIC differential interference contrast - DMSO dimethyl sulfoxide - FITC fluorescein isothiocyanate - MBS m-maleimidobenzoyl N-hydroxy-succinimide ester - PBS phosphate-buffered saline - SCAB subcortical actin bundle     

Cytoskeletal Architecture of Dermal Chromatophores of the Freshwater Teleost Oryzias latipes

Cytoskeletal construction of dermal chromatophores of Orgzias latipes was studied by immunofluorescence microscopy. A microtubule system was most prominent in melanophores where a large number of microtubules emanated from the center of the cell. Xanthophores had an arrangement basically similar to that of melanophores, though the radial pattern became more irregular in the peripheral region where intersecting wavy microtubules were quite frequent. Oval-shaped leucophores exhibited the least-developed microtubule system, where the limited number of microtubules formed a loose basket-like architecture. Intermediate filaments were ubiquitously present in all types of chromatophores and were found to be vimentin-immunoreactive. Examination of doubly-labeled cells indicated that vimentin filaments had similar distribution patterns with microtubules. Orderly arranged bundles of actin filaments were found only in xanthophores, while in melanophores and xanthophores, actin expression was diffuse without displaying a conspicuous filamentous organization. Colchicine treatment induced depolymerization of microtubules and retraction of dendrites in varying degrees in cells in culture and in situ. Melanophores in culture are very sensitive to the treatment while xanthophores appeared to be more resistant in respect to the maintenance of cell morphology.     

Sensitivity of Fibroblasts and Their Cytoskeletons to Substratum Topographies: Topographic Guidance and Topographic Compensation by Micromachined Grooves of Different Dimensions

Fibroblasts alter their shape, orientation, and direction of movement to align with the direction of micromachined grooves, exhibiting a phenomenon termed topographic guidance. In this study we examined the ability of the microtubule and actin microfilament bundle systems, either in combination with or independently from each other, to affect alignment of human gingival fibroblasts on sets of micromachined grooves of different dimensions. To assess specifically the role of microtubules and actin microfilament bundles, we examined cell alignment, over time, in the presence or absence of specific inhibitors of microtubules (colcemid) and actin microfilament bundles (cytochalasin B). Using time-lapse videomicroscopy, computer-assisted morphometry and confocal microscopy of the cytoskeleton we found that the dimensions of the grooves influenced the kinetics of cell alignment irrespective of whether cytoskeletons were intact or disturbed. Either an intact microtubule or an intact actin microfilament-bundle system could produce cell alignment with an appropriate substratum. Cells with intact microtubules aligned to smaller topographic features than cells deficient in microtubules. Moreover, cells deficient in microtubules required significantly more time to become aligned. An unexpected finding was that very narrow 0.5-μm-wide and 0.5-μm-deep grooves aligned cells deficient in actin microfilament bundles (cytochalasin B-treated) better than untreated control cells but failed to align cells deficient in microtubules yet containing microfilament bundles (colcemid treated). Thus, the microtubule system appeared to be the principal but not sole cytoskeletal substratum-response mechanism affecting topographic guidance of human gingival fibroblasts. This study also demonstrated that micromachined substrata can be useful in dissecting the role of microtubules and actin microfilament bundles in cell behaviors such as contact guidance and cell migration without the use of drugs such as cytochalasin and colcemid.