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     

Distribution of actin bundles in Bowman's capsule of rat kidney

In this study we define the distribution of actin bundle arrangement in Bowman's capsule of rat renal corpuscles. Parietal cells of Bowman's capsule were examined by conventional light microscopy, electron microscopy and confocal microscopy. Within each parietal cell individual actin bundles are arranged in a parallel fashion running the length of the cell. Computer reconstructions obtained using confocal microscopy clearly show the lengths of actin bundles to be arranged, on a capsule level, end-to-end, at angles and perpendicular to bundles in adjacent cells. The bundles stain positively for non-muscle myosin and vinculin. The presence and arrangement of actin bundles in parietal cells is consistent with a role in reinforcing capsule structure.     

Massive actin bundle couples macrocilia to muscles in the ctenophore Bero?

Macrocilia are thick compound ciliary organelles arising individually from elongated epithelial cells on the lips of beroid ctenophores. A giant wedge-shaped bundle of microfilaments extends 25-30 microns from the base of each macrocilium to the lower end of the cell, terminating at a junction with an underlying smooth muscle cell. The broad end of the microfilament bundle is anchored to the macrocilium by striated rootlet fibers that extend from the basal bodies into the bundle and are linked to the microfilaments by periodic bridges. Fluorescence microscopy of rhodamine-phalloidin stained intact tissue, dissociated macrociliary cells, and Triton/glycerol-isolated bundles shows that the microfilaments contain actin. The microfilaments run generally parallel to the long axis of the bundle but are not highly ordered. Filaments decorated with myosin S1 show a uniform polarity with arrowheads pointing away from the tapered membrane-associated end of the bundle. No variations in bundle length (nor changes in rootlet periodicity) were observed in tissue fixed under conditions of calcium activation. Isolated bundles did not contract in Mg-ATP, even though detached macrocilia underwent reactivated beating and sliding disintegration. Macrocilia are used to bite through food organisms or transport prey into the stomach. The actin filament bundles probably play a supporting role as a structural linker between macrocilia and subepithelial muscle fibers and may serve as intracellular tendons to mechanically coordinate the motor activities of macrocilia and muscles during prey ingestion.     

Distribution of endothelin receptor subtypes ETA and ETB in the rat kidney.

The endothelin (ET) receptor system is markedly involved in the regulation of renal function under both physiological and pathophysiological conditions. The present study determined the detailed cellular localization of both ET receptor subtypes, ET(A) and ET(B), in the vascular and tubular system of the rat kidney by immunofluorescence microscopy. In the vascular system we observed both ET(A) and ET(B) receptors in the media of interlobular arteries and afferent and efferent arterioles. In interlobar and arcuate arteries, only ET(A) receptors were present on vascular smooth muscle cells. ET(B) receptor immunoreactivity was sparse on endothelial cells of renal arteries, whereas there was strong labeling of peritubular and glomerular capillaries as well as vasa recta endothelium. ET(A) receptors were evident on glomerular mesangial cells and pericytes of descending vasa recta bundles. In the renal tubular system, ET(B) receptors were located in epithelial cells of proximal tubules and inner medullary collecting ducts, whereas ET(A) receptors were found in distal tubules and cortical collecting ducts. Distribution of ET(A) and ET(B) receptors in the vascular and tubular system of the rat kidney reported in the present study supports the concept that both ET receptor subtypes cooperate in mediating renal cortical vasoconstriction but exert differential and partially antagonistic effects on renal medullary function.     

Phagocytosis of E. coli by renal tubular epithelia

Despite significant advances in our understanding or renal tubular cell function, the in vivo handling of E. coli by renal tubules has not been previously investigated. The present studies were, therefore, designed to study this aspect of nephron function. Live and dead E. coli and vehicle alone were microinjected into the proximal tubular lumen of a single nephron of rats, and the microinjected tubules were morphologically studied at one-half, two, four, and six hours after. The bacteria initially contacted the luminal cell membrane. The luminal cell membrane adjacent to the bacteria subsequently invaginated, and both live and dead E. coli eventually became internalized into the tubular epithelial cytoplasm. Since dead E. coli are unlikely to invade the cells, their intracytoplasmic localization is a result of tubular epithelial phagocytosis. Similar microinjections of dead E. coli together with rat erythrocytes revealed a preferential phagocytosis of dead E. coli. Examination of the microinjected nephron with dead E. coli 48 hours after also demonstrated a development of microscopic interstitial nephritis surrounding the microinjected tubule. In conclusion, the renal tubular epithelia of the proximal and distal segments of rat nephron have phagocytic potential for E. coli which are further capable of inducing an inflammatory reaction around the microinjected tubule.     

Evidence for particulate guanylate cyclase in rat kidney after stimulation by atrial natriuretic factor. An ultracytochemical study

Summary Cytochemical localization of particulate guanylate cyclase (GC) in rat kidney, after stimulation with atrial natriuretic factor (ANF), was studied by electron microscopy. In the renal corpuscle GC reaction product was localized on podocytes. Other segments of the nephron that showed ultracytochemical evidence of GC activity were the proximal convoluted tubule, the thick ascending limb of the loop of Henle and the collecting tubule. All GC positivity was associated with plasma membranes. Samples incubated in basal conditions (without ANF) did not reveal any GC reaction product. These results indicate that ANF is a strong activator of particulate GC. Our data also suggests that, through the enzyme, ANF acts directly on epithelial cells of tubules where Na⁺ reabsorption occurs. This is in agreement with the hypothesis that ANF has a direct tubular effect on natriuresis.     

Comparison of actin-filament bundles in myoid cells and Sertoli cells of the rat,golden hamster and mouse

Testes of adult rats, golden hamsters and mice were fixed with paraformaldehyde. Seminiferous tubules were then isolated by collagenase dissociation, stained with fluorescent phallotoxin, and viewed in a confocal laser microscope to observe actin filaments. Bundles of actin filaments in the myoid cells, especially in the rat, were arranged at right angles to each other in relation to the longitudinal axis of the tubule. In the hamster, circumferentially directed bundles were more frequent than longitudinally directed bundles. The actin bundles in the mouse were thinner than those in the rat and hamster, and their lattice network was less prominent. Nuclei of the myoid cells were elliptical and their short diameters were parallel to the long axis of the seminiferous tubules in the animals examined. Areas of myoid cells and of basal junctional portions of Sertoli cells were measured and compared in all animals studied. There were significant differences in the areas among the three species. The golden hamster showed the largest value for myoid-cell area, and the mean value for Sertoli-cell area was highest in the mouse.     

Quantitative immunogold localization of Na, K-ATPase along rat nephron.

Ultrastructural localization of Na, K-ATPase alpha-subunit along rat nephron segments was investigated quantitatively by immunogold electron microscopy on LR-White ultrathin sections using affinity-purified antibody against alpha-subunit of the enzyme. Ultrathin sections were incubated with the antibody at a saturation level and the number of gold particles bound per micron of the plasma membrane (particle density) of the tubular epithelial cells from the proximal tubule to the collecting duct was determined. In all the tubular epithelial cells, gold particles were located exclusively on the basolateral surface, and no significant binding of gold particles to the apical surface was observed. Distribution of gold particles on the basolateral membranes was quite heterogeneous; lateral membranes and infolded basal membranes were highly labeled, whereas the basal membranes which are in direct contact with the basal lamina were scarcely labeled. The average particle density on the basal surface was highest in the distal straight tubule cells (11.4 units), very high in the distal convoluted tubule cells (9.8 units), intermediate in the proximal tubule cells (3.3 units), in the connecting tubule cells (4.3 units), and in the principal cells of the collecting duct (5.6-3.8 units), low in the thin limb of Henle's loop (1.0 unit), and at the control level in the intercalated cells in the connecting and collecting duct. The relative number of gold particles/mm nephron segment and the relative number of gold particles in the various nephron segments were calculated using quantitative morphological data. The estimated distribution profile of the former was in good agreement with the Na, K-ATPase activity profile in rat nephron, which was determined biochemically with a microenzymatic method.     

GABA-immunoreactive structures in rat kidney.

We examined the distribution of gamma-aminobutyric acid-like immunoreactivity (GABA-LI) in the rat kidney by light and electron microscopy. In vibratome sections, GABA-LI was present in both the renal medulla and cortex. The inner stripe of the outer medulla was most heavily and almost homogeneously labeled, whereas GABA-LI in the cortex was mainly confined only to some tubules. GABA-positive structures involved the epithelial cells of the thin and the thick ascending limbs of the loop of Henle, the connecting tubules, and the collecting ducts. In GABA-positive connecting tubules and collecting ducts the immunoreactivity was present in the cytoplasm of about half of the epithelial cells. As revealed by electron microscopy, the labeled cells in the collecting tubules were the light (principal) cells. No GABA-LI occurred in neuronal structures. These findings are consistent with the presence of a non-neuronal GABA system in the rat kidney. Furthermore, the specific distribution of GABA in the tubular epithelium suggests a functional significance of this amino acid in tubular transport processes.     

Ultrastructural markers of renal tubular transport in rats under physiological conditions

Mitochondria of the proximal and distal tubules which are in different configurational states of epithelial cells and their surface--volume relationship of intercellular spaces and basal infolded channels were evaluated in rats. The evaluation was performed with stereological methods. The studies were carried out on 5 rats under physiological conditions using electron microscopy. Mitochondria within the proximal and distal tubules were found to occur in transitional states close to the orthodox state. However, mitochondria within the proximal tubules were in a higher energy state, closer to the orthodox state when compared with those within the distal tubules. Surface--volume parameters of intercellular spaces and basal infolded channels were unexpectedly higher than the relation to active ion transport as well as indiscernible permeability of the distal tubular basement membrane.     

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.     

Increase in actin contents and elongation of apical projections in retinal pigmented epithelial cells during development of the chicken eye

The structural and biochemical changes of cytoskeletal components of retinal pigmented epithelial cells were studied during the development of chicken eyes. When the cytoskeletal components of the pigmented epithelial cells from various stages of development were examined by SDS PAGE, actin contents in the cells markedly increased between the 15-d-old and hatching stages. Immunofluorescence microscopy showed that chicken pigmented epithelial cells have two types of actin bundles. One is the circumferential bundle associated with the zonula adherens region as previously reported (Owaribe, K., and H. Masuda, 1982, J. Cell Biol., 95:310-315). The other is the paracrystalline bundle forming the core of the apical projections. The increase in actin contents after the 15-d-old stage is accompanied by the formation and elongation of core filaments of apical projections in the cells. During this period the apical projections extend into extracellular space among outer and inner segments of photoreceptor cells. Accompanying this change is an elongation of the paracrystalline bundles of actin filaments in the core of the projection. By electron microscopy, the bundles decorated with muscle heavy meromyosin showed unidirectional polarity, and had transverse striations with approximately 12-nm intervals, as determined by optical diffraction of electron micrographs. Since the shape of these bundles was not altered in the presence or absence of Ca2+, they seemed not to have villin-like proteins. Unlike the circumferential bundles, the paracrystalline bundles did not contract when exposed to Mg-ATP. These observations indicate that the paracrystalline bundles are structurally and functionally different from the circumferential actin bundles.     

Rho controls actin cytoskeletal assembly in renal epithelial cells during ATP depletion and recovery

Actincytoskeletal disruption is a hallmark of ischemic injury and ATPdepletion in a number of cell types, including renal epithelial cells.We manipulated Rho GTPase signaling by transfection and microinjectionin LLC-PK proximal tubule epithelial cells and observed actincytoskeletal organization following ATP depletion or recovery byconfocal microscopy and quantitative image analysis. ATP depletionresulted in disruption of stress fibers, cortical F-actin, and apicalactin bundles. Constitutively active RhoV14 prevented disruption ofstress fibers and cortical F-actin during ATP depletion and enhancedthe rate of stress fiber reassembly during recovery. Conversely, theRho inhibitor C3 or dominant negative RhoN19 prevented recovery ofF-actin assemblies upon repletion. Actin bundles in the apicalmicrovilli and cytosolic F-actin were not affected by Rho signaling.Assembly of vinculin and paxillin into focal adhesions was disrupted byATP depletion, and constitutively active RhoV14, although protectingstress fibers from disassembly, did not prevent dispersion of vinculinand paxillin, resulting in uncoupling of stress fiber and focaladhesion assembly. We propose that ATP depletion causes Rhoinactivation during ischemia and that recovery of normalcellular architecture and function requires Rho.

     

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.     

Reorganization of actin filament bundles in living fibroblasts

We investigated how actin bundles assemble, disassemble, and reorganize during cell movement. Living chick embryonic fibroblasts were microinjected with actin molecules that had been fluorescently labeled with tetramethylrhodamine. We found that the fluorescent analogue of actin can be used successfully by both existing and newly formed cellular structures. Using time-lapse photography coupled to image- intensified fluorescence microscopy, we were able to detect various patterns of reorganization in motile cells. Assembly of stress fibers occurred near both the leading and the trailing ends of the cell. The initial structure appeared as discrete spots that subsequently extended into stress fibers. The extension occurred unidirectionally. The site of initiation near the leading edge remained stationary relative to the substrate during subsequent cell advancement. However, the orientation of the fiber could change according to the direction of cell movement. In addition, existing stress fibers could merge or fragment. The shortening of stress fibers can occur from either end of the fiber. Shortening from the proximal end (centrifugal shortening) was accompanied by a decrease in fluorescence intensity, as if the bundle were disassembling, and usually led to the total disappearance of the bundle. Shortening from the distal end (centripetal shortening), on the other hand, is usually accompanied by an increase in fluorescence intensity at the distal end of the bundle, as if this end had pulled loose from its attachment and retracted toward the center of the cell. Besides stress fibers, arc-like actin bundles have also been detected in spreading cells. These observations can explain how the organization of actin bundles coordinates with cell movement, and how stress fibers reach a highly regular pattern in static cells.     

Banding and polarity of actin filaments in interphase and cleaving cells

Heavy meromyosin (HMM) decoration of actin filaments was used to detect the polarity of microfilaments in interphase and cleaving rat kangaroo (PtK2) cells. Ethanol at -20 degrees C was used to make the cells permeable to HMM followed by tannic acid-glutaraldehyde fixation for electron microscopy. Uniform polarity of actin filaments was observed at cell junctions and central attachment plaques with the HMM arrowheads always pointing away from the junction or plaque. Stress fibers were banded in appearance with their component microfilaments exhibiting both parallel and antiparallel orientation with respect to one another. Identical banding of microfilament bundles was also seen in cleavage furrows with the same variation in filament polarity as found in stress fibers. Similarly banded fibers were not seen outside the cleavage furrow in mitotic cells. By the time that a mid-body was present, the actin filaments in the cleavage furrow were no longer in banded fibers. The alternating dark and light bands of both the stress fibers and cleavage furrow fibers are approximately equal in length, each measuring approximately 0.16 micrometer. Actin filaments were present in both bands, and individual decorated filaments could sometimes be traced through four band lengths. Undecorated filaments, 10 nm in diameter, could often be seen within the light bands. A model is proposed to explain the arrangement of filaments in stress fibers and cleavage furrows based on the striations observed with tannic acid and the polarity of the actin filaments.     

Immunocytochemical localization of cathepsin H in rat kidney

Summary Light and electron microscopic localization of cathepsin H in rat kidney was studied using post-embedding immunocytochemical techniques. For ligh microscopy, Epon sections of the kidney were stained by immunoenzyme method after removal of Epon and for electron microscopy, ultrathin sections of the Lowicryl K4M-embedded material were labeled by protein A-gold (pAg) technique. By light microscopy, fine granular staining was found in throughout the nephron, but the staining intensity considerably varied. The strongest staining was noted in the S1 segment of the proximal tubules followed by the S2 and S3 segments and the medullary collecting tubules. The glomeruli, the distal tubules, and the cortical collecting tubules were weakly stained. By electron microscopy, a gold label was found exclusively in lysosomes, which showed various sizes and labeling intensity. The results were quite consistent with the light microscopic results. The labeling intensity tended to increase as the matrix of lysosomes was condensed. Quantitative analysis of the labeling density of lysosomes demonstrated that the highest labeling density is found in the S1 segment of the proximal tubules and the labeling density of other renal segments is significantly low levels. The results indicate that a main site for cathepsin H in rat kidney is the S1 segment of the proximal tubules.     

Histochemical evaluation of mouse and rat kidneys with lectin-horseradish peroxidase conjugates

Paraffin sections of mouse and rat kidney were stained with a battery of ten lectin-horseradish peroxidase conjugates and lectin binding was correlated with the ultrastructural distribution of periodate-reactive sugar residues as determined by the periodic acid-thiocarbohydrazide-silver proteinate technique. Various segments of the uriniferous tubule in both species showed differential affinity for labelled lectins. Significant differences were also evident between comparable tubular segments in mouse and rat kidneys. Neutral glycoconjugates containing terminal beta-galactose and terminal alpha-N-acetylgalactosamine were prevalent on the luminal surface of the proximal convoluted tubule in the rat, but alpha-N-acetylgalactosamine was absent in this site in the mouse. In both species, terminal N-acetylglucosamine was abundant in the brush border of proximal straight tubules but absent in proximal convolutions. Fucose was demonstrated in both proximal and distal segments of mouse kidney tubules but only in the distal nephron and collecting ducts in the rat. Lectin staining revealed striking heterogeneity in the structure and distribution of cellular glycoconjugates. Such cellular heterogeneity was previously unrecognizable with earlier histochemical methods. The marked cellular heterogeneity observed with several lectin-conjugates in distal convoluted tubules and collecting ducts of both species raises a prospect that lectins can provide specific markers for intercalated and principal cells in the mammalian kidney. Glycoconjugates containing terminal sialic acid and penultimate beta-galactose were present on vascular endothelium in both rodent kidneys, as were terminal alpha-galactose residues; but both species lacked reactivity for Ulex europeus I lectin in contrast to human vascular endothelial cells. The constant binding pattern of lectin conjugates allows convenient and precise differentiation of renal tubular segments and should prove valuable in the study of changes in kidney morphology promoted by experimental manipulation or pathologic changes.     

Alpha-actinin and actin in the outer retina: a double immunoelectron microscopic study

Actin has many diverse functions in the outer retina. To help elucidate its organization in this area, we have investigated the extent of its association with the actin cross-linking protein alpha-actinin. Ultrathin sections of chicken retina were double-immunolabelled with monospecific antibodies against actin and alpha-actinin. The highest relative amount of alpha-actinin to actin label was measured in the adherens junctions between the individual retinal pigmented epithelial (RPE) cells and between the photoreceptor and Mueller cells; in the photoreceptor myoid; and in the RPE basal microvilli. The lowest amount was in the Mueller cell microvilli, the RPE apical processes, and in the photoreceptor ellipsoid. It is likely that the areas containing the highest ratio of alpha-actinin to actin labelling are where the actin filaments are most highly cross-linked into bundles and linked to the plasma membrane by alpha-actinin. Actin filaments terminate in these areas, and, except for the myoid region, they are involved in cell-cell or cell-substrate adherens junctions.