Microscopic anatomy of the thin-walled vessels leaving the heart of the lobster Homarus americanus: anterior median artery

Abstract. The anterior median artery is an unpaired vessel that leaves the anterior end of the lobster ( Homarus americanus ) heart and supplies hemolymph to the brain, the antennae, and the eyestalks. This vessel has a trilaminar organization, consisting of a tunica interna with elastic fibrils, a tunica intermedia represented by a bilayered cell mass, and a tunica externa with collagen fibrils. The exposed ends of the medial cells in the tunica intermedia exhibit small, diffuse bundles of microfilaments that are penetrated by microtubules. These bundles have a circumferential or a slightly oblique orientation relative to the lumen of the vessel. The precise role of the microfilaments is unresolved. If the irregularly shaped bundles are static structures, they might contribute to the non-linear elasticity of the artery. Alternatively, if they generate force, a coordinated contraction of the medial cells might reduce the luminal diameter of the artery and, thus, retard hemolymph flow. Microfilaments of the medial cells anchor to subplasmalemmal filamentous mats, some of which are integral to intermediate junctions and some of which are associated with unbounded cell membranes (hemi-intermediate junctions). Contraction of the microfilament-bearing cells would have to occur without the benefit of nervous innervation or the participation of communicating (gap) junctions. If cell contractility is confirmed, a reclassification of the anterior median artery, from capacitance vessel to resistance vessel, is in order, and the bilayered cell mass in the tunica intermedia would likely qualify as the first unstriated muscle found in crustaceans.     

Isolation and Contraction of the Stress Fiber

Stress fibers were isolated from cultured human foreskin fibroblasts and bovine endothelial cells, and their contraction was demonstrated in vitro. Cells in culture dishes were first treated with a low-ionic-strength extraction solution and then further extracted using detergents. With gentle washes by pipetting, the nucleus and the apical part of cells were removed. The material on the culture dish was scraped, and the freed material was forced through a hypodermic needle and fractionated by sucrose gradient centrifugation. Isolated, free-floating stress fibers stained brightly with fluorescently labeled phalloidin. When stained with anti-α-actinin or anti-myosin, isolated stress fibers showed banded staining patterns. By electron microscopy, they consisted of bundles of microfilaments, and electron-dense areas were associated with them in a semiperiodic manner. By negative staining, isolated stress fibers often exhibited gentle twisting of microfilament bundles. Focal adhesion–associated proteins were also detected in the isolated stress fiber by both immunocytochemical and biochemical means. In the presence of Mg-ATP, isolated stress fibers shortened, on the average, to 23% of the initial length. The maximum velocity of shortening was several micrometers per second. Polystyrene beads on shortening isolated stress fibers rotated, indicating spiral contraction of stress fibers. Myosin regulatory light chain phosphorylation was detected in contracting stress fibers, and a myosin light chain kinase inhibitor, KT5926, inhibited isolated stress fiber contraction. Our study demonstrates that stress fibers can be isolated with no apparent loss of morphological features and that they are truly contractile organelle.     

A histological study on the coronary artery of the indigenous black Bengal goat in Bangladesh.

The coronary artery of the black Bengal goat was studied by light microscopy. The wall of the coronary artery consisted of the tunica intima, tunica media and tunica externa. The tunica intima consisted of a single layer of flattened endothelium. The tunica media was well-developed and composed of mainly of smooth muscle cells together with some fine elastic fibers. The tunica externa consisted of predominant collagen fibers, and some elastic fibers and smooth muscle cells. Elastic fibers in the tunica externa formed a circular arrangement around the tunica media. Sex differences were not observed. The media with well-developed smooth muscle cells may be responsible for changes in functional physiological conditions of the heart.     

Organization of rat mesenteric artery after removal of cells or extracellular matrix components

Summary Rat mesenteric arteries, perfusion fixed in relaxed or contracted conditions, were digested with acid and elastase, bleach (sodium hypochlorite), or alkali to selectively remove collagen, elastin, or cells. Scanning electron microscopy was used to study the three-dimensional organization of the remaining cells or extracellular components. Smooth muscle cells of the tunica media were elongated and circumferentially oriented. Superior mesenteric artery cells had an irregular surface with numerous projections and some ends were forked. Small mesenteric artery cells were spindle shaped with longitudinal surface ridges, and showed extensive corrugations upon contraction. Elastin was present both as laminae and as an interconnected fibrous meshwork. Collagen was arranged in an irregular network of individual fibrils and small bundles of fibrils that formed nests around the cells in both arteries. This irregular arrangement persisted, with no apparent reordering or loss of order, upon contraction. The lack of an ordered arrangement or specialized organization at the cell ends suggests mechanical coupling of the cells to elastin or collagen throughout the length of the cell, allowing for force transmission in a number of directions. The tunica media is thus a composite material consisting of cells, elastin, and collagen. The isotropic network of fibers is well suited for transmitting the shearing forces placed on it by contraction of smooth muscle cells and by pressure-induced loading.     

Fine structure of the glomerular basement membrane of the rat kidney visualized by high-resolution scanning electron microscopy

Summary The fine structure of the glomerular basement membrane (GBM) of the rat kidney was studied by means of high resolution scanning electron microscopy. Specimens were taken from kidneys perfused with paraformaldehyde, freeze-fractured and then processed with conductive staining. The fractured surface of glomerular tufts exhibited the inner and outer surface of the GBM uncovered by endothelial and epithelial cells. The lamina densa was composed of densely packed granular material together with scattered fibrils. The laminae rarae interna and externa were composed of a meshwork that showed some structural heterogeneities. The meshwork composing the lamina rara interna contained 5-to 9-nm-thick fibrils, had pores 11–30 nm wide, and was associated with granular material except in those places that corresponded with endothelial fenestrae. The meshwork of the lamina rara externa was made up of 6- to 11-nm-thick fibrils, and had smaller pores under the foot processes (10–24 nm wide) than those near the filtration slits (16–32 nm wide). In addition to the meshwork, the lamina rara interna contained microfibrils that were arranged differently depending on the topography of the capillary wall: scattered fibrils had no predominant orientation at the convex side, circumferential bundles lay at the concave side of the peripheral capillary wall, and had a circumferential arrangement in the paramesangial wall.     

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.     

Development of terminal filaments and ovariole envelopes in Thermobia domestica (Insecta, Zygentoma) larvae

The 3rd instar female larvae of Thermobia domestica have five pairs of gonad primordia, each enclosed within a basal lamina (tunica propria). At the end of the 3rd instar some somatic cells scattered on the outer surface of the lamina are seen. During the 4th larval instar the gonad primordia start to form the ovarioles. Each ovariole is elongated and polarized, having anterior and posterior ends. The anterior group of outer somatic cells proliferate to form the terminal filament. At the 6th larval stage the ovarioles are already formed. The terminal filament is separated from the germarium by a thick basal lamina (transverse septum). There are three types of cell building the terminal filament. 1/Basal cells with numerous fingerlike projections; 2/Cells with electron lucent cytoplasm and large nuclei, and 3/Cells with darker cytoplasm containing bundles of fibers and more compact nuclei. The outer surface of the filament is covered by a thick, fibrous basal lamina. The somatic cells that in the previous stages were scattered on the tunica propria as distinct cells, in the 6th larval stage form a cellular envelope (tunica externa). This envelope is formed by a layer of flat cells, and contains numerous tracheae.     

The autonomous innervation of the buffalo (Bubalus bubalis) testis. An immunohistochemical study

The innervation pattern in the buffalo testis was determined by using histochemical and immunohistochemical methods. Nerves were concentrated in the tunica albuginea and septula testis, and did not show an uniform distribution. The tunica albuginea at the lateral and medial sides and at the free border of the testis is most densely innervated than at the epididymal border. At the cranial pole thick nerve bundles were observed between albugineal vessels and muscle bundles. Rare parenchymal nerves were found in perivascular position between seminiferous tubules and their occurrence is confined to lobules at the cranial and caudal testicular poles. An intense NPY immunoreactivity occurred in nerve bundles and in solitary varicose fibres. Nerves were concentrated in the tunica albuginea at the lateral and medial side and at the free border of the testis, and in the lobules at the cranial and caudal testicular poles. Sub P immunoreactivity was occasionally detected in some thicker nerve bundles and solitary fibers, in the tunica albuginea and in the wall of blood vessels, showing a similar distribution but less intensity and density than NPY immunoreactivity. TH immunoreactivity stained nerve fibers in the buffalo testis with a distribution pattern similar to that obtained with general neuronal markers. The histochemical reaction for AchE was negative, so cholinergic fibers cannot be detected in the buffalo testis. The histochemical NADPHd reaction stained rare nitrergic nerve bundles and solitary fibers. The majority of NADPHd activity was confined to the vascular endothelium, and rarely to the interstitial Leydig cells, whereas the Sertoli and germ cells did not show any reaction.     

Actin cytoskeleton of extraembryonic endoderm and teratocarcinoma-derived endoderm cells

Distinct F-actin- and myosin-containing stress fibers were observed in situ in many endoderm cells of parietal yolk sacs from 11-day mouse embryos. In visceral endoderm (VE) such fibers were not seen, and F-actin was concentrated in the cell periphery. Correspondingly, in electron microscopy ventral cell membrane-associated bundles of microfilaments were revealed in the periphery of parietal endoderm (PE) cells but not in VE cells. Both PE and VE cells formed stress fibers in primary cultures. Undifferentiated F9 embryonal carcinoma cells formed only short actin spikes and fibrils irrespective of growth substratum. In PE-like derivatives of F9 cells, on the other hand, distribution of F-actin was markedly affected by the growth substratum: They formed distinct stress fibers when plated on fibronectin but did not when plated on gelatin. Similarly, in teratocarcinoma-derived PE cells (PYS-2) adhesion to fibronectin induced the formation of distinct bundles of F-actin and plaques of vinculin. The results suggest that the susceptibility of teratocarcinoma cell actin cytoskeleton to the influence of molecular composition of surrounding matrix is developmentally regulated. On the other hand, the reason for the presence of stress fibers in PE cells and for their absence in VE cells is unclear.     

Ultrastructure of the swim bladder of the goldfish,Carassius auratus

Summary The swim bladder of the cyprinid Carassius auratus (goldfish) is a two-chambered organ connected to the esophagus by a pneumatic duct. The anterior chamber is lined by a single type of squamous epithelial cell. Two types of epithelial cells are present in the posterior chamber. Flattened cells with differences in the electron density of the cytoplasm line most of the chamber. Darker cells generally contain large amounts of glycogen. Cuboidal epithelial cells also occur in the posterior chamber. A glandular layer external to the muscularis in the posterior chamber is composed of large cells containing little glycogen, an extensive Golgi apparatus, and numerous mitochondria with single large granules. Capillaries and nerves are present in large numbers in this layer. Blood vessels form micro-retia mirabilia in the submuscular layer external to the glandular layer. Vessels are of two distinct types with wide lumina and flattened endothelium characterizing the venous vessels. Arterial vessels have smaller lumina, thick endothelial cells with prominent pinocytotic vesicles, and surrounding pericytes. Collagen is present in three forms in this swim bladder — large tactoids in the tunica externa of the anterior chamber, smaller tactoids in the lamina propria of the posterior chamber, and small fibrils in all other areas.Supported by a Young Investigator Pulmonary Research Grant # 1 R23 HL 19593-01 and by HL 23338-01 from the National Institutes of Health     

Ultrastructure and some other properties of inverted thyroid follicles in suspension culture

Separated thyroid follicles in suspension culture invert in 5% serum. In some, the inversion is not complete in that a small normal follicle persists completely in the interior of an inverted follicle. In inverted follicles the lumens are distended and electron lucent. The bounding epithelial cells are stretched, have relatively few microvilli on the surface toward the medium but they have bundles of oriented microfilaments usually located near the lumen. The cells are connected together by tight junctions. When inverted follicles are punctured, the lumen shrinks, the cells retract and become cuboidal and microvilli reappear. Microfilaments persist at the luminal surface but no longer in oriented bundles. No appreciable extracellular matrix is present at the basal cell surface in contact with the lumen, but matrix is occasionally observed between cells. Since bundles of microfilaments like stress fibers are observed in the cells in suspension culture, the presence of stress fibers in cells in monolayer culture is probably not dependent on attachment but might be a reflection of the spreading of the attached cells.     

Immunofluorescent and ultrastructural studies of polygonal microfilament networks in respreading non-muscle cells

Respreading gerbil fibroma cells (CCL146) have been found to display cytoplasmic actin-based polygonal fiber networks 10 h after replating (stage III of respreading according to Vasiliev & Gelfand, [1]). The networks have been analyzed by immunofluorescence and electron microscopy. The foci, sites of actin, α-actinin and filamin distribution, are condensed meshworks of microfilaments attached to the inner surface of the plasma membrane. The interconnecting fibers, sites of uniform actin distribution and complementary periodicities of α-actinin and myosin, are bundles of parallel microfilaments with periodic dense bodies. Heavy meromyosin (HMM) labelling of the microfilaments in the foci and interconnecting bundles confirm that they contain actin. In addition, approx. 70% of the microfilaments associated with an individual focus have a uniform polarity relative to it (arrowheads pointing away) suggesting that they have their origin there. Our results support earlier conclusions [2] that polygonal networks are structural intermediates responsible for organizing contractile proteins of the cortical microfilament layer into stress fibers.     

Evidence for the existence of substance P in the prepubertal rat ovary. II. Immunocytochemical localization

Nerve fibers containing substance P (SP) were localized in ovaries from juvenile and peripubertal rats by immunofluorescence. These fibers were closely associated with the theca externa of antral follicles, as well as being in the interstitial tissue and within the tunica adventitia of small blood vessels, mostly arterioles. Consistently, the greatest amount of SP immunoreactivity was observed surrounding the ovarian vasculature. Substance P was not detected in cells or within the corpora lutea (CL). Additionally, the peripubertal animals seemed to have a greater concentration of ovarian SP than the juvenile animals. Possible functional roles for this peptide in the ovary are discussed.     

Stress fibers in the mesenteric mesothelial cells of the large intestine of the bullfrog,Rana catesbeiana

Summary Actin-containing cytoplasmic fibers were visualized in the mesenteric mesothelial cells of the large intestine of bullfrog tadpoles by rhodamine-phalloidin staining of en face preparations of mesothelial cells. These fibers were concurrently stained by immunofluorescence using antibodies to myosin or -actinin. Electron microscopy showed the presence of bundles of microfilaments in the basal cytoplasm of the cells. Such fibers in the mesothelial cells may be comparable to the stress fibers present in cultured cells. The mesothelial cells initially formed axially oriented stress fibers when they changed from a rhombic to a slender spindle-like shape. On the other hand, stress fibers disappeared as cells transformed from elongated to polygonal shapes during the period of metamorphic climax. Expression of stress fibers in these cells appears to be related to the degree of tension loaded on the mesentery, which may be generated by mesenteric winding. These stress fibers in the mesothelial cells may serve to regulate cellular transformation. They may also help to maintain cellular integrity by strengthening the cellular attachment to subepithelial tissue against tensile stress exerted on the mesentery.     

The distribution of actin in non-muscle cells : The use of actin antibody in the localization of actin within the microfilament bundles of mouse 3T3 cells

Living mouse 3T3 cells display a complex array of fibrous structures which are visible with phase contrast, Nomarski and polarized light optics. When cells are fixed and stained for indirect immunofluorescence with actin antibody, the same fibers show intense fluorescence indicating that they contain actin. Electron microscopy reveals that these fibrous structures consist of submembranous bundles of microfilaments located primarily on the attached side of the cells. The results are discussed in terms of the intracellular localization of a possible submembranous contractile system involved in motile activities such as cell locomotion.     

A multi-modular tensegrity model of an actin stress fiber

Stress fibers are contractile bundles in the cytoskeleton that stabilize cell structure by exerting traction forces on the extracellular matrix. Individual stress fibers are molecular bundles composed of parallel actin and myosin filaments linked by various actin-binding proteins, which are organized end-on-end in a sarcomere-like pattern within an elongated three-dimensional network. While measurements of single stress fibers in living cells show that they behave like tensed viscoelastic fibers, precisely how this mechanical behavior arises from this complex supramolecular arrangement of protein components remains unclear. Here we show that computationally modeling a stress fiber as a multi-modular tensegrity network can predict several key behaviors of stress fibers measured in living cells, including viscoelastic retraction, fiber splaying after severing, non-uniform contraction, and elliptical strain of a puncture wound within the fiber. The tensegrity model can also explain how they simultaneously experience passive tension and generate active contraction forces; in contrast, a tensed cable net model predicts some, but not all, of these properties. Thus, tensegrity models may provide a useful link between molecular and cellular scale mechanical behaviors and represent a new handle on multi-scale modeling of living materials.     

Developmental stage dependent expression of the endothelial stress fibers and organization of fibronectin fibrils in the aorta of chick embryos

Organizational relationships between endothelial stress fibers and fibronectin fibrils in the developing chick abdominal aorta, from 5th day embryos to 3rd day young chicks, were studied with immunofluorescence and electron microscopy. Stress fibers, axially aligned parallel to the longitudinal cell axis, were expressed in the largely elongated endothelial cells, in embryos older than 8th day of incubation. Fibronectin fibrils in the aortic basal lamina, changed its organizational pattern from the network-like form to the straight bundles arranged parallel to the vessel's longitudinal axis after 9th day of incubation. Such axial alignment was dominant in the matrix beneath the elongated cells containing stress fibers, suggesting the existence of stress fibers may possibly modify the fibronectin's organizational pattern. The vinculin-containing dense plaque, which shaped like as the adhesion plaque in the cultured cells, was located at the ends of or lateral associating sites of stress fibers in embryos older than 8th day stage. The expression of stress fibers, as well as the formation of stress fiber's end plaques, may closely relate to the alignment between the stress fiber and fibronectin fibrils in the extracellular matrix.     

Smooth muscle cells in the walls of ovarian follicles in the Japanese quail

Summary The walls of pre-ovulatory follicles of the Japanese quail were examined at the ultrastructural level for the presence of cells displaying the typical morphological features of smooth muscle cells. These characteristics were found in the cells of the chordae, the tunica albuginea, and the theca externa. Small, elongated cells, containing microfilaments, were observed in the theca of prelampbrush follicles localized in the ovarian cortex. These thecal cells were considered as the putative precursors of the thecal smooth muscle cells of the pre-ovulatory follicle. The difference between the smooth muscle cells of the pre-ovulatory follicle and those in the wall of the most recent post-ovulatory follicle is the contracted state of the latter, which is most evident in the cells of the theca externa. It can be concluded that the cells of the theca externa are smooth muscle cells which are mainly contracted during the ovulatory process. A comparison was made with other vertebrate species.     

Functional morphology of the pars intermedia of the rat hypophysis as revealed with the electron microscope

Summary The pars intermedia of the hypophysis of normal and experimental rats was studied by electron microscopy. Observations of the hypophysis at various intervals following formalin induced stress or adrenalectomy indicate the existence of a functional relationship between the posterior lobe, the pars intermedia, and the adrenal cortex.Glandular cells of the normal pars intermedia are divided into two types, i. e., the light and dark cells. The former type dominates in number and is characterized by a large amount of cytoplasm filled with clear vesicles 250–350 m in diameter. Dark secretory granules smaller than 300 m are few in number and restricted to the Golgi region.After a single injection of formalin, the clear vesicles of the light cell dimmish and dark secretory granules varying in opacity increase in number. Transition from dark granules to clear vesicles is suggested. Three to five days after adrenalectomy, the light cells contain an abundance of moderately dense vesicles which are smaller than the larger more electron lucent vesicles of the normal light cells. The moderately dense vesicles are about 200 m in diameter and are extremely abundant filling the entire cytoplasm of the light cells 7 days after adrenalectomy.Bundles of unmyelinated nerve fibers are often observed in the pars intermedia, and a typical neuroglandular synapse was found in the pars intermedia of a sham-operated animal suggesting neural control of the secretion process of pars intermedia cells.The author wishes to express his hearty thanks to Dr. K. Kurosumi for his guidance throughout this work.     

Myosin subfragment binding for the localization of actin-like microfilaments in cultured cells. A light and electron microscope study

Fluorescein-labeled heavy meromyosin subfragment-1 (F-S-1) has been purified by ion exchange chromatography and characterized in terms of its ability to bind specifically to actin. F-S-1 activates the Mg++-adenosine triphosphatase activity of rabbit skeletal muscle actin and decorates actin as shown by negative stains and thin sections of rabbit actin and rat embryo cell microfilament bundles, respectively. Binding of F-S-1 to cellular structures is prevented by pyrophosphate and by competition with excess unlabeled S-1. The F-S-1 is used in light microscope studies to determine the distribution of actin-containing structures in wnterphase and mitotic rat embryo and rat kangaroo cells. Interphase cells display the familiar pattern of fluorescent stress fibers. Chromosome-to-pole fibers are fluorescent in mitotic cells. The glycerol extraction procedures employed provide an opportunity to examine cells prepared in an identical manner by light and electron microscopy. The latter technique reveals that actin-like microfilaments are identifiable in spindles of glycerinated cells before and after addition of S-1 or HMM. In some cases, microfilaments appear to be closely associated with spindle microtubles. Comparison of the light and electron microscope results aids in the evaluation of the fluorescent myosin fragment technique and provides further evidence for possible structural and functional roles of actin in the mitotic apparatus.