Tropomyosin assembly intermediates in the control of microfilament system turnover

Tropomyosin is a coiled-coil alpha-helical protein, which self-associates in a head-to-tail fashion along polymers of actin to produce thin filaments. Mammalian non-muscle cells express a large number of tropomyosin isoforms, which are differentially regulated during embryogenesis and associated with specialized actin microfilament ensembles in cells. The function of tropomyosin in specifying form and localization of these subcellular structures, and the precise mechanism(s) by which they carry out their functions, is unclear. This paper reports that, while the major fraction of non-muscle cell tropomyosin resides in actin thin filaments of the cytomatrix, the soluble part of the cytoplasm contains tropomyosins in the form of actin-free multimers, which are isoform specific and of high molecular weight (MW(app) 180,000-250,000). Stimulation of motile cells with growth factors induces a rapid, actin polymerization-dependent outgrowth of lamellipodia and filopodia. Concomitantly, the levels of tropomyosin isoform-specific multimers decrease, suggesting their involvement in actin thin filament formation. Malignant tumor cells have drastically altered levels and composition of tropomyosin isoform-specific multimers as well as tropomyosin in the cytomatrix.     

High-voltage electron microscopy of whole,critical-point dried plant cells

Summary The lower epidermis ofSelaginella Helvetica leaves has numerous chloroplasts. In the diffuse light of the plant's normal habitat these are distributed over the inner wall of the cell, while in bright sunlight they move to the lateral walls. High voltage electron microscopy of whole critical-point dried cells shows that in the diffuse-light position the chloroplasts are connected by bundles of tightly-packed parallel filaments; these are distinct from, but seem to interconnect with, the filaments of the cytomatrix. In thin sections these appear as conventional microfilament bundles, while staining with rhodamineconjugated phalloidin implies that they are composed of actin. In bright light, when the chloroplasts have moved to the lateral walls, these microfilament bundles completely disappear, while filaments of the cytomatrix system remain attached to the chloroplasts. These results suggest that the function of the microfilament bundles may be to anchor the chloroplasts as much as to move them, and that the cytomatrix system may play a part in the movement; it is possible that actin microfilament bundles may actually dissociate into separate filaments within the cytomatrix. Staining of cryo-sections with FITC-labelled antitubulin reveals a typical cortical pattern of microtubules which appears to play no part in chloroplast motility.Abbreviations EDTA ethylenediaminetetra-acetic acid - EM electron microscopy - FITC fluorescein-iso-thiocyanate - HVEM high voltage electron microscopy - PIPES piperazine-NN-bis-2-ethanesulphonic acid     

Isolation and monolayer culture of guinea pig pancreatic duct epithelial cells

Summary Monolayers of cultured epithelial cells have been prepared from fragments of guinea pig pancreatic excretory ducts isolated by a simple procedure employing collagenase digestion and manual selection, through which virtually all of the ductal system can be recovered. The isolated fragments were cultured in enriched Waymouth's medium on extracellular matrices of various composition and thickness, including: thin (<5 μm) and thick (0.5 mm) layers of rat tail collagen; thin layers of human placental collagen; thin layers of Matrigel (a reconstituted basement membrane material); uncoated tissue culture plastic; and the cellulose ester membranes of Millipore Millicells. Cells spread rapidly from duct fragments cultured on uncoated plastic or on plastic coated with thin layers of rat tail collagen or human placental collagen and formed epithelial monolayers. However, these cells were squamous and lacked the abundant basolateral membrane amplification and apical microvilli characteristic of freshly isolated duct epithelial cells. Cells did not spread from duct fragments cultured on Matrigel. In contrast, when fragments of pancreatic ducts were explanted onto either a thick layer of rat tail collagen or onto Millicell membranes, cells readily spread and formed confluent monolayers of cuboidal epithelial cells characterized by abundant mitochondria, apical microvilli, and basolateral plasma membrane elaboration. These results demonstrate that different forms of extracellular matrix modulate the growth and differentiation of pancreatic duct epithelial cells, and that culture on a permeable substrate markedly enhances the maintenance of differentiated characteristics in this cell type. The monolayers formed on Millicell membranes should provide a useful model system for physiologic analysis of the regulation of electrolyte secretion by this epithelium. This research was supported by grants DK32994 and DK35912 from the National Institutes of Health, Bethesda, MD.     

中国芸香科植物叶分泌囊比较解剖学研究

利用整体透明、石腊和薄切片方法对芸香科22属,40种和2变种植物叶分泌囊的形态结构和分 布进行了比较研究。成熟分泌囊都由鞘细胞和一层上皮细胞围绕圆形腔隙构成,上皮细胞扁平,细胞壁 薄、完整,故分泌囊属裂生方式发生。鞘细胞1～5层,不同种类的层数有变化,个别种缺乏。内层鞘细 胞为扁平的薄壁细胞,外层的细胞壁较厚。分泌囊的形态结构、着生位置和分布密度等在不同属或不同 种间存在一定差异。根据分泌囊在叶中的分布位置和形态结构特点,可将其划分为:叶缘齿缝分泌囊, 叶肉分泌囊和两者混合型。叶肉分泌囊又可分海绵组织分泌囊和栅栏组织分泌囊。在此基础上对该科各类型分泌囊的形态演化关系以及各亚科或各属间的亲缘关系进行了探讨。     

Comparative aspects of structural organization of astrocytes of the first layer of the human and rat cerebral cortex

Importance of study of astrocytes for fundamental biology and medicine is due their key role in formation of the brain barrier system. On taking into consideration the controversial data on structure of the mammalian neocortex superficial layers, of great actuality are the comparative studies of the structural and cytochemical organization of astrocytes in human and in the laboratory animals used in the experimental studies connected with modeling of brain diseases and traumas. The goal of the present work was to study structural organization of astrocytes in the human and rat neocortical layer I. The work was on the autopsy and experimental material from Wistar rats. Astrocytes were revealed immunocytochemically by using antibodies to GFAP, vimentin and nestin. The preparations were examined with aid of light and confocal laser microscopy. No significant difference in the sizes of perinuclear areas were established between the rat and human astrocytes. In the majority of cortex regions, the specter of proteins forming intermediate filaments in these cells was identical. However, there were essential differences revealed in organization of the superficial glial bordering lamina (SGBL). The human SGBL is formed by interlacing of thin processes in the layer I processes, whereas the rat SGBL is represented by specialized astrocytes spread along the cortical surface and connected with the wide-blade processes. The human layer I astrocytes have translaminar processes passing via several cortical layers, whereas in rats such processes are located within the limits of one layer. The revealed differences in the astrocyte structural organization should be taken into account when interpreting results of experimental studies carried out on rats and extrapolating these results to human.     

Enhanced Stability and Thickness‐Independent Oxygen Evolution Electrocatalysis of Heterostructured Anodes with Buried Epitaxial Bilayers

Achieving high oxygen evolution reaction (OER) activity while maintaining performance stability is a key challenge for designing perovskite structure oxide OER catalysts, which are often unstable in alkaline environments transforming into an amorphous phase. While the chemical and structural transformation occurring during electrolysis at the electrolyte–catalyst interface is now regarded as a crucial factor influencing OER activity, here, using La_0.7Sr_0.3CoO_3?δ (LSCO) as an active OER catalyst, the critical influence of buried layers on the oxidation current stability in nanoscopically thin, chemically and structurally evolving, catalyst layers is revealed. The use of epitaxial thin films is demonstrated to engineer both depletion layer widths and chemical stability of the catalyst support structure resulting in heterostructured anodes that maintain facile transport kinetics across the electrolyte–anode interface for atomically thin (2–3 unit cells) LSCO catalyst layers and greatly enhanced oxidation current stability as the perovskite structure OER catalysts chemically and structurally transform. This work opens up an approach to design robust and active heterostructured anodes with dynamically evolving ultrathin OER electrocatalyst layers for future green fuel technologies such as conformal coatings of high‐density 3D anode topologies for water splitting.     

Cell separation in the buffy coat

One of the most rapid methods to determine cell counts in whole blood is by way of layer thickness measurements of the buffy coat. The purpose of this study was to determine the separation and purity of blood cells in the different layers of the buffy coat. Blood samples were centrifuged at 10,000 g in microhematocrit tubes with an inserted float to expand the buffy coat region. Whole blood from normal laboratory individuals separates by density into four regions: platelets, a layer of lymphocyte and monocytes, granulocytes and erythrocytes. A thin band of highly swollen red cells was discovered between the buffy coat layers of many normal volunteers and patients. Stereological analysis of electron micrographs showed that mixing of formed elements within the layers is less than 2% with the exception of some erythrocytes, which can make up a higher volume fraction in the lymphocyte/monocyte and granulocyte layers. The red cell column contains about 95.7% erythrocytes and is depleted of platelets and leukocytes. In approximately 5% of hospital blood samples, the granulocyte-erythrocyte interface was feathered and undetectable, and a significantly higher volume fraction of red cells was found among the granulocytes. Cell mass density determinations indicate that the erythrocytes in these abnormal granulocyte layers have a lowered mass density, overlapping with that of the granulocytes.     

Surface arrays on the cell wall of Spirillum metamorphum.

A complex and easily disrupted arrangement of macromolecules was present on the outer (lipopolysaccharide) membrane of the cell wall of Spirillum metamorphum. Separation of the arrays from the cell and spontaneous reassembly into regularly structured complexes usually occurred during preparation for electron microscopy. Freeze etchings, thin sections, and optical diffraction analysis of negatively stained fragments indicated that they consisted of two sets of a thin layer which was studied with 3-nm particles arranged in a loose (OL). The OSL consisted of a hexagonal arrangement of 8-nm disks and the OL of a thin layer which was studied with 3-nm particles arranged in a loose rectangular manner. The OSL of reassembled fragments displayed numerous broken delta-linkers between units and a center-to-center spacing of half the expected distance, which suggests that an interdigitation of two OSL arrays had occurred. The observations combined with freeze etchings and thin sections of whole cells suggested a possible reassembly mechanism. The normal surface arrangement of these layers on cells was thought to consist of the OL overlying one set of OSL which was loosely adherent to a thin amorphous backing layer.     

Ultrastructural localization of lectin-binding sites in different basement membranes

Summary In the present work we localized binding sites for the lectins WGA, RCA I, con A and SBA at the ultrastructural levels in morphologically different basement membranes. These different basement membranes included (a) thin ones, for example, tubular basement membrane of the mouse kidney which separates epithelial cell layers from mesenchymal cells and glomerular basement membrane which separates epithelial cells from other epithelial cells, (b) thick multilayered ones, for example, Reichert's membrane which is built up during the embryonic development of rodents and as an example of a pathologically thickened basement membrane, the basement membrane of the Engelbreth-Holm-Swarm (EHS) sarcoma. We were able to show that, in contrast to the thick multilayered basement membranes, the thin ones showed a strong positive SBA-binding pattern. Thick basement membranes otherwise revealed very strong labelling with the lectins WGA and RCA I. Our findings lead us to conclude that thin and thick basement membranes differ markedly in the quality and quantity of the carbohydrates which they contain.     

Comparative aspects of structural organization of astrocytes of the layer i of the human and rat brain cortex

Importance of study of astrocytes for fundamental biology and medicine is due to their key role in formation of the brain barrier system. On taking into consideration the controversial data on structure of the mammalian neocortex superficial layers, of great actuality are the comparative studies of the structural and cytochemical organization of astrocytes in human and in the laboratory animals used in the experimental studies connected with modeling of brain diseases and traumas. The goal of the present work was to study structural organization of astrocytes in the human and rat neocortical layer I. The work was carried out on the autopsy and experimental material from Wistar rats. Astrocytes were revealed immunocytochemically by using antibodies to GFAP, vimentin and nestin. The preparations were examined with aid of light and confocal laser microscopy. No significant difference in the sizes of perinuclear areas were established between the rat and human astrocytes. In the majority of cortex regions, the spectrum of intermediate filaments-forming proteins in these cells was identical. However, there were essential differences revealed in organization of the superficial glial limiting membrane (SGLM). The human SGLM is formed by interlacing of thin processes in the layer I processes, whereas the rat SGLM is represented by specialized astrocytes spread along the cortical surface and connected with the wide-blade processes. The human layer I astrocytes have translaminar processes passing via several cortical layers, whereas in rats such processes are located within the limits of one layer. The revealed differences in the astrocyte structural organization should be taken into account when interpreting results of experimental studies carried out on rats and extrapolating these results to human.     

Sperm chemotaxis in marine species is optimal at physiological flow rates according theory of filament surfing

Sperm of marine invertebrates have to find eggs cells in the ocean. Turbulent flows mix sperm and egg cells up to the millimeter scale; below this, active swimming and chemotaxis become important. Previous work addressed either turbulent mixing or chemotaxis in still water. Here, we present a general theory of sperm chemotaxis inside the smallest eddies of turbulent flow, where signaling molecules released by egg cells are spread into thin concentration filaments. Sperm cells ‘surf’ along these filaments towards the egg. External flows make filaments longer, but also thinner. These opposing effects set an optimal flow strength. The optimum predicted by our theory matches flow measurements in shallow coastal waters. Our theory quantitatively agrees with two previous fertilization experiments in Taylor-Couette chambers and provides a mechanistic understanding of these early experiments. ‘Surfing along concentration filaments’ could be a paradigm for navigation in complex environments in the presence of turbulent flow.     

Alterations in flightin phosphorylation inDrosophila flight muscles are associated with myofibrillar defects engendered by actin and myosin heavy-chain mutant alleles

Flightin is a 20-kD myofibrillar protein found in the stretch-activated flight muscles ofDrosophila melanogaster. Nine of the eleven isoelectric variants of flightin are generatedin vivo by multiple phosphorylations. The accumulation of these isoelectric variants is affected differently by mutations that eliminate thick filaments or thin filaments. Mutations in the myosin heavy-chain gene that prevent thick filament assembly block accumulation of all flightin variants except N1, the unphosphorylated precursor, which is present at much reduced levels. Mutations in the flight muscle-specific actin gene that block actin synthesis and prevent thin filament assembly disrupt the temporal regulation of flightin phosphorylation, resulting in premature phosphorylation and premature accumulation of flightin phosphovariants. Cellular fractionation of fibers that are devoid of thin filaments show that flightin remains associated with the thick filamentrich cytomatrix. These results suggest that flightin is a structural component of the thick filaments whose regulated phosphorylation is dependent upon the presence of thin filaments.This work was supported by National Science Foundation Grant IBN-9253045.     

Giant reticulospinal synapse in lamprey: molecular links between active and periactive zones

Deciphering the function of synaptic release sites is central to understanding neuronal communication. Here, we review studies of the lamprey giant reticulospinal synapse, a model that can be used to dissect synaptic vesicle trafficking at single release sites. The presynaptic axon is large and contains active zones that are spatially separated from each other. During activity, synaptic vesicle membrane is shuttled between the active zone and the periactive zone at which endocytosis occurs. Recent studies have shown that the periactive zone contains an actin-rich cytomatrix that expands during synaptic activity. This cytomatrix has been implicated in multiple functions that include (1) activity-dependent trafficking of proteins between the synaptic vesicle cluster and the periactive zone, (2) synaptic vesicle endocytosis, and (3) the movement of newly formed synaptic vesicles to the vesicle cluster. The actin cytomatrix thus provides a link between the active zone and the periactive zone; this link appears to be critical for sustained cycling of synaptic vesicles.This work was supported by Swedish Research Council grants (K2004-33X-11287-10A, LB; K2005-32X-13473-06A, OS).     

ELECTRON MICROSCOPY OF THE AVIAN RENAL GLOMERULUS

Electron microscopy of sections of chicken glomeruli shows them to possess a large central cell mass, occupying the hilum and the centre of the glomerulus, and continuous with the adventitia of the afferent and efferent arterioles. The glomerular capillaries form a much simpler system than in mammals and are spread over the surface of the central cell mass. Between the capillaries the mass is limited externally by the major component of the glomerular capillary basement membrane, which continues over the surface of the mass from one capillary to the next. Projections of the central cell mass characteristically form the support for glomerular capillaries, and smaller knobs of the central mass may project actually into the lumen of the capillaries, but always carry a layer of endothelial cytoplasm before them. They are never in direct contact with blood. The basement membrane of the glomerular capillary loop has a central dense layer and two lateral less dense layers as in mammals. The central dense layer is continuous with similar appearing dense material in the intercellular spaces of the adventitiae of the arterioles, and also with that of the central cell mass. The two less dense layers can also be traced into direct continuity with the less dense regions of this intercellular substance. The endothelial cytoplasm is spread as a thin sheet over the inner surface of the capillary basement membrane, and shows scattered "pores" resembling those described in mammals. Epithelial cells with interlacing pedicels are at least as prominent as those in mammals. Bowman's capsular membrane also possesses three layers similar to but less wide than those of the capillary basement membrane, and all three layers can be traced into continuity with the dark and light regions of the intercellular material of the adventitial cells of the arterioles, and beyond them with that of the central cell mass. At the hilum Bowman's capsular membrane also fuses with the capillary basement membrane.     

Morphogenesis of the antenna of the male silkmoth, Antheraea polyphemus. I. The leaf-shaped antenna of the pupa from diapause to apolysis

The antenna of the male silkmoth Antheraea polyphemus is a featherlike structure consisting of a central stem and ca. 120 side branches, which altogether carry about 70,000 olfactory sensilla. We investigate the development during the pupal phase. At the end of diapause, the antennal rudiment consists of a leaf-shaped, one-layered epidermal sac. It is supplied with oxygen via a central main trachea, which gives off numerous thin side branches. These are segmentally arranged into bundles which run to the periphery of the antennal blade. When the epidermis retracts from the pupal cuticle (apolysis; stage 1), it consists of cells which are morphologically uniform. The epidermal cells form a network of long, irregular basal protrusions (epidermal feet), which crisscross the antennal lumen. During the first day post-apolysis (stage 2), the antennal epidermis differentiates into alternating thick 'sensillogenic' and thin 'non-sensillogenic' areas arranged in stripes which run in parallel to the tracheal bundles. Numerous dark, elongated cells, which might be the sensillar stem cells, are scattered in the sensillogenic epithelium. A number of very early sensilla has been found at the distal edges of the sensillogenic stripes in positions which later will be occupied by sensilla chaetica. The whole antennal blade is enveloped by the transparent ecdysial membrane, consisting of the innermost layers of the pupal cuticle which are detached during apolysis.     

Thermo-responsive PNiPAAm-g-PEG films for controlled cell detachment

A series of graft copolymers consisting of either poly(N-isopropylacrylamide) (PNiPAAm) or poly(N,N-diethylacrylamide) (PDEAAm) as a thermo-responsive component in the polymer backbone and poly(ethyleneglycol) (PEG) were immobilized as thin films and cross-linked on a fluoropolymer substrate using low-pressure argon plasma treatment. The surface-immobilized hydrogels exhibit a transition from partially collapsed to completely swollen, which is in the range of 32-35 degrees C and corresponds to the lower critical solution temperature of the soluble polymers. The hydrogels were used as cell carriers in culture experiments with L929 mouse fibroblast cells to probe for cell adhesion, proliferation, and temperature-dependent detachment of cell layers. The fibroblast cells adhere, spread, and proliferate on the hydrogel layers at 37 degrees C and become completely detached after reducing the temperature by 3 K. The cell release characteristics were further correlated to the swelling and collapsing behavior of the hydrogel films and the polymer solutions as measured in PBS solution and RPMI cell cultivation medium. It could be shown that, long before the swelling has completed upon temperature reduction, the cells detach. This can be attributed to the large content of PEG present in the hydrogel, which weaken the cell adhesion strength to the hydrogel layers.     

Visualization of the three-dimensional distribution of collagen fibrils over preovulatory follicles in the hamster

Before an oocyte can escape from a preovulatory follicle, the apical wall must thin to the point of rupture. Although numerous layers of cells are present, it is the collagen fibrils in the theca externa that provide most of the strength to the developing follicle. The three-dimensional distribution and integrity of these fibrils over a follicle cannot be appreciated with standard used methods such as examination of thin sections by transmission electron microscopy In this paper we describe a technique that removes cells superficial to the collagen fibrils so that their distribution may be examined by scanning electron microscopy. On the third day of the hamster's 4-day estrous cycle, bundles of fibrils pass from intrafollicular areas and ascend follicles. Approximately halfway up the follicle wall, the bundles fan out and form a meshwork of fibrils which covers the apex. As the time of ovulation approaches, the number of layers of fibrils decreases over the apex until a tear forms in the weakened matrix. Experimental results demonstrating that the meshwork is composed of collagen fibrils are presented. The usefulness of this technique in visualizing the collagen content in preovulatory follicles is discussed as well as factors that may aid in weakening this layer so that follicle rupture may occur.     

Assembly of active zone precursor vesicles: obligatory trafficking of presynaptic cytomatrix proteins Bassoon and Piccolo via a trans-Golgi compartment

Neurotransmitter release from presynaptic nerve terminals is restricted to specialized areas of the plasma membrane, so-called active zones. Active zones are characterized by a network of cytoplasmic scaffolding proteins involved in active zone generation and synaptic transmission. To analyze the modes of biogenesis of this cytomatrix, we asked how Bassoon and Piccolo, two prototypic active zone cytomatrix molecules, are delivered to nascent synapses. Although these proteins may be transported via vesicles, little is known about the importance of a vesicular pathway and about molecular determinants of cytomatrix molecule trafficking. We found that Bassoon and Piccolo co-localize with markers of the trans-Golgi network in cultured neurons. Impairing vesicle exit from the Golgi complex, either using brefeldin A, recombinant proteins, or a low temperature block, prevented transport of Bassoon out of the soma. Deleting a newly identified Golgi-binding region of Bassoon impaired subcellular targeting of recombinant Bassoon. Overexpressing this region to specifically block Golgi binding of the endogenous protein reduced the concentration of Bassoon at synapses. These results suggest that, during the period of bulk synaptogenesis, a primordial cytomatrix assembles in a trans-Golgi compartment. They further indicate that transport via Golgi-derived vesicles is essential for delivery of cytomatrix proteins to the synapse. Paradigmatically this establishes Golgi transit as an obligatory step for subcellular trafficking of distinct cytoplasmic scaffolding proteins.     

The micromorphology of the nauplius eye of the estuarine calanoid copepod, Sulcanus conflictus Nicholls (Crustacea)

The nauplius eye consists of one median and two lateral ocelli, each within a pigment cup. The three pigment cups are made up from two multi-nucleate pigment cells: each cell forming one lateral cup and half of the median cup. The three cups are lined on the insides by tapetal cells which contain layers of reflectile crystals. Each of the ocelli contains six sensory cells which protrude from the rims of the pigment cups and the protruding parts are sheathed by the conjunctiva cells. The whole eye is enveloped by a thin membrane which also sheaths the proximal parts of the five nerve bundles that leave the eye. All the sensory cells of the lateral ocelli are similar and have rhabdomeric microvilli on the terminal end, and contain phaosomes and a multitude of other organelles and cytoplasmic inclusions. The complex median ocellus contains a superior group of three retinular cells, linked by interdigitating processes, and an inferior group consisting of a large central cell enclosed in two cup-shaped peripheral retinular cells. A two-tiered rhabdome arrangement exists, with a rather complex inferior rhabdome set made up of a central rhabdomere and two hemi-annulate rhabdomeres. The cytoplasm of the retinular cells of the median ocellus lack phaosomes but instead contain double-walled tubular elements, possibly formed by the inpushings of microvilli into adjacent cells. The possible functional significance of the unique arrangement seen in the median ocellus is discussed. The retinular cells are of the inverse type. There are no efferent nerve fibres from the brain nor any nervous connection between the lateral and the median ocelli.     

Cytological characteristics of the gonocytes in the course of their migration in early rat embryos]

The work was carried out in 23 rat embryos from 9,5 to the 11th day of development. In 9,5-day embryos the primary sex cells are localized in the mesenchyma of the allantois and in the intestinal entoderm. Later they migrate either with the blood flow or on the surface of cellular layers towards gonad germs which are reached by the 11th day of the intrauterine development. In the course of this process there occur structural and cytochemical changes in gonocyte nuclei. The nucleolus is replaced to the periphery of the nucleus, around it there appears a rim intensively stained with methylene green. Chromatin has a shape of thin threads. These changes of the nuclei seem to be associated with an increased synthesis of r-DNA and hence with the synthesis of substances having the role to prevent the somatic differentiation of primary sex cells in the course of migration.