Collagen Columns Research Articles

Characterization of the Column and Autocellular Junctions That Define the Vasculature of Gill Lamellae

Novel adhesion junctions have been characterized that are formed at the interface between pillar cells and collagen columns, both of which are essential constituents of the gill lamellae in fish. We termed these junctions the "column junction" and "autocellular junction" and determined their molecular compositions by immunofluorescence microscopy using pufferfish. We visualized collagen columns by concanavalin A staining and found that the components of integrin-mediated cell-matrix adhesion, such as talin, vinculin, paxillin, and fibronectin, were concentrated on plasma membranes surrounding collagen columns (column membranes). This connection is analogous to the focal adhesion of cultured mammalian cells, dense plaque of smooth muscle cells, and myotendinous junction of skeletal muscle cells. We named this connection the "column junction." In the cytoplasm near the column, actin fibers, actinin, and a phosphorylated myosin light chain of 20 kDa are densely located, suggesting the contractile nature of pillar cells. The membrane infoldings surrounding the collagen columns were found to be connected by the autocellular junction, whose components are highly tyrosine-phosphorylated and contain the tight junction protein ZO-1. This study represents the first molecular characterization and fluorescence visualization of the column and autocellular junctions involved in both maintaining structural integrity and the hemodynamics of the branchial lamellae.

Fluorescence Visualization of Branchial Collagen Columns Embraced by Pillar Cells

A collagen column is a structure of the extracellular matrix that helps to maintain the flatness and width of gill lamella. Collagen columns are unique in that they are enfolded by plasma membrane of pillar cells that form two-dimensional vascular networks between parallel sheets of respiratory epithelia. Despite their unique structure and fundamental importance in the physiology of aquatic animals, little is known about their properties and molecular components, owing to the lack of detection methods. In this study we demonstrated that collagen columns can be visualized by staining with fluorescence-labeled concanavalin A (ConA), a lectin that specifically recognizes the trimannoside core of N-glycosylated proteins and histidine-tagged green fluorescent protein (His(6)-Xpress-GFP), a fluorescent substrate for transglutaminase. We constructed a three-dimensional image of a pillar cell and visualized the spatial relationship between collagen columns and contractile apparatuses within the pillar cell body. This manuscript contains online supplemental material at (www.jhc.org). Please visit this article online to view these materials.

FHL5, a novel actin-binding protein, is highly expressed in eel gill pillar cells and responds to wall tension.

Supporting evidence for the contractile nature of fish branchial pillar cells was provided by demonstrating the presence of actin fibers and a novel four-and-a-half LIM (FHL) protein in which expression is specific for contractile tissues and sensitive to the tension applied to the pillar cell. When eel gill sections were stained with rhodamine-phalloidin, a selective fluorescent probe for fibrous actin, a strong bundle-like staining was observed around collagen columns in pillar cells, suggesting the presence of abundant actin fibers. A cDNA clone encoding a novel member of the actin-binding FHL family, FHL5, was isolated from a subtracted cDNA library of eel gill. Northern analysis revealed that FHL5 mRNA is highly expressed only in gills, heart, and skeletal muscle. In gills, FHL5 was found to be confined to pillar cells by immunohistochemistry. Confocal fluorescence microscopy showed that FHL5 is present in both cytosol and nucleus; within the cytosol, a large portion of FHL5 is colocalized with the phalloidin-positive actin bundles. Furthermore, transfection of myogenic C2C12 cells with FHL5 cDNA demonstrated, in addition to its interaction with actin stress fibers, a nuclear shuttling activity of FHL5. The mRNA and protein levels were found to be elevated on 1) transfer of eels from seawater to freshwater, 2) volume expansion by infusion of isotonic dextran-saline, and 3) constriction of gill vasculature by bolus injection of endothelin-1. These results suggest contractile nature of pillar cells and a role of FHL5 in maintaining the integrity and regulating the dynamics of pillar cells.

A monoclonal antibody recognizing a complex tubular structure in rainbow trout pillar cells

Pillar cells are important to maintain the structural integrity of the fish gills. This study describes a new monoclonal antibody (mab 25) which detects a complex tubular structure of rainbow trout Oncorhynchus mykiss gill pillar cells. This mab was selected and established after immunization of mice with density gradient separated trout mononuclear cells. In primary screening using a cell enzyme‐linked immunoabsorbant assay (ELISA) the corresponding hybridoma supernatant reacted with viable and fixed trout leukocytes. In immunofluorescence analysis mab 25 showed a distinct reaction with a prominent snow chain like tubular structure in pillar cells. It consists of seven to 11 concave turned tubuli of about 5 μm length forming a circle of about 7 μm. Reactivity of mab 25 with fibrous structures was observed in cryostat sections of gill, pseudobranchia, spleen, striated muscle and heart muscle. mab 25 recognized a protein with a molecular weight of about 58 kDa as determined by radioimmunoprecipitation. This structure appears to be related to elements which have been described as membrane enclosed collagen columns by electronmicroscopic analysis and is reminiscent of patterns labelled with anti‐human cytokeratin antibodies. mab 25 may be an important tool to study the impact of environmental and physiological conditions on the structural integrity of this complex structure in trout gill pillar cells.

Collagen immobilised on silica derivatives as a new stationary phase for HPLC.

Synthesis of new stationary phases containing covalently bound collagen has been described. Commercially available soluble collagen and the silica derivatives, aminopropylsilica (APS) and diol-silica, were used for the experiment. The products of synthesis were subjected to elemental analysis and to a 13C-NMR analysis to prove the presence of the covalently bound protein on the support's surface. The stationary phases were packed into the columns and introduced into an HPLC system. A series of diversified test compounds was analysed to elucidate the retention mechanism operating on the collagen column by means of the quantitative structure-retention relationship (QSRR) analysis. Chromatographic analysis of a series of selected compounds was performed for which human skin permeation data were available. Quantitative structure-activity relationship (QSAR) analysis was done to model the human skin permeation by employing of the chromatographic data determined on the collagen column. The newly obtained collagen phases were demonstrated to possess distinctive retention properties due to a combination of specific (polar) and hydrophobic solute-stationary phase interactions. The normal-phase retention mechanism seemed to prevail on the collagen phases. For the set of test solutes available the interactions with collagen appear to be of secondary importance for their ability to permeate human skin compared to their hydrophobicity and binding to keratin. None the less, the collagen columns may be of value to complete the chromatographic model of human skin permeation.

Catch in the Primary Spines of the Sea Urchin Eucidaris tribuloides: A Brief Review and a New Interpretation

Previous models of reversible catch in echinoid spines, as a property of muscle or of collagen, are briefly reviewed and discussed. This brief review offers a new interpretation of catch in primary spines of Eucidaris tribuloides, viewing the collagen and small muscles of the catch ligament working together as a variable-length tendon. In the model presented, changes in ligament length when out of catch are accommodated by sliding of discontinuous, interdigitating and cross-link-stabilized columns of collagen fibrils, the muscle layer external to the ligament effecting spine movement. Catch is viewed as a consequence of contraction of small muscles inserted on the collagen columns within the ligament. Ligament shortening tightens the profuse (ca. 30,000/mm2) and highly ordered collagen insertion loops within the stereoms of the spine base and test, and catch results from the multiplicative effect of these friction sites in series. New data are presented on novel structural cross-links between collagen fibrils. The cross-links stabilize the ligament columns. The central ligament in Eucidaris plays a purely passive mechanical role in maintaining the alignment of the spine-test articulation. It contains no muscle and neither contracts nor undergoes catch; its insertions are simple, unlike the complex stereom insertions of the main ligament.

Gill ultrastructure of the Pacific hagfish Eptatretus stouti.

At the gross anatomical level, hagfish gills show unusual features not seen in any other fish gills. Our study was undertaken to determine if peculiarities also characterize the microscopic anatomy and ultrastructure of hagfish gills. To the contrary, branchial respiratory lamellae of Pacific hagfish were found to resemble the lamellae of lampreys, elasmobranchs, and teleosts, often down to the finest subcellular details. As in other fish, hagfish lamellae are lined by epithelium containing pavement cells with organelles indicative of a secretory function, basal cells showing undifferentiated cell features, and branchial ionocytes. The ionocytes are identical to chloride cells of teleosts in cytostructure, distribution, and abundance. There are pillar and marginal capillaries in hagfish gill lamellae. Pillar cells contain bundles of 5-nm microfilaments, and they associate with collagen columns as in other fish. Hagfish pillar cells do exhibit odd features, however: They cluster (groups of up to nine were seen), and their extracellular collagen columns are rarer than in other fish gills (averaging only two columns per three pillar cells). Other special features of hagfish gills are the following: lipid droplets and smooth endoplasmic reticulum are well developed in all cell types; pavement cells secrete a lipomucous product (stains with periodic acid-Schiff, Alcian blue, and Sudan black B); and goblet cells are absent. The presence of "chloride cells" in hagfish is puzzling, as hagfish body fluids are iso-osmotic to seawater and there is no need to osmoregulate for sodium chloride; the ionocytes contain carbonic anhydrase, suggesting a function in acid/base regulation.

Correlation between cell substrate attachment in vitro and cell surface heparan sulfate affinity for fibronectin and collagen.

Heparan sulfate glycosaminoglycan, isolated from the cell surface of nonadhering murine myeloma cells (P3X63-Ag8653), does not bind to plasma fibronectin, but binds partially to collagen type I, as assayed by affinity chromatography with proteins immobilized on cyanogen bromide-activated Sepharose 4B. Identical results were obtained when myeloma heparan sulfate was cochromatographed, on the same fibronectin and collagen columns, with cell surface heparan sulfates collagen columns, with cell surface heparan sulfates from adhering Swiss mouse 3T3 and SV3T3 cells. These latter heparan sulfates do, however, bind to both fibronectin and collagen, as reported earlier (Stamatoglou, S.C., and J.M. Keller, 1981, Biochim. Biophys. Acta., 719:90-97). Cell adhesion assays established that hydrated collagen substrata can support myeloma cell attachment, but fibronectin cannot. Saturation of the heparan sulfate binding sites on the collagen substrata with heparan sulfate or heparin, prior to cell inoculation, abolished the ability to support cell adhesion, whereas chondroitin 4 sulfate, chondroitin 6 sulfate, and hyaluronic acid had no effect.

Molecular distribution within collagen gel columns

Purified collagen columns with collagen concentrations of 5 and 10% were constructed by modification of the procedure for the 1% gel as described by Shaw and Schy. The resulting columns were calibrated by elution of various tracers, and the observations compared with those of teh 1% collagen gel column. The data were fitted to the Ogston gel model as formulated by Laurent and Killander, but the model was found to be not fully applicable. The thermodynamic treatment of Hjertén was applied to the date with more satisfactory agreement.

Morphology of the gills of larval and parasitic adult sea lamprey, Petromyzon marinus L.

The general morphology of the gills is similar in larval (ammocoetes) and parasitic adult sea lampreys, Petromyzon marinus, despite different methods of ventilation necessitated by their feeding habits. The gill lamellae are supported by randomly-distributed pillar cells which enclose blood spaces and collagen columns. The distribution of these cells in lampreys is different from that of higher fishes and it may be inefficient for respiratory exchange. The presence of cytoplasmic microfilaments suggests that these cells have the ability to reduce the lamellar blood spaces through contraction. Marginal channels at the tips of the lamellae are lined only by endothelial cells. The thickness of the water-blood pathway in lampreys falls within the range described for higher fishes, with the most efficient gas exchange likely occuring at the lamellar tips where only a single layer of epithelial cells is present. The abrupt increase in height of the epithelium near the lamellar bases in adults, compared to the gradual transition in height along the lamellae in ammocoetes, is perhaps reflective of higher oxygen requirements during the parasitic stage. The consistent appearance of wide, lateral intercellular spaces within the respiratory epithelium of lampreys indicates possible involvement of these spaces in transport. Mucous secretion appears to be an important function of the superficial platelet cells in ammocoetes. "Mitochondria-rich" and "mitochondria-poor" superficial cells are observed in both ammocoetes and adults, with the mitochondria-rich cells more prevalent toward the lamellar bases. The possibility that at least some of these cells may be involved in absorption is discussed. Mitochondria-rich cells in the interlamellar region are morphologically different in ammocoetes and adults but all possess an abundance of smooth endoplasmic reticulum and hence resemble "chloride cells" of higher fishes. The similarity of these cells in the parasitic adult lamprey to chloride cells of marine fishes may reflect the potential of the adult lamprey to osmoregulate in salt water. A scarcity of these cells in ammocoetes and their resemblance to chloride cells in freshwater fishes may reflect the restriction of larval lampreys to a freshwater habitat.

Contractile filamentous material in the pillar cells of fish gills.

ABSTRACT The secondary lamellae of the gill filaments are the sites of gas exchange in fish gills. They are mainly composed of 2 epithelial sheets joined together by pillar cells. These cells are characterized by collagen columns contained in infoldings of the cell membrane and oriented perpendicular to the epithelial sheets. The gill is the first organ to which the blood flows from the heart and within the secondary lamellae it flows through channels between the pillar cells. The presence in the pillar cells of fine cytoplasmic filaments situated parallel to the collagen columns has now been observed in many fishes and the hypothesis has been advanced that they constitute a contractile system. This paper describes how gill filaments were treated in a way similar to that used for other non-muscular cells in order to demonstrate in situ the presence of contractile proteins of the actomyosin type. Gill filaments were extracted in glycerol-containing solutions of low ionic strength, and then incubated with and without ATP. After incubation with ATP examination in the electron microscope showed that in the vicinity of the collagen columns, the pillar cells contain clusters of disordered thin filaments intermingled with spindle-shaped needles. This structure is characteristic of muscle actomyosin, as well as actomyosin-like proteins extracted from cells of non-muscular origin and fixed in a contracted state. It is deduced that the thin cytoplasmic filaments surrounding the collagen columns consist of an actomyosin-like con-tractile protein. On incubation of extracted gill filaments without ATP, the expected negativeresult was obtained, i.e. the filaments retain their orientation parallel to the columns. The function of the contractile filaments within the pillar cells is discussed in relation to the control of blood flow through the secondary lamellae and the reduction of pressure drop during the flow of blood across the gills of fish.