scholarly journals Interactions between actin filaments and between actin filaments and membranes in quick-frozen and deeply etched hair cells of the chick ear.

1982 ◽  
Vol 95 (1) ◽  
pp. 249-261 ◽  
Author(s):  
N Hirokawa ◽  
L G Tilney
Keyword(s):  
Hair Cells ◽  
Actin Filaments ◽  
Apical Cell ◽  
Apical Surface ◽  
Contractile Ring ◽  
Vestibular Organ ◽  
Apical Cell Membrane ◽  
Cuticular Plate ◽  
Quick Freezing ◽  
Two Populations

Replicas of the apical surface of hair cells of the inner ear (vestibular organ) were examined after quick freezing and rotary shadowing. With this technique we illustrate two previously undescribed ways in which the actin filaments in the stereocilia and in the cuticular plate are attached to the plasma membrane. First, in each stereocilium there are threadlike connectors running from the actin filament bundle to the limiting membrane. Second, many of the actin filaments in the cuticular plate are connected to the apical cell membrane by tiny branched connecting units like a "crow's foot." Where these "feet" contact the membrane there is a small swelling. These branched "feet" extend mainly from the ends of the actin filaments but some connect the lateral surfaces of the actin filaments as well. Actin filaments in the cuticular plate are also connected to each other by finer filaments, 3 nm in thickness and 74 +/- 14 nm in length. Interestingly, these 3-nm filaments (which measure 4 nm in replicas) connect actin filaments not only of the same polarity but of opposite polarities as documented by examining replicas of the cuticular plate which had been decorated with subfragment 1 (S1) of myosin. At the apicolateral margins of the cell we find two populations of actin filaments, one just beneath the tight junction as a network, the other at the level of the zonula adherens as a ring. The latter which is quite substantial is composed of actin filaments that run parallel to each other; adjacent filaments often show opposite polarities, as evidenced by S1 decoration. The filaments making up this ring are connected together by the 3-nm connectors. Because of the polarity of the filaments this ring may be a "contractile" ring; the implications of this is discussed.

scholarly journals Preliminary biochemical characterization of the stereocilia and cuticular plate of hair cells of the chick cochlea.

1989 ◽  
Vol 109 (4) ◽  
pp. 1711-1723 ◽  
Author(s):  
M S Tilney ◽  
L G Tilney ◽  
R E Stephens ◽  
C Merte ◽  
D Drenckhahn ◽  
...  
Keyword(s):  
Hair Cells ◽  
Actin Filaments ◽  
Biochemical Characterization ◽  
Cell Types ◽  
Sensory Epithelium ◽  
Tectorial Membrane ◽  
Uncharacterized Protein ◽  
Proteolytic Fragment ◽  
Cuticular Plate ◽  
Filament Bundles

The sensory epithelium of the chick cochlea contains only two cell types, hair cells and supporting cells. We developed methods to rapidly dissect out the sensory epithelium and to prepare a detergent-extracted cytoskeleton. High salt treatment of the cytoskeleton leaves a "hair border", containing actin filament bundles of the stereocilia still attached to the cuticular plate. On SDS-PAGE stained with silver the intact epithelium is seen to contain a large number of bands, the most prominent of which are calbindin and actin. Detergent extraction solubilizes most of the proteins including calbindin. On immunoblots antibodies prepared against fimbrin from chicken intestinal epithelial cells cross react with the 57- and 65-kD bands present in the sensory epithelium and the cytoskeleton. It is probable that the 57-kD is a proteolytic fragment of the 65-kD protein. Preparations of stereocilia attached to the overlying tectorial membrane contain the 57- and 65-kD bands. A 400-kD band is present in the cuticular plate. By immunofluorescence, fimbrin is detected in stereocilia but not in the hair borders after salt extraction. The prominent 125 A transverse stripping pattern characteristic of the actin cross-bridges in a bundle is also absent in hair borders suggesting fimbrin as the component that gives rise to the transverse stripes. Because the actin filaments in the stereocilia of hair borders still remain as compact bundles, albeit very disordered, there must be an additional uncharacterized protein besides fimbrin that cross-links the actin filaments together.

scholarly journals Actin filaments, stereocilia, and hair cells of the bird cochlea. I. Length, number, width, and distribution of stereocilia of each hair cell are related to the position of the hair cell on the cochlea.

1983 ◽  
Vol 96 (3) ◽  
pp. 807-821 ◽  
Author(s):  
L G Tilney ◽  
J C Saunders
Keyword(s):  
Hair Cell ◽  
Surface Area ◽  
Hair Cells ◽  
Actin Filaments ◽  
Hexagonal Lattice ◽  
Frequency Discrimination ◽  
Sensory Epithelium ◽  
Scanning Microscopy ◽  
Apical Surface ◽  
Length Width

Located on the sensory epithelium of the sickle-shaped cochlea of a 7- to 10-d-old chick are approximately 5,000 hair cells. When the apical surface of these cell is examined by scanning microscopy, we find that the length, number, width, and distribution of the stereocilia on each hair cell are predetermined. Thus, a hair cell located at the distal end of the cochlea has 50 stereocilia, the longest of which are 5.5 microns in length and 0.12 microns in width, while those at the proximal end number 300 and are maximally 1.5 microns in length and 0.2 micron in width. In fact, if we travel along the cochlea from its distal to proximal end, we see that the stereocilia on successive hair cells gradually increase in number and width, yet decrease in length. Also, if we look transversely across the cochlea where adjacent hair cells have the same length and number of stereocilia (they are the same distance from the distal end of the cochlea), we find that the stereocilia of successive hair cells become thinner and that the apical surface area of the hair cell proper, not including the stereocilia, decreases from a maximum of 80 microns2 to 15 microns2. Thus, if we are told the length of the longest stereocilium on a hair cell and the width of that stereocilium, we can pinpoint the position of that hair cell on the cochlea in two axes. Likewise, if we are told the number of stereocilia and the apical surface of a hair cell, we can pinpoint the location of that cell in two axes. The distribution of the stereocilia on the apical surface of the cell is also precisely determined. More specifically, the stereocilia are hexagonally packed and this hexagonal lattice is precisely positioned relative to the kinocilium. Because of the precision with which individual hair cells regulate the length, width, number, and distribution of their cell extensions, we have a magnificent object with which to ask questions about how actin filaments that are present within the cell are regulated. Equally interesting is that the gradient in stereociliary length, number, width, and distribution may play an important role in frequency discrimination in the cochlea. This conclusion is amplified by the information presented in the accompanying paper (Tilney, L.G., E.H. Egelman, D.J. DeRosier, and J.C. Saunders, 1983, J. Cell Biol., 96:822-834) on the packing of actin filaments in this stereocilia.

scholarly journals Vesicular transport of cationized ferritin by the epithelium of the rat choroid plexus.

1981 ◽  
Vol 89 (1) ◽  
pp. 131-139 ◽  
Author(s):  
B Van Deurs ◽  
F Von Bülow ◽  
M Møller
Keyword(s):  
Choroid Plexus ◽  
Vesicular Transport ◽  
Apical Cell ◽  
Multivesicular Bodies ◽  
Apical Surface ◽  
Cationized Ferritin ◽  
Epithelial Surface ◽  
Apical Cell Membrane ◽  
Choroidal Epithelium ◽  
Limited Degree

We have studied the transport of ferritin that was internalized by coated micropinocytic vesicles at the apical surface of the choroid plexus epithelium in situ. After ventriculocisternal perfusion of native ferritin (NF) or cationized ferritin (CF), three routes followed by the tracers are revealed: (a) to lysosomes, (b) to cisternal compartments, and (c) to the basolateral cell surface. (a) NF is micropinocytosed to a very limited degree and appears in a few lysosomal elements whereas CF is taken up in large amounts and can be followed, via endocytic vacuoles and light multivesicular bodies, to dark multivesicular bodies and dense bodies. (b) Occasionally, CF particles are found in cisterns that may represent GERL or trans-Golgi elements, whereas stacked Golgi cisterns never contain CF. (c) Transepithelial vesicular transport of CF is distinctly revealed. The intercellular spaces of the epithelium, below the apical tight junctions, contain numerous clusters of CF particles, often associated with surface-connected, coated vesicles. Vesicles in the process of exocytosis of CF are also present at the basal epithelial surface, whereas connective tissue elements below the epithelium are unlabeled. Our conclusion is that fluid and solutes removed from the cerebrospinal fluid by endocytosis either become sequestered in the lysosomal apparatus of the choroidal epithelium or are transported to the basolateral surface. However, our results do not indicate any significant recycling via Golgi complexes of internalized apical cell membrane.

scholarly journals Glycoconjugates on corneal epithelial surface: effect of neuraminidase treatment.

1989 ◽  
Vol 37 (8) ◽  
pp. 1215-1224 ◽  
Author(s):  
L D Hazlett ◽  
P Mathieu
Keyword(s):  
Plasma Membranes ◽  
Surface Effect ◽  
Apical Cell ◽  
Apical Surface ◽  
Corneal Surface ◽  
Epithelial Surface ◽  
Neuraminidase Treatment ◽  
Apical Cell Membrane ◽  
Corneal Epithelial ◽  
Adult Mice

The purpose of this study was to develop a procedure to quantitatively examine corneal epithelial apical cell membrane-associated glycoconjugates. Saccharide moieties on young, mature, and aged corneal epithelial cells were detected and localized in corneas of immature and adult mice by using colloidal gold-labeled lectins and transmission electron microscopy (TEM). In general, dense binding to the corneal epithelial apical surface cell membranes with wheat germ agglutinin (WGA) was seen in the adult, whereas the immature cornea bound less WGA-gold. Neuraminidase digestion decreased binding of the conjugate on epithelial plasma membranes of young and mature cells in adult cornea. Lectin-gold binding was decreased in the immature cornea on mature and aged cells. WGA-gold binding after neuraminidase was elevated on young cells of immature and on aged cells of adult animals. No binding of peanut agglutinin (PNA) or horse gram agglutinin (DBA) to the corneal epithelial surface was seen in animals of either age. After neuraminidase digestion, PNA binding sites were exposed only on the adult corneal surface. These data suggest that a terminal trisaccharide sequence, sialic acid-galactose beta(1----3)-N-acetylgalactosamine, is present at the adult corneal surface but is absent or at undetectable levels at the corneal surface of the immature animal. These data may be of significance in light of the dissimilar pattern of P. aeruginosa recognition and binding to the immature vs adult corneal epithelium.

scholarly journals Unconventional Myosins in Inner-Ear Sensory Epithelia

1997 ◽  
Vol 137 (6) ◽  
pp. 1287-1307 ◽  
Author(s):  
Tama Hasson ◽  
Peter G. Gillespie ◽  
Jesus A. Garcia ◽  
Richard B. MacDonald ◽  
Yi-dong Zhao ◽  
...  
Keyword(s):  
Inner Ear ◽  
Hair Cells ◽  
Actin Filaments ◽  
Structural Integrity ◽  
Myosin Vi ◽  
Cortical Actin ◽  
Unconventional Myosin ◽  
Myosin Isozymes ◽  
Cuticular Plate ◽  
Myosin Viia

To understand how cells differentially use the dozens of myosin isozymes present in each genome, we examined the distribution of four unconventional myosin isozymes in the inner ear, a tissue that is particularly reliant on actin-rich structures and unconventional myosin isozymes. Of the four isozymes, each from a different class, three are expressed in the hair cells of amphibia and mammals. In stereocilia, constructed of cross-linked F-actin filaments, myosin-Iβ is found mostly near stereociliary tips, myosin-VI is largely absent, and myosin-VIIa colocalizes with crosslinks that connect adjacent stereocilia. In the cuticular plate, a meshwork of actin filaments, myosin-Iβ is excluded, myosin-VI is concentrated, and modest amounts of myosin-VIIa are present. These three myosin isozymes are excluded from other actin-rich domains, including the circumferential actin belt and the cortical actin network. A member of a fourth class, myosin-V, is not expressed in hair cells but is present at high levels in afferent nerve cells that innervate hair cells. Substantial amounts of myosins-Iβ, -VI, and -VIIa are located in a pericuticular necklace that is largely free of F-actin, squeezed between (but not associated with) actin of the cuticular plate and the circumferential belt. Our localization results suggest specific functions for three hair-cell myosin isozymes. As suggested previously, myosin-Iβ probably plays a role in adaptation; concentration of myosin-VI in cuticular plates and association with stereociliary rootlets suggest that this isozyme participates in rigidly anchoring stereocilia; and finally, colocalization with cross-links between adjacent stereocilia indicates that myosin-VIIa is required for the structural integrity of hair bundles.

scholarly journals Differential Infection of Polarized Epithelial Cell Lines by Sialic Acid-Dependent and Sialic Acid-Independent Rotavirus Strains

2001 ◽  
Vol 75 (23) ◽  
pp. 11834-11850 ◽  
Author(s):  
Max Ciarlet ◽  
Sue E. Crawford ◽  
Mary K. Estes
Keyword(s):  
Sialic Acid ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Cell Lines ◽  
Apical Cell ◽  
Paracellular Permeability ◽  
Apical Surface ◽  
Apical Cell Membrane ◽  
Basolateral Side ◽  
Epithelial Cell Lines

ABSTRACT Infection of epithelial cells by some animal rotaviruses, but not human or most animal rotaviruses, requires the presence ofN-acetylneuraminic (sialic) acid (SA) on the cell surface for efficient infectivity. To further understand how rotaviruses enter susceptible cells, six different polarized epithelial cell lines, grown on permeable filter membrane supports containing 0.4-μm pores, were infected apically or basolaterally with SA-independent or SA-dependent rotaviruses. SA-independent rotaviruses applied apically or basolaterally were capable of efficiently infecting both sides of the epithelium of all six polarized cell lines tested, while SA-dependent rotaviruses only infected efficiently through the apical surface of five of the polarized cell lines tested. Regardless of the route of virus entry, SA-dependent and SA-independent rotaviruses were released almost exclusively from the apical domain of the plasma membrane of polarized cells before monolayer disruption or cell lysis. The transepithelial electrical resistance (TER) of cells decreased at the same time, irrespective of whether infection with SA-independent rotaviruses occurred apically or basolaterally. The TER of cells infected apically with SA-dependent rotaviruses decreased earlier than that of cells infected basolaterally. Rotavirus infection decreased TER before the appearance of cytopathic effect and cell death and resulted in an increase in the paracellular permeability to [3H]inulin as a function of loss of TER. The presence of SA residues on either the apical or basolateral side was determined using a Texas Red-conjugated lectin, wheat germ agglutinin (WGA), which binds SA residues. WGA bound exclusively to SA residues on the apical surface of the cells, confirming the requirement for SA residues on the apical cell membrane for efficient infectivity of SA-dependent rotaviruses. These results indicate that the rotavirus SA-independent cellular receptor is present on both sides of the epithelium, but SA-dependent and SA-independent rotavirus strains infect polarized epithelial cells by different mechanisms, which may be relevant for pathogenesis and selection of vaccine strains. Finally, rotavirus-induced alterations of the epithelial barrier and paracellular permeability suggest that common mechanisms of pathogenesis may exist between viral and bacterial pathogens of the intestinal tract.

Cytoskeleton of intestinal goblet cells: role of actin filaments in baseline secretion

1990 ◽  
Vol 259 (6) ◽  
pp. G991-G997 ◽  
Author(s):  
M. G. Oliver ◽  
R. D. Specian
Keyword(s):  
Plasma Membrane ◽  
Goblet Cell ◽  
Actin Filaments ◽  
Cellular Response ◽  
Apical Cell ◽  
Goblet Cells ◽  
Apical Plasma Membrane ◽  
Apical Surface ◽  
Cytochemical Localization

Although microtubules appear necessary to maintain mucin granule transport in intestinal goblet cells, the role of microfilaments in mucus secretion is unknown. To determine the functional significance of microfilaments in goblet cell secretion, fluorescent cytochemistry of microfilaments and autoradiographic studies on granule movement were performed on rabbit intestinal goblet cells, with and without the actin depolymerizing agents, cytochalasin D (cyto D), and dihydro-cytochalasin B (dihydro B). In normal goblet cells, cytochemical localization of F-actin with NBD-phallacidin demonstrated their restriction to the apical surface of the goblet cell. Visualization of the goblet cell apical surface by electron microscopy revealed the presence of a thin layer of cytoplasm overlying the granule mass. Treatment with cyto D and dihydro B eliminated NBD-phallacidin staining of the apical cell surface. Quantitative analysis of baseline granule translocation demonstrated that treatment with cyto D and dihydro B resulted in dramatic acceleration of granule movement through goblet cells. This cellular response results from an increase in baseline secretion and facilitation of secretion of newly synthesized mucins, not stimulation of an accelerated secretory event. These data imply that actin filaments fulfill a barrier function in baseline secretion by hindering granule access to the plasma membrane; once the granule contacts the plasma membrane, exocytosis occurs. Secretion is balanced by the translocation of subjacent granules. In contrast, an accelerated secretory event is not triggered by plasma membrane access alone; this event requires a regulatory signal. We hypothesize that, unlike accelerated secretion, baseline secretion is constitutive, with exocytosis limited solely by the physical constraint of secretory granule access to the apical plasma membrane.

scholarly journals The organization of actin filaments in the stereocilia of cochlear hair cells.

1980 ◽  
Vol 86 (1) ◽  
pp. 244-259 ◽  
Author(s):  
L G Tilney ◽  
D J Derosier ◽  
M J Mulroy
Keyword(s):  
Hair Cells ◽  
Actin Filaments ◽  
Optical Diffraction ◽  
Careful Examination ◽  
Intestinal Epithelial ◽  
Cochlear Hair Cells ◽  
Cuticular Plate ◽  
Detergent Extraction ◽  
Alligator Lizard ◽  
Paracrystalline Array

Within each tapering stereocilium of the cochlea of the alligator lizard is a bundle of actin filaments with > 3,000 filaments near the tip and only 18-29 filaments at the base where the bundle enters into the cuticular plate; there the filaments splay out as if on the surface of a cone, forming the rootlet. Decoration of the hair cells with subfragment 1 of myosin reveals that all the filaments in the stereocilia, including those that extend into the cuticular plate forming the rootlet, have unidirectional polarity, with the arrowheads pointing towards the cell center. The rest of the cuticular plate is composed of actin filaments that show random polarity, and numerous fine, 30 A filaments that connect the rootlet filaments to each other, to the cuticular plate, and to the membrane. A careful examination of the packing of the actin filaments in the stereocilia by thin sectin and by optical diffraction reveals that the filaments are packed in a paracrystalline array with the crossover points of all the actin helices in hear-perfect register. In transverse sections, the actin filaments are not hexagonally packed but, rather, are arranged in scalloped rows that present a festooned profile. We demonstrated that this profile is a product of the crossbridges by examining serial sections, sections of different thicknesses, and the same stereocilium at two different cutting angles. The filament packing is not altered by fixation in different media, removal of the limiting membrane by detergent extraction, or incubation of extracted hair cells in EGTA, EDTA, and Ca++ and ATP. From our results, we conclude that the stereocilia of the ear, unlike the brush border of intestinal epithelial cells, are not designed to shorten, nor do the filaments appear to slide past one another. In fact, the stereocilium is like a large, rigid structure designed to move as a lever.

scholarly journals Retargeting the Coxsackievirus and Adenovirus Receptor to the Apical Surface of Polarized Epithelial Cells Reveals the Glycocalyx as a Barrier to Adenovirus-Mediated Gene Transfer

2000 ◽  
Vol 74 (13) ◽  
pp. 6050-6057 ◽  
Author(s):  
Raymond J. Pickles ◽  
Jill A. Fahrner ◽  
JenniElizabeth M. Petrella ◽  
Richard C. Boucher ◽  
Jeffrey M. Bergelson
Keyword(s):  
Gene Transfer ◽  
Epithelial Cells ◽  
Basolateral Membrane ◽  
Apical Cell ◽  
Airway Epithelial Cells ◽  
Apical Surface ◽  
Apical Cell Membrane ◽  
Human Airway ◽  
Coxsackievirus And Adenovirus Receptor ◽  
Adenovirus Receptor

ABSTRACT Lumenal delivery of adenovirus vectors (AdV) results in inefficient gene transfer to human airway epithelium. The human coxsackievirus and adenovirus receptor (hCAR) was detected by immunofluorescence selectively at the basolateral surfaces of freshly excised human airway epithelial cells, suggesting that the absence of apical hCAR constitutes a barrier to adenovirus-mediated gene delivery in vivo. In transfected polarized Madin-Darby canine kidney cells, wild-type hCAR was expressed selectively at the basolateral membrane, whereas hCAR lacking the transmembrane and/or cytoplasmic domains was expressed on both the basolateral and apical membranes. Cells expressing apical hCAR still were not efficiently transduced by AdV applied to the apical surface. However, after the cells were treated with agents that remove components of the apical surface glycocalyx, AdV transduction occurred. These results indicate that adenovirus can infect via receptors located at the apical cell membrane but that the glycocalyx impedes interaction of AdV with apical receptors.

scholarly journals The structure of the cuticular plate, an in vivo actin gel.

1989 ◽  
Vol 109 (6) ◽  
pp. 2853-2867 ◽  
Author(s):  
D J DeRosier ◽  
L G Tilney
Keyword(s):  
Hair Cells ◽  
Actin Filaments ◽  
Test Tube ◽  
Intestinal Epithelial ◽  
Thin Sections ◽  
Terminal Web ◽  
Cuticular Plate ◽  
Quantitative Analyses ◽  
Alligator Lizard

The cuticular plate is a network of actin filaments found in hair cells of the cochlea. In the alligator lizard, it consists of rootlets, emanating from the stereocilia, and of cross-connecting actin filaments that anchor these rootlets. In thin sections, this network displays striking patches of 650 +/- 110-A striae. By quantitative analyses of the images, the mystery of the striae can be explained. They are due in part to the rootlets which are sets of flat ribbons of actin filaments. The ribbons in each set are separated by approximately 650 A. Numerous whiskers 30 A in diameter extend from each ribbon's face, interconnecting adjacent ribbons. The nonrootlet filaments, except at the margins of the cell, occur primarily as single filaments. Like the ribbons, they are bristling with whiskers. The patches of striae are explained by ribbons and filaments held at a 650-A separation by the whiskers that project from them. A simple model for regions of bewhiskered filaments is a box crammed full of randomly oriented test-tube brushes. A thin slice through the box will show regions of dark lines or striae due to the wire backbones of the brushes separated from one another by the bristle length. Using the computer instead of test-tube brushes, we have been able to model quantitatively the filament distribution and pattern of striae seen in the cuticular plate of the lizard. The organization of actin filaments we have deduced from our simulations differs from that found in macrophages or in the terminal web of intestinal epithelial cells.

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