scholarly journals Three different actin filament assemblies occur in every hair cell: each contains a specific actin crosslinking protein.

1991 ◽  
Vol 112 (4) ◽  
pp. 641-651 ◽  
Author(s):  
D Drenckhahn ◽  
K Engel ◽  
D Höfer ◽  
C Merte ◽  
L Tilney ◽  
...  
Keyword(s):  
Actin Filament ◽  
Hair Cells ◽  
Actin Filaments ◽  
Supramolecular Assembly ◽  
Spatial Proximity ◽  
Cross Linking ◽  
Supporting Cells ◽  
Cuticular Plate ◽  
Auditory Organ ◽  
Close Spatial Proximity

The apex of hair cells of the chicken auditory organ contains three different kinds of assemblies of actin filaments in close spatial proximity. These are (a) paracrystals of actin filaments with identical polarity in stereocilia, (b) a dense gellike meshwork of actin filaments forming the cuticular plate, and (c) a bundle of parallel actin filaments with mixed polarities that constitute the circumferential filament belt attached to the cytoplasmic aspect of the zonula adhaerens (ZA). Each different supramolecular assembly of actin filaments contains a specific actin filament cross-linking protein which is unique to that particular assembly. Thus fimbrin appears to be responsible for paracrystallin packing of actin filaments in stereocillia; an isoform of spectrin resides in the cuticular plate where it forms the whisker-like crossbridges, and alpha actinin is the actin crosslinking protein of the circumferential ZA bundle. Tropomyosin, which stabilizes actin filaments, is present in all the actin filament assemblies except for the stereocilia. Another striking finding was that myosin appears to be absent from the ZA ring and cuticular plate of hair cells although present in the ZA ring of supporting cells. The abundance of myosin in the ZA ring of the surrounding supporting cells means that it may be important in forming a supporting tensile cellular framework in which the hair cells are inserted.

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 The F-actin bundler α-actinin Ain1 is tailored for ring assembly and constriction during cytokinesis in fission yeast

2016 ◽  
Vol 27 (11) ◽  
pp. 1821-1833 ◽  
Author(s):  
Yujie Li ◽  
Jenna R. Christensen ◽  
Kaitlin E. Homa ◽  
Glen M. Hocky ◽  
Alice Fok ◽  
...  
Keyword(s):  
Fission Yeast ◽  
Actin Filament ◽  
Actin Filaments ◽  
Biochemical Properties ◽  
Ring Formation ◽  
Cross Linking ◽  
Contractile Ring ◽  
Mixed Polarity ◽  
Dividing Cells

The actomyosin contractile ring is a network of cross-linked actin filaments that facilitates cytokinesis in dividing cells. Contractile ring formation has been well characterized in Schizosaccharomyces pombe, in which the cross-linking protein α-actinin SpAin1 bundles the actin filament network. However, the specific biochemical properties of SpAin1 and whether they are tailored for cytokinesis are not known. Therefore we purified SpAin1 and quantified its ability to dynamically bind and bundle actin filaments in vitro using a combination of bulk sedimentation assays and direct visualization by two-color total internal reflection fluorescence microscopy. We found that, while SpAin1 bundles actin filaments of mixed polarity like other α-actinins, SpAin1 has lower bundling activity and is more dynamic than human α-actinin HsACTN4. To determine whether dynamic bundling is important for cytokinesis in fission yeast, we created the less dynamic bundling mutant SpAin1(R216E). We found that dynamic bundling is critical for cytokinesis, as cells expressing SpAin1(R216E) display disorganized ring material and delays in both ring formation and constriction. Furthermore, computer simulations of initial actin filament elongation and alignment revealed that an intermediate level of cross-linking best facilitates filament alignment. Together our results demonstrate that dynamic bundling by SpAin1 is important for proper contractile ring formation and constriction.

scholarly journals Bundling of actin filaments by alpha-actinin depends on its molecular length.

1990 ◽  
Vol 110 (6) ◽  
pp. 2013-2024 ◽  
Author(s):  
R K Meyer ◽  
U Aebi
Keyword(s):  
Smooth Muscle ◽  
Actin Filament ◽  
Actin Filaments ◽  
Actin Binding ◽  
Molar Ratio ◽  
Cross Linking ◽  
Actin Bundle ◽  
Actin Bundles ◽  
Chicken Gizzard ◽  
Molecular Length

Cross-linking of actin filaments (F-actin) into bundles and networks was investigated with three different isoforms of the dumbbell-shaped alpha-actinin homodimer under identical reaction conditions. These were isolated from chicken gizzard smooth muscle, Acanthamoeba, and Dictyostelium, respectively. Examination in the electron microscope revealed that each isoform was able to cross-link F-actin into networks. In addition, F-actin bundles were obtained with chicken gizzard and Acanthamoeba alpha-actinin, but not Dictyostelium alpha-actinin under conditions where actin by itself polymerized into disperse filaments. This F-actin bundle formation critically depended on the proper molar ratio of alpha-actinin to actin, and hence F-actin bundles immediately disappeared when free alpha-actinin was withdrawn from the surrounding medium. The apparent dissociation constants (Kds) at half-saturation of the actin binding sites were 0.4 microM at 22 degrees C and 1.2 microM at 37 degrees C for chicken gizzard, and 2.7 microM at 22 degrees C for both Acanthamoeba and Dictyostelium alpha-actinin. Chicken gizzard and Dictyostelium alpha-actinin predominantly cross-linked actin filaments in an antiparallel fashion, whereas Acanthamoeba alpha-actinin cross-linked actin filaments preferentially in a parallel fashion. The average molecular length of free alpha-actinin was 37 nm for glycerol-sprayed/rotary metal-shadowed and 35 nm for negatively stained chicken gizzard; 46 and 44 nm, respectively, for Acanthamoeba; and 34 and 31 nm, respectively, for Dictyostelium alpha-actinin. In negatively stained preparations we also evaluated the average molecular length of alpha-actinin when bound to actin filaments: 36 nm for chicken gizzard and 35 nm for Acanthamoeba alpha-actinin, a molecular length roughly coinciding with the crossover repeat of the two-stranded F-actin helix (i.e., 36 nm), but only 28 nm for Dictyostelium alpha-actinin. Furthermore, the minimal spacing between cross-linking alpha-actinin molecules along actin filaments was close to 36 nm for both smooth muscle and Acanthamoeba alpha-actinin, but only 31 nm for Dictyostelium alpha-actinin. This observation suggests that the molecular length of the alpha-actinin homodimer may determine its spacing along the actin filament, and hence F-actin bundle formation may require "tight" (i.e., one molecule after the other) and "untwisted" (i.e., the long axis of the molecule being parallel to the actin filament axis) packing of alpha-actinin molecules along the actin filaments.

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 The actin filament content of hair cells of the bird cochlea is nearly constant even though the length, width, and number of stereocilia vary depending on the hair cell location.

1988 ◽  
Vol 107 (6) ◽  
pp. 2563-2574 ◽  
Author(s):  
L G Tilney ◽  
M S Tilney
Keyword(s):  
Hair Cell ◽  
Actin Filament ◽  
Cross Sections ◽  
Hair Cells ◽  
Actin Filaments ◽  
Membrane Surface ◽  
Scanning Electron Micrographs ◽  
Length Width ◽  
Filament Content ◽  
Filament Bundle

By direct counts off scanning electron micrographs, we determined the number of stereocilia per hair cell of the chicken cochlea as a function of the position of the hair cell on the cochlea. Micrographs of thin cross sections of stereociliary bundles located at known positions on the cochlea were enlarged and the total number of actin filaments per stereocilium was counted and recorded. By comparing the counts of filament number with measurements of actin filament bundle width of the same stereocilium, we were able to relate actin filament bundle width to filament number with an error margin (r2) of 16%. Combining this data with data already published or in the process of publication from our laboratory on the length and width of stereocilia, we were able to calculate the total length of actin filaments present in stereociliary bundles of hair cells located at a variety of positions on the cochlea. We found that stereociliary bundles of hair cells contain 80,000-98,000 micron of actin filament, i.e., the concentration of actin is constant in all hair cells with a range of values that is less than our error in measurement and/or biological variation, the greatest variation being in relating the diameters of the stereocilia to filament number. We also calculated the membrane surface needed to cover the stereocilia of hair cells located throughout the cochlea. The values (172-192 micron 2) are also constant. The implications of our observation that the total amount of actin is constant even though the length, width, and number of stereocilia per hair cell vary are discussed.

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 Actin filament destruction by osmium tetroxide

1978 ◽  
Vol 77 (3) ◽  
pp. 837-852 ◽  
Author(s):  
P Maupin-Szamier ◽  
TD Pollard
Keyword(s):  
Actin Filament ◽  
Actin Filaments ◽  
Sodium Phosphate ◽  
Osmium Tetroxide ◽  
Time Span ◽  
Cross Linking ◽  
Muscle Actin ◽  
Viscosity Change ◽  
Sodium Phosphate Buffer ◽  
Sulfur Containing

We have studied the destruction of purified muscle actin filaments by osmium tetroxide (OsO4) to develop methods to preserve actin filaments during preparation for electron microscopy. Actin filaments are fragmented during exposure to OsO4. This causes the viscosity of solutions of actin filaments to decrease, ultimately to zero, and provides a convenient quantitative assay to analyze the reaction. The rate of filament destruction is determined by the OsO4 concentration, temperature, buffer type and concentration, and pH. Filament destruction is minimized by treatment with a low concentration of OsO4 in sodium phosphate buffer, pH 6.0, at 0 degrees C. Under these conditions, the viscosity of actin filament solutions is stable and actin filaments retain their straight, unbranched structure, even after dehydration and embedding. Under more severe conditions, the straight actin filaments are converted into what look like the microfilament networks commonly observed in cells fixed with OsO4. Destruction of actin filaments can be inhibited by binding tropomyosin to the actin. Cross-linking the actin molecules within a filament with glutaraldehyde does not prevent their destruction by OsO4. The viscosity decrease requires the continued presence of free OsO4. During the time of the viscosity change, OsO4 is reduced and the sulfur-containing amino acids of actin are oxidized, but little of the osmium is bound to the actin. Over a much longer time span, the actin molecules are split into discrete peptides.

scholarly journals Isoforms of α-Actinin from Cardiac, Smooth, and Skeletal Muscle Form Polar Arrays of Actin Filaments

2000 ◽  
Vol 149 (3) ◽  
pp. 635-646 ◽  
Author(s):  
Kenneth A. Taylor ◽  
Dianne W. Taylor ◽  
Fred Schachat
Keyword(s):  
Actin Filament ◽  
Actin Filaments ◽  
Polar Filament ◽  
Relative Orientation ◽  
Lipid Monolayer ◽  
Cross Linking ◽  
Internal Diameter ◽  
Vertebrate Muscle ◽  
Filament Bundles ◽  
Positively Charged

We have used a positively charged lipid monolayer to form two-dimensional bundles of F-actin cross-linked by α-actinin to investigate the relative orientation of the actin filaments within them. This method prevents growth of the bundles perpendicular to the monolayer plane, thereby facilitating interpretation of the electron micrographs. Using α-actinin isoforms isolated from the three types of vertebrate muscle, i.e., cardiac, skeletal, and smooth, we have observed almost exclusively cross-linking between polar arrays of filaments, i.e., actin filaments with their plus ends oriented in the same direction. One type of bundle can be classified as an Archimedian spiral consisting of a single actin filament that spirals inward as the filament grows and the bundle is formed. These spirals have a consistent hand and grow to a limiting internal diameter of 0.4–0.7 μm, where the filaments appear to break and spiral formation ceases. These results, using isoforms usually characterized as cross-linkers of bipolar actin filament bundles, suggest that α-actinin is capable of cross-linking actin filaments in any orientation. Formation of specifically bipolar or polar filament arrays cross-linked by α-actinin may require additional factors that either determine the filament orientation or restrict the cross-linking capabilities of α-actinin.

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.

Coagulation factor Va is an actin filament binding and cross-linking protein

1995 ◽  
Vol 73 (1-2) ◽  
pp. 105-112 ◽  
Author(s):  
Emilia Furmaniak-Kazmierczak ◽  
Michael E. Nesheim ◽  
Graham P. Côté
Keyword(s):  
Actin Filament ◽  
Light Chain ◽  
Actin Filaments ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Actin Binding ◽  
Cross Linking ◽  
Factor V ◽  
Proteolytic Activation ◽  
Factor Va

Bovine coagulation cofactor factor Va is shown to bind to filaments of skeletal muscle actin with a dissociation constant of 40–50 nM in the presence of 50 mM NaCl. At saturation, approximately one molecule of factor Va was bound for every two actin molecules. The binding of factor Va to F-actin was inhibited by increasing ionic strength, being approximately 20-fold weaker at 150 mM NaCl. Addition of factor Va dramatically increased both the low-speed sedimentation and the low-shear viscosity of actin filament solutions, indicating that factor Va cross-links actin filaments. Factor Va also bound to actin filaments saturated with myosin. The isolated 74-kilodalton light chain of factor Va displayed actin binding and cross-linking properties indistinguishable from those of intact factor Va. The procofactor factor V bound weakly to F-actin, indicating that proteolytic activation is required to uncover the actin binding sites within the light chain domain. Actin filaments had only a slight inhibitory effect on the prothombinase activity of the factor Va – factor Xa – phospholipid complex. Since high concentrations of actin filaments can be exposed to the circulation when cells are damaged, the interaction of factor Va with actin may be of physiological relevance.Key words: blood coagulation, factor V, actin.

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