scholarly journals Actin filaments elongate from their membrane-associated ends

1981 ◽  
Vol 90 (2) ◽  
pp. 485-494 ◽  
Author(s):  
LG Tilney ◽  
EM Bonder ◽  
DJ DeRosier
Keyword(s):  
Plasma Membrane ◽  
Actin Filament ◽  
Actin Filaments ◽  
The Other ◽  
Dense Material ◽  
Thin Sections ◽  
Vacuole Membrane ◽  
Filament Bundles ◽  
Filament Bundle

In limulus sperm an actin filament bundle 55 mum in length extends from the acrosomal vacuole membrane through a canal in the nucleus and then coils in a regular fashion around the base of the nucleus. The bundle expands systematically from 15 filaments near the acrosomal vacuole to 85 filaments at the basal end. Thin sections of sperm fixed during stages in spermatid maturation reveal that the filament bundle begins to assemble on dense material attached to the acrosomal vacuole membrane. In micrographs fo these early stages in maturation, short bundles are seen extending posteriorly from the dense material. The significance is that these short, developing bundles have about 85 filaments, suggesting that the 85-filament end of the bundle is assembled first. By using filament bundles isolated and incubated in vitro with G actin from muscle, we can determine the end "preferred" for addition of actin monomers during polymerization. The end that would be associated with the acrosomal vacuole membrane, a membrane destined to be continuous with the plasma membrane, is preferred about 10 times over the other, thicker end. Decoration of the newly polymerized portions of the filament bundle with subfragment 1 of myosin reveals that the arrowheads point away from the acrosomal vacuole membrane, as is true of other actin filament bundles attached to membranes. From these observations we conclude that the bundle is nucleated from the dense material associated with the acrosomal vacuole and that monomers are added to the membrane-associated end. As monomers are added at the dense material, the thick first-made end of the filament bundle is pushed down through the nucleus where, upon reaching the base of the nucleus, it coils up. Tapering is brought about by the capping of the peripheral filaments in the bundle.

The capactins, a class of proteins that cap the ends of actin filaments

1982 ◽  
Vol 299 (1095) ◽  
pp. 263-273 ◽  
Keyword(s):  
Skeletal Muscle ◽  
Actin Filament ◽  
Actin Filaments ◽  
Muscle Cells ◽  
Preliminary Evidence ◽  
The Other ◽  
Cellular Regulation ◽  
Filament Assembly ◽  
Filament Bundles

A number of proteins that bind specifically to the barbed ends of actin filaments in a cytochalasin-like manner have been purified to various degrees from a variety of muscle and non-muscle cells and tissues. Preliminary evidence also indicates that proteins that interact with the pointed ends of filaments are present in skeletal muscle. Because of their ability to cap one or the other end of an actin filament, we have designated this class of proteins as the ‘capactins’. On the basis of their effect on actin filament assembly and interaction in vitro , we propose that the capactins play important roles in cellular regulation of actin-based cytoskeletal and contractile functions. Our finding that the disappearance of actin filament bundles in virally transformed fibroblasts can be correlated with an increase in capactin activity in the extracts of these cells is consistent with this hypothesis.

scholarly journals F-actin bundles in Drosophila bristles are assembled from modules composed of short filaments.

1996 ◽  
Vol 135 (5) ◽  
pp. 1291-1308 ◽  
Author(s):  
L G Tilney ◽  
P Connelly ◽  
S Smith ◽  
G M Guild
Keyword(s):  
Actin Filament ◽  
Actin Filaments ◽  
Modular Design ◽  
Thin Sections ◽  
Actin Bundle ◽  
Actin Bundles ◽  
Phalloidin Staining ◽  
Filament Bundles ◽  
End To End ◽  
As If

The actin bundles in Drosophila bristles run the length of the bristle cell and are accordingly 65 microns (microchaetes) or 400 microns (macrochaetes) in length, depending on the bristle type. Shortly after completion of bristle elongation in pupae, the actin bundles break down as the bristle surface becomes chitinized. The bundles break down in a bizarre way; it is as if each bundle is sawed transversely into pieces that average 3 microns in length. Disassembly of the actin filaments proceeds at the "sawed" surfaces. In all cases, the cuts in adjacent bundles appear in transverse register. From these images, we suspected that each actin bundle is made up of a series of shorter bundles or modules that are attached end-to-end. With fluorescent phalloidin staining and serial thin sections, we show that the modular design is present in nondegenerating bundles. Decoration of the actin filaments in adjacent bundles in the same bristle with subfragment 1 of myosin reveals that the actin filaments in every module have the same polarity. To study how modules form developmentally, we sectioned newly formed and elongating bristles. At the bristle tip are numerous tiny clusters of 6-10 filaments. These clusters become connected together more basally to form filament bundles that are poorly organized, initially, but with time become maximally cross-linked. Additional filaments are then added to the periphery of these organized bundle modules. All these observations make us aware of a new mechanism for the formation and elongation of actin filament bundles, one in which short bundles are assembled and attached end-to-end to other short bundles, as are the vertical girders between the floors of a skyscraper.

scholarly journals Structural organization of actin in the sea urchin egg cortex: microvillar elongation in the absence of actin filament bundle formation

1982 ◽  
Vol 93 (1) ◽  
pp. 24-32 ◽  
Author(s):  
DA Begg ◽  
LI Rebhun ◽  
H Hyatt
Keyword(s):  
Sea Urchin ◽  
Actin Filament ◽  
Actin Filaments ◽  
Calcium Ionophore ◽  
Ionophore A23187 ◽  
Acid Activation ◽  
Cytoplasmic Ph ◽  
Sea Urchin Egg ◽  
Filament Bundles ◽  
Filament Bundle

We have investigated the relationship between the formation of actin filament bundles and the elongation of microvilli (MV) after fertilization in sea urchin eggs. In a previous study (1979, J Cell Biol. 83:241-248) we demonstrated that increased pH induced the formation of actin filaments in isolated sea urchin egg cortices with the concomitant elongation of MV. On the basis of these results we suggested that increased cytoplasmic pH after fertilization causes a reorganization of cortical actin, which in turn provides the force for MV elongation. To test this hypothesis, we compared the morphology of microvilli in eggs activated with and without the release of fertilization acid. Activation of eggs in normal sea water with the calcium ionophore A23187 causes the release of fertilization acid and the elongation of MV containing core bundles of actin filaments. Eggs activated with A23187 in NA(+)-free water do not undergo normal fertilization acid release but develop elongated, flaccid MV. These MV contain an irregular network of actin filaments rather than the parallel bundles of filaments found in normal MV. The addition of 40 mM NaCl to these eggs results in the release of H(+) and the concomitant conversion of flaccid MV to erect MV containing typical core bundles of actin filaments. Identical results are obtained when 10 mM NH(4)Cl is substituted for NaCl. The induction of cytoplasmic alkalinization in unactivated eggs with NH(4)Cl does not cause either MV elongation or the formation of actin filament bundles . These results suggest that: (a) the elongation of MV is stimulated by a rise in intracellular free Ca(++) concentration; (b) actin filament bundle formation is triggered by an increase in cytoplasmic pH; and (c) the formation of actin filament bundles is not necessary for MV elongation but is required to provide rigid support for MV.

scholarly journals Palladin Is a Regulator of Actin Filament Bundles at the Ectoplasmic Specialization in Adult Rat Testes

Endocrinology ◽  
2013 ◽  
Vol 154 (5) ◽  
pp. 1907-1920 ◽  
Author(s):  
Xiaojing Qian ◽  
Dolores D. Mruk ◽  
Elissa W. P. Wong ◽  
Pearl P. Y. Lie ◽  
C. Yan Cheng
Keyword(s):  
Actin Filament ◽  
Actin Filaments ◽  
Plasma Membranes ◽  
Permeability Barrier ◽  
Protein Distribution ◽  
Filament Bundles ◽  
Ectoplasmic Specialization ◽  
Blood Testis Barrier

Abstract In rat testes, the ectoplasmic specialization (ES) at the Sertoli-Sertoli and Sertoli-spermatid interface known as the basal ES at the blood-testis barrier and the apical ES in the adluminal compartment, respectively, is a testis-specific adherens junction. The remarkable ultrastructural feature of the ES is the actin filament bundles that sandwiched in between the cisternae of endoplasmic reticulum and apposing plasma membranes. Although these actin filament bundles undergo extensive reorganization to switch between their bundled and debundled state to facilitate blood-testis barrier restructuring and spermatid adhesion/transport, the regulatory molecules underlying these events remain unknown. Herein we report findings of an actin filament cross-linking/bundling protein palladin, which displayed restrictive spatiotemporal expression at the apical and the basal ES during the epithelial cycle. Palladin structurally interacted and colocalized with Eps8 (epidermal growth factor receptor pathway substrate 8, an actin barbed end capping and bundling protein) and Arp3 (actin related protein 3, which together with Arp2 form the Arp2/3 complex to induce branched actin nucleation, converting bundled actin filaments to an unbundled/branched network), illustrating its role in regulating actin filament bundle dynamics at the ES. A knockdown of palladin in Sertoli cells in vitro with an established tight junction (TJ)-permeability barrier was found to disrupt the TJ function, which was associated with a disorganization of actin filaments that affected protein distribution at the TJ. Its knockdown in vivo also perturbed F-actin organization that led to a loss of spermatid polarity and adhesion, causing defects in spermatid transport and spermiation. In summary, palladin is an actin filament regulator at the ES.

scholarly journals Formation of actin filament bundles in the ring canals of developing Drosophila follicles.

1996 ◽  
Vol 133 (1) ◽  
pp. 61-74 ◽  
Author(s):  
L G Tilney ◽  
M S Tilney ◽  
G M Guild
Keyword(s):  
Actin Filament ◽  
Actin Filaments ◽  
Actin Binding ◽  
Nurse Cells ◽  
Ring Canal ◽  
Contractile Ring ◽  
Thin Sections ◽  
Development Stages ◽  
Ring Canals ◽  
Filament Bundles

Growing the intracellular bridges that connect nurse cells with each o ther and to the developing oocyte is vital for egg development. These ring canals increase from 0.5 microns in diameter at stage 2 to 10 microns in diameter at stage 11. Thin sections cut horizontally as you would cut a bagel, show that there is a layer of circumferentially oriented actin filaments attached to the plasma membrane at the periphery of each canal. By decoration with subfragment 1 of myosin we find actin filaments of mixed polarities in the ring such as found in the "contractile ring" formed during cytokinesis. In vertical sections through the canal the actin filaments appear as dense dots. At stage 2 there are 82 actin filaments in the ring, by stage 6 there are 717 and by stage 10 there are 726. Taking into account the diameter, this indicates that there is 170 microns of actin filaments/canal at stage 2 (pi x 0.5 microns x 82), 14,000 microns at stage 9 and approximately 23,000 microns at stage 11 or one inch of actin filament! The density of actin filaments remains unchanged throughout development. What is particularly striking is that by stages 4-5, the ring of actin filaments has achieved its maximum thickness, even though the diameter has not yet increased significantly. Thereafter, the diameter increases. Throughout development, stages 2-11, the canal length also increases. Although the density (number of actin filaments/micron2) through a canal remains constant from stage 5 on, the actin filaments appear as a net of interconnected bundles. Further information on this net of bundles comes from studying mutant animals that lack kelch, a protein located in the ring canal that has homology to the actin binding protein, scruin. In this mutant, the actin filaments form normally but individual bundles that comprise the fibers of the net are not bound tightly together. Some bundles enter into the ring canal lumen but do not completely occlude the lumen. all these observations lay the groundwork for our understanding of how a noncontractile ring increases in thickness, diameter, and length during development.

scholarly journals Drosophila singed, a fascin homolog, is required for actin bundle formation during oogenesis and bristle extension.

1994 ◽  
Vol 125 (2) ◽  
pp. 369-380 ◽  
Author(s):  
K Cant ◽  
B A Knowles ◽  
M S Mooseker ◽  
L Cooley
Keyword(s):  
Sea Urchin ◽  
Actin Filament ◽  
Nurse Cell ◽  
Structural Integrity ◽  
Molar Ratio ◽  
Cell Nuclei ◽  
Cytoplasmic Actin ◽  
Filament Bundles ◽  
Filament Bundle

Drosophila singed mutants were named for their gnarled bristle phenotype but severe alleles are also female sterile. Recently, singed protein was shown to have 35% peptide identity with echinoderm fascin. Fascin is found in actin filament bundles in microvilli of sea urchin eggs and in filopodial extensions in coelomocytes. We show that Drosophila singed is required for actin filament bundle formation in the cytoplasm of nurse cells during oogenesis; in severe mutants, the absence of cytoplasmic actin filament bundles allows nurse cell nuclei to lodge in ring canals and block nurse cell cytoplasm transport. Singed is also required for organized actin filament bundle formation in the cellular extension that forms a bristle; in severe mutants, the small disorganized actin filament bundles lack structural integrity and allow bristles to bend and branch during extension. Singed protein is also expressed in migratory cells of the developing egg chamber and in the socket cell of the developing bristle, but no defect is observed in these cells in singed mutants. Purified, bacterially expressed singed protein bundles actin filaments in vitro with the same stoichiometry reported for purified sea urchin fascin. Singed-saturated actin bundles have a molar ratio of singed/actin of approximately 1:4.3 and a transverse cross-banding pattern of 12 nm seen using electron microscopy. Our results suggest that singed protein is required for actin filament bundle formation and is a Drosophila homolog of echinoderm fascin.

scholarly journals Movement of the actin filament bundle in Mytilus sperm: a new mechanism is proposed

1987 ◽  
Vol 104 (4) ◽  
pp. 981-993 ◽  
Author(s):  
LG Tilney ◽  
Y Fukui ◽  
DJ DeRosier
Keyword(s):  
Plasma Membrane ◽  
Actin Filament ◽  
Cell Elongation ◽  
Actin Filaments ◽  
Blue Mussel ◽  
Optical Diffraction ◽  
Acrosomal Reaction ◽  
Filament Bundle

An actin filament bundle approximately 2-5 microns in length is present in the sperm of the blue mussel, Mytilus. In unfired sperm this bundle extends from the midpiece through a canal in the center of the nucleus to terminate on the membrane limiting the inside of the cone-shaped acrosomal vacuole. The bundle is composed of 45-65 actin filaments which are hexagonally packed and regularly cross-bridged together to form an actin paracrystal so well ordered that it has six nearly equal faces. Upon induction of the acrosomal reaction, a needle-like process is formed in a few seconds. Within this process is the actin filament bundle which appears unchanged in filament number and packing as determined by optical diffraction methods. Using fluorescein-conjugated phalloidin we were able to establish that the bundle does not change length but instead is projected anteriorly out of the midpiece and nuclear canal like an arrow. Existing mechanisms to explain this extension cannot apply. Specifically, the bundle does not increase in length (no polymerization), does not change its organization (no change in actin twist), does not change filament number (no filament sliding), and cannot move by myosin (wrong polarity). Thus we are forced to look elsewhere for a mechanism and have postulated that at least a component of this movement, or cell elongation, is the interaction of the actin filament bundle with the plasma membrane.

scholarly journals Actin filaments, stereocilia, and hair cells of the bird cochlea. II. Packing of actin filaments in the stereocilia and in the cuticular plate and what happens to the organization when the stereocilia are bent.

1983 ◽  
Vol 96 (3) ◽  
pp. 822-834 ◽  
Author(s):  
L G Tilney ◽  
E H Egelman ◽  
D J DeRosier ◽  
J C Saunder
Keyword(s):  
Actin Filament ◽  
Hair Cells ◽  
Actin Filaments ◽  
Fine Tuning ◽  
Thin Sections ◽  
The Cross ◽  
Diffraction Patterns ◽  
Cross Bridges ◽  
Different Parts ◽  
Filament Bundle

A comparison of hair cells from different parts of the cochlea reveals the same organization of actin filaments; the elements that vary are the length and number of the filaments. Thin sections of stereocilia reveal that the actin filaments are hexagonally packed and from diffraction patterns of these sections we found that the actin filaments are aligned such that the crossover points of adjacent actin filaments are in register. As a result, the cross-bridges that connect adjacent actin filaments are easily seen in longitudinal sections. The cross-bridges appear as regularly spaced bands that are perpendicular to the axis of the stereocilium. Particularly interesting is that, unlike what one might predict, when a stereocilium is bent or displaced, as might occur during stimulation by sound, the actin filaments are not compressed or stretched but slide past one another so that the bridges become tilted relative to the long axis of the actin filament bundle. In the images of bent bundles, the bands of cross-bridges are then tilted off perpendicular to the stereocilium axis. When the stereocilium is bent at its base, all cross-bridges in the stereocilium are affected. Thus, resistance to bending or displacement must be property of the number of bridges present, which in turn is a function of the number of actin filaments present and their respective lengths. Since hair cells in different parts of the cochlea have stereocilia of different, yet predictable lengths and widths, this means that the force needed to displace the stereocilia of hair cells located at different regions of the cochlea will not be the same. This suggests that fine tuning of the hair cells must be a built-in property of the stereocilia. Perhaps its physiological vulnerability may result from changes of stereociliary structure.

scholarly journals Organization of actin in the leading edge of cultured cells: influence of osmium tetroxide and dehydration on the ultrastructure of actin meshworks.

1981 ◽  
Vol 91 (3) ◽  
pp. 695-705 ◽  
Author(s):  
J V Small
Keyword(s):  
Electron Microscopy ◽  
Actin Filament ◽  
Actin Filaments ◽  
Osmium Tetroxide ◽  
Cultured Cells ◽  
Leading Edge ◽  
Cultured Fibroblasts ◽  
Cell Biol ◽  
Filament Bundles

The ordered structure of the leading edge (lamellipodium) of cultured fibroblasts is readily revealed in cells extracted briefly in Triton X-100-glutaraldehyde mixtures, fixed further in glutaraldehyde, and then negatively stained for electron microscopy. By this procedure, the leading edge regions show a highly organised, three-dimensional network of actin filaments together with variable numbers of radiating actin filament bundles or microspikes. The use of Phalloidin after glutaraldehyde fixation resulted in a marginal improvement in filament order. Processing of the cytoskeletons though the additional steps generally employed for conventional electron microscopy resulted in a marked deterioration or complete disruption of the order of the actin filament networks. In contrast, the actin filaments of the stress fiber bundles were essentially unaffected. Thus, postfixation in osmium tetroxide (1% for 7 min at room temperature) transformed the networks to a reticulum of kinked fibers, resembling those produced by the exposure of muscle F-actin to OsO4 in vitro (P. Maupin-Szamier and T. D. Pollard. 1978. J. Cell Biol. 77:837--852). While limited exposure to OsO4 (0.2+ for 20 min at 0 degrees C) obviated this destruction, dehydration in acetone or ethanol, with or without post-osmication, caused a further and unavoidable disordering and aggregation of the meshwork filaments. The meshwork regions of the leading edge then showed a striking resemblance to the networks hitherto described in critical point-dried preparations of cultured cells. I conclude that much of the "microtrabecular lattice" described by Wolosewick and Porter (1979. J. Cell Biol. 82:114--139) in the latter preparations constitutes actin meshworks and actin filament arrays, with their associated components, that have been distorted and aggregated by the preparative procedures employed.

scholarly journals The polymerization of actin: II. how nonfilamentous actin becomes nonrandomly distributed in sperm: evidence for the association of this actin with membranes

1976 ◽  
Vol 69 (1) ◽  
pp. 51-72 ◽  
Author(s):  
LG Tilney
Keyword(s):  
Plasma Membrane ◽  
Nuclear Envelope ◽  
Thin Section ◽  
Actin Filaments ◽  
Early Stage ◽  
Mature Sperm ◽  
Vacuole Membrane ◽  
Other Substances ◽  
Associated Proteins ◽  
Basal Half

At an early stage in spermiogenesis the acrosomal vacuole and other organelles including ribosomes are located at the basal end of the cell. From here actin must be transported to its future location at the anterior end of the cell. At no stage in the accumulation of actin in the periacrosomal region is the actin sequestered in a membrane-bounded compartment such as a vacuole or vesicle. Since filaments are not present in the periacrosomal region during the accumulation of the actin even though the fixation of these cells is sufficiently good to distinguish actin filaments in thin section, the actin must accumulate in the nonfilamentous state. The membranes in the periacrosomal region, specifically a portion of the nuclear envelope and the basal half of the acrosomal vacuole membrane, become specialized morphologically in advance of the accumulation of actin in this region. My working hypothesis is that the actin in combination with other substances binds to these specialized membranes and to itself and thus can accumulate in the periacrosmoal region by being trapped on these specialized membranes. Diffusion would then be sufficient to move these substances to this region. In support of this hypothesis are experiments in which I treated mature sperm with detergents, glycols, and hypotonic media, which solubilize or lift away the plasma membrane. The actin and its associated proteins remain attached to these specialized membranes. Thus actin can be nonrandomly distributed in cells in a nonfilamentous state presumably by its association with specialized membranes.

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