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L G Tilney

Publications and source records attributed to L G Tilney.

At least 91 records · Page 5Linked to original sources
PubMed1981

Actin filaments elongate from their membrane-associated ends.

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.

Actins↗
PubMed1980

Actin in the inner ear: the remarkable structure of the stereocilium.

The actin filament bundle in the stereocilium of the cochlear hair cell has liquid transverse order, but paracrystalline axial order. A single bonding rule, which determines how a cross bridging protein links neighbouring filaments, accounts for this unusual structure. Such liquid order is possible for bundles of other filamentous proteins.

Actins↗
PubMed1980

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

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.

Actins↗
PubMed1980

Actin, microvilli, and the fertilization cone of sea urchin eggs.

Sea urchin eggs and oocytes at the germinal vesicle stage were fixed at various times after insemination, and thin sections were examined. Actin filaments can first be found in the cortical cytoplasm 1 min after insemination, and by 2 min enormous numbers of filaments are present. At these early stages, the filaments are only occasionally organized into bundles, but one end of many filaments contacts the plasma membrane. By 3 min, and even more dramatically by 5 min after insemination, the filaments become progressively more often found in bundles that lie parallel to the long axis of the microvilli and the fertilization cones. By 7 min, the bundles of filaments in the cone are maximally pronounced, with virtually all the filaments lying parallel to one another. Decoration of the filaments with subfragment 1 of myosin shows that, in both the microvilli and the cones, the filaments are unidirectionally polarized with the arrowheads pointing towards the cell center. The efflux of H+ from the eggs was measured as a function of time after insemination. The rapid phase of H+ efflux occurs at the same time as actin polymerization. From these results it appears that the formation of bundles of actin filaments in microvilli and in cones is a two-step process, involving actin polymerization to form filaments, randomly oriented but in most cases having one end in contact with the plasma membrane, followed by the zippering together of the filaments by macromolecular bridges.

Actins↗
PubMed1979

Membrane events in the acrosomal reaction of Limulus sperm. Membrane fusion, filament-membrane particle attachment, and the source and formation of new membrane surface.

The membranes of Limulus (horseshoe crab) sperm were examined before and during the acrosomal reaction by using the technique of freeze-fracturing and thin sectioning. We focused on three areas. First, we examined stages in the fusion of the acrosomal vacuole with the cell surface. Fusion takes place in a particle-free zone which is surrounded by a circlet of particles on the P face of the plasma membrane and an underlying circlet of particles on the P face of the acrosomal vauole membrane. These circlets of particles are present before induction. Up to nine focal points of fusion occur within the particle-free zone. Second, we describe a system of fine filaments, each 30 A in diameter, which lies between the acrosomal vacuole and the plasma membrane. These filaments change their orientation as the vacuole opens, a process that takes place in less than 50 ms. Membrane particles seen on the P face of the acrosomal vacuole membrane change their orientation at the same time and in the same way as do the filaments, thus indicating that the membrane particles and filaments are probably connected. Third, we examined the source and the point of fusion of new membrane needed to cover the acrosomal process. This new membrane is almost certainly derived from the outer nuclear envelope and appears to insert into the plasma membrane in a particle-free area adjacent to an area rich in particles. The latter is the region where the particles are probably connected to the cytoplasmic filaments. The relevance of these observations in relation to the process of fertilization of this fantastic sperm is discussed.

Acrosome↗
PubMed1979

Polymerization of actin. VI. The polarity of the actin filaments in the acrosomal process and how it might be determined.

The polarity of the actin filaments which assemble from the nucleating body or actomere of Thyone and Pisaster sperm was determined using myosin subfragment 1 decoration. The polarity was found to be unidirectional with the arrowheads pointing towards the cell center. When polymerization is induced at low temperature with concentrations of actin near the critical concentration for polymerization, elongation of filaments occurs preferentially off the apical end. If the sperm are induced to undergo the acrosomal reaction with an ionophore, the polarity of the actin filaments attached to the actomere is the same as that already described, but the filaments which polymerize parallel to, but peripheral to, those extending from the actomere are randomly polarized. These randomly polarized filaments appear to result from spontaneous nucleation. When sperm are induced to undergo the acrosomal reaction with eggs, the polarity of the actin filaments is also unidirectional with the arrowheads pointing towards the cell center. From these results we conclude: (a) that the actomere, by nucleating the polymerization of actin filaments, controls the polarity of the actin filaments in the acrosomal process, (b) that the actomere recognizes a surface of the actin monomer that is different from that surface recognized by the dense material attached to membranes, and (c) that egg myosin could not act to pull the sperm into the egg. Included is a discussion of how the observation that monomers add largely to one end of a decorated filament in vitro relates to these in vivo observations.

Acrosome↗
PubMed1978

Polymerization of actin. IV. Role of Ca++ and H+ in the assembly of actin and in membrane fusion in the acrosomal reaction of echinoderm sperm.

When Pisaster, Asterias, or Thyone sperm are treated with the ionophore A23187 or X537A, an acrosomal reaction similar but not identical to a normal acrosomal reaction is induced in all the sperm. Based upon the response of the sperm, the acrosomal reaction consists of a series of temporally related steps. These include the fusion of the acrosomal vacuole with the cell surface, the polymerization of the actin, the alignment of the actin filaments, an increase in volume, an increase in the limiting membrane, and changes in the shape of the nucleus. In this report, we have concentrated on the first two steps in this sequence. Although fusion of the acrosomal vacuole with the cell surface requires Ca++, we found that the polymerization of actin instead appears to be dependent upon an increase in intracellular pH. This conclusion was reached by applying to sperm A23187, X537A, or nigericin, ionophores which all carry H+ at high affinity, yet vary in their affinity for other cations. When sperm are suspended in isotonic NaCl, isotonic KCl, calcium-free seawater, or seawater, all at pH 8.0, and the ionophore is added, the actin polymerizes explosively and an efflux of H+ from the cell occurs. However, if the pH, of the external medium is maintained at 6.5, the presumed intracellular pH, no effect is observed. And, finally, if egg jelly is added to sperm (the natural stimulus for the acrosomal reaction) at pH 8.0, H+ is also released. On the basis of these observations and those presented in earlier papers in this series, we conclude that a rise in intracellular pH induces the actin to disassociate from its binding proteins. Now it can polymerize.

Acrosome↗
PubMed1978

Polymerization of actin. V. A new organelle, the actomere, that initates the assembly of actin filaments in Thyone sperm.

Between the acrosomal vacuole and the nucleus is a cup of amorphous material (profilactin) which is transformed into filaments during the acrosomal reaction. In the center of this cup in untreated Thyone sperm is a dense material which I refer to as the actomere; it is composed of 20-25 filaments embedded in a dense matrix. To visualize the substructure of the actomere, the profilactin around it must be removed. This is achieved either by demembranating the sperm with Triton X-100 and then raising the pH to 8.0, or by adding inophores to intact sperm at pH 8.0. Under these conditions, the actomere remains as a unit while the rest of the profilactin is solubilized or polymerized. When demembranated sperm are incubated under conditions in which the actin should polymerize, filaments grow from the end of the actomere: the actomere thus appears to behave as a nucleating body. This observation is strengthened by experiments in which untreated sperm are incubated in seawater or isotonic NaCl at pH 7.0 and the ionophore X537A is added; in this case, only a partial polymerization of the actin occurs and the acrosomal vacuole does not fuse with the cell surface. The actin filaments that do form, however, are attached to the apical end of the actomere. In fact, the elongating filaments push their way into and frequently through the acrosomal vacuole. Thus, it appears that the sperm organizes the actin filaments by controlling their nucleation. My model is that the cell controls the ammount of unbound actin such that it is slightly above the critical concentration for polymerization. Then, spontaneous nucleation is unfavored and polymerization would proceed from existing nuclei such as the actomer.

Acrosome↗
PubMed1977

Origin of the membrane for the acrosomal process: is actin complexed with membrane precursors?

The formation of the acrosomal process requires an increase of 40-150% in the plasma membrane limiting the cell body. In freeze fracture replicas and thin sections this new membrane behaves as a typical unit membrane. Since in sperm there are no vesicles or surface projections which could account for the increase in surface, we suggest that the new membrane is generated from precursors stored with the actin in the periacrosomal region. Consistent with this suggestion is the observation that compounds such as didansyl cysteine and N phenyl-1-napthylamine which fluoresce when they enter a hydrophobic environment stain the periacrosomal material.

Acrosome↗
PubMed1977

Movement generated by interactions between the dense material at the ends of microtubles and non-actin-containing microfilaments in Sticholonche zanclea.

Axopods of the planktonic protozoan, Sticholonche, are used as oars to propel the organism through seawater. Within each axopod is an orgainzed array of microtubules which inserts into a dense material that assumes the form of the head of a hip joint. This material, in turn, articulates on the surface of the nucleus. Microfilaments, 20-30 A in diameter, connect the dense material associated with the microtubules to the surface of the nucleus, and they move the axopod by their contractions. The active phase of the movement may take as little as about 0.04 s and the recovery phase may take between 0.2 and 0.4 s. The microfilaments are not actin, as based on: (a) their small diameter, (b) the lack of decoration with heavy meromyosin, and (c) their ability to coil, spiral or fold during contraction. By the use of Thorotrast, we were able to demonstrate that the cell surface is deeply infolded, extending all the way to the hip joint. Here, there is a specialized membrane system that resembles the diad in skeletal muscle. From cytochemical tests and the use of ionophores and chelators, there is some evidence that the motile process may be controlled by calcium. This study demonstrates that, in at least one system, microtubules can be moves by contractile microfilaments attached to the dense material at there tips.

Animals↗
PubMed1976

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

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 sequested in a membrane-bounded compartment such as a vacuole or vesicle. Since filaments are not present in the periacrsomoal 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.

Acrosome↗
PubMed1976

The polymerization of actin. III. Aggregates of nonfilamentous actin and its associated proteins: a storage form of actin.

When echinoderm sperm are treated with the detergent Triton X-100 at pH 6.4 in 10 mM phosphate buffer, the membranes are solubilized, but the actin which is located in the periacrosomal region remains as a phase-dense cup. These cups can be isolated free from the flagella and chromatin and can be solubilized by increasing the pH to 8.0 and by changing the ionic strength and type of buffer used. Since the actin does not exist in the "F" state in unreacted sperm, and since the actin remains as a unit that does not diffuse away, it must be present in the mature sperm in a bound or storage state. The actin is, in fact, associated with a pair of proteins whose mol wt are 250,000 and 230,000. When the isolated cups are digested with trypsin, these high molecular weight proteins are digested, thereby liberating the actin. The actin will polymerize if heavy meromyosin or subfragment 1 is added to a preparation of isolated cups. Evidence is presented that this pair of high molecular weight proteins is similar in molecular weight and properties to erythrocyte spectrin. Attempts at transforming the storage form of actin in the cup into filaments were only moderately successful. The best conditions for filament formation involve incubating the cup in ATP and divalent salts. Careful examination of these cups reveals that the actin polymerized preferentially on either end of oriented filaments that already exist in the cup, indicating that self-nucleation is inefficacious. I conclude that the actin can exist in the storage form by its association with spectrin-like molecules and that the actin in this state polymerizes preferentially onto existing filaments.

Actins↗
PubMed1976

Actin filament-membrane attachment: are membrane particles involved?

The association of actin filaments with membranes is an important feature in the motility of nonmuscle cells. We investigated the role of membrane particles in the attachment of actin filaments to membranes in those systems in which the attachment site can be identified. Freeze fractures through the end-on attachment site of the acrosomal filament bundles in Mytilus (mussel) and Limulus (horseshoe crab) sperm and the attachment site of the microvillar filament bundles in the brush border of intestinal epithelial cells were examined. There are no particles on the P face of the membrane at these sites in the sperm systems and generally none at these sites in microvilli. In microvilli, the actin filaments are also attached along their lengths to the membrane by bridges. When the isolated brush border is incubated in high concentrations of Mg++ (15 mM), the actin filaments form paracrystals and, as a result, the bridges are in register (330 A period). Under these conditions, alignment of the particles on the P face of the membrane into circumferential bands also occurs. However, these bands are generally separated by 800-900 A, indicating that all the bridges cannot be directly attached to membrane particles. Thus membrane particles are not directly involved in the attachment of actin filaments to membranes.

Acrosome↗
PubMed1975

Substructural analysis of the microtubule and its polymorphic forms.

Microtubules are composed of 13 protofilaments which are aligned parallel to the long axis of the tubule, as can be seen in thin sections of material fixed in the presence of tannic acid. Evidence is presented based upon Markham rotations of the isolated flagellar axoneme that the bridges (spoke, nexin, dynein) must connect to certain specified protofilaments. By means of geometric considerations it is concluded that the bridges in a Centrohelida and Raphidiophrys also connect certain specified protofilaments. Two polymorphic forms of microtubules were also examined using tannic acid in the fixative, a 320 A tubule produced in Echinosphaerium by treatment with low temperature, and a vinblastine-induced microtubule crystal. In both forms, the arrangement of the subunits is not in the form of linear protofilaments. The vinblastine crystal in transverse section is made up of hexagonally packed tubules approximately 320 A in diameter. The wall of each tubule in turn is composed of two parallel protofilaments which spiral upwards, giving the appearance of a barber pole. The center-to-center separation of adjacent protofilaments is 180 A. We conclude that the tubulin heterodimer lies parallel to the axis of the protofilaments, exposing the colchicine binding site.

Animals↗
PubMed1975

Actin filaments in the acrosomal reaction of Limulus sperm. Motion generated by alterations in the packing of the filaments.

When Limulus sperm are induced to undergo the acrosomal reaction, a process, 50 mum in length, is generated in a few seconds. This process rotates as it elongates; thus the acrosomal process literally screws through the jelly of the egg. Within the process is a bundle of filaments which before induction are coiled up inside the sperm. The filament bundle exists in three stable states in the sperm. One of the states can be isolated in pure form. It is composed of only three proteins whose molecular weights (mol wt) are 43,000, 55,000, and 95,000. The 43,000 mol wt protein is actin, based on its molecular weight, net charge, morphology, G-F transformation, and heavy meromyosin (HMM) binding. The 55,000 mol wt protein is in equimolar ratio to actin and is not tubulin, binds tenaciously to actin, and inhibits HMM binding. Evidence is presented that both the 55,000 mol wt protein and the 95,000 mol wt protein (possibly alpha-actinin) are also present in Limulus muscle. Presumably these proteins function in the sperm in holding the actin filaments together. Before the acrosomal reaction, the actin filaments are twisted over one another in a supercoil; when the reaction is completed, the filaments lie parallel to each other and form an actin paracrystal. This change in their packing appears to give rise to the motion of the acrosomal process and is under the control of the 55,000 mol wt protein and the 95,000 mol wt protein.

Actins↗
PubMed1975

Actin in erythrocyte ghosts and its association with spectrin. Evidence for a nonfilamentous form of these two molecules in situ.

Actin was isolated from erythrocyte ghosts. It is identical to muscle actin in its molecular weight, net charge, ability to polymerize into filaments with the double helical morphology, and its decoration with heavy meromyosin (HMM). when erythrocyte ghosts are incubated in 0.1 mM EDTA, actin and spectrin are solubilized. Spectrin has a larger molecular weight than muscle myosin. When salt is added to the EDTA extract, a branching filamentous polymer is formed. However, when muscle actin and the EDTA extract are mixed together in the presence of salt, the viscosity achieved is less than the viscosity of the solution if spectrin is omitted. Thus, spectrin seems to inhibit the polymerization of actin. If the actin is already polymerized, the addition of spectrin increases the viscosity of the solution, presumably by cross-linking the actin filaments. The addition of HMM of trypsin to erythrocyte ghosts results in filament formation in situ. These agents apparently act by detaching erythrocyte actin from spectrin, thereby allowing the polmerization of one or both proteins to occur. Since filaments are not present in untreated erythrocyte ghosts, we conclude that erythrocyte actin and spectrin associate to form an anastomosing network beneath the erythrocyte membrane. This network presumably functions in restricting the lateral movement of membrane-penetrating particles.

Actins↗