A change in twist of actin provides the force for the extension of the acrosomal process in limulus sperm: the false-discharge reaction

One of the most spectacular motions is the generation of the acrosomal process in the limulus sperm. On contact with the egg, the sperm generates a 60-mum-long process that literally drills its way through the jelly surrounding the egg. This irresversible reaction takes only a few seconds. We suggested earlier that this motion is driven by a change in twist of the actin filaments comprising the acrosomal process. In this paper we analyze the so-called false discharge, a reversible reaction, in which the acrosomal filament bundle extends laterally from the base of the sperm and not anteriorly from the apex. Unlike the true discharge, which is straight, the false discharge is helical. Before extension, the filament bundle is coiled about the base of the sperm. In the coil, the bundle is not smoothly bent but consists of arms (straight segments) and elbows (corners) so that the coil looks like a 14-sided polygon. The extension of the false discharge works as follows: starting at the base of the bundle, the filaments change their twist which concomitantly changes the orientations of the elbows relative to each other; that is, in the coil, the elbows all like in a common plane, but after the change in twist, the plane of each elbow is rotated to be perpendicular to that of its neighbors. This change transforms the bundle from a compact coil into an extended left- handed helix. Because the basal end of the bundle is unconstrained, the extension is lateral. The true discharge works the same way but starts at the apical end of the bundle. The apical end, however, is constrained by its passage through the nuclear canal, which directs the extention anteriorly. Unlike the false discharge, during the true discharge the elbows are melted out, making the reaction irreversible. This study shows that rapid movement can be regenerated by actin without myosin and gives us insight into the molecular mechanism.     

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

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.     

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.     

Actin-like filaments in the acrosomal apparatus of spermatozoa of a sea urchin

The acrosomal apparatus of a sea urchin, Echinocardium cordatum, consists of an acrosomal vesicle and a post-acrosomal rod. The rod is 2.5 μm long and extends from the acrosomal vesicle to the bottom of a nuclear invagination. The rod consists of a bundle of longitudinally disposed, 60 Å thick, actin-like filaments which bind heavy meromyosin to form arrowhead complexes. The actin-like filaments may have a dual function in the fertilization process: (1) extension of the acrosomal process through the egg investments; (2) incorporation of the sperm nucleus.     

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.     

Three-dimensional reconstruction of an actin bundle

We present the three-dimensional structure of an actin filament bundle from the sperm of Limulus. The bundle is a motile structure which by changing its twist, converts from a coiled to an extended form. The bundle is composed of actin plus two auxiliary proteins of molecular masses 50 and 60 kD. Fraying the bundle with potassium thiocyanate created three classes of filaments: actin, actin plus the 60-kD protein, and actin plus both the auxiliary proteins. We examined these filaments by transmission electron microscopy and scanning transmission electron microscopy (STEM). Three-dimensional reconstructions from electron micrographs allowed us to visualize the actin subunit and the 60- and 50-kD subunits bound to it. The actin subunit appears to be bilobed with dimensions 70 X 40 X 35 A. The inner lobe of the actin subunit, located at 20 A radius, is a prolate ellipsoid, 50 X 25 A; the outer actin lobe, at 30 A radius, is a 35-A-diam spheroid. Attached to the inner lobe of actin is the 60-kD protein, an oblate spheroid, 55 X 40 A, at 50 A radius. The armlike 50-kD protein, at 55 A radius, links the 60-kD protein on one of actin's twin strands to the outer lobe of the actin subunit on the opposite strand. We speculate that the 60-kD protein may be a bundling protein and that the 50-kD protein may be responsible for the change in twist of the filaments which causes extension of the bundle.     

Organization of Actin Filaments in the Axial Rod of Abalone Sperm Revealed by Quick Freeze Technique

An axial rod in abalone ( Haliotis discus ) sperm is a structure composed of a bundle of actin filaments, which elongates anteriorly to form the acrosomal process during the acrosome reaction. The ultrastructure of the actin filament bundle constituting the axial rod was examined using quick freeze technique followed by either freeze-substitution or deep-etch electron microscopy. Thin sections of quick freeze and freeze-substituted sperm revealed that the actin filaments in the axial rod are hexagonally packed in a paracrystalline array through its almost entire length with an average center-to-center spacing of 12 nm. Periodic transverse bands were also observed across the actin filament bundle, which may reflect the cross-bridges interconnecting the adjacent filaments. Quick-freeze deep-etch analysis provided the three-dimensional view of the axial rod. Actin filaments exhibiting 5.5–6 nm spaced striations were observed to run in parallel with each other inside the axial rod. The existence of cross-bridging structures was also displayed between adjacent filaments. These results suggest that the actin filaments in the axial rod are probably held together by regularly spaced cross-bridges to form a well ordered hexagonally packed bundle, and also cross-linked by fibrous structure to the lateral inner acrosomal membrane which closely surrounds the anterior half of the actin filament bundle.     

Structure of actin-containing filaments from two types of non-muscle cells

Bundles of actin-containing filaments from the acrosomal process of horseshoe crab sperm and from sea urchin egg contain a second protein having a molecular weight of about 55,000. Electron micrographs of these filamentous bundles show features reminiscent of paracrystalline arrays of actin except that bundles from the sea urchin egg have distinctive transverse bands every 110 Å. From optical diffraction patterns of the micrographs, we deduced very similar models for both structures. The models consist of hexagonal arrays of actin filaments cross-linked by the second protein. The pattern of transverse bands in bundles derived from the sea urchin eggs is accounted for by postulating that the second protein is bonded to actin only at positions where cross-linking can occur, rather than being bonded to every actin. The helical symmetry of the actin requires that the bonding contacts involved in the cross-linking be slightly different at different positions along the length of the bundle. The technique of image reconstruction was used to obtain a three-dimensional map of the bundles from the acrosomal process.     

Crystallographic conformers of actin in a biologically active bundle of filaments

Actin carries out many of its cellular functions through its filamentous form; thus, understanding the detailed structure of actin filaments is an essential step in achieving a mechanistic understanding of actin function. The acrosomal bundle in the Limulus sperm has been shown to be a quasi-crystalline array with an asymmetric unit composed of a filament with 14 actin-scruin pairs. The bundle in its true discharge state penetrates the jelly coat of the egg. Our previous electron crystallographic reconstruction demonstrated that the actin filament cross-linked by scruin in this acrosomal bundle state deviates significantly from a perfect F-actin helix. In that study, the tertiary structure of each of the 14 actin protomers in the asymmetric unit of the bundle filament was assumed to be constant. In the current study, an actin filament atomic model in the acrosomal bundle has been refined by combining rigid-body docking with multiple actin crystal structures from the Protein Data Bank and constrained energy minimization. Our observation demonstrates that actin protomers adopt different tertiary conformations when they form an actin filament in the bundle. The scruin and bundle packing forces appear to influence the tertiary and quaternary conformations of actin in the filament of this biologically active bundle.     

Sperm Bundles in the Seminal Vesicle of the Crematogaster victima (Smith) Adult Males (Hymenoptera: Formicidae)

This study establishes the presence of spermatodesm in the seminal vesicles of sexually mature males of Crematogaster victima (Smith). In this species, the spermatozoa are maintained together by an extracellular matrix in which the acrosomal regions are embedded. This characteristic has not yet been observed in any other Aculeata. However, the sperm morphology in this species is similar to that described for other ants. The spermatozoa measure on average 100 μm in length, and the number of sperm per bundle is up to 256. They are composed of a head formed by the acrosome and nucleus; this is followed by the flagellum, which is formed by the centriolar adjunct, an axoneme with a 9?+?9?+?2 microtubule pattern, two mitochondrial derivatives, and two accessory bodies. The acrosome is formed by the acrosomal vesicle and perforatorium. The nucleus is filled with compact chromatin with many areas of thick and non-compacted filaments. Both mitochondrial derivatives have the same shape and diameters. The presence of sperm bundles in sexually mature males differentiates C. victima from other ants; however, the similarities in the sperm ultrastructure support the monophyly of this insect group.     

Bending stiffness of a crystalline actin bundle

The acrosomal process of the sperm of the horseshoe crab (Limulus polyphemus) is a unique crystalline actin bundle, consisting of multiple actin filaments cross-linked by the actin-bundling protein, scruin. For successful fertilization, the acrosomal bundle must penetrate through a 30 microm thick jelly coat surrounding the egg and thus it must be sufficiently stiff. Here, we present two measurements of the bending stiffness of a single crystalline bundle of actin. Results from these measurements indicate that the actin:scruin composite bundle has an average elastic modulus of 2 GPa, which is similar to that of a single actin filament, and a bending stiffness that is more than two orders of magnitude larger than that of a bundle of uncross-linked actin filaments due to stiffening by the scruin matrix.     

Three-dimensional structure of a single filament in the Limulus acrosomal bundle: scruin binds to homologous helix-loop-beta motifs in actin

Frozen, hydrated acrosomal bundles from Limulus sperm were imaged with a 400 kV electron cryomicroscope. Segments of this long bundle can be studied as a P1 crystal with a unit cell containing an acrosomal filament with 28 actin and 28 scruin molecules in 13 helical turns. A novel computational procedure was developed to extract single columns of superimposed acrosomal filaments from the distinctive crystallographic view. Helical reconstruction was used to generate a three-dimensional structure of this computationally isolated acrosomal filament. The scruin molecule is organized into two domains which contact two actin subunits in different strands of the same actin filament. A correlation of Holmes' actin filament model to the density in our acrosomal filament map shows that actin subdomains 1, 2, and 3 match the model density closely. However, actin subdomain 4 matches rather poorly, suggesting that interactions with scruin may have altered actin conformation. Scruin makes extensive interactions with helix-loop-beta motifs in subdomain 3 of one actin subunit and in subdomain 1 of a consecutive actin subunit along the genetic filament helix. These two actin subdomains are structurally homologous and are closely spaced along the actin filament. Our model suggests that scruin, which is derived from a tandemly duplicated gene, has evolved to bind structurally homologous but non-identical positions across two consecutive actin subunits.     

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.     

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.     

Crystallographic analysis of acrosomal bundle from Limulus sperm

The acrosomal process of Limulus sperm contains a bundle of filaments composed of actin and a 102 kDa protein in a 1:1 molar ratio. The structure of the bundle in true discharge was investigated by electron cryomicroscopy, X-ray scattering and crystallographic image analysis. A bundle can be characterized as a quasi-crystal with continuously varying views along the bundle axis. Each segment of the bundle is found to obey the symmetry of space group P1, with a = b = 147 A, c = 762 A, alpha = 90 degrees, beta = 90.6 degrees, gamma = 120 degrees. A unit cell contains a helical repeat of the filament with a selection rule following that of an actin filament. A 24 A projection map based on the h0l view was reconstructed after averaging 5300 unit cells from six electron images. Filaments in this projection are well separated and clearly display a 21 screw symmetry. This screw symmetry results from the helical parameters of the bundle filament and is found to be a non-crystallographic symmetry element present in the unit cell. Our structural analysis has led to the proposal that the assembly of a stable bundle with a defined maximum diameter can be controlled by the crystallographic packing of the twisted filaments.     

Conformational transitions of schizophyllan in aqueous alkaline solution

The conformational transitions of schizophyllan were studied in aqueous alkaline solutions by high-sensitivity differential scanning calorimetry (DSC) and optical rotation measurements. The temperature of half completion for reversible intramolecular conformational transition determined by DSC, centered at 7.4°C in water, increases to 37.2°C at 0.01M KOH with increasing alkaline concentration. The transition enthalpy per mole of the polysaccharide repeating unit is 2.62 ± 0.23 kJ mol⁻¹ independent of the alkaline concentration. The cooperative unit size for the transition decreases with increasing alkaline concentration. Optical rotation was measured as a function of pH at 25 and 60°C. A sharp decrease in optical rotation was observed at pH = 13, which is ascribed to the triple helix-coil transition. From data obtained by DSC and optical rotation measurements, in combination with results reported previously, a phase diagram for the conformation of schizophyllan as a function of temperature and pH is proposed. The irreversibility of the triple helix to single coil transition, induced by strong alkali, was investigated as a function of polymer concentration by gel permeation chromatography and electron microscopy. The renatured samples at polymer concentrations < 1.0 mg/mL, which are prepared by dissolution in 0.25M KOH followed by neutralization with HCl, are observed as a mixture of globular, linear, and circular structures, and larger aggregates with less-defined morphology by electron microscopy. Higher concentrations lead to increased proportions of multichain clusters (aggregates). Subsequent annealing of the renatured samples at 115–120°C increases the proportion of circular species. The change in molecular weight distribution of samples that accompanies the renaturation and annealing mentioned above can be well interpreted in terms of the proportion of species having different morphology as observed by electron microscopy. © 1996 John Wiley & Sons, Inc.     

Calcium Regulation of an Actin Spring

Calcium is essential for many biological processes involved in cellular motility. However, the pathway by which calcium influences motility, in processes such as muscle contraction and neuronal growth, is often indirect and complex. We establish a simple and direct mechanochemical link that shows how calcium quantitatively regulates the dynamics of a primitive motile system, the actin-based acrosomal bundle of horseshoe crab sperm. The extension of this bundle requires the continuous presence of external calcium. Furthermore, the extension rate increases with calcium concentration, but at a given concentration, we find that the volumetric rate of extension is constant. Our experiments and theory suggest that calcium sequentially binds to calmodulin molecules decorating the actin filaments. This binding leads to a collective wave of untwisting of the actin filaments that drives bundle extension.     

The variable twist of actin and its modulation by actin-binding proteins

Previous studies demonstrated that actin filaments have variable twist in which the intersubunit angles vary by approximately +/- 10 degrees within a filament. In this work we show that this variability was unchanged when different methods were used to prepare filaments for electron microscopy. We also show that actin-binding proteins can modulate the variability in twist. Three preparations of actin filaments were photographed in the electron microscope: negatively stained filaments, replicas of rapidly frozen, etched filaments, and frozen hydrated filaments. In addition, micrographs of actin + tropomyosin + troponin (thin filaments), of actin + myosin S1 (decorated filaments), and of filaments frayed from the acrosomal process of Limulus sperm (Limulus filaments) were obtained. We used two independent methods to measure variable twist based on Fourier transforms of single filaments. The first involved measuring layer line intensity versus filament length and the second involved measuring layer line position. We measured a variability in the intersubunit angle of actin filaments of approximately 12 degrees independent of the method of preparation or of measurement. Thin filaments have 15 degrees of variability, but the increase over pure actin is not statistically significant. Decorated filaments and Limulus filaments, however, have significantly less variability (approximately 2 and 1 degree, respectively), indicating a torsional stiffening relative to actin. The results from actin alone using different preparative methods are evidence that variable twist is a property of actin in solution. The results from actin filaments in the presence of actin-binding proteins suggest that the angular variability can be modulated, depending on the biological function.     

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.     

polymerization of actin: v.a new organelle, the actomere, that initiates 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 ionophores 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 amount 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 actomere.