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.     

A change in the twist of the actin-containing filaments occurs during the extension of the acrosomal process in Limulus sperm

The spectacular extension of the acrosomal process in Limulus sperm is effected by a bundle of actin-containing filaments with apparently no contribution from myosin. The bundle is coiled about the base of the sperm and, upon reaction, unwinds and extends out of the anterior end of the sperm with a screwing motion. We have analyzed the structure of the bundle in the coil and following its discharge. Optical diffraction studies of electron micrographs show a difference in the twist of the filaments in the two forms. The filaments in the coil have a twist of 0.23 ° per subunit more than that in the true discharge. As the signal to extend moves down the coil, the filaments change their twist and the bundle straightens. The coupling of these two movements produces the screwing motion. In the coil, the filaments wind around the axis of the bundle. As the filaments change their twist, the winding is undone. From freeze-fracture replicas we determined the hand of the winding of filaments in the coil and, in thin sections, we were able to determine the number of turns the filaments make for each loop of the coil. From these data we were able to predict the hand and amount of rotation during the discharge. From movie film sequences we could determine only the amount of rotation and found it to be 0.25 ° ± 0.05 ° per subunit discharged. This is in reasonable agreement with the expected value of 0.23 ° ± 0.05 ° per subunit. We propose that it is the change in twist of the actin filaments themselves that is responsible for the generation of force for the extension of the acrosomal process.     

Organization of an actin filament-membrane complex. Filament polarity and membrane attachment in the microvilli of intestinal epithelial cells

The association of actin filaments with membranes is now recognized as an important parameter in the motility of nonmuscle cells. We have investigated the organization of one of the most extensive and highly ordered actin filament-membrane complexes in nature, the brush border of intestinal epithelial cells. Through the analysis of isolated, demembranated brush borders decorated with the myosin subfragment, S1, we have determined that all the microvillar actin filaments have the same polarity. The S1 arrowhead complexes point away from the site of attachment of actin filaments at the apical tip of the microvillar membrane. In addition to the end-on attachment of actin filaments at the tip of the microvillus, these filaments are also connected to the plasma membrane all along their lengths by periodic (33 nm) cross bridges. These bridges were best observed in isolated brush borders incubated in high concentrations of Mg++. Their visibility is attributed to the induction of actin paracrystals in the filament bundles of the microvilli. Finally, we present evidence for the presence of myosinlike filaments in the terminal web region of the brush border. A model for the functional organization of actin and myosin in the brush border is presented.     

ISOLATION AND REACTIVATION OF THE AXOSTYLE : Evidence for a Dynein-like ATPase in the Axostyle

The contractile axostyle is a ribbon-shaped organelle present in certain species of flagellates found in the hindgut of wood eating insects. This organelle propagates an undulatory wave whose motion, like flagella and cilia, is related to microtubules. Unlike the axoneme of cilia and flagella, however, the axostyle is composed of singlet microtubules linked together in parallel rows. Axostyles were isolated from Cryptocercus gut protozoa with Triton X-100. Normal motility of the isolated axostyle could be restored with adenosine triphosphate (ATP); the specific conditions necessary for this reactivation were essentially identical with those reported for the reactivation of isolated flagella or whole sperm. ATPase activity of the isolated axostyle was comparable to the values reported for ciliary or flagellar axonemes. The axostyle was reasonably specific for ATP. Most of the proteins of the isolated axostyle comigrated with proteins of the ciliary axoneme on sodium dodecyl sulfate (SDS) polyacrylamide gels (i e. equivalent molecular weights). These included the following: the higher molecular weight component of dynein, tubulin, linkage protein (nexin), and various secondary proteins. Evidence for dynein in the axostyle is presented and a model proposed to explain how repeated propagated waves can be generated.     

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.     

HOW MICROTUBULE PATTERNS ARE GENERATED : The Relative Importance of Nucleation and Bridging of Microtubules in the Formation of the Axoneme of Raphidiophrys

The axonemes of Raphidiophrys converge near the center of the cell in an electron-opaque material, the centroplast. In order to establish whether this material acts not only to nucleate the microtubules which form the axonemes but also to give the axoneme its characteristic pattern, the microtubules were disassembled with low temperature and stages in their reformation were studied. It was shown that even though the microtubules appear to be nucleated from the centroplast, pattern formation first appeared at a distance from the centroplast. Thus, the axonemal pattern could not be attributed to any prepattern in the centroplast. Rather, the pattern appears to arise by specific interactions between tubules brought about by bridges. It was concluded that each tubule could bind to a maximum of four other tubules and that once one bridge attached to a tubule it specified the binding positions of the others, thus giving the characteristic axonemal pattern of Raphidiophrys.     

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.     

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.     

Microtubules in the formation and development of the primary mesenchyme in Arbacia punctulata. II. An experimental analysis of their role in development and maintenance of cell shape

To experimentally test the suggestion made in the preceding paper that the microtubules are involved in cell shape development during the formation and differentiation of the primary mesenchyme, we applied to the embryos two types of agents which affect cytoplasmic microtubules: (a) colchicine and hydrostatic pressure, which cause the microtubules to disassemble, and (b) D₂O, which tends to stabilize them. When the first type of agent is applied to sea urchin gastrulae, the development of the primary mesenchyme ceases, the microtubules disappear, and the cells tend to spherulate. With D₂O development also ceases, but the tubules appear "frozen," and the cell asymmetries persist unaltered. These agents appear to block development by primarily interfering with the sequential disassembly and/or reassembly of microtubules into new patterns. The microtubules, therefore, appear to be influential in the development of cell form. On the other hand through a careful analysis of the action of these agents and others on both intra- and extracellular factors, we concluded that the microtubules do rather little for the maintenance of cell shape in differentiated tissues.     

Time-resolved fluoroimmunoassay of potato virus M with monoclonal antibodies

Mouse monoclonal antibodies (MAbs) specific for potato virus M (PVM) were prepared and the properties of three of them were studied. MAb M4C1 is IgG2b, it binds with high affinity to PVM coat protein, to purified virus preparations and recognises PVM in infected potato leaves and tubers. MAb M6D5 is IgG2a and also reacts with PVM coat protein, purified PVM and with PVM in potato leaf and tuber extracts. In double-antibody sandwich ELISA (DAS ELISA) MAbs M4C1 and M6D5 reacted with all 17 PVM isolates tested. MAb M7 is IgG2b and recognises PVM only in indirect dot ELISA on nitrocellulose filters and viral coat protein on Western blots. MAbs against PVM were used as capture antibodies and europium-labelled MAbs as conjugates in time-resolved fluoroimmunoassay (EuTRFIA). The standard EuTRFIA curve of PVM detection is approximately linear over a range of PVM concentrations from 0.5 ng/ml to 1000 ng/ml. The lowest PVM concentration detectable in EuTRFIA was 0.5 ng/ml and correspondingly 6 ng/ml in DAS ELISA. The use of the europium chelate label allows PVM detection in potato leaf and tuber sap at dilutions greater than 10^--⁴ with very low background fluorescence. EuTRFIA with MAbs, with either one or two incubations is about 10–20 times more sensitive for PVM detection than is DAS ELISA. PVM and PVX, mixed with healthy potato tuber sap, were simultaneously tested in a single sample at concentrations lower than 10 ng/ml by double-label TRFIA using europium-labelled MAbs to PVM and samarium-labelled MAbs to PVX.