Jasplakinolide reversibly disrupts actin filaments in suspension-cultured tobacco BY-2 cells

Summary. Jasplakinolide is potentially a useful pharmacological tool for the study of actin organization and dynamics in living cells, since it induces actin polymerization in vitro and, unlike phalloidin, is membrane permeative. In the present work, the effect of jasplakinolide on the actin cytoskeleton of living suspension-cultured Nicotiana tabacum ‘Bright Yellow 2’ cells was investigated. Actin filaments in the living cells were disrupted by jasplakinolide. The effect of jasplakionlide on the actin cytoskeleton was concentration and time dependent. When cells were treated with a moderate concentration (150 nM) of jasplakinolide, cortical actin filaments were disrupted preferentially, whereas actin aggregated at the perinuclear region. With concentrations higher than 400 nM and exposure times longer than 30 min, actin filaments in the cell disappeared completely. The effect of jasplakinolide on the actin cytoskeleton was reversible even at high concentration. Actin bundles appeared first in the perinuclear region within 5 min, and the cortical actin array was reestablished in 15 min, suggesting that actin filaments might be organized at this region. Received July 31, 2001 Accepted December 14, 2001     

Actin Stabilization by Jasplakinolide Affects the Function of Bone Marrow-Derived Late Endothelial Progenitor Cells

Background

Bone marrow-derived endothelial progenitor cells (EPCs), especially late EPCs, play a critical role in endothelial maintenance and repair, and postnatal vasculogenesis. Although the actin cytoskeleton has been considered as a modulator that controls the function and modulation of stem cells, its role in the function of EPCs, and in particular late EPCs, remains poorly understood.

Methodology/Principal Finding

Bone marrow-derived late EPCs were treated with jasplakinolide, a compound that stabilizes actin filaments. Cell apoptosis, proliferation, adhesion, migration, tube formation, nitric oxide (NO) production and endothelial NO synthase (eNOS) phosphorylation were subsequently assayed in vitro. Moreover, EPCs were locally infused into freshly balloon-injured carotid arteries, and the reendothelialization capacity was evaluated after 14 days. Jasplakinolide affected the actin distribution of late EPCs in a concentration and time dependent manner, and a moderate concentration of (100 nmol/l) jasplakinolide directly stabilized the actin filament of late EPCs. Actin stabilization by jasplakinolide enhanced the late EPC apoptosis induced by VEGF deprivation, and significantly impaired late EPC proliferation, adhesion, migration and tube formation. Furthermore, jasplakinolide attenuated the reendothelialization capacity of transplanted EPCs in the injured arterial segment in vivo. However, eNOS phosphorylation and NO production were increased in late EPCs treated with jasplakinolide. NO donor sodium nitroprusside (SNP) rescued the functional activities of jasplakinolide-stressed late EPCs while the endothelial NO synthase inhibitor L-NAME led to a further dysfunction induced by jasplakinolide in late EPCs.

Conclusions/Significance

A moderate concentration of jasplakinolide results in an accumulation of actin filaments, enhancing the apoptosis induced by cytokine deprivation, and impairing the proliferation and function of late EPCs both in vitro and in vivo. NO donor reverses these impairments, suggesting the role of NO-related mechanisms in jasplakinolide-induced EPC downregulation. Actin cytoskeleton may thus play a pivotal role in regulating late EPC function.     

Rate of treadmilling of actin filaments in vitro

Actin filaments capped at the barbed ends were formed by polymerizing monomeric actin onto a gelsolin-actin complex. The rate of depolymerization and polymerization of the pointed ends was determined by diluting gelsolin-capped actin filaments into various concentrations of monomeric actin. Under the conditions of the experiments (100 mM-KCl, 2 mM-MgCl2 at 37 degrees C) the rate constant of dissociation of subunits both from a shortening and a lengthening filament was found to be 0.21 s-1. As the rate of dissociation of subunits from the slow pointed end determines the rate of treadmilling, it is concluded that actin filaments treadmill with a rate of about 2 micron/h.     

Effects of CapZ, an actin capping protein of muscle, on the polymerization of actin

We have studied the interaction of CapZ, a barbed-end actin capping protein from the Z line of skeletal muscle, with actin. CapZ blocks actin polymerization and depolymerization (i.e., it "caps") at the barbed end with a Kd of approximately 0.5-1 nM or less, measured by three different assays. CapZ inhibits the polymerization of ATP-actin onto filament ends with ATP subunits slightly less than onto ends with ADP subunits, and onto ends with ADP-BeF3- subunits about as much as ends with ADP subunits. No effect of CapZ is seen at the pointed end by measurements either of polymerization from acrosomal processes or of the critical concentration for polymerization at steady state. CapZ has no measureable ability to sever actin filaments in a filament dilution assay. CapZ nucleates actin polymerization at a rate proportional to the first power of the CapZ concentration and the 2.5 power of the actin concentration. No significant binding is observed between CapZ and rhodamine-labeled actin monomers by fluorescence photobleaching recovery. These new experiments are consistent with but do not distinguish between three models for nucleation proposed previously (Cooper & Pollard, 1985). As a prelude to the functional studies, the purification protocol for CapZ was refined to yield 2 mg/kg of chicken breast muscle in 1 week. The activity is stable in solution and can be lyophilized. The native molecular weight is 59,600 +/- 2000 by equilibrium ultracentrifugation, and the extinction coefficient is 1.25 mL mg-1 cm-1 by interference optics. Polymorphism of the alpha and beta subunits has been detected by isoelectric focusing and reverse-phase chromatography. CapZ contains no phosphate (less than 0.1 mol/mol).     

Jasplakinolide, an actin stabilizing agent, alters anaphase chromosome movements in crane-fly spermatocytes

We added jasplakinolide to anaphase crane-fly spermatocytes and determined its effects on chromosome movement. Previous work showed that the actin depolymerizing agents cytochalasin D or latrunculin B blocked or slowed chromosome movements. We studied the effects of jasplakinolide, a compound that stabilizes actin filaments. Jasplakinolide had the same effect on movements of each half- bivalent in a separating pair of half-bivalents, but different half-bivalent pairs in the same cell often responded differently, even when the concentrations of jasplakinolide varied by a factor of two. Jasplakinolide had no effect on about 20% of the pairs, but otherwise caused movements to slow, or to stop, or, rarely, to accelerate. When cells were kept in jasplakinolide, stopped pairs eventually resumed movement; slowed pairs did not change their speeds. Confocal microscopy indicated that neither the distributions of spindle actin filaments nor the distributions of spindle microtubules were altered by the jasplakinolide. It is possible that jasplakinolide binds to spindle actin and blocks critical binding sites, but we suggest that jasplakinolide affects anaphase chromosome movement by preventing actin-filament depolymerization that is necessary for anaphase to proceed. Overall, our data indicate that actin is involved in one of the redundant mechanisms cells use to move chromosomes.     

Nucleation limits the lengths of actin filaments assembled by formin

Formins stimulate actin polymerization by promoting both filament nucleation and elongation. Because nucleation and elongation draw upon a common pool of actin monomers, the rate at which each reaction proceeds influences the other. This interdependent mechanism determines the number of filaments assembled over the course of a polymerization reaction, as well as their equilibrium lengths. In this study, we used kinetic modeling and in vitro polymerization reactions to dissect the contributions of filament nucleation and elongation to the process of formin-mediated actin assembly. We found that the rates of nucleation and elongation evolve over the course of a polymerization reaction. The period over which each process occurs is a key determinant of the total number of filaments that are assembled, as well as their average lengths at equilibrium. Inclusion of formin in polymerization reactions speeds filament nucleation, thus increasing the number and shortening the lengths of filaments that are assembled over the course of the reaction. Modulation of the elongation rate produces modest changes in the equilibrium lengths of formin-bound filaments. However, the dependence of filament length on the elongation rate is limited by the number of filament ends generated via formin’s nucleation activity. Sustained elongation of small numbers of formin-bound filaments, therefore, requires inhibition of nucleation via monomer sequestration and a low concentration of activated formin. Our results underscore the mechanistic advantage for keeping formin’s nucleation efficiency relatively low in cells, where unregulated actin assembly would produce deleterious effects on cytoskeletal dynamics. Under these conditions, differences in the elongation rates mediated by formin isoforms are most likely to impact the kinetics of actin assembly.     

Guardians of the actin monomer

Actin is a universal force provider in eukaryotic cells. Biological processes harness the pressure generated from actin polymerization through dictating the time, place and direction of filament growth. As such, polymerization is initiated and maintained via tightly controlled filament nucleation and elongation machineries. Biological systems integrate force into their activities through recruiting and activating these machineries. In order that actin function as a common force generating polymerization motor, cells must maintain a pool of active, polymerization-ready monomeric actin, and minimize extemporaneous polymerization. Maintenance of the active monomeric actin pool requires the recycling of actin filaments, through depolymerization, nucleotide exchange and reloading of the polymerization machineries, while the levels of monomers are constantly monitored and supplemented, when needed, via the access of a reserve pool of monomers and through gene expression. Throughout its monomeric life, actin needs to be protected against gratuitous nucleation events. Here, we review the proteins that act as custodians of monomeric actin. We estimate their levels on a tissue scale, and calculate the implied concentrations of each actin complex based on reported binding affinities. These estimations predict that monomeric actin is rarely, if ever, alone. Thus, the guardians keep the volatility of actin in check, so that its explosive power is only released in the controlled environments of the nucleation and polymerization machineries.     

Yeast actin patches are networks of branched actin filaments

Cortical actin patches are the most prominent actin structure in budding and fission yeast. Patches assemble, move, and disassemble rapidly. We investigated the mechanisms underlying patch actin assembly and motility by studying actin filament ultrastructure within a patch. Actin patches were partially purified from Saccharomyces cerevisiae and examined by negative-stain electron microscopy (EM). To identify patches in the EM, we correlated fluorescence and EM images of GFP-labeled patches. Patches contained a network of actin filaments with branches characteristic of Arp2/3 complex. An average patch contained 85 filaments. The average filament was only 50-nm (20 actin subunits) long, and the filament to branch ratio was 3:1. Patches lacking Sac6/fimbrin were unstable, and patches lacking capping protein were relatively normal. Our results are consistent with Arp2/3 complex-mediated actin polymerization driving yeast actin patch assembly and motility, as described by a variation of the dendritic nucleation model.     

Polylysine induces an antiparallel actin dimer that nucleates filament assembly: crystal structure at 3.5-A resolution

An antiparallel actin dimer has been proposed to be an intermediate species during actin filament nucleation. We now show that latrunculin A, a marine natural product that inhibits actin polymerization, arrests polylysine-induced nucleation at the level of an antiparallel dimer, resulting in its accumulation. These dimers, when composed of pyrene-labeled actin subunits, give rise to a fluorescent excimer, permitting detection during polymerization in vitro. We report the crystallographic structure of the polylysine-actin-latrunculin A complex at 3.5-A resolution. The non-crystallographic contact is consistent with a dimeric structure and confirms the antiparallel orientation of its subunits. The crystallographic contacts reveal that the mobile DNase I binding loop of one subunit of a symmetry-related antiparallel actin dimer is partially stabilized in the interface between the two subunits of a second antiparallel dimer. These results provide a potential explanation for the paradoxical nucleation of actin filaments that have exclusively parallel subunits by a dimer containing antiparallel subunits.     

Interaction of thymosin beta 4 with muscle and platelet actin: implications for actin sequestration in resting platelets.

Quantitative measurements of the interactions of T beta 4 with muscle actin suggest that its only physiological role is monomer sequestration. T beta 4 forms a 1:1 complex with monomeric actin under physiological salt conditions. Its Kd for actin is not affected by calcium. T beta 4 binds only to actin monomers and not to filament ends or alongside the filament. T beta 4-actin complexes do not elongate actin filaments at either the barbed or the pointed end, and, unlike actobindin, T beta 4 does not specifically suppress the nucleation of polymerization. We assessed the fraction of monomeric actin that can be sequestered by T beta 4 in resting platelets. This was done on the basis of (a) its Kd of 0.4-0.7 microM for platelet actin, which had been prepared by a newly devised simpler method, and (b) the values for the concentrations of monomeric actin and of T beta 4 which we measured as 280 and 560 microM, respectively. Using the higher Kd value of 0.7 microM, the T beta 4-complexed actin is calculated to be between 70 and 240 microM, depending on the steady-state free G-actin concentration. This may vary from 0.1 to 0.5 microM, the critical concentrations for uncapped and for fully barbed-end-capped actin filaments. If the Kd in the platelet is the same as in vitro, most of the sequestered actin would be bound to T beta 4 if more than 95% of the actin filaments are capped at their barbed ends in resting platelets.     

Structure of a Longitudinal Actin Dimer Assembled by Tandem W Domains: Implications for Actin Filament Nucleation

Actin filament nucleators initiate polymerization in cells in a regulated manner. A common architecture among these molecules consists of tandem WASP homology 2 domains (W domains) that recruit three to four actin subunits to form a polymerization nucleus. We describe a low-resolution crystal structure of an actin dimer assembled by tandem W domains, where the first W domain is cross-linked to Cys374 of the actin subunit bound to it, whereas the last W domain is followed by the C-terminal pointed end-capping helix of thymosin β4. While the arrangement of actin subunits in the dimer resembles that of a long-pitch helix of the actin filament, important differences are observed. These differences result from steric hindrance of the W domain with intersubunit contacts in the actin filament. We also determined the structure of the first W domain of Vibrio parahaemolyticus VopL cross-linked to actin Cys374 and show it to be nearly identical with non-cross-linked W-Actin structures. This result validates the use of cross-linking as a tool for the study of actin nucleation complexes, whose natural tendency to polymerize interferes with most structural methods. Combined with a biochemical analysis of nucleation, the structures may explain why nucleators based on tandem W domains with short inter-W linkers have relatively weak activity, cannot stay bound to filaments after nucleation, and are unlikely to influence filament elongation. The findings may also explain why nucleation-promoting factors of the Arp2/3 complex, which are related to tandem-W-domain nucleators, are ejected from branch junctions after nucleation. We finally show that the simple addition of the C-terminal pointed end-capping helix of thymosin β4 to tandem W domains can change their activity from actin filament nucleation to monomer sequestration.     

Stochastic severing of actin filaments by actin depolymerizing factor/cofilin controls the emergence of a steady dynamical regime

Actin dynamics (i.e., polymerization/depolymerization) powers a large number of cellular processes. However, a great deal remains to be learned to explain the rapid actin filament turnover observed in vivo. Here, we developed a minimal kinetic model that describes key details of actin filament dynamics in the presence of actin depolymerizing factor (ADF)/cofilin. We limited the molecular mechanism to 1), the spontaneous growth of filaments by polymerization of actin monomers, 2), the ageing of actin subunits in filaments, 3), the cooperative binding of ADF/cofilin to actin filament subunits, and 4), filament severing by ADF/cofilin. First, from numerical simulations and mathematical analysis, we found that the average filament length, 〈L〉, is controlled by the concentration of actin monomers (power law: 5/6) and ADF/cofilin (power law: −2/3). We also showed that the average subunit residence time inside the filament, 〈T〉, depends on the actin monomer (power law: −1/6) and ADF/cofilin (power law: −2/3) concentrations. In addition, filament length fluctuations are ∼20% of the average filament length. Moreover, ADF/cofilin fragmentation while modulating filament length keeps filaments in a high molar ratio of ATP- or ADP-P_i versus ADP-bound subunits. This latter property has a protective effect against a too high severing activity of ADF/cofilin. We propose that the activity of ADF/cofilin in vivo is under the control of an affinity gradient that builds up dynamically along growing actin filaments. Our analysis shows that ADF/cofilin regulation maintains actin filaments in a highly dynamical state compatible with the cytoskeleton dynamics observed in vivo.     

Effect of jasplakinolide on the growth, encystation, and actin cytoskeleton of Entamoeba histolytica and Entamoeba invadens

The effect of jasplakinolide. an actin-polymerizing and filament-stabilizing drug, on the growth, encystation, and actin cytoskeleton of Entamoeba histolytica and Entamoeba invadens was examined. Jasplakinolide inhibited the growth of E. histolytica strain HM-1:IMSS and E. invadens strain IP-1 in a concentration-dependent manner, the latter being more resistant to the drug. The inhibitory effect of jasplakinolide on the growth of E. histolytica trophozoites was reversed by removal of the drug after exposure to 1 microM for 1 day. Encystation of E. invadens as induced in vitro was also inhibited by jasplakinolide. Trophozoites exposed to jasplakinolide in encystation medium for 1 day did not encyst after removal of the drug, whereas those exposed to the drug in growth medium for 7 days did encyst without the drug. The process of cyst maturation was unaffected by jasplakinolide. Large round structures were formed in trophozoites of both amoebae grown with jasplakinolide; these were identified as F-actin aggregates by staining with fluorescent phalloidin. Accumulation in trophozoites of both amoebae of actin aggregates was observed after culture in jasplakinolide. Also, E. invadens cysts formed from trophozoites treated with jasplakinolide contained the actin aggregate. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis revealed that the jasplakinolide treatment led to an increase in the proportion of F-actin associated with formation of the aggregate. The results suggest that aggregates are formed from the cortical flow of F-actin filaments, and that these filaments would normally be depolymerized but are artificially stabilized by jasplakinolide binding.     

F actin assembly modulated by villin: Ca++-dependent nucleation and capping of the barbed end

We have studied the mechanism of Ca++-dependent restriction of actin filament length by villin, one of the major actin-associated proteins of intestinal microvilli microfilament bundles. Villin acts, even at a ratio of 1 to 1000 with respect to actin, very efficiently as a Ca++-dependent nucleation factor on actin assembly. This gives rise to unidirectional assembly, with the morphologically defined "barbed" end of the resulting filament being capped. Consequently, at steady state treadmilling of actin monomers through the filament is inhibited. Increase of the villin-to-actin ratio enhances the number of nucleated filaments necessarily shorter in length. This results finally in nonsedimentable F actin and a low molecular weight complex of one villin and three monomeric actins, which itself is a potent nucleator. Thus restriction of actin assembly by villin is not due to a direct inhibition of assembly but arises as the consequence of strongly enhanced nucleation followed by unidirectional elongation at the pointed end of the nucleated filaments. In addition, in the presence of Ca++-villin, but not the villin-actin complex, seems able to "break" or "sever" preformed F actin filaments. Thus a variety of cellular phenomena-nucleation, unidirectional assembly, filament end capping, nonpolymerizable actin and F actin bundles-can be observed in vitro in a two-protein component system modulated by the concentration of free Ca++.     

Inhibition of the Arp2/3 complex-nucleated actin polymerization and branch formation by tropomyosin

The actin filament network immediately under the plasma membrane at the leading edge of rapidly moving cells consists of short, branched filaments, while those deeper in the cortex are much longer and are rarely branched. Nucleation by the Arp2/3 complex activated by membrane-bound factors (Rho-family GTPases and PIP(2)) is postulated to account for the formation of the branched network. Tropomyosin (TM) binds along the sides of filaments and protects them from severing proteins and pointed-end depolymerization in vitro. Here, we show that TM inhibits actin filament branching and nucleation by the Arp2/3 complex activated by WASp-WA. Tropomyosin increases the lag at the outset of polymerization, reduces the concentration of ends by 75%, and reduces the number of branches by approximately 50%. We conclude that TM bound to actin filaments inhibits their ability to act as secondary activators of nucleation by the Arp2/3 complex. This is the first example of inhibition of branching by an actin binding protein. We suggest that TM suppresses the nucleation of actin filament branches from actin filaments in the deep cortex of motile cells. Other abundant actin binding proteins may also locally regulate the branching nucleation by the Arp2/3 complex in cells.     

Exogenous nucleation sites fail to induce detectable polymerization of actin in living cells

Most nonmuscle cells are known to maintain a relatively high concentration of unpolymerized actin. To determine how the polymerization of actin is regulated, exogenous nucleation sites, prepared by sonicating fluorescein phalloidin-labeled actin filaments, were microinjected into living Swiss 3T3 and NRK cells. The nucleation sites remained as a cluster for over an hour after microinjection, and caused no detectable change in the phase morphology of the cell. As determined by immunofluorescence specific for endogenous actin and by staining cells with rhodamine phalloidin, the microinjection induced neither an extensive polymerization of endogenous actin off the nucleation sites, nor changes in the distribution of actin filaments. In addition, the extent of actin polymerization, as estimated by integrating the fluorescence intensities of bound rhodamine phalloidin, did not appear to be affected. To determine whether the nucleation sites remained active after microinjection, cells were first injected with nucleation sites and, following a 20-min incubation, microinjected with monomeric rhodamine-labeled actin. The rhodamine-labeled actin became extensively associated with the nucleation sites, suggesting that at least some of the nucleation activity was maintained, and that the endogenous actin behaved in a different manner from the exogenous actin subunits. Similarly, when cells containing nucleation sites were extracted and incubated with rhodamine-labeled actin, the rhodamine-labeled actin became associated with the nucleation sites in a cytochalasin-sensitive manner. These observations suggest that capping and inhibition of nucleation cannot account for the regulation of actin polymerization in living cells. However, the sequestration of monomers probably plays a crucial role.     

Cofilin, a protein in porcine brain that binds to actin filaments and inhibits their interactions with myosin and tropomyosin

Cofilin, a 21 000 molecular weight protein of porcine brain, reacts stoichiometrically with actin in a 1:1 molar ratio. Upon binding of cofilin, the fluorescence of pyrene-labeled actin under polymerizing conditions is changed into the monomer form, irrespective of whether cofilin is added to actin before or after polymerization. Cofilin decreases the viscosity of actin filaments but increases the light-scattering intensity of the filaments. The centrifugation assay and the DNase I inhibition assay demonstrate that cofilin binds to actin filaments in a 1:1 molar ratio of cofilin to actin monomer in the filament and that cofilin increases the monomeric actin to a limited extent (up to 1.1-1.5 microM monomer) in the presence of physiological concentrations of Mg2+ and KCl. Cofilin is also able to bind to monomeric actin, as demonstrated by gel filtration. Electron microscopy showed that actin filaments are shortened and slightly thickened in the presence of cofilin. No bundle formation was observed in the presence of various concentrations of cofilin. The gel point assay using an actin cross-linking protein and the nucleation assay also suggested that cofilin shortens the actin filaments and hence increases the filament number. Cofilin blocks the binding of tropomyosin to actin filaments. Tropomyosin is dissociated from actin filaments by the binding of cofilin to actin filaments. Cofilin was found to inhibit the superprecipitation of actin-myosin mixtures as well as the actin-activated myosin ATPase. All these results suggest that cofilin is a new type of actin-associated protein.     

Evolutionarily divergent, unstable filamentous actin is essential for gliding motility in apicomplexan parasites

Apicomplexan parasites rely on a novel form of actin-based motility called gliding, which depends on parasite actin polymerization, to migrate through their hosts and invade cells. However, parasite actins are divergent both in sequence and function and only form short, unstable filaments in contrast to the stability of conventional actin filaments. The molecular basis for parasite actin filament instability and its relationship to gliding motility remain unresolved. We demonstrate that recombinant Toxoplasma (TgACTI) and Plasmodium (PfACTI and PfACTII) actins polymerized into very short filaments in vitro but were induced to form long, stable filaments by addition of equimolar levels of phalloidin. Parasite actins contain a conserved phalloidin-binding site as determined by molecular modeling and computational docking, yet vary in several residues that are predicted to impact filament stability. In particular, two residues were identified that form intermolecular contacts between different protomers in conventional actin filaments and these residues showed non-conservative differences in apicomplexan parasites. Substitution of divergent residues found in TgACTI with those from mammalian actin resulted in formation of longer, more stable filaments in vitro. Expression of these stabilized actins in T. gondii increased sensitivity to the actin-stabilizing compound jasplakinolide and disrupted normal gliding motility in the absence of treatment. These results identify the molecular basis for short, dynamic filaments in apicomplexan parasites and demonstrate that inherent instability of parasite actin filaments is a critical adaptation for gliding motility.     

Structure of an F-actin trimer disrupted by gelsolin and implications for the mechanism of severing

Stable oligomers of filamentous actin were obtained by cross-linking F-actin with 1,4-N,N'-phenylenedimaleimide and depolymerization with excess segment-1 of gelsolin. Segment-1-bound and cross-linked actin oligomers containing either two or three actin subunits were purified and shown to nucleate actin assembly. Kinetic assembly data from mixtures of monomeric actin and the actin oligomers fit a nucleation model where cross-linked actin dimer or trimer reacts with an actin monomer to produce a competent nucleus for filament assembly. We report the three-dimensional structure of the segment-1-actin hexamer containing three actin subunits, each with a tightly bound ATP. Comparative analysis of this structure with twelve other actin structures provides an atomic level explanation for the preferential binding of ATP by the segment-1-complexed actin. Although the structure of segment-1-bound actin trimer is topologically similar to the helical model of F-actin (1), it has a distorted symmetry compared with that of the helical model. This distortion results from intercalation of segment-1 between actin protomers that increase the rise per subunit and rotate each of the actin subunits relative to their positions in F-actin. We also show that segment-1 of gelsolin is able to sever actin filaments, although the severing activity of segment-1 is significantly lower than full-length gelsolin.     

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.