C-terminal fragments of thymopoietin favor formation of F-actin bundles: Electron microscopic data

Electron microscopy showed that PEP₃₃, a synthetic peptide corresponding to the C-terminal part of the thymic hormone thymopoietin, favors bundling of F-actin filaments in the presence of 0.1 M KCl. The structure of PEP₃₃ aggregates located within bundles between actin filaments is very similar to the structure of aggregates visible in preparations of pure PEP₃₃. No changes were observed in the structure of G-actin in the presence of PEP₃₃. A similar though weaker bundling effect was also detected for PEP₅, or thymopentin, a fragment of PEP₃₃. This peptide favors the formation of bundles of actin filaments of small size. The possible role of aggregation of actin filaments under the action of thymopoietin peptides is discussed in the light of the fact that the systematic release of thymopoietin from thymus leads to a phenomenon characteristic of the serious neuromuscular disease myasthenia gravis.     

Simultaneous interaction of actin with alpha-actinin and calponin

Interaction of calponin and alpha-actinin with actin was analyzed by means of cosedimentation and electron microscopy. G-actin was polymerized in the presence of calponin, alpha-actinin, or both of these actin-binding proteins (ABPs). The single and bundled actin filaments were separated, and the stoichiometry of ABPs and actin in both types of filaments was determined. Binding of calponin to the single or bundled actin filaments was not dependent on the presence of alpha-actinin and did not displace alpha-actinin from actin. In the presence of calponin, however, less alpha-actinin was bound to the bundled actin filaments, and the binding of alpha-actinin was accompanied by a partial decrease in the calponin/actin stoichiometry in the bundles of actin filaments. Calponin had no influence on the binding of alpha-actinin to the single actin filaments. The structure of actin bundles formed in the presence of the two ABPs differed from that formed in the presence of either one singly. We conclude that calponin and alpha-actinin can coexist on actin and that nearly each actin monomer can bind one of these ABPs.     

Interrelation between the spatial disposition of actin filaments and microtubules during the differentiation of tracheary elements in culturedZinnia cells

Summary Changes in the spatial relationship between actin filaments and microtubules during the differentiation of tracheary elements (TEs) was investigated by a double staining technique in isolatedZinnia mesophyll cells. Before thickening of the secondary wall began to occur, the actin filaments and microtubules were oriented parallel to the long axis of the cell. Reticulate bundles of microtubules and aggregates of actin filaments emerged beneath the plasma membrane almost simultaneously, immediately before the start of the deposition of the secondary wall. The aggregates of actin filaments were observed exclusively between the microtubule bundles. Subsequently, the aggregates of actin filaments extended preferentially in the direction transverse to the long axis of the cell, and the arrays of bundles of microtubules which were still present between the aggregates of actin filaments became transversely aligned. The deposition of the secondary walls then took place along the transversely aligned bundles of microtubules.Disruption of actin filaments by cytochalasin B produced TEs with longitudinal bands of secondary wall, along which bundles of microtubules were seen, while TEs produced in the absence of cytochalasin B had transverse bands of secondary wall. These results indicate that actin filaments play an important role in the change in the orientation of arrays of microtubules from longitudinal to transverse. Disruption of microtubules by colchicine resulted in dispersal of the regularly arranged aggregates of actin filaments, but did not inhibit the formation of the aggregates itself, suggesting that microtubules are involved in maintaining the arrangement of actin filaments but are not involved in inducing the formation of the regularly arranged aggregates of actin filaments.These findings demonstrate that actin filaments cooperate with microtubules in controlling the site of deposition of the secondary wall in developing TEs.Abbreviations DMSO dimethylsulfoxide - EGTA ethyleneglycolbis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - FITC fluorescein isothiocyanate - MSB microtubule-stabilizing buffer - PBS phosphate buffered saline - PIPES piperazine-N,N-bis(2-ethanesulfonic acid) - TE tracheary element     

Bundling of actin filaments by aorta caldesmon is not related to its regulatory function

Ca2+-sensitive thin filaments from vascular smooth muscle were disassembled into their constituent proteins, actin, tropomyosin and caldesmon. Caldesmon bound to both actin and to actin-tropomyosin and inhibited actin-tropomyosin activation of skeletal muscle myosin MgATPase. It also promoted the aggregation of actin or actin-tropomyosin into parallel aligned bundles. Quantitative electron microscopy measurements showed that with 1.1 microM actin-tropomyosin, 1.6 +/- 0.5% (n = 3) of the filaments were in bundles. At 0.073 microM, caldesmon inhibited MgATPase activity by 50%, whereas bundling was 3.0 +/- 1.3% (n = 4). At 0.37 microM caldesmon, MgATPase inhibition was 83% while 28.1 +/- 6.9% (n = 4) of filaments were in bundles. Experiments at 4.4 microM in which MgATPase and bundling were measured in the same samples gave similar results. Small bundles of 2-3 filaments showed the most frequent occurrence at 1.1 microM actin. At 4.4 microM actin the most common bundle size was 3-5 filaments, with the occasional occurrence of large bundles consisting of up to 120 filaments. The incidence of bundling was the same in the presence and absence of tropomyosin. Thus caldesmon can induce the formation of actin bundles but this property bears no relationship to its inhibition of MgATPase activity.     

The two actin-binding regions on the myosin heads of cardiac muscle

In the presence of myosin S1 or myosin heads, actin filaments tend to form bundles. The biological meaning of the bundling of actin filaments has been unclear. In this study, we found that the cardiac myosin heads can form the bundles of actin filaments more rapidly than can skeletal S1, as monitored by light scattering and electron microscopy. Moreover, the actin bundles formed by cardiac S1 were found to be more stable against mechanical agitation. The distance between actin filaments in the bundles was approximately 20 nm, which is comparable to the length of a myosin head and two actin molecules. This suggests the direct binding of S1 tails to the adjacent actin filament. The "essential" light chain of cardiac myosin could be cross-linked to the actin molecule in the bundle. When monomeric actin molecules were added to the bundle, the bundles could be dispersed into individual filaments. The three-dimensional structure of the dispersed actin filaments was reconstructed from electron cryo-microscopic images of the single actin filaments dispersed by monomer actin. We were able to demonstrate that cardiac myosin heads bind to two actin molecules: one actin molecule at the conventional actin-binding region and the other at the essential light-chain-binding region. This capability of cardiac myosin heads to bind two actin molecules is discussed in view of lower ATPase activity and slower shortening velocity than those of skeletal ones.     

Supramolecular forms of actin from amoebae of Dictyostelium discoideum.

Actin purified from amoebae of Dictyostelium discoideum polymerizes into filaments at 24 degrees upon addition of KCl, as judged by a change in optical density at 232 nm and by electron microscopy. The rate and extent of formation of this supramolecular assembly and the optimal KCl concentrations (0.1 M) for assembly are similar to those of striated muscle actin. The apparent equilibrium constant for the monomer-polymer transition is 1.3 muM for both Dictyostelium and muscle actin. Although assembly of highly purified Dictyostelium actin monomers into individual actin filaments resembles that of muscle actin, Dictyostelium actin but not muscle actin was observed to assemble into two-dimensional nets in 10 mM CaCl2. The Dictyostelium actin also forms filament bundles which are 0.1 mum in diameter and which assemble in the presence of 5 mM MgCl2. These bundles formed from partially purified Dictyostelium actin preparations but not from highly purified preparations, suggesting that their formation may depend on the presence of another component. These actin bundles reconstituted in vitro resemble the actin-containing bundles found in situ by microscopy in many non-muscle cells.     

The contractile basis of ameboid movement. II. Structure and contractility of motile extracts and plasmalemma-ectoplasm ghosts

The role of calcium and magnesium-ATP on the structure and contractility in motile extracts of Amoeba proteus and plasmalemma-ectoplasm "ghosts" of Chaos carolinensis has been investigated by correlating light and electron microscope observations with turbidity and birefringence measurements. The extract is nonmotile and contains very few F-actin filaments and myosin aggregates when prepared in the presence of both low calcium ion and ATP concentrations at an ionic strength of I = 0.05, pH 6.8. The addition of 1.0 mM magnesium chloride, 1.0 mM ATP, in the presence of a low calcium ion concentration (relaxation solution) induced the formation of some fibrous bundles of actin without contracting, whereas the addition of a micromolar concentration of calcium in addition to 1.0 mM magnesium-ATP (contraction solution) (Taylor, D. L., J. S. Condeelis, P. L. Moore, and R. D. Allen. 1973. J. Cell Biol. 59:378-394) initiated the formation of large arrays of F-actin filaments followed by contractions. Furthermore, plasmalemma-ectoplasm ghosts prepared in the relaxation solution exhibited very few straight F-actin filaments and myosin aggregates. In contrast, plasmalemmaectoplasm ghosts treated with the contraction solution contained many straight F-actin filaments and myosin aggregates. The increase in the structure of ameba cytoplasm at the endoplasm-ectoplasm interface can be explained by a combination of the transformation of actin from a less filamentous to a more structured filamentous state possibly involving the cross-linking of actin to form fibrillar arrays (see above-mentioned reference) followed by contractions of the actin and myosin along an undetermined distance of the endoplasm and/or ectoplasm.     

Binding of actin filaments to connectin

The binding of actin filaments to connectin, a muscle elastic protein, was investigated by means of turbidity and sedimentation measurements and electron microscopy. In the presence of less than 0.12 M KCl at pH 7.0, actin filaments bound to connectin. Long actin filaments formed bundles. Short actin filaments also aggregated into irregular bundles or a meshwork, and were frequently attached perpendicularly to long bundles. The binding of F-actin to connectin was saturated at an equal weight ratio (molar ratio, 50 : 1), as determined by a cosedimentation assay. Larger amounts of sonicated short actin filaments appeared to bind to connectin than intact F-actin. Myosin S1-decorated actin filaments did not bind to connectin. The addition of S1 to connectin-induced actin bundles resulted in partial disaggregation. Thus, connectin does not appear to interfere with actin-myosin interactions, since myosin S1 binds to actin more strongly than connectin.     

Formation of two-dimensional complexes of F-actin and crosslinking proteins on lipid monolayers: demonstration of unipolar alpha-actinin-F-actin crosslinking.

A method is described for forming two-dimensional (2-D) paracrystalline complexes of F-actin and bundling/gelation proteins on positively charged lipid monolayers. These arrays facilitate detailed structural studies of protein interactions with F-actin by eliminating superposition effects present in 3-D bundles. Bundles of F-actin have been produced using the glycolytic enzymes aldolase and glyceraldehyde-3-phosphate dehydrogenase, the cytoskeletal protein erythrocyte adducin as well as smooth muscle alpha-actinin from chicken gizzard. All of the 2-D bundles formed contain F-actin with a 13/6 helical structure. F-actin-aldolase bundles have an interfilament spacing of 12.6 nm and a superlattice arrangement of actin filaments that can be explained by expression of a local twofold axis in the neighborhood of the aldolase. Well ordered F-actin-alpha-actinin 2-D bundles have an interfilament spacing of 36 nm and contain crosslinks 33 nm in length angled approximately 25-35 degrees to the filament axis. Images and optical diffraction patterns of these bundles suggest that they consist of parallel, unipolar arrays of actin filaments. This observation is consistent with an actin crosslinking function at adhesion plaques where actin filaments are bound to the cell membrane with uniform polarity.     

Overexpression of cofilin stimulates bundling of actin filaments, membrane ruffling, and cell movement in Dictyostelium

Cofilin is a low molecular weight actin-modulating protein whose structure and function are conserved among eucaryotes. Cofilin exhibits in vitro both a monomeric actin-sequestering activity and a filamentous actin-severing activity. To investigate in vivo functions of cofilin, cofilin was overexpressed in Dictyostelium discoideum cells. An increase in the content of D. discoideum cofilin (d-cofilin) by sevenfold induced a co-overproduction of actin by threefold. In cells over-expressing d-cofilin, the amount of filamentous actin but not that of monomeric actin was increased. Overexpressed d-cofilin co-sedimented with actin filaments, suggesting that the sequestering activity of d- cofilin is weak in vivo. The overexpression of d-cofilin increased actin bundles just beneath ruffling membranes where d-cofilin was co- localized. The overexpression of d-cofilin also stimulated cell movement as well as membrane ruffling. We have demonstrated in vitro that d-cofilin transformed latticework of actin filaments cross-linked by alpha-actinin into bundles probably by severing the filaments. D. discoideum cofilin may sever actin filaments in vivo and induce bundling of the filaments in the presence of cross-linking proteins so as to generate contractile systems involved in membrane ruffling and cell movement.     

Actin-caldesmon-myosin-subfragment-1 ternary complex viewed by electron microscopy. Competitive actin binding region for caldesmon and myosin subfragment-1

An earlier electron microscopic study using different caldesmon forms complexed with actin revealed that the aggregates produced display regular periodic striation after antibody labeling of the 35-kDa caldesmon fragment. This approach provides further evidence that a caldesmon fragment, even as small as 15 kDa, can induce actin filaments to assemble into bundles. The observed difference in the compactness of these structures, depending on the use of the 15-kDa fragment instead of the 35-kDa fragment, suggests the existence of more than one actin-binding site in the caldesmon molecule. In this study, the caldesmon-induced process of F-actin association was investigated in the presence of skeletal myosin subfragment-1, using light-scattering methods, cosedimentation experiment and electron microscopic techniques. We show that the actin-caldesmon association is partially destabilized in the presence of subfragment-1 and this leads to a ternary complex formation. Immunogold labelling of the actin filaments still reveals the presence of caldesmon within this structure. This latter result strengthens the hypothesis that actin has a site(s) able to bind both caldesmon and myosin subfragment-1, as detected by recent NMR observations. This evidence is discussed with respect to the regulatory function of caldesmon during smooth muscle contraction.     

Partial purification and characterization of an actin-bundling protein, band 4.9, from human erythrocytes

Band 4.9 (a 48,000-mol-wt polypeptide) has been partially purified from human erythrocyte membranes. In solution, band 4.9 polypeptides exist as trimers with an apparent molecular weight of 145,000 and a Stokes radius of 50 A. Electron microscopy shows that the protein is a three-lobed structure with a radius slightly greater than 50 A. When gel-filtered rabbit muscle actin is polymerized in the presence of band 4.9, actin bundles are generated that are similar in appearance to those induced by "vinculin" or fimbrin. The bundles appear brittle and when they are centrifuged small pieces of filaments break off and remain in the supernatant. At low band 4.9 to actin molar ratios (1:30), band 4.9 lowers the apparent steady-state low-shear falling ball viscosity by sequestering filaments into thin bundles; at higher ratios, the bundles become thicker and obstruct the ball's movement leading to an apparent increase in steady-state viscosity. Band 4.9 increases the length of the lag phase and decreases the rate of elongation during actin polymerization as measured by high-shear Ostwald viscometry or by the increase in the fluorescence of pyrene-labeled actin. Band 4.9 does not alter the critical actin monomer concentration. We hypothesize that band 4.9, together with actin, erythrocyte tropomyosin, and spectrin, forms structures in erythroid precursor cells analogous to those formed by fimbrin, actin, tropomyosin, and TW 260/240 in epithelial brush borders. During erythroid development and enucleation, the actin filaments may depolymerize up to the membrane, leaving a membrane skeleton with short stubs of actin bundled by band 4.9 and cross-linked by spectrin.     

The actin filament severing protein actophorin promotes the formation of rigid bundles of actin filaments crosslinked with alpha-actinin

The actin filament severing protein, Acanthamoeba actophorin, decreases the viscosity of actin filaments, but increases the stiffness and viscosity of mixtures of actin filaments and the crosslinking protein alpha-actinin. The explanation of this paradox is that in the presence of both the severing protein and crosslinker the actin filaments aggregate into an interlocking meshwork of bundles large enough to be visualized by light microscopy. The size of these bundles depends on the size of the containing vessel. The actin filaments in these bundles are tightly packed in some areas while in others they are more disperse. The bundles form a continuous reticulum that fills the container, since the filaments from a particular bundle may interdigitate with filaments from other bundles at points where they intersect. The same phenomena are seen when rabbit muscle aldolase rather than alpha-actinin is used as the crosslinker. We propose that actophorin promotes bundling by shortening the actin filaments enough to allow them to rotate into positions favorable for lateral interactions with each other via alpha-actinin. The network of bundles is more rigid and less thixotropic than the corresponding network of single actin filaments linked by alpha-actinin. One explanation may be that alpha-actinin (or aldolase) normally in rapid equilibria with actin filaments may become trapped between the filaments increasing the effective concentration of the crosslinker.     

Characteristic structures of actin gels induced with hepatic actinogelin or with chicken gizzard alpha-actinin: implication for their function

We studied the properties of actinogelin, a Ca2+-regulated actin cross-linking protein isolated from Ehrlich tumor cells or rat liver. Chicken gizzard alpha-actinin was used as a Ca2+-insensitive control. Actinogelin, which has very high gelation activity under low Ca2+ conditions, was found using electron microscopic or fluorescence studies to induce formation of a characteristic structure in which actin filaments and bundles radiate to (or converge from) all directions from spot-like core structures. A similar structure was induced with actinogelin, even in the presence of 0.7 saturation of tropomyosin. No such structure was detected with actinogelin under high Ca2+ conditions, and only a few were found with gizzard alpha-actinin. Because reconstituted structures are similar to those observed intracellularly, actinogelin may be important in the formation of similar microfilament organization in the cells. It seems also important that these structures are reconstituted with only two purified protein components, i.e., actinogelin and actin. Immunocompetition studies showed that actinogelin and gizzard alpha-actinin partially shared antigenicity, and their molecular shape and peptide maps were similar. Their amino acid compositions [Kuo et al., 1982], subunit and domain structures, and binding sites on actin [Mimura and Asano, 1987] are also very similar. Therefore, it is concluded that actinogelin belongs to alpha-actinin superfamily proteins. Furthermore, the presence of functionally different subfamilies concerned with Ca2+ sensitivity, gelation-efficiency, and others is discussed. Actinogelin, which induces networks of actin filaments, may be classified as high gelation type.     

Actin filament bundling by fimbrin is important for endocytosis, cytokinesis, and polarization in fission yeast

Through the coordinated action of diverse actin-binding proteins, cells simultaneously assemble actin filaments with distinct architectures and dynamics to drive different processes. Actin filament cross-linking proteins organize filaments into higher order networks, although the requirement of cross-linking activity in cells has largely been assumed rather than directly tested. Fission yeast Schizosaccharomyces pombe assembles actin into three discrete structures: endocytic actin patches, polarizing actin cables, and the cytokinetic contractile ring. The fission yeast filament cross-linker fimbrin Fim1 primarily localizes to Arp2/3 complex-nucleated branched filaments of the actin patch and by a lesser amount to bundles of linear antiparallel filaments in the contractile ring. It is unclear whether Fim1 associates with bundles of parallel filaments in actin cables. We previously discovered that a principal role of Fim1 is to control localization of tropomyosin Cdc8, thereby facilitating cofilin-mediated filament turnover. Therefore, we hypothesized that the bundling ability of Fim1 is dispensable for actin patches but is important for the contractile ring and possibly actin cables. By directly visualizing actin filament assembly using total internal reflection fluorescence microscopy, we determined that Fim1 bundles filaments in both parallel and antiparallel orientations and efficiently bundles Arp2/3 complex-branched filaments in the absence but not the presence of actin capping protein. Examination of cells exclusively expressing a truncated version of Fim1 that can bind but not bundle actin filaments revealed that bundling activity of Fim1 is in fact important for all three actin structures. Therefore, fimbrin Fim1 has diverse roles as both a filament "gatekeeper" and as a filament cross-linker.     

Affinity of alpha-actinin for actin determines the structure and mechanical properties of actin filament gels.

Proteins that cross-link actin filaments can either form bundles of parallel filaments or isotropic networks of individual filaments. We have found that mixtures of actin filaments with alpha-actinin purified from either Acanthamoeba castellanii or chicken smooth muscle can form bundles or isotropic networks depending on their concentration. Low concentrations of alpha-actinin and actin filaments form networks indistinguishable in electron micrographs from gels of actin alone. Higher concentrations of alpha-actinin and actin filaments form bundles. The threshold for bundling depends on the affinity of the alpha-actinin for actin. The complex of Acanthamoeba alpha-actinin with actin filaments has a Kd of 4.7 microM and a bundling threshold of 0.1 microM; chicken smooth muscle has a Kd of 0.6 microM and a bundling threshold of 1 microM. The physical properties of isotropic networks of cross-linked actin filaments are very different from a gel of bundles: the network behaves like a solid because each actin filament is part of a single structure that encompasses all the filaments. Bundles of filaments behave more like a very viscous fluid because each bundle, while very long and stiff, can slip past other bundles. We have developed a computer model that predicts the bundling threshold based on four variables: the length of the actin filaments, the affinity of the alpha-actinin for actin, and the concentrations of actin and alpha-actinin.     

Ca2+-calmodulin-dependent polymerization of actin by myelin basic protein

The interaction between myelin basic protein (MBP) and G-actin was studied under nonpolymerizing conditions, i.e.,2mM HEPES, pH 7.5, 0.1 mM CaCl2 and 0.2 mM ATP. Fluorescence studies using pyrenyl-actin and the measurements of ATP hydrolysis rate show that MBP induces changes in the structure of the actin monomer similar to those occurring during polymerization by salt. Electron microscope observations of the MBP-G-actin complex reveal the presence of filamentous structures which appear as separate filaments or as bundles of filaments in lateral association. These filaments are polar as visualized by attachment of heavy meromyosin. The biochemical data together with electron microscope observations suggest that the binding of MBP to G-actin under non-polymerizing conditions induces an interaction between actin monomers leading to the formation of filamentous structures which may be similar to F-actin filaments. The effects of MBP on G-actin can be reversed by calmodulin in the presence of Ca2+.     

Actin filament turnover regulated by cross-linking accounts for the size, shape, location, and number of actin bundles in Drosophila bristles

Drosophila bristle cells are shaped during growth by longitudinal bundles of cross-linked actin filaments attached to the plasma membrane. We used confocal and electron microscopy to examine actin bundle structure and found that during bristle elongation, snarls of uncross-linked actin filaments and small internal bundles also form in the shaft cytoplasm only to disappear within 4 min. Thus, formation and later removal of actin filaments are prominent features of growing bristles. These transient snarls and internal bundles can be stabilized by culturing elongating bristles with jasplakinolide, a membrane-permeant inhibitor of actin filament depolymerization, resulting in enormous numbers of internal bundles and uncross-linked filaments. Examination of bundle disassembly in mutant bristles shows that plasma membrane association and cross-bridging adjacent actin filaments together inhibits depolymerization. Thus, highly cross-bridged and membrane-bound actin filaments turn over slowly and persist, whereas poorly cross-linked filaments turnover more rapidly. We argue that the selection of stable bundles relative to poorly cross-bridged filaments can account for the size, shape, number, and location of the longitudinal actin bundles in bristles. As a result, filament turnover plays an important role in regulating cytoskeleton assembly and consequently cell shape.     

Heavy meromyosin induces sliding movements between antiparallel actin filaments

Suzuki et al. [Biochemistry 28, 6513-6518 (1989)] have shown that, when F-actin is mixed with inert high polymer, a large number of actin filaments closely align in parallel with overlaps to form a long and thick bundle. The bundle may be designated non-polar, as the constituent filaments are random in polarity (Suzuki et al. 1989). I prepared non-polar bundles of F-actin using methylcellulose (MC) as the high polymer, exposed them to heavy meromyosin (HMM) in the presence of ATP under a light microscope, and followed their morphological changes in the continuous presence of MC. It was found that bundles several tens of micrometers long contracted to about one-third the initial length, while becoming thicker, in half a minute after exposure to HMM. Subsequently, each bundle was split longitudinally into several bundles in a stepwise manner, while the newly formed ones remained associated together at one of the two ends. The product, an aster-like assembly of actin bundles, was morphologically quiescent; that is, individual bundles never contracted upon second exposure to HMM and ATP, although they were still longer than the F-actin used. Bundles in this state consisted of filaments with parallel polarity as examined by electron microscopy. This implies that non-polar bundles were transformed into assemblies of polar bundles with ATP hydrolysis by HMM. Importantly, myosin subfragment-1 caused neither contraction nor transformation. These results are interpreted as follows. In the presence of ATP, the two-headed HMM molecule was able to cross-bridge antiparallel actin filaments, as well as parallel ones.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure of helical RecA-DNA complexes. II. Local conformational changes visualized in bundles of RecA-ATP gamma S filaments

Complexes of RecA-DNA filaments, formed in the presence of a non-hydrolyzable ATP analog, ATP gamma S, aggregate together into regular bundles in the presence of Mg2+. Electron micrographs of several different forms of RecA-double-stranded DNA bundles have been analyzed: bundles of six supercoiled filaments at two different concentrations of Mg2+, and bundles of three supercoiled filaments at a single concentration of Mg2+. The bundles are all characterized by a regular left-handed supercoiling of the component filaments arising from the non-integral number of RecA subunits per turn of the RecA helix in these aggregates, about 6.15 units/turn. When single-stranded DNA is used instead of double-stranded DNA, regular aggregates composed of many filaments are formed. These aggregates do not supercoil, consistent with a symmetry of the component filaments of close to 6.0 units/turn. These different structures have provided a strong confirmation of the analysis of isolated RecA filaments. Since different RecA protomers within the component filaments of these aggregates are in different environments, they have provided a direct view of different conformations that RecA subunits may adopt within the same filament as a result of nonequivalent contacts. The conformational changes we have visualized are quite large, with apparent movements of mass over distances greater than 2 nm. The RecA-mediated strand exchange reaction is a highly dynamic process, which involves both the unwinding and stretching of DNA, in addition to the physical movement of DNA strands. It is quite likely, therefore, that the different conformations of RecA subunits seen in these aggregates represent different states of RecA during its enzymatic strand exchange activity.