Effect of bilayer morphology on the subgel phase formation

We examined how morphology of bilayer assemblies affects the kinetics of the subgel phase formation in dimyristoylphosphatidylglycerol (DMPG) bilayers, which change their morphology depending on NaCl concentration. Quantitative analysis of the kinetics revealed that in flat sheet-like structures (bilayer sheets) the subgel phase forms in a simple two-state manner with the relaxation time of about 3 min at -10 degrees C while in vesicles it forms much slower under a multi-step process. Freeze-etch electron microscopic observations suggested that the kinetics of the subgel phase formation is directly correlated with the morphology of bilayer assemblies. It is likely that the bilayer sheet structure is more favorable to the subgel phase formation in DMPG bilayers than the vesicular structure.     

Microstructural polymorphism in bovine brain galactocerebroside and its two major subfractions.

Aqueous suspensions of either brain galactocerebrosides or its subfraction consisting of alpha-hydroxyacyl galactocerebrosides are mainly composed of vesicles or granular lipid with occasional multilamellar sheets. In aqueous media the other subfraction consisting of non-hydroxyacyl galactocerebrosides forms some helical structures, but most of the lipid remains as granules or vesicles. It is demonstrated that thermal cycling of non-hydroxyacyl galactocerebrosides in polar nonaqueous solvents can greatly enhance the degree of conversion to helical ribbons about 100 nm in diameter. These structures appear to be a stable dehydrated crystalline form of this lipid and are morphologically similar to helical microstructures produced by a few synthetic lipids. On the other hand, similar treatment of unfractionated bovine brain cerebroside and its alpha-hydroxy fatty acyl subfraction quantitatively produces straight needles that appear to be cochleate cylinders. While their dimensions depend on formation conditions, a typical suspension has uniform particles with diameters close to 100 nm and lengths variable from one to a few hundred micrometers. This is the first report demonstrating the quantitative formation of crystalline high axial ratio microstructures from complex mixtures of natural lipids. The different microstructures formed by the two components appear related to the various forms of lipid deposits occurring in lipid storage diseases. The similarity of these "synthetic" microstructures to biological structures in which they are found (such as myelin and intestinal brush border microvilli) strengthens the possibility that galactocerebrosides have a role in stabilizing cylindrical biological structures.     

Formation of helical protein assemblies of IgG and transducin on varied lipid tubules

Helical protein arrays on lipid tubules are valuable assemblies for studying protein structure and protein-lipid interactions through electron microscopy and crystallography. We describe conditions for forming such arrays from two proteins, IgG and transducin, the photoreceptor G protein, using a variety of lipid surfaces. Anti-dinitrophenyl (DNP) IgG arrays formed on DNP-phosphatidylethanolamine (DNP-PE) mixed with either galactosyl-ceramide lipids or phosphatidylcholine (PC) display different pH sensitivities and dimensions, yet have similar helical symmetries. DNP-PE/PC mixtures formed small crystals and large well-ordered flattened tubules. The peripheral membrane protein transducin (G(t)) formed helical arrays either on a mixture of cationic and neutral lipids or on residual photoreceptor lipids. Despite differences in lipid composition, the G(t) arrays have similar structures and show similar sensitivity to activation and variations in ionic environment. G(t) activation causes the helical assemblies to collapse to small vesicles, a process resembling the vesiculation of activated dynamin-lipid tubules. In a preliminary three-dimensional reconstruction, the hapten-bound IgG appears to make two contacts to the central lipid tubule, presumably via the F(ab) domains. The ability to generate a three-dimensional reconstruction without tilts illustrates one advantage of helical structures for two-dimensional crystallography, especially for visualizing protein-lipid interactions.     

pH-dependent formation of membranous cytoplasmic body-like structure of ganglioside G(M1)/bis(monoacylglycero)phosphate mixed membranes

Membrane structures of the mixtures of ganglioside G(M1) and endosome specific lipid, bis (monoacylglycero) phosphate (BMP, also known as lysobisphosphatidic acid) were examined at various pH conditions by freeze-fracture electron microscopy and small-angle x-ray scattering. At pH 8.5-6.5, a G(M1)/BMP (1:1 mol/mol) mixture formed small vesicular aggregates, whereas the mixture formed closely packed lamellar structures under acidic conditions (pH 5.5, 4.6) with the lamellar repeat distance of 8.06 nm. Since BMP alone exhibits a diffuse lamellar structure at a broad range of pH values and G(M1) forms a micelle, the results indicate that both G(M1) and BMP are required to produce closely stacked multilamellar vesicles. These vesicles resemble membranous cytoplasmic bodies in cells derived from patients suffering from G(M1) gangliosidosis. Similar to G(M1) gangliosidosis, cholesterol was trapped in BMP vesicles in G(M1)- and in a low pH-dependent manner. Studies employing different gangliosides and a G(M1) analog suggest the importance of sugar chains and a sialic acid of G(M1) in the pH-dependent structural change of G(M1)/BMP membranes.     

Backbone-modified amylin derivatives: implications for amyloid inhibitor design and as template for self-assembling bionanomaterials.

This report reviews our approach to the design, synthesis and structural/morphological analysis of backbone-modified amylin(20-29) derivatives. Depending on the position in the peptide backbone and the type of amide bond isostere/modification, the amylin(20-29) peptides behave either as inhibitors of amyloid fibril formation, which are able to retard amyloid formation of native amylin(20-29), or as templates for the formation of self-assembled supramolecular structures. Molecular fine-tuning of the hydrogen-bond accepting/donating properties allows the control over the morphology of the supramolecular aggregation motifs such as helical ribbons and tapes, ribbons progressing to closed peptide nanotubes, (twisted) lamellar sheets or amyloid fibrils.     

Stereo images of vesicular stomatitis virus assembly.

Viral assembly was studied by viewing platinum replicas of cytoplasmic and outer plasma membrane surfaces of baby hamster kidney cells infected with vesicular stomatitis virus. Replicas of the cytoplasmic surface of the basilar plasma membrane revealed nucleocapsids forming bullet-shaped tight helical coils. The apex of each viral nose cone was anchored to the membrane and was free of uncoiled nucleocapsid, whereas tortuous nucleocapsid was attached to the base of tightly coiled structures. Using immunoelectron microscopy, we identified the nucleocapsid (N) viral protein as a component of both the tight-coil and tortuous nucleocapsids, whereas the matrix (M) protein was found only on tortuous nucleocapsids. The M protein was not found on the membrane. Using immunoreagents specific for the viral glycoprotein (G protein), we found that the amount of G protein per virion varied. The G protein was consistently localized at the apex of viral buds, whereas the density of G protein on the shaft was equivalent to that in the surrounding membrane. These observations suggest that G-protein interaction with the nucleocapsid via its cytoplasmic domain may be necessary for the initiation of viral assembly. Once contact is established, nucleocapsid coiling proceeds with nose cone formation followed by formation of the helical cylinder. M protein may function to induce a nucleocapsid conformation favorable for coiling or may cross-link adjacent turns in the tight coil or both.     

Modeling of peptides connecting the ligand‐binding and transmembrane domains in the GluR2 glutamate receptor

Ligand‐gated Glutamate receptors (GluR) mediate synaptic signals in the nervous system. Ionotropic GluRs of AMPA type, the subject of this study, are tetrameric assemblies of monomer subunits, each of which is constructed in a modular fashion from functional subdomains. The extracellular ligand‐binding domain (LBD) changes its conformation upon binding of an agonist ligand followed by opening of a transmembrane (TM) ion channel. Peptides connecting the LBD and TM domains facilitate gating of the channel, and their structure and composition are important for the receptor functioning. In this study, we used replica exchange molecular dynamics (REMD) simulations to model S1M1 and S2M3 connecting peptides of the GluR2 receptor in two implicit solvents, water and interfacial water/lipid medium characterized by lower polarity. Propensity of these peptides to form helical structures was analyzed using helicity measure derived from the free energy of the simulated ensembles of structures. The S1M1 and S2M3 connecting peptides were not helical in our simulations in both dielectric environments in the absence of the rest of the protein. The structures of the LBD fragment with known high‐resolution α‐helical structure and of the TM3 helix were successfully predicted in the simulations, which in part validate our results. The S2M3 peptide, which is important in gating, formed a well‐defined coil structure and salt‐bridges with the S2 domain. The S1M1 peptide formed a loop structure via formation of internal salt‐bridges. Potential implications of these structures on function of the receptor are discussed. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

Incorporation of cholesterol in sphingomyelin- phosphatidylcholine vesicles has profound effects on detergent-induced phase transitions

Vesicle <--> micelle transitions are important phenomena during bile formation and intestinal lipid processing. The hepatocyte canalicular membrane outer leaflet contains appreciable amounts of phosphatidylcholine (PC) and sphingomyelin (SM), and both phospholipids are found in the human diet. Dietary SM enrichment inhibits intestinal cholesterol absorption. We therefore studied detergent-induced vesicle --> micelle transitions in SM-PC vesicles. Phase transitions were evaluated by spectrophotometry and cryotransmission electron microscopy (cryo-TEM) after addition of taurocholate (3-7 mM) to SM-PC vesicles (4 mM phospholipid, SM/PC 40%/60%, without or with 1.6 mM cholesterol). After addition of excess (5-7 mM) taurocholate, SM-PC vesicles were more sensitive to micellization than PC vesicles. As shown by sequential cryo-TEM, addition of equimolar (4 mM) taurocholate to SM-PC vesicles induced formation of open vesicles, then (at the absorbance peak) fusion of bilayer fragments into large open structures (around 200 nm diameter) coexisting with some multilamellar or fused vesicles and thread-like micelles and, finally, transformation into an uniform picture with long thread-like micelles. Incorporation of cholesterol in the SM/PC bilayer changed initial vesicular shape from spherical into ellipsoid and profoundly increased detergent resistance. Disk-like micelles and multilamellar vesicles, and then extremely large vesicular structures, were observed by sequential cryo-TEM under these circumstances, with persistently increased absorbance values by spectrophotometry. These findings may be relevant for bile formation and intestinal lipid processing. Inhibition of intestinal cholesterol absorption by dietary SM enrichment may relate to high resistance against bile salt-induced micellization of intestinal lipids in presence of the sphingolipid.     

Functional reconstitution of the nicotinic acetylcholine receptor by CHAPS dialysis depends on the concentrations of salt, lipid, and protein.

The detergent CHAPS was found to be the preferable surfactant for the efficient purification and reconstitution of the Torpedo californica nicotinic acetylcholine receptor (AChR). The main result is that the incorporation of the AChR proteins into lipid vesicles by CHAPS dialysis was strongly dependent on the salt and protein concentrations. As monitored by sucrose gradients, by electron microscopy, and by agonist-induced lithium ion flux, the best reconstitution yields were obtained in 0.5 M NaCl at a protein concentration of 0.5 g/L and in 0.84 M NaCl at 0.15 g/L protein. Electron micrographs of receptor molecules, which were incorporated into vesicles, showed single, nonaggregated dimer (M(r) = 580,000) and monomer (M(r) = 290,000) species. CHAPS dialysis at NaCl concentrations less than 0.5 M largely reduced the receptor incorporation concomitant with protein aggregation. Electron micrographs of these preparations revealed large protein sheets or ribbons not incorporated into vesicles. The analysis of static and dynamic light scattering demonstrated that the detergent-solubilized AChR molecules aggregate at low lipid contents (less than or equal to 500 phospholipids/AChR dimer), independent of the salt concentration. AChR proteins eluted from an affinity column with a solution containing 8 mM CHAPS (but no added lipid) still contained 130 +/- 34 tightly bound phospholipids per dimer. The aggregates (about 10 dimers on the average) could be dissociated by readdition of lipid and, interestingly, also by increasing the CHAPS concentration up to 15 mM. This value is much higher than the CMC of CHAPS = 4.0 +/- 0.4 mM, which was determined by surface tension measurements. The data clearly suggest protein-micelle interactions in addition to the association of monomeric detergents with proteins. Furthermore, the concentration of the (free) monomeric CHAPS at the vesicle-micelle transformation in 0.5 M NaCl ([Dw]c = 3.65 mM) was higher than in 50 mM NaCl ([Dw]c = 2.8 mM). However, it is suggested that the main effect of high salt concentrations during the reconstitution process is an increase of the fusion (rate) of the ternary protein/lipid/CHAPS complexes with mixed micelles or with vesicular structures, similar to the salt-dependent fusion of vesicles.     

EDTA-induced self-assembly of cationic lipid-DNA multilayers near a monolayer-covered air-water interface

The presence of EDTA in the suspending buffer can induce the formation of multilayer structures from a mixture of the cationic lipid 3beta[N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol and the zwitterionic 'helper' lipid 1, 2-dimyristoyl-sn-glycero-3-phosphocholine with DNA. The resulting structures consist of stacks of alternating sheets of lipid bilayer with intercalated DNA. In the absence of EDTA, only a single layer of DNA adsorbs to the lipid membrane. The buffer composition therefore influences the morphology of the lipid-aggregate/DNA assembly, which was not known to date.     

An apolipoprotein AI mimetic peptide: membrane interactions and the role of cholesterol

The 18-amino acid amphipathic helical peptide Ac-DWFKAFYDKVAEKFKEAF-NH(2) promotes the separation of cholesterol from the phospholipid, resulting in the formation of cholesterol crystallites, even at mole fractions of cholesterol as low as 0.3. The peptide exerts a greater degree of penetration into membranes of pure phosphatidylcholine in the absence of cholesterol than into bilayers of phosphatidylcholine and cholesterol. The circular dichroism spectrum of the peptide in buffer indicates that it self-associates, leading to the formation of structures with higher helical content. However, in the presence of lipid, the peptide remains helical over a larger concentration range. The peptide undergoes a thermal transition on heating. Cholesterol has little effect on the secondary structure of the peptide; however, increased Trp emission intensity in the absence of cholesterol indicates a deeper penetration of the helix upon removal of cholesterol from the membrane. The results with these model systems demonstrate changes in peptide-lipid interactions that may be related to the observed biological properties of this peptide.     

Cysteine mutants of human apolipoprotein A-I: a study of secondary structural and functional properties

Apolipoprotein A-I(Milano) (A-I(M)) (R173C), a natural mutant of human apolipoprotein A-I (apoA-I), and five other cysteine variants of apoA-I at residues 52 (S52C), 74 (N74C), 107 (K107C), 129 (G129C), and 195 (K195C) were generated. Cysteine residues were incorporated in each of the various helices at the same helical wheel position as for the substitution in A-I(M). The secondary structural properties of the monomeric mutants, their abilities to bind lipid and to promote cholesterol efflux from THP-1 macrophages, and the possibility of antiperoxidation were investigated. Results showed that the alpha helical contents of all of the cysteine mutants were similar to that of wild-type apoA-I (wtapoA-I). The cysteine variant of A-I(M) at residue 173 [A-I(M)(R173C)] exhibited weakened structural stability, whereas A-I(G129C) a more stable structure than wtapoA-I. A-I(G129C) and A-I(K195C) exhibited significantly impaired capabilities to bind lipid compared with wtapoA-I. A-I(K107C) possessed a higher capacity to promote cholesterol efflux from macrophages than wtapoA-I, and A-I(M)(R173C) and A-I(K195C) exhibited an impaired efflux capability. Neither A-I(M)(R173C) nor any other cysteine mutant could resist oxidation against lipoxygenase. In summary, in spite of the similar mutant position on the helix, these variants exhibited different structural features or biological activities, suggesting the potential influence of the local environment of mutations on the whole polypeptide chain.     

What can AlphaFold do for antimicrobial amyloids?

Amyloids, protein, and peptide assemblies in various organisms are crucial in physiological and pathological processes. Their intricate structures, however, present significant challenges, limiting our understanding of their functions, regulatory mechanisms, and potential applications in biomedicine and technology. This study evaluated the AlphaFold2 ColabFold method's structure predictions for antimicrobial amyloids, using eight antimicrobial peptides (AMPs), including those with experimentally determined structures and AMPs known for their distinct amyloidogenic morphological features. Additionally, two well-known human amyloids, amyloid-β and islet amyloid polypeptide, were included in the analysis due to their disease relevance, short sequences, and antimicrobial properties. Amyloids typically exhibit tightly mated β-strand sheets forming a cross-β configuration. However, certain amphipathic α-helical subunits can also form amyloid fibrils adopting a cross-α structure. Some AMPs in the study exhibited a combination of cross-α and cross-β amyloid fibrils, adding complexity to structure prediction. The results showed that the AlphaFold2 ColabFold models favored α-helical structures in the tested amyloids, successfully predicting the presence of α-helical mated sheets and a hydrophobic core resembling the cross-α configuration. This implies that the AI-based algorithms prefer assemblies of the monomeric state, which was frequently predicted as helical, or capture an α-helical membrane-active form of toxic peptides, which is triggered upon interaction with lipid membranes.     

Formation of double-walled microtubules and multilayered tubulin sheets by basic proteins

Some basic proteins enable microtubule protein to form special assembly products in vitro, known as double-walled microtubules. Using histones (H1, core histones) as well as the human encephalitogenic protein to induce the formation of double-walled microtubules, we made the following electron microscopic observations: (1) Double-walled microtubules consist of an "inner" microtubule which is covered by electron-dense material, apparently formed from the basic protein, and by a second tubulin wall. (2) The tubulin of the second wall seems to be arranged as protofilaments, surrounding the inner microtubule in a helical or ring-like manner. (3) The surface of double-walled microtubules lacks the projections of microtubule-associated proteins, usually found on microtubules. (4) In the case of protofilament ribbons (incomplete microtubules), H1 binds exclusively to their convex sides that correspond to the surface of microtubules. Zn2+-induced tubulin sheets, consisting in contrast to microtubules of alternately arranged protofilaments, are covered by H1 on both surfaces. Furthermore, multilayered sheet aggregates appeared. The results indicate that the basic proteins used interact only with that protofilament side which represents the microtubule surface. In accordance with this general principle, models on the structure of double-walled microtubules and multilayered tubulin sheets were derived.     

The orientation effect of gramicidin A on bicelles and Eu3+ -doped bicelles as studied by solid-state NMR and FT-IR spectroscopy

We have explored the effect of gramicidin A (gA) on bicelle (Bic) orientation in the absence and presence of Eu(3+) by (31)P and (2)H NMR at different DMPC/gA ratios. FT-IR spectroscopy was used to assess the lipid chain ordering and verify the transmembrane peptide conformation. Our results show a time-dependent flipping of the bilayer normal alignment at high temperatures and high proportion of gA. The results are explained by both the diamagnetic susceptibility anisotropy of the beta(6.3) helical peptides and viscosity of the lipid mixture. The concentration effect of gramicidin on Bic/Eu(3+) is compared to that on Eu(3+)-doped DMPC liposomes. The Bic/Eu(3+) system is no longer oriented in the presence of gA and adopts a vesicular morphology while the peptide incorporation induces the formation of ellipsoidal DMPC/Eu(3+) assemblies aligned with their normal parallel to the magnetic field. The difference is explained in terms of lipid chain disorder and size of the bilayers.     

The formation of helical tubular vesicles by binary monolayers containing a nickel-chelating lipid and phosphoinositides in the presence of basic polypeptides

Binary lipid monolayers consisting of equimolar proportions of a phosphoinositide and a nickel-chelating lipid formed helical tubular vesicular structures, which appeared to be induced and/or stabilized by myelin basic protein (MBP). Another basic polypeptide, poly-L-lysine, had a similar effect but not to as great a degree as MBP; the proteins thus appeared to act as polycations. Although, the nickel-chelating lipid is a synthetic product, other endogenous divalent cations such as Zn(2+), as well as phosphoinositides, are integral and dynamic components of the myelin sheath in vivo. There, comparable helical tubular structures might represent a means for sequestration of these lipids into domains of high local concentration, perhaps in regions where the membrane is greatly curved.     

VSV transmembrane domain (TMD) peptide promotes PEG-mediated fusion of liposomes in a conformationally sensitive fashion

Helical instability induced by gly residues in the transmembrane domain (TMD) of G protein, the fusion protein of vesicular stomatitis virus (VSV), was speculated to aid in the later steps of the fusion process, because G protein with ala's substituted for the two TMD gly's was inactive (Cleverley, D. Z., and Lenard, J. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 3425-30). Here we examine the conformations of synthetic peptides corresponding to fusion-active (GGpep) and inactive (AApep; G's replaced by A's) TMDs by CD spectroscopy, and then their effects on the kinetics of poly (ethyleneglycol) (PEG)-mediated fusion of small unilamellar vesicles. GGpep and AApep both assumed history-dependent, non-interconvertible ordered structures. Both peptides were largely helical under all conditions if derived from trifluoroethanol solutions, and aggregated in a beta-sheet form if derived from acetonitrile solutions. In solvent, detergents or lipid bilayers, GGpep showed a greater range of secondary structural features than did AApep. The two peptides had large but different effects on PEG-mediated fusion. Both enhanced the rate but not the extent of lipid mixing. AApep significantly inhibited the extent of fusion pore formation while GGpep had no effect. The initial rate of fusion was enhanced 6-fold by GGpep and less than 2-fold by AApep. Addition of 5 mol % hexadecane overrode all peptide-induced effects. We suggest that both GGpep and hexadecane promote pore formation by stabilizing the nonlamellar structures in fusion intermediates or initial small pores. AApep, which had fewer nonhelical features in its CD spectrum than GGpep, actually inhibited fusion pore formation.     

Lipid microdomain formation: characterization by infrared spectroscopy and ultrasonic velocimetry

We demonstrate the use of vibrational infrared spectroscopy applied to characterize lipid microdomain sizes derived from a model raft-like system consisting of nonhydroxy galactocerebroside, cholesterol, and dipalmitoylphosphatidylcholine components. The resulting spectroscopic correlation field components of the lipid acyl chain CH₂ methylene deformation modes, observed when lipid multilamellar assemblies are rapidly frozen from the liquid crystalline state to the gel phase, indicate the existence of lipid microdomains on a scale of several nanometers. The addition of cholesterol disrupts the glycosphingolipid selectively but perturbs the di-saturated chain phospholipid matrix. Complementary acoustic velocimetry measurements indicate that the microdomain formation decreases the total volume adiabatic compressibilities of the multilamellar vesicle assemblies. The addition of cholesterol, however, disrupts the galactocerebroside domains, resulting in a slight increase in the lipid assemblies’ total adiabatic compressibility. The combination of these two physical approaches offers new insight into microdomain formation and their properties in model bilayer systems.     

Structures and mode of membrane interaction of a short alpha helical lytic peptide and its diastereomer determined by NMR, FTIR, and fluorescence spectroscopy.

The interaction of many lytic cationic antimicrobial peptides with their target cells involves electrostatic interactions, hydrophobic effects, and the formation of amphipathic secondary structures, such as alpha helices or beta sheets. We have shown in previous studies that incorporating approximately 30%d-amino acids into a short alpha helical lytic peptide composed of leucine and lysine preserved the antimicrobial activity of the parent peptide, while the hemolytic activity was abolished. However, the mechanisms underlying the unique structural features induced by incorporating d-amino acids that enable short diastereomeric antimicrobial peptides to preserve membrane binding and lytic capabilities remain unknown. In this study, we analyze in detail the structures of a model amphipathic alpha helical cytolytic peptide KLLLKWLL KLLK-NH2 and its diastereomeric analog and their interactions with zwitterionic and negatively charged membranes. Calculations based on high-resolution NMR experiments in dodecylphosphocholine (DPCho) and sodium dodecyl sulfate (SDS) micelles yield three-dimensional structures of both peptides. Structural analysis reveals that the peptides have an amphipathic organization within both membranes. Specifically, the alpha helical structure of the L-type peptide causes orientation of the hydrophobic and polar amino acids onto separate surfaces, allowing interactions with both the hydrophobic core of the membrane and the polar head group region. Significantly, despite the absence of helical structures, the diastereomer peptide analog exhibits similar segregation between the polar and hydrophobic surfaces. Further insight into the membrane-binding properties of the peptides and their depth of penetration into the lipid bilayer has been obtained through tryptophan quenching experiments using brominated phospholipids and the recently developed lipid/polydiacetylene (PDA) colorimetric assay. The combined NMR, FTIR, fluorescence, and colorimetric studies shed light on the importance of segregation between the positive charges and the hydrophobic moieties on opposite surfaces within the peptides for facilitating membrane binding and disruption, compared to the formation of alpha helical or beta sheet structures.     

Myosin II filament assemblies in the active lamella of fibroblasts: their morphogenesis and role in the formation of actin filament bundles

The morphogenesis of myosin II structures in active lamella undergoing net protrusion was analyzed by correlative fluorescence and electron microscopy. In rat embryo fibroblasts (REF 52) microinjected with tetramethylrhodamine-myosin II, nascent myosin spots formed close to the active edge during periods of retraction and then elongated into wavy ribbons of uniform width. The spots and ribbons initially behaved as distinct structural entities but subsequently aligned with each other in a sarcomeric-like pattern. Electron microscopy established that the spots and ribbons consisted of bipolar minifilaments associated with each other at their head-containing ends and arranged in a single row in an "open" zig-zag conformation or as a "closed" parallel stack. Ribbons also contacted each other in a nonsarcomeric, network-like arrangement as described previously (Verkhovsky and Borisy, 1993. J. Cell Biol. 123:637-652). Myosin ribbons were particularly pronounced in REF 52 cells, but small ribbons and networks were found also in a range of other mammalian cells. At the edge of the cell, individual spots and open ribbons were associated with relatively disordered actin filaments. Further from the edge, myosin filament alignment increased in parallel with the development of actin bundles. In actin bundles, the actin cross-linking protein, alpha-actinin, was excluded from sites of myosin localization but concentrated in paired sites flanking each myosin ribbon, suggesting that myosin filament association may initiate a pathway for the formation of actin filament bundles. We propose that zig-zag assemblies of myosin II filaments induce the formation of actin bundles by pulling on an actin filament network and that co-alignment of actin and myosin filaments proceeds via folding of myosin II filament assemblies in an accordion-like fashion.