Registration of the rod is not critical for the phosphorylation-dependent regulation of smooth muscle myosin.

A recent report has suggested that the interaction between the head and the rod region of smooth muscle myosin at S2 is important for the phosphorylation-mediated regulation of myosin motor activity [Trybus, K. M., Freyzon, Y., Faust, L. Z., and Sweeney, H. L. (1997) Proc. Natl. Acad. Sci. U.S.A. 74, 48-52]. To investigate whether specific amino acid residues at S2 or whether the registration of the 7-residue/28-residue repeat appearing in the alpha-helical coiled-coil structure of the rod are critical for such an interaction, two smooth muscle myosin mutants were constructed in which the N-terminal sequences of S2 were deleted to various extents. One mutant contained a deletion of 71 residues at the position immediately C-terminal to the invariant proline (Pro849) linking the S1 domain directly to the downstream sequence of the rod, while in another mutant, 53 residues were deleted at a position 56 residues downstream of Pro849. Despite these alterations which change the registration of both the 28-residue repeat and the 7-residue repeat found in myosin rod sequence, both myosin mutants showed a stable double-headed structure by electron microscopic observation. Both the actin-activated ATPase activity and the actin translocating activity of the mutants were completely regulated by the phosphorylation of the regulatory light chain. The actin sliding velocity of the two mutant myosins was the same as the wild-type recombinant myosin. Furthermore, the head configuration critical for myosin filament formation (extended or folded) was unchanged in either mutant. These results indicate that neither the specific amino acid residues nor the registration of the amino acid repeat in S2 is critical for the head configuration. These results indicate that neither a specific amino acid sequence at the head-rod junction nor the rod sequence registration is critical for the regulation of smooth muscle myosin.     

Role of the C-terminal extremities of the smooth muscle myosin heavy chains: implication for assembly properties.

The two light meromyosin isoforms from rabbit smooth muscle were prepared as recombinant proteins in Escherichia coli. These species which differed only by their C-terminal extremity showed the same circular dichroism spectra and endotherms in measurements of differential scanning calorimetry. Their solubility properties were different at pH 7.0 in the absence of monovalent salts. Their paracrystals formed at low pH differed by their aspect and number. These data suggest a role for the C-terminal extremity of myosin heavy chains in the assembly of myosin molecules in filaments and consequently in the contractility of smooth muscles.     

Interaction between vertebrate skeletal and uterine muscle myosins and light meromyosins

The specific contributions of this work may be summarized as follows: (a) No hybridization of uterine and skeletal myosin occurs at pH 6.0 although previous studies have shown that hybridization does occur at pH 6.5 (B. Kaminer et al. 1976. J. Mol. Biol. 100:379-386) or 7.0 (T. Pollard. 1975. J. Cell Biol. 67:93-104) (b) Hybridization of uterine and skeletal light meromyosins (LMM) occurs at pH 7.0 but not at pH 6.0, which is analogous to the hybridization of myosins. (c) In hybridized paracrystals there is a uniform distribution of both uterine and skeletal LMM molecules because all the paracrystals have only one axial repeat pattern. This makes it highly likely that in hybridized filaments the two myosins are also uniformly distributed throughout the filaments. (d) The 14-nm repeat of white bands observed in paracrystals of uterine LMM formed at pH 6.0, compared with the 14-nm repeat of dark bands observed with skeletal LMM under the same conditions, probably reflects differences in surface charge density along the different LMM molecules.     

Electron microscopic analysis of tropomyosin paracrystals.

Negatively stained images of divalent cation-induced tropomyosin paracrystals show polymorphic patterns which are almost bipolar. Although these bipolar forms are naturally due to antiparallel arrays of molecules, the precise molecular arrangements have not been clarified yet except in the case of one type of these polymorphic paracrystals by Stewart and McLachlan [(1976) J. Mol. Biol. 103, 251--269]. In the previous paper we showed that the lead-induced polar paracrystal is a parallel and in-register array of tropomyosin molecules. Moreover, we have made it possible to locate a given residue on the staining pattern. By overlapping two photographic transparencies of the polar paracrystal antiparallel, directly observed images of polymorphic bipolar paracrystals could be synthesized photographically with fidelity. The overlap length between N-terminals of antiparallel pairs of molecules could be easily determined without any assumptions. Next, we considered the stabilizing forces involved in the morphogenesis of such polymorphic paracrystals. The cation-bridged attractive forces already proposed by some groups were insufficient to account for the stability of some specific forms of tropomyosin paracrystals. From the primary amino acid sequence of tropomyosin, we calculated the changes of repulsive forces between the basic residues with changes of molecular overlap length between the N-terminals of antiparallel pairs. By setting the values of charge appropriately, we could account well for the stability of the polymorphic structures observed by electron microscopy.     

The myosin filament. XIII. The sensitivity of LMM assembly to Mg.ATP

An LMM fragment (Mr 62,000) of myosin has been prepared which has aggregation properties that are sensitive to the presence of Mg.ATP. Aggregation of the LMM by reducing the ionic strength in the presence of 1 mM Mg.ATP produces non-periodic aggregates which gradually rearrange to paracrystals with a 43 nm axial repeat pattern. This fragment includes the C-terminal end of the myosin rod starting at residue 1376. Therefore, at least one of the Mg.ATP binding sites responsible for this effect is located somewhere along this region of the myosin rod. Although assembly of the rod fragment of myosin into paracrystals does not show sensitivity to Mg.ATP, assembly of intact myosin molecules to form filaments does show sensitivity to Mg.ATP. For myosin filaments, assembly initially gives a broad distribution around a mean length of 1.5 microns, which sharpens around the mean length with time. The rearrangement of the LMM rods and intact myosin molecules both induced by the presence of Mg.ATP are probably related. These findings highlight the complexity of the cooperative interactions between different portions of the myosin molecule that are involved in determining the assembly properties of the intact molecule.     

In vitro reconstitution of recombinant lamin A and a lamin A mutant lacking the carboxy-terminal tail

Xenopus lamin A and a lamin A mutant lacking the complete 280 amino acid long carboxy-terminal tail were expressed in bacteria and purified from inclusion bodies. Electron microscopic analysis of lamin A dimers revealed that the carboxy-terminal 280 amino acids correspond to the globular domain seen in rotary-shadowed wild-type lamin and that the rodlike domain consists of the short non-helical amino terminus and the alpha-helical region. During reconstitution lamin A dimers first formed polar head to tail aggregates which then associated laterally resulting in paracrystals with periodic repeats of 25 nm. In the mutant, the longitudinal and lateral association of dimers had not been influenced, however, periodic repeats were absent in the filament bundles formed. Thus our data clearly demonstrate that carboxy-terminal tails are localized in light-stained regions of negatively stained paracrystals and that they are responsible for the alternating light dark staining of paracrystals. Fibrils, 2 to 3 nm thick, were a common structural element of paracrystals and filament bundles.     

Peptidoglycan cross-linking in Staphylococcus aureus. An apparent random polymerisation process

The peptidoglycan of Staphylococcus aureus contains relatively short glycan chains and is highly cross-linked via its peptide chains. The material from wild-type (strain H) and mutants H28, H4B and MR-1 was freed from the teichoic-acid-linked component and then hydrolysed by Chalaropsis muramidase to yield disaccharide-repeating units of the glycan with attached peptides either non-cross-linked (monomer) or joined to similar units by one (dimer), two (trimer) or more (oligomer) peptide cross links. The resulting fragments were separated by high-resolution HPLC so that distinguishable components as large as nonamer could be identified. Extrapolation showed that, in S. aureus H, H28 and MR-1, oligomers at least as large as eicosamer formed part of the smooth distribution of oligomer fragments, whereas in strain H4B (PBP4-) the maximum size was around dodecamer. The oligomer distribution profile was related to the polymerization theories of Flory, which allow a distinction to be made between a monomer addition model, whereby each oligomer can only be synthesized by the addition of a single monomer unit to its next lower homologue, and a random addition model, in which an oligomer can be formed by linkage of any combination of its constituent smaller units. In S. aureus close approximation to the random addition model for oligomer synthesis and hence for peptidoglycan cross-linking was observed, both in PBP4+ and PBP4- mutants. The implications for secondary cross-linking in S. aureus cell wall formation are inescapable, although the possibility of an endopeptidase/transpeptidase providing later modification of the peptidoglycan is not completely ruled out.     

The structure of spindle-shaped paracrystals of light meromyosin

Light meromyosin paracrystals have been studied by electron microscopy combined with optical diffraction in order to understand how the tails of the myosin molecules might pack in the backbone of muscle thick filaments. The forms of paracrystal investigated were all spindle-shaped structures with an axial periodicity of either 43 nm or 14.3 nm or hybrids involving aspects of both repeats. Transverse sections show that they are not smooth but polygonal in outline. Analysis of the band patterns in negatively stained specimens indicates that the molecular arrangement in the paracrystals involves both parallel and antiparallel interactions. A parallel axial displacement of the molecules by 43 nm is intrinsic to all forms of paracrystal investigated. The principal antiparallel overlap between molecules appears to be one of 84 nm, and it is suggested that an antiparallel dimer is the structural unit in the paracrystals. The role of the interactions leading to these displacements in the formation of the thick filament backbone is discussed.     

Light meromyosin paracrystal formation

Studies of paracrystal formation by column purified light meromyosin (LMM) prepared in a variety of ways led to the following conclusions: (a) different portions of the myosin rod may be coded for different stagger relationships. This was concluded from observations that paracrystals with different axial repeat periodicities could be obtained either with LMM framents of different lengths prepared with the same enzyme, or with LMM fragments of identical lengths but prepared with different enzymes. (b) Paracrystals with a 14-nm axial repeat periodicity are most likely formed by the aggregation of sheets with a 44-nm axial repeat within the sheets which are staggered by 14 nm. All of the axial repeat patterns expected from one sheet or aggregates of more than one sheet, on this basis, were observed in the same electron micrograph. (c) C-protein binding probably occurs preferentially to LMM molecules related in some specific way. This was concluded from the observation that the same axial repeat pattern was obtained in paracrystals formed from different LMM preparations in the presence of C-protein, regardless of differences in the axial repeat obtained in the absence of C-protein. (d) Nucleic acid is responsible for the 43-nm axial repeat patterns observed in paracrystals formed by the ethanol-resistant fraction of LMM. In the absence of nuclei acid, paracrystals with a 14nm axial repeat are obtained. (e) The 43-nm axial repeat pattern observed with the ethanol-resistant fraction of LMM is different for LMM preparations obtained by trypsin and papain digestions.     

Effect of H-protein on the formation of myosin filaments and light meromyosin paracrystals

H-protein is a component of the thick filaments of skeletal myofibrils. Its effects on the assembly of myosin into filaments and on the formation of light meromyosin (LMM) paracrystals at low ionic strength have been investigated. H-protein reduced the turbidities of myosin filament and LMM paracrystal suspensions. Electron microscopic observation showed that the appearances of the filaments prepared in the presence and absence of H-protein were different. The filament length was not substantially changed by H-protein, but the diameter of the myosin filament was markedly reduced. H-protein bound to LMM and co-sedimented with it at low ionic strength upon centrifugation. Two types of paracrystals, spindle-shaped and sheet-like, were observed in LMM suspensions. H-protein altered the structure of the LMM paracrystals, especially the spindle-shaped ones. The thickness of the spindle-shaped paracrystals was reduced when H-protein was present during LMM paracrystal formation. On the other hand, periodic features along the long axis of the sheet-like paracrystals were retained even at high ratios of H-protein to LMM. However, there were fewer sheet-like paracrystals in the LMM suspensions containing H-protein than in the control. These results suggest that H-protein interferes with self-association of myosin molecule into filaments due to its binding to the tail portion of the myosin. However, H-protein does not have a length-determining effect on the formation of myosin filaments.     

Impaired protein folding, dimer formation, and heterotetramer assembly cause intra- and extracellular instability of a Y283C mutant of the A subunit for coagulation factor XIII.

Factor XIII (XIII) is a heterotetramer consisting of two catalytic A subunits (XIIIA) and two noncatalytic B subunits (XIIIB). We examined the molecular mechanisms of a Y283C mutation which had previously been identified in a patient with XIIIA deficiency. The recombinant Y283C protein was labile when expressed in MEG-01 cells, which can endogenously synthesize XIIIA. We also included two other mutants, G562R and I464stop, previously characterized in a non-XIIIA-producing cell line. All these mutants exhibited decreased thermostability and resistance against proteolytic digestion when compared with the wild-type. Gel-filtration analysis revealed that the mutants were in monomer form, while the wild-type formed a dimer. These results were consistent with the prediction by molecular modeling that the mutant molecules would be misfolded. Although assembly of a heterotetramer with XIIIB was demonstrated for Y283C, its binding ability was 10% that of the wild-type. No complex formation was observed for the G562R or I464stop mutants. The wild-type was stabilized in plasma by complex formation with XIIIB, resulting in an increased resistance against proteolytic digestion. In contrast, the mutants were unstable in plasma even in the presence of XIIIB. Thus, impaired folding, dimer formation, and heterotetramer assembly of the mutant XIIIAs lead to both intra- and extracellular instability, which must be responsible for XIIIA deficiency in the patient.     

Arthrin: a new actin-like protein in insect flight muscle

There are one or more proteins of 50,000 to 60,000 Mr in the thin filaments of insect flight muscle. A protein of 55,000 Mr has been isolated from insect fibrillar flight muscle and called arthrin. Despite its higher molecular weight, arthrin is in many ways like actin. The amino acid composition of arthrin was similar to that of actin. There were similarities in the peptides produced by digesting the denatured proteins and mild digestion of polymerized proteins cleaved similar-sized fragments from arthrin and actin. Polymerized arthrin activated the Mg2+ ATPase of myosin to the same extent as actin and the ATPase was regulated by rabbit or Lethocerus troponin and tropomyosin. Arthrin did not itself act as troponin-T. Electron microscopy of negatively stained specimens showed that arthrin and actin filaments were similar in structure and that arthrin could be decorated by rabbit subfragment-1 to form normal-looking arrowheads. Arthrin formed paracrystals at an optimum concentration of MgCl2 (25 mM) that was somewhat lower than the optimum for actin paracrystals. Optical diffraction showed that the structure of the paracrystals was similar to those formed from actin. The mass of arthrin and actin filaments relative to phage fd was measured by scanning transmission electron microscopy; the relative mass of arthrin and actin was 1.33, in agreement with molecular weight estimations. Therefore arthrin has the properties of a heavy form of actin. The proportion of actin, arthrin and troponin-T in Lethocerus myofibrils was six moles of actin to one mole of arthrin and one mole of troponin-T. The function of arthrin is not known.     

Molecular interactions in paracrystals of a fragment corresponding to the alpha-helical coiled-coil rod portion of glial fibrillary acidic protein: evidence for an antiparallel packing of molecules and polymorphism related to intermediate filament structure

We have expressed in Escherichia coli a fragment of c-DNA that broadly corresponds to the alpha-helical coiled-coil rod section of glial fibrillary acidic protein (GFAP) and have used the resultant protein to prepare paracrystals in which molecular interactions can be investigated. An engineered fragment of mouse GFAP c-DNA was inserted into a modified version of the E. coli expression vector pLcII, from which large quantities of a lambda cII-GFAP rod fusion protein were prepared. A protein fragment corresponding to the GFAP rod was then obtained by proteolysis with thrombin. Paracrystals of this material were produced using divalent cations (Mg, Ca, Ba) in the presence of a chaotrophic agent such as thiocyanate. These paracrystals showed a number of polymorphic patterns that were based on a fundamental pattern that had dyad symmetry and an axial repeat of 57 nm. Analysis of both positive and negative staining patterns showed that this fundamental pattern was consistent with a unit cell containing two 48-nm-long molecules in an antiparallel arrangement with their NH2 termini overlapping by approximately 34 nm. More complicated patterns were produced by stacking the fundamental pattern with staggers of approximately 1/5, 2/5, and 1/2 the axial repeat. The molecular packing the unit cell was consistent with a range of solution studies on intermediate filaments that have indicated that a molecular dimer (i.e., a tetramer containing four chains or two coiled-coil molecules) is an intermediate in filament assembly. Moreover, these paracrystals allow the molecular interactions involved in the tetramer to be investigated in some detail.     

Paramyosin gene (unc-15) of Caenorhabditis elegans. Molecular cloning, nucleotide sequence and models for thick filament structure

Paramyosin is a major structural component of thick filaments isolated from many invertebrate muscles. The Caenorhabditis elegans paramyosin gene (unc-15) was identified by screening with specific antibodies an "exon-expression" library containing lacZ/nematode gene fusions. Short probes recovered from the library were used to identify bacteriophage lambda and cosmid clones that encompass the entire paramyosin (unc-15) gene. From these clones, numerous subclones containing epitopes reacting with anti-paramyosin sera were obtained, providing strong evidence that the initial cloned fragment was, in fact, derived from the structural gene for paramyosin. The complete nucleotide sequence of a 12 x 10(3) base-pair region spanning the gene was obtained. The gene is composed of ten short exons encoding a protein of 866 [corrected] amino acid residues. Paramyosin is highly similar to residues 267 to 1089 of myosin heavy chain rods. For most of its length, paramyosin appears to form an alpha-helical coiled-coil and shows the expected heptad repeat of hydrophobic amino acid residues and the 28-residue repeat of charged amino acids characteristic of myosin heavy chain rods. However, paramyosin differs from myosin in having non-helical extensions at both the N and C termini and an additional "skip" residue that interrupts the 28-residue repeat. The distribution of charges along the length of the paramyosin rod is also significantly different from that of myosin heavy chain rods. Potential charge-mediated interactions between paramyosin rods and between paramyosin and myosin rods were calculated using a model successfully applied previously to the analysis of the myosin rod sequences. Myosin rods aligned in parallel show optimal charge-charge interactions at multiples of 98 residue staggers (i.e. at axial displacements of multiples of 143 A). Paramyosin rods, in contrast, appear to interact optimally at parallel staggers of 493 residues (i.e. at axial displacements of 720 A) but show only weak interaction peaks at 98 or 296 residues. Similar calculations suggest optimal interactions between paramyosin molecules and myosin rods and in their anti-parallel alignments. The implications of these results for the structure of the bare zone and the assembly of nematode thick filaments are discussed.     

Interaction of tropomyosin with troponin components.

1. The TN-T and TN-I components of troponin both interact with tropomyosin and cause its precipitation in 0.1 M KC1 at neutral pH. The precipitate contains both end-to-end and side-by-side aggregates of tropomyosin molecules. 2. The TN-T and TN-I components change the band pattern of tropomyosin paracrystals formed in MgC1(2) solutions, although in different ways. TN-T causes the formation of hexagonal net structures, double-stranded net or paracrystals which result from the collapse of the double-stranded net. TN-I at pH 7.9 causes the formation of paracrystals with a 400 A periodic band pattern and a 200 A repeat. The same band pattern can also be seen in tropomyosin paracrystals formed at pH values below 6.0. 3. The TN-C component does not precipitate tropomyosin in 0.1 M KC1. The aggregates of tropomyosin obtained with either TN-T or TN-I can be solubilized by the addition of TN-C. No interaction of TN-C was observed with tropomyosin paracrystals formed in the presence of MgC12.     

Two regions of the tail are necessary for the isoform-specific functions of nonmuscle myosin IIB

To function in the cell, nonmuscle myosin II molecules assemble into filaments through their C-terminal tails. Because myosin II isoforms most likely assemble into homo-filaments in vivo, it seems that some self-recognition mechanisms of individual myosin II isoforms should exist. Exogenous expression of myosin IIB rod fragment is thus expected to prevent the function of myosin IIB specifically. We expected to reveal some self-recognition sites of myosin IIB from the phenotype by expressing appropriate myosin IIB rod fragments. We expressed the C-terminal 305-residue rod fragment of the myosin IIB heavy chain (BRF305) in MRC-5 SV1 TG1 cells. As a result, unstable morphology was observed like MHC-IIB(-/-) fibroblasts. This phenotype was not observed in cells expressing BRF305 mutants: 1) with a defect in assembling, 2) lacking N-terminal 57 residues (N-57), or 3) lacking C-terminal 63 residues (C-63). A myosin IIA rod fragment ARF296 corresponding to BRF305 was not effective. However, the chimeric ARF296, in which the N-57 and C-63 of BRF305 were substituted for the corresponding regions of ARF296, acquired the ability to induce unstable morphology. We propose that the N-57 and C-63 of BRF305 are involved in self-recognition when myosin IIB molecules assemble into homo-filament.     

Critical regions for assembly of vertebrate nonmuscle myosin II

Myosin II molecules assemble and form filaments through their C-terminal rod region, and the dynamic filament assembly-disassembly process of nonmuscle myosin II molecules is important for cellular activities. To estimate the critical region for filament formation of vertebrate nonmuscle myosin II, we assessed the solubility of a series of truncated recombinant rod fragments of nonmuscle myosin IIB at various concentrations of NaCl. A C-terminal 248-residue rod fragment (Asp 1729-Glu 1976) was shown by its solubility behavior to retain native assembly features, and two regions within it were found to be necessary for assembly: 35 amino acid residues from Asp 1729 to Thr 1763 and 39 amino acid residues from Ala 1875 to Ala 1913, the latter containing a sequence similar to the assembly competence domain (ACD) of skeletal muscle myosin. Fragments lacking either of the two regions were soluble at any NaCl concentration. We referred to these two regions as nonmuscle myosin ACD1 (nACD1) and nACD2, respectively. In addition, we constructed an alpha-helical coiled-coil model of the rod fragment, and found that a remarkable negative charge cluster (termed N1) and a positive charge cluster (termed P2) were present within nACD1 and nACD2, respectively, besides another positive charge cluster (termed P1) in the amino-terminal vicinity of nACD2. From these results, we propose two major electrostatic interactions that are essential for filament formation of nonmuscle myosin II: the antiparallel interaction between P2 and N1 which is essential for the nucleation step and the parallel interaction between P1 and N1 which is important for the elongation step.     

Local migration of myosin in F-actin plus ATP solution on the boundary of a diffusion cell.

The diffusion phenomena of myosin (myosin A, H-meromyosin or subfragment-1) in F-actin plus ATP solutions were investigated. The upper part of the diffusion cell was filled with F-actin plus ATP, and the lower part was filled with F-actin, ATP, and myosin, then both parts were brought into contact so that a boundary of the two solutions was formed and the diffusion of myosin in F-actin plus ATP solutions started. The diffusion pattern was observed with a schlieren lens system. When almost all the ATP in the lower part of the cell had been consumed by actomyosin, a hyper-sharp schlieren pattern appeared near the boundary. On analyzing this pattern, it was found that a local fast migration of proteins was occurring. Simple Brownian motion of myosin molecules could not explain the hyper-sharp phenomenon. This phenomenon occurred in ther pesence of Mg2+ or Ca2+, but very little in the presence of EDTA. Although it is well known that the superprecipitation of myosin B suspension occurs only at physiological ionic strength, this phenomenon occurred over a relatively wide range of ionic strengths. The molecular mechanism of this phenomenon is discussed in relation to the basic mechanism of the interaction between myosin and F-actin.     

Structure of magnesium paracrystals of α-tropomyosin

The molecular packing of magnesium paracrystals of α-tropomyosin has been examined by electron microscopy. Previous work (Caspar et al., 1969) had shown that these structures are composed of antiparallel arrays of molecules and we have now studied the relative positions of the molecules by matching the banding patterns of paracrystals positively stained with uranyl acetate to the sequence of the molecule. The overlap between the C-termini of the molecules in the unit cell is 175 ± 2 residues and the overlap of the N-termini lies in the range 107 to 122 residues. In the long overlap region (between C-termini), and probably also in the short overlap region, the molecular packing is such that the periodic zones of negative charge present in the sequence (Stewart & McLachlan, 1975) lie opposite one another. We propose that magnesium bridges between opposing negative charges contribute strongly to the stability of the structure. We confirm earlier work (Stewart, 1975b) on the absolute orientation of the molecules in the paracrystal: the troponin binding site on tropomyosin is approximately 130 Å from the C-terminus, and Cys190 is within 10 to 15 Å units of the C-C dyad.     

Comparative studies on the structure and aggregative properties of the myosin molecule. III. The in vitro aggregative properties of the lobster myosin molecule.

The solubility of rabbit skeletal and lobster abdominal muscle myosin has been studied in monovalent salt solutions as a function of pH (over the range 4.75 to 8.5) and ionic strength (50-500 mM). Rabbit skeletal muscle myosin was found to precipitate over a narrower pH range than the lobster abdominal muscle myosin but at equivalent pH values and ionic strengths the former exhibited greater solubility. Comparison of the solubility of rabbit myosin, per se with that of light meromyosin and lobster myosin with its equivalent proteolytically produced fragment (fraction B1) showed that both rod fragments were more soluble than their parent molecules. Under conditions of low solubility (low ionic strength and pH) the quantitiy of protein in solution remained essentially constant with increasing total protein, thus suggesting that the aggregation phenomenon is of a phase transition type. Examination of the aggregates by electron microscopy revealed that rabbit myosin formed classical, elongate, spindle-shaped filaments similar to those previously observed by others. In contrast lobster myosin only formed short, dumbbell-shaped filaments 0.2-0.3 mum long. Consideration of the pH ranges over which aggregation occurred suggests that protonation of histidine residues may be involved in rabbit myosin filament formation while for lobster myosin, aggregation may involve protonation of epsilon-amino or guanidino groups. The possible relationship between the distribution of these groups along the rod portion of the myosin molecule and the formation of elongate filaments has been explored.