Substructures in the core of thick filaments: arrangement and number in relation to the paramyosin content of insect flight muscles

Transverse sections (100-140 nm thick) of solid myosin filaments of the flight muscles of the honeybee, Apis mellifica, the fleshfly, Phormia terrae-novae and the waterbug, Lethocerus uhleri, were photographed in a JEM-200 electron microscope at 200 kV. The images were digitized and computer processed by rotational filtering. The power spectra of the images of each of these filaments showed six-fold symmetry for the outer wall region and three-fold symmetry for the inner wall region. Images of the honeybee additionally showed three-fold symmetry for the center of the filament. Considering both paramyosin content of the myosin filaments and the results of the rotational filtering, we suggest the existence of 3 paramyosin strands in the myosin filaments of the fleshfly, 6 paramyosin strands in the honeybee filaments and 5 strands in the myosin filaments of the waterbug. In the case of the honeybee, the 3 paramyosin strands of the inner wall are positioned directly opposite the myosin subfilaments, while the 3 strands of the center seem to be arranged opposite the gaps between the myosin subfilaments. The paramyosin filaments of the fleshfly wobble between 2 myosin subfilaments, without loosing their three-fold symmetry arrangement in the inner wall. The 3 paramyosin strands in the inner wall of the waterbug myosin filaments are either arranged opposite the myosin subfilaments or opposite the gaps between the subfilaments. Finally, we were able to generate a 3-dimensional reconstruction of the myosin filament of the honeybee, showing the parallel arrangement of both, myosin subfilaments and paramyosin strands, relative to the long filament axis.     

The myosin filament. X. Observation of nine subfilaments in transverse sections

The molecular packing of the subfilaments in muscle thick filaments has been investigated by electron microscopy. Thin (80-100 nm) transverse sections of vertebrate skeletal muscle were cut, and 129 electron microscope images of thick filaments from 15 different areas including seven to ten images in each area were analyzed by computer image processing. The transverse sections were limited to the portion of the filaments between the bare zone and the C-protein bearing region. Of the 129 images, six were discarded because they were structurally disrupted, 17 did not show evidence for the presence of subfilaments from the autocorrelation function, and four did not show evidence for three-fold rotational symmetry from the power spectrum. The remaining 102 filaments all showed evidence for three-fold rotational symmetry, consistent with other available evidence (Pepe, 1982). From the analysis of these images by rotational filtering, we have found that the vertebrate skeletal myosin filament is made up of nine subfilaments and that the image appears to have trigonal symmetry. Of these subfilaments, six are arranged with a center-to-center spacing of about 4 nm and the other three on the surface of the filament are distorted from this arrangement. Three additional densities, which together with the other nine, correspond to the pattern of 12 densities previously observed in more highly selected images (Stewart et al., 1981; Pepe and Drucker, 1972) were observed in 5% of the images. Another pattern of nine subfilaments peripherally arranged around the circumference of the filament was observed occasionally. This latter image may represent the organization of the subfilaments in the bare zone region of the filament, resulting from sampling of individual filaments displaced longitudinally relative to the other filaments in the A-band.     

Invertebrate myosin filament: Parallel subfilament arrangement in the wall of solid filaments from the honeybee, Apis mellifica

Transverse serial sections (100-140 nm thick) of solid myosin filaments of the honeybee, Apis mellifica, were photographed in a JEM-200 electron microscope at 200 kV. The images were digitized and computer processed by rotational filtering. 87% of the myosin filaments showed 6-fold symmetry in their power spectra, confirming the results of earlier works (Beinbrech et al., 1988, 1991). To determine if the subfilaments were arranged parallel to the filament backbone, two methods were used. First, the three images of each myosin filament in the three serial sections were superimposed. 85% of the resulting images showed a strong peak for 6-fold symmetry and the averaged images showed 6 pairs of subfilaments, which gives evidence for parallel arrangement of the subfilaments relative to the filament axis. This result was confirmed by the second method in which a 3-dimensional reconstruction was made. An average image was made from the images of the same 17 myosin filaments from each of the three sections. The data for the 3-dimensional reconstruction were collected by tracing the outlines of the structures in the three successive sections. The resulting stereo image shows a parallel arrangement of the subfilaments.     

The myosin filament XIV backbone structure.

The substructure of the thick filaments of chemically skinned chicken pectoralis muscle was investigated by electron microscopy. Images of transverse sections of the myosin filaments were determined to have threefold symmetry by cross-correlation analysis, which gives an unbiased determination of the rotational symmetry of the images. Resolution, using the phase residual test (Frank et al. 1981. Science [Wash. DC]. 214:1353-1355), was found to be between 3.2 and 3.6 nm. Three arrangements of nine subfilaments in the backbone were found in all regions of the filament at ionic strengths of 20 and 200 mM. In the average images of two of these, there were three dense central subfilaments and three pairs of subfilaments on the surface of the thick filament. In the average image of the third arrangement, all of the protein mass of the nine subfilaments was on the surface of the filament with three of them showing less variation in position than the others. A fourth arrangement appearing to be transitional between two of these was seen often at 200 mM ionic strength and only rarely at 20 mM. On average, the myosin subfilaments were parallel to the long axis of the filament. The different arrangements of subfilaments appear to be randomly distributed among the filaments in a transverse section of the A-band. Relative rotational orientations with respect to the hexagonal filament lattice, using the three densest subfilaments as reference showed a major clustering (32%) of filaments within one 10 degrees spread, a lesser clustering (15%) at 90 degrees to the first, and the remainder scattered thinly over the rest of the 120 degrees range. There was no obvious pattern of distribution of the two predominant orientations that could define a superlattice in the filament lattice.     

The invertebrate myosin filament: subfilament arrangement of the solid filaments of insect flight muscles.

Transverse sections (approximately 140 nm thick) of solid myosin filaments of the flight muscles of the fleshfly, Phormia terrae-novae, the honey bee, Apis mellifica, and the waterbug, Lethocerus uhleri, were photographed in a JEM model 200A electron microscope at 200 kV. The images were digitized and computer processed by rotational filtering. In each of these filaments it was found that the symmetry of the core and the wall was not the same. The power spectra of the images showed sixfold symmetry for the wall and threefold symmetry for the core of the filaments. The images of the filaments in each muscle were superimposed according to the sixfold center of the wall. These averaged images for all three muscles showed six pairs of subunits in the wall similar to those found in the wall of tubular filaments. From serial sections of the fleshfly filaments, we conclude that the subunits in the wall of the filaments represent subfilaments essentially parallel to the long axis of the filament. In each muscle there are additional subunits in the core, closely related to the subunits in the wall. Evaluation of serial sections through fleshfly filaments suggests that the relationship of the three subunits observed in the core to those in the wall varies along the length of the filaments. In waterbug filaments there are three dense and three less dense subunits for a total of six all closely related to the wall. Bee filaments have three subunits related to the wall and three subunits located eccentrically in the core of the filaments. The presence of core subunits can be related to the paramyosin content of the filaments.     

Subfilament organization in myosin filaments of the fast abdominal muscles of the lobster, Homarus americanus

The myosin filaments of the fast abdominal muscle of the lobster are about 2.7 microns long with a diameter of about 20 nm. They have a low density core in transverse sections except for a short portion in the middle of the filaments about 140 nm in length which is solid. In the solid region the diameter of the filaments is 25 nm. The wall of the filaments is made up of 12 subfilaments arranged in six pairs in a single layer around the wall. The spacing between the subfilaments of a pair is 3.4 nm and the spacing between successive pairs is 8.4 nm. An extra density is present on the inner surface of the wall of the filament along the entire length of the tubular portion of the filament. This density is always adherent to the wall and in serial transverse sections of the same filament its position changes from section to section without any apparent pattern to the change. No structural organization could be detected in this extra density.     

Substructure and accessory proteins in scallop myosin filaments

Native myosin filaments from scallop striated muscle fray into subfilaments of approximately 100-A diameter when exposed to solutions of low ionic strength. The number of subfilaments appears to be five to seven (close to the sevenfold rotational symmetry of the native filament), and the subfilaments probably coil around one another. Synthetic filaments assembled from purified scallop myosin at roughly physiological ionic strength have diameters similar to those of native filaments, but are much longer. They too can be frayed into subfilaments at low ionic strength. Synthetic filaments share what may be an important regulatory property with native filaments: an order-disorder transition in the helical arrangement of myosin cross-bridges that is induced on activation by calcium, removal of nucleotide, or modification of a myosin head sulfhydryl. Some native filaments from scallop striated muscle carry short "end filaments" protruding from their tips, comparable to the structures associated with vertebrate striated muscle myosin filaments. Gell electrophoresis of scallop muscle homogenates reveals the presence of high molecular weight proteins that may include the invertebrate counterpart of titin, a component of the vertebrate end filament. Although the myosin molecule itself may contain much of the information required to direct its assembly, other factors acting in vivo, including interactions with accessory proteins, probably contribute to the assembly of a precisely defined thick filament during myofibrillogenesis.     

Organization of Actin Filaments in the Axial Rod of Abalone Sperm Revealed by Quick Freeze Technique

An axial rod in abalone ( Haliotis discus ) sperm is a structure composed of a bundle of actin filaments, which elongates anteriorly to form the acrosomal process during the acrosome reaction. The ultrastructure of the actin filament bundle constituting the axial rod was examined using quick freeze technique followed by either freeze-substitution or deep-etch electron microscopy. Thin sections of quick freeze and freeze-substituted sperm revealed that the actin filaments in the axial rod are hexagonally packed in a paracrystalline array through its almost entire length with an average center-to-center spacing of 12 nm. Periodic transverse bands were also observed across the actin filament bundle, which may reflect the cross-bridges interconnecting the adjacent filaments. Quick-freeze deep-etch analysis provided the three-dimensional view of the axial rod. Actin filaments exhibiting 5.5–6 nm spaced striations were observed to run in parallel with each other inside the axial rod. The existence of cross-bridging structures was also displayed between adjacent filaments. These results suggest that the actin filaments in the axial rod are probably held together by regularly spaced cross-bridges to form a well ordered hexagonally packed bundle, and also cross-linked by fibrous structure to the lateral inner acrosomal membrane which closely surrounds the anterior half of the actin filament bundle.     

Helical reconstruction of frozen-hydrated scallop myosin filaments.

Native myosin filaments from scallop striated muscle that have been rapidly frozen in relaxing solutions appear to be well preserved in vitreous ice. Electron micrographs of samples at -177 degrees C were recorded with an electron dose of 10 e/A2 at 1.5 microns defocus. After filament images were straightened by spline-fitting, several transforms showed well-defined layer-lines arising from the helical structure of the filament. A set of 17 near-meridional layer-lines has been collected and corrected for background and for phase and amplitude contrast functions. Preliminary helical reconstructions from this still incomplete data set reveal aspects of structure that were not apparent from earlier analysis of negatively stained filaments from scallop muscle. Individual pear-shaped myosin heads now appear to be well resolved from each other and from the filament backbone. The two heads of each myosin molecule appear to be splayed apart axially. The reconstructions also reveal that the filament backbone has a polygonal shape in cross-section, and that it appears to contain seven peripherally located subfilaments.     

Arrangement of myosin heads in relaxed thick filaments from frog skeletal muscle

The distribution of myosin heads on the surface of frog skeletal muscle thick filaments has been determined by computer processing of electron micrographs of isolated filaments stained with tannic acid and uranyl acetate. The heads are arranged in three strands but not in a strictly helical manner and so the structure has cylindrical symmetry. This accounts for the "forbidden" meridional reflections seen in diffraction patterns. Each layer-line therefore represents the sum of terms of Bessel orders 0, +/- 3, +/- 6, +/- 9 and so on. These terms interact so that, unlike a helical object without terms from overlapping Bessel orders, as the azimuth is changed, the amplitude on a layer-line at a particular radius varies substantially and its phase does not alter linearly. Consequently, a three-dimensional reconstruction cannot be produced from a single view. We have therefore used tilt series of three individual filaments to decompose the data on layer-lines 0 to 6 into terms of Bessel orders up to +/- 9 using a least-squares procedure. These data had a least-squares residual of 0.32 and enabled a three-dimensional reconstruction to be obtained at a nominal resolution of 6 nm. This showed, at a radius of about 10 nm, three strands of projecting morphological units with three units spaced along each strand every 42.9 nm axially. We have identified these units with pairs of myosin heads. Successive units along a strand are perturbed axially, azimuthally and radially from the positions expected if the structure was perfectly helical. This may simply be a consequence of steric restrictions in packing the heads on the thick filament surface, but could also reflect an underlying non-helical arrangement of myosin tails, which would be consistent with the thick filament shaft being constructed from three subfilaments in which the tails were arranged regularly. There was also material at a radius of about 6 nm spaced 42.9 nm axially, which we tentatively identified with accessory proteins. The filament shaft had a pronounced pattern of axial staining.     

Paramyosin content and thick filament structure in insect muscles

Summary The myosin filaments of the flight muscles of the locust Locusta migratoria, the cockchafer Melolontha melolontha and the femur muscles of L. migratoria have solid centers. Those of the flight muscles of the housefly Musca domestica and Drosophila melanogaster are tubular. Electron micrographs of myofibrils of the fleshfly Phormia terrae-novae contain both filament types within one sarcomere and suggest the existence of 4 cross-bridges per crown.Estimates of the ratios of myosin to paramyosin and of myosin to actin on sodium dodecyl sulphate-polyacrylamide gels yielded paramyosin contents of 9% of the thick filament mass for the solid and 2.6% for the tubular filaments (3.8% for P. terrae-novae). Based on the myosinactin ratios up to 6 myosin dimers per crown could be calculated.The molar ratio of actin to arthrin on SDS gels was found to be 3.37 for native and extracted myofibrils of flight muscles from P. terrae-novae. Arthrin is also present in isolated actin filaments suggesting that it is localized in or on the thin filaments. If we assume that it is constituent part of the helices of the thin filaments the number of myosin dimers per crown can be diminished to 4.5, considerably closer to the values obtained by evaluation of electron micrographs.Dedicated to Prof. Dr. Bernhard Rensch on his 85^th birthday     

Structure of short thick filaments from Limulus muscle

Shortened Limulus thick filaments, isolated from stimulated muscle, are structurally similar to long filaments, isolated from unstimulated muscle, except for length. Both have 3-fold screw symmetry with a helical repeat at approximately 43 nm, axial spacing of 14.5 nm between successive crowns of crossbridges and 4-fold rotational symmetry as estimated from the Bessel argument, by analysis of optical transforms of electron micrograph negatives of negatively stained samples. Both short and long filaments also have similar radii for the location of their crossbridges, thus similar diameters. Equal numbers of subunits/helical strand are also apparent on images of metal-shadowed long and short filaments. Since these data argue against molecular reorganization during filament shortening, it is suggested that the change in length of Limulus thick filaments may occur by reversible disaggregation of constituent protein molecules.     

低分子量神经丝蛋白（NF—L）的体外组装

Neurofilaments were isolated from bovine spinal cords by ultra-speed centrifugation and examined by negative staining. The neurofilament triplet proteins: NF-L, NF-M and NF-H were purified by DE-52 anion exchange chromatography in the presence of 6 mol/L urea. The reassembly of NF-L under controlled conditions was studied. NF-L can reassemble into 10 nm width filaments within 60 minutes at physiological condition of around 0.15 mol/L NaCl, 2 mmol/L MgCl2, neutral pH(pH 6.8) and 37 degrees C. In 6 mol/L urea, NF-L was examined as 12 nm-diameter particle by low angle rotary shadowing. When dialyzed against reassembly buffer for 20 minutes, some irregular filaments were formed. Further dialyzed for another 40 minutes, the long smooth filaments appeared. Some filaments were unraveled at the end regions, where existed 2-4 subfilaments. Four subfilaments were more often observed. That is to say, the 10 nm-width filament was composed of 4 subfilaments. While dialyzed against the alkaline buffer containing 0.15 mol/L NaCl, NF-L reconstituted into 45-180 nm-long, 10 nm-width filaments, which were not able to elongate into long filaments.     

Intermediate (skeletin) filaments in heart Purkinje fibers. A correlative morphological and biochemical identification with evidence of a cytoskeletal function

Cow Purkinje fibers contain a population of free cytoplasmic filaments which consistently differ in ultrastructural appearance from actin and myosin filaments, irrespective of preparation technique. The fixation and staining techniques, however, influenced the filament diameter, which was found to be 7.4--9.5 nm for filaments in plastic-embedded material, and 7.0 nm in cryo-sectioned material, thus intermediate as compared to actin and myosin filaments. Cross-sectional profiles suggested that the intermediate-sized filaments are composed of four subfilaments. To provide a basis for further biochemical investigations on the filaments, extraction procedures were carried out to remove other cell organelles. Electron microscopy showed that undulating bundles of intermediate filaments converging towards desmosomes still remained, after the extractions, together with Z-disk material. In spite of the extensive extraction, the shape of the individual cells and the assemblies of cell bundles remained intact. This confirms that the intermediate filaments of cow Purkinje fibers together with desmosomes do in fact have a cytoskeletal function. On account of (a) the cytoskeletal function of the filaments, (b) the similarities to the smooth muscle "100-A filament" protein subunit skeletin, and (c) the inadequate and confusing existing terminology, we suggest that the filaments be named "skeletin filaments."     

MgATP specifically controls in vitro self-assembly of vertebrate skeletal myosin in the physiological pH range

The appearances in the electron microscope of rat and rabbit skeletal muscle myosin filaments and rod aggregates, formed in the presence of variable amounts of MgATP, were compared at different pH values. It is shown that small amounts of MgATP, similar to those sufficient to trigger the dissociation of the actomyosin complex, were able to modify the geometry of myosin filaments profoundly in the physiological pH range, whereas the conformation of rod aggregates remained unchanged even in the presence of high concentrations of MgATP. Myosin filaments formed in the absence of MgATP displayed the classical spindle-shaped conformation and variable diameters at all pH values, whereas myosin filaments formed in the presence of MgATP in the physiological pH range had constant diameters, similar to those of natural thick filaments. These filaments of constant diameter frayed, rapidly and reversibly, into two types of subfilaments with respective diameters of 4 to 5 nm and 9 to 10 nm, when the pH of the medium was raised above 7.2. Spindle-shaped myosin filaments and rod aggregates remained unchanged by such small changes in pH. It was possible to change the conformation of preformed spindle-shaped filaments by simply adding MgATP to the medium, but this reaction was slow and took several hours to be completed. Relatively high concentrations of MgATP, similar to those in the living cell, increased the solubility of both myosin filaments and rod aggregates in the alkaline pH range (pH greater than or equal to 7.0). Low pH values (less than or equal to 6.5) and excess free Mg2+ (greater than or equal to 6 to 7 mM) abolished both the specific effect of MgATP on myosin filament conformation and its solubilizing effect on both myosin filaments and rod aggregates. The degree of purity of the myosin preparations and the level of phosphorylation of the LC-2 light chains did not influence filament behaviour noticeably and rat and rabbit myosins behaved similarly.     

Rigor crossbridges are double-headed in fast muscle from crayfish

The structure of rigor crossbridges was examined by comparing rigor crossbridges in fast muscle fibers from glycerol-extracted abdominal flexor muscle of crayfish with those in "natively decorated" thin filaments from the same muscle. Natively decorated thin filaments were obtained by dissociating the backbone of the myosin filaments of rigor myofibrils in 0.6 M KCl. Intact fibers were freeze-fractured, deep-etched, and rotary shadowed; isolated filaments were either negatively stained or freeze dried and rotary shadowed. The crossbridges on the natively decorated actin maintain the original spacing and the disposition in chevrons and double chevrons for several hours, indicating that no rearrangement of the actomyosin interactions occurs. Thus the crossbridges of the natively decorated filaments were formed within the geometrical constraints of the intact myofibril. The majority of crossbridges in the intact muscle have a triangular shape indicative of double-headed crossbridge. The triangular shape is maintained in the isolated filaments and negative staining resolves two heads in a single crossbridge. In the isolated filaments, crossbridges are attached at uniform acute angles. Unlike those in insect flight muscle (Taylor et al., 1984), lead and rear elements of the double chevron may be both double-headed. Deep-etched images reveal a twisted arrangement of subfilaments in the backbone of the thick filament.     

Oblique section 3-D reconstruction of relaxed insect flight muscle reveals the cross-bridge lattice in helical registration.

In this work we examined the arrangement of cross-bridges on the surface of myosin filaments in the A-band of Lethocerus flight muscle. Muscle fibers were fixed using the tannic-acid-uranyl-acetate, ("TAURAC") procedure. This new procedure provides remarkably good preservation of native features in relaxed insect flight muscle. We computed 3-D reconstructions from single images of oblique transverse sections. The reconstructions reveal a square profile of the averaged myosin filaments in cross section view, resulting from the symmetrical arrangement of four pairs of myosin heads in each 14.5-nm repeat along the filament. The square profiles form a very regular right-handed helical arrangement along the surface of the myosin filament. Furthermore, TAURAC fixation traps a near complete 38.7 nm labeling of the thin filaments in relaxed muscle marking the left-handed helix of actin targets surrounding the thick filaments. These features observed in an averaged reconstruction encompassing nearly an entire myofibril indicate that the myosin heads, even in relaxed muscle, are in excellent helical register in the A-band.     

The myosin filament: V. Intermediate voltage electron microscopy and optical diffraction studies of the substructure

Using a 200 kV electron microscope (JEM 200 A), thick (up to 0.4 μm) crosssections of the myosin filaments of vertebrate striated muscle were studied. It was found that: (a) with increasing section thickness the cross-sectional profiles of the shaft of the filament were increasingly more triangular and in sections 0.4 μm thick each apex of the triangle was clearly blunted. This unique cross-sectional profile is predicted by the model proposed by Pepe (1966,1967) in which 12 parallel structural units are packed to form a triangular profile with a structural unit missing at each apex of the triangle. (b) With increasing section thickness the substructure of the myosin filament was enhanced, with the best substructure visible in sections 0.2 μm to 0.3 μm thick. This strongly supports parallel alignment of structural units in the shaft of the filament as proposed by Pepe (1966,1967). (c) The substructure spacing, determined by optical diffraction from electron micrographs of cross-sections of individual myosin filaments or groups of filaments is about 4 nm. (d) The different optical diffraction patterns observed from individual myosin filaments can be explained if the projection of each structural unit in the plane of the section has an elongated profile. With a substructure spacing of 4 nm an elongated cross-sectional profile could be produced by having two myosin molecules per structural unit. Models drawn with two myosin molecules per structural unit in the model proposed by Pepe (1966,1967) gave optical diffraction patterns similar to those observed from individual filaments. (e) The different optical diffraction patterns observed from individual myosin filaments can be explained if the elongated profiles in each structural unit are similarly oriented but with the orientation changing along the length of the filament. The change in orientation per unit length of the filament must be small enough to maintain an elongated profile for the projection of the structural unit in the plane of the sections 0.3 μm thick. All of these observations and conclusions strongly support the model for the myosin filament proposed by Pepe (1966,1967).     

Three-Dimensional Structure of the M-region (Bare Zone) of Vertebrate Striated Muscle Myosin Filaments by Single-Particle Analysis

The rods of anti-parallel myosin molecules overlap at the centre of bipolar myosin filaments to produce an M-region (bare zone) that is free of myosin heads. Beyond the M-region edges, myosin molecules aggregate in a parallel fashion to yield the bridge regions of the myosin filaments. Adjacent myosin filaments in striated muscle A-bands are cross-linked by the M-band. Vertebrate striated muscle myosin filaments have a 3-fold rotational symmetry around their long axes. In addition, at the centre of the M-region, there are three 2-fold axes perpendicular to the filament long axis, giving the whole filament dihedral 32-point group symmetry. Here we describe the three-dimensional structure obtained by a single-particle analysis of the M-region of myosin filaments from goldfish skeletal muscle under relaxing conditions and as viewed in negative stain. This is the first single-particle reconstruction of isolated M-regions. The resulting three-dimensional reconstruction reveals details to about 55 Å resolution of the density distribution in the five main nonmyosin densities in the M-band (M6′, M4′, M1, M4 and M6) and in the myosin head crowns (P1, P2 and P3) at the M-region edges. The outermost crowns in the reconstruction were identified specifically by their close similarity to the corresponding crown levels in our previously published bridge region reconstructions. The packing of myosin molecules into the M-region structure is discussed, and some unidentified densities are highlighted.     

THE ULTRASTRUCTURE OF FLAGELLAR FIBRILS

The tips of rat sperm tails were slightly frayed by mechanical agitation, thus exposing the fibrils, which were then studied by electron microscopy after negative staining. Only the fibrils survived this treatment. Each fibril proved to be a cylinder with a hollow core. The walls of the cylinders were made up of 10 longitudinally oriented filaments. The filaments had a markedly beaded appearance, with a repeating period of 88 A. The filament thickness (bead width) was approximately 35 to 40 A. Beads of neighboring filaments were in register with each other so that cross-linking bound the filaments together to complete the wall structure of each fibril. The center-to-center spacing from one filament to the next was 55 to 60 A. The periodicity and the diameters of the filaments make it unlikely that the filaments are related to either actin or myosin. From the way the fibrils kinked, it can be inferred that they possessed considerable mechanical strength. It is consistent with present knowledge that fibrils of the mitotic apparatus may have the same basic structure as the flagellar fibrils. Under some circumstances, pairs of fibrils separated from one another along their length, except at their extreme tips. It was apparent that there was special bridging material to be found there. In other preparations, however, the paired fibrils remained together, indicating a powerful coupling mechanism.