Interfilament Spacing Research Articles

Do cross-bridges contribute to the tension during stretch of passive muscle?

The tension rise during stretch of passive skeletal muscle is biphasic, with an initial steep rise, followed by a subsequent more gradual change. The initial rise has been interpreted as being due to the presence of numbers of long-term, stable cross-bridges in resting muscle fibres. A point of weakness with the cross-bridge interpretation is that the initial stiffness reaches its peak value at muscle lengths beyond the optimum for myofilament overlap. To explain this result it has been suggested that despite the reduced overlap at longer lengths, the closer interfilament spacing and a higher sensitivity of the myofilaments to Ca2+ allows more stable cross-bridges to form. Recently the stretch responses of passive muscle have been re-examined and it has been suggested that it is not necessary to invoke cross-bridge mechanisms at all. Explanations based on a viscous resistance to interfilament sliding and mechanical properties of the elastic filaments, the gap filaments, were thought to adequately account for the observed tension changes. However, an important property of passive muscle, the dependence of stretch responses on the immediate history of contraction and length changes, thixotropy, cannot be explained simply in terms of viscous and viscoelastic properties. The review discusses the cross-bridge interpretation of muscle thixotropy and the relationship of passive stiffness to filament resting tension and latency relaxation. It is proposed that cross-bridges can exist in three states; one, responsible for the resting stiffness, requires resting levels of calcium. When, during activation, calcium levels rise, cross-bridges enter a low-force, high-stiffness state, signalled by latency relaxation, before they move to the third, force-generating state. It is concluded that, compared with viscoelastic models, a cross-bridge-based explanation of passive muscle properties is better able to accommodate the currently known facts although, as new information becomes available, this view may need to be revised.

Neurofilament phosphorylation and axon diameter in the squid giant fibre system

Electron energy loss spectroscopic analysis of squid giant axons in a phosphorus energy window yielded bright signals, which were shown to originate from highly phosphorylated neurofilaments. The frequency and distribution of these signals were analysed at defined intervals in cross-sections of the giant axon, starting from its origin in the stellate ganglion and extending distally along the stellar nerve. The analysis revealed a proximodistal gradient of increasing neurofilament phosphorylation. Within the stellate ganglion and for some distance beyond, the increase in frequency of signals correlated with the widening of the neurofilament meshwork and the radial growth of the axon. This agrees with the hypothesis that neurofilament phosphorylation regulates axon calibre by affecting interfilament spacing. In distal axon domains where the axon diameter diminished, contrary to expectations, the spacing of signals increased and the signals were significantly larger. Hyperphosphorylation apparently compensated for a diminishing supply of neurofilament protein. Contrary to predictions, the presynaptic terminal of the giant synapse contained a distinct and highly phosphorylated neurofilament meshwork. We conclude that the growth of the axon diameter is a function of neurofilament phosphorylation, interfilament spacing and neurofilament density. A mature and highly phosphorylated neurofilament cytoskeleton completely filled the presynaptic terminal of the giant synapse.

Changes in Interfilament Spacing Mimic the Effects of Myosin Regulatory Light Chain Phosphorylation in Rabbit Psoas Fibers

The modulatory effect of myosin regulatory light chain phosphorylation in mammalian skeletal muscle, first documented as posttetanic potentiation of twitch tension, was subsequently shown to enhance the expression and development of tension at submaximal levels of activating calcium. Structural analyses demonstrated that thick filaments with phosphorylated myosin regulatory light chains appeared disordered: they lost the near-helical, periodic arrangement of myosin head characteristic of the relaxed state. We suggested that disordered heads may be more mobile than ordered heads and are likely to spend more time close to their binding sites on thin filaments. In this study we determined that the physiological effects of phosphorylation could be mimicked by decreasing the lattice spacing between the thick and the thin filaments, either by osmotic compression with dextran or by increasing the sarcomere length of permeabilized rabbit psoas fibers. Phosphorylation of regulatory light chains by incubation of permeabilized fibers with myosin light chain kinase and calmodulin, followed by low levels of activating calcium, potentiated tension development at resting or lower sarcomere lengths in the absence of dextran but had no additional effect on tension potentiation or development in fibers with decreased lattice spacing due to either osmotic compression or increased sarcomere length.

Entropic exclusion by neurofilament sidearms: a mechanism for maintaining interfilament spacing.

A long-range repulsive force near isolated neurofilaments was detected by exclusion of large molecules and by direct force measurements with atomic force microscopy. Adsorption of isolated native neurofilaments to a solid substrate in a high-salt solution (170 mM NaCl), in the presence of coisolating contaminants, shows that the contaminants are excluded from a zone that extends from 50-100 nm from the core of the filament. Force-distance measurements by AFM show the presence of a weak repulsive force that extends >50 nm from the core of the filament; this repulsive force is absent in homopolymers of neurofilament L or trypsinized native filaments that lack the long sidearms present in native filaments. These results suggest that neurofilament sidearms form an entropic brush, thereby providing a mechanism for maintaining interfilament spacing.

Bacterial templating of ordered macrostructures in silica and silica-surfactant mesophases

The synthesis of inorganic frameworks with specified and organized pore networks is of potential importance in catalysis1,2, separation technology3 and biomaterials engineering4,5. Ordered arrangements of porous channels have been produced in silica-based materials by post-synthetic removal of surfactant templates from inorganic–organic mesostructures6,7. The resulting pore sizes are commensurate with the packing dimensions of the organic molecules, and are currently limited to length scales of up to 10nm. Here we show how a bacterial superstructure, consisting of a thread of coaligned multicellular filaments of Bacillus subtilis8,9, can be used to extend the length scale of inorganic materials patterning. We produce ordered macroporous fibres of either amorphous silica or ordered mesoporous silica6,7 (MCM-41) by template-directed mineralization of the interfilament spaces followed by removal of organic material by heating to 600°C. The inorganic macrostructures consist of a macroporous framework of 0.5-μm-wide channels with curved walls of either silica or mesoporous silica, 50 to 200 nm in thickness. The formation of ordered pores in the MCM-41 replica on both the mesoscopic and macroscopic length scales illustrates how supramolecular and supercellular templates might be combined for the fabrication of inorganic materials with structural hierarchy.

Structure and periodicities of cross-bridges in relaxation, in rigor, and during contractions initiated by photolysis of caged Ca2+

Ultra-rapid freezing and electron microscopy were used to directly observe structural details of frog muscle fibers in rigor, in relaxation, and during force development initiated by laser photolysis of DM-nitrophen (a caged Ca2+). Longitudinal sections from relaxed fibers show helical tracks of the myosin heads on the surface of the thick filaments. Fibers frozen at approximately 13, approximately 34, and approximately 220 ms after activation from the relaxed state by photorelease of Ca2+ all show surprisingly similar cross-bridge dispositions. In sections along the 1,1 lattice plane of activated fibers, individual cross-bridge densities have a wide range of shapes and angles, perpendicular to the fiber axis or pointing toward or away from the Z line. This highly variable distribution is established very early during development of contraction. Cross-bridge density across the interfilament space is more uniform than in rigor, wherein the cross-bridges are more dense near the thin filaments. Optical diffraction (OD) patterns and computed power density spectra of the electron micrographs were used to analyze periodicities of structures within the overlap regions of the sarcomeres. Most aspects of these patterns are consistent with time resolved x-ray diffraction data from the corresponding states of intact muscle, but some features are different, presumably reflecting different origins of contrast between the two methods and possible alterations in the structure of the electron microscopy samples during processing. In relaxed fibers, OD patterns show strong meridional spots and layer lines up to the sixth order of the 43-nm myosin repeat, indicating preservation and resolution of periodic structures smaller than 10 nm. In rigor, layer lines at 18, 24, and 36 nm indicate cross-bridge attachment along the thin filament helix. After activation by photorelease of Ca2+, the 14.3-nm meridional spot is present, but the second-order meridional spot (22 nm) disappears. The myosin 43-nm layer line becomes less intense, and higher orders of 43-nm layer lines disappear. A 36-nm layer line is apparent by 13 ms and becomes progressively stronger while moving laterally away from the meridian of the pattern at later times, indicating cross-bridges labeling the actin helix at decreasing radius.

Particle Capture by the Gills of Dreissena polymorpha: Structure and Function of Latero-frontal Cirri.

Microscopic techniques were used to examine the role of gill cirri in particle capture by Dreissena polymorpha. The latero-frontal cirri, formed from two fused ciliary plates, consist of about 40 pairs of cilia. Each cilium in the plate contains a typical 9 + 2 axoneme in the fused region of the cirrus, but the structure of the axoneme in the long, free ciliary tips is reduced. The cilia in a cirrus are graded in length, with the shortest cilia positioned frontally. The cirral cilia move in unison, allowing the cirrus to move from a flexed position with its tip arched over the front of the gill filament, to an extended position with the cirrus projected in the plane of the latero-frontal cell and extending across the interfilament space. In the latter position, the free ciliary tips of opposing and neighboring cirri form a "trap" (net) with a spacing of about 0.5 μm. Observations with laser confocal microscopy indicated that these structures can physically trap particles <1 μm in diameter. Particles captured by the extended cirri are moved to the frontal surface of the gill, where the cirri are swept by the lateralmost frontal cilia. During cirral movement the shift from extended to flexed position is, in part, achieved by the base of the cirrus pivoting at a hinge region. Morphologically, the hinge region shows axonemal specializations that consist of electron-dense plates and other structures of undefined function that may be important in the overall movement of the cirrus. In addition to trapping by cirri, we also observed particles moving in the water currents, particularly in the frontal current located over the apical surface of the filament, suggesting that some particles are captured in these water currents without being physically trapped. Probably, therefore, trapping by the cirri and establishment of water currents by the filaments both participate in the interception of particles by Dreissena polymorpha.

Neurofilament spacing, phosphorylation, and axon diameter in regenerating and uninjured lamprey axons

It has been postulated that phosphorylation of the carboxy terminus sidearms of neurofilaments (NFs) increases axon diameter through repulsive electrostatic forces that increase sidearm extension and interfilament spacing. To evaluate this hypothesis, the relationships among NF phosphorylation, NF spacing, and axon diameter were examined in uninjured and spinal cord-transected larval sea lampreys (Petromyzon marinus). In untransected animals, axon diameters in the spinal cord varied from 0.5 to 50 μm. Antibodies specific for highly phosphorylated NFs labeled only large axons (>10 μm), whereas antibodies for lightly phosphorylated NFs labeled medium-sized and small axons more darkly than large axons. For most axons in untransected animals, diameter was inversely related to NF packing density, but the interfilament distances of the largest axons were only 1.5 times those of the smallest axons. In addition, the lightly phosphorylated NFs of the small axons in the dorsal columns were widely spaced, suggesting that phosphorylation of NFs does not rigidly determine their spacing and that NF spacing does not rigidly determine axon diameter. Regenerating neurites of giant reticulospinal axons (GRAs) have diameters only 5–10% of those of their parent axons. If axon caliber is controlled by NF phosphorylation via mutual electrostatic repulsion, then NFs in the slender regenerating neurites should be lightly phosphorylated and densely packed (similar to NFs in uninjured small caliber axons), whereas NFs in the parent GRAs should be highly phosphorylated and loosely packed. However, although linear density of NFs (the number of NFs per micrometer) in these slender regenerating neurites was twice that in their parent axons, they were highly phosphorylated. Following sectioning of these same axons close to the cell body, axon-like neurites regenerated ectopically from dendritic tips. These ectopically regenerating neurites had NF linear densities 2.5 times those of uncut GRAs but were also highly phosphorylated. Thus, in the lamprey, NF phosphorylation may not control axon diameter directly through electrorepulsive charges that increase NF sidearm extension and NF spacing. It is possible that phosphorylation of NFs normally influences axon diameter through indirect mechanisms, such as the slowing of NF transport and the formation of a stationary cytoskeletal lattice, as has been proposed by others. Such a mechanism could be overridden during regeneration, when a more compact, phosphorylated NF backbone might add mechanical stiffness that promotes the advance of the neurite tip within a restricted central nervous system environment. © 1996 Wiley-Liss, Inc.

Time dependence of ac losses in multifilamentary NbTi superconductors due to proximity coupling

The ac losses of NbTi multifilamentary superconducting wires with a copper matrix were measured using a sensitive calorimetric method. The wire samples, which have filament diameters in the micrometer range and interfilament spacings of a few tenths of a micrometer, exhibit a strong proximity effect. The measured power loss at a constant ac field amplitude and frequency showed an unexpected time dependence on a very long time scale of hours, compared to the ac field cycle time being in the millisecond range. The measured loss vs time curves were found to depend sensitively on amplitude and frequency of the ac field, and in the way the ac field was switched on. The effect can be rather large, changes of losses by up to 60% were found. Losses can increase or decrease with time depending on the structure of the sample. As an independent check losses were also measured by a magnetization method. The results were in very good agreement with the calorimetric measurement. The effect of time dependent losses is qualitatively explained by the slow decay of proximity currents which are induced when the ac field is switched on. They create a time dependent bias magnetization, which influences the ac losses.

Osmotic compression of skinned cardiac and skeletal muscle bundles: Effects on force generation, Ca 2+ sensitivity and Ca 2+ binding

Length-dependence of myofilament Ca2+ sensitivity is now considered to be an important component of the steep relationship between active force and sarcomere length along the ascending limb of the cardiac force-length curve. Studies with skinned cardiac muscle preparations have demonstrated that Ca(2+)-troponin C affinity is significantly increased as sarcomere length is increased over the range 1.7-2.3 microns. Increase in sarcomere length is accompanied by a reduction in interfilament spacing. In skinned fiber preparations from both cardiac and skeletal muscle osmotic compression of the filament lattice enhances myofilament Ca2+ sensitivity. This study was undertaken to evaluate the hypothesis that a change in filament separation may contribute to the length-dependent activation seen in cardiac muscle. Moderate reduction in interfilament spacing caused by exposure to Dextran T-500 (5-10%) produced an increase in force generation in both maximally activated and partially activated preparations of skinned bovine ventricular muscle. With fiber bundles of mean sarcomere length 1.7 microns the addition of 5% Dextran T-500 produced an increase in Ca2+ sensitivity of about 0.25 pCa units and a significant increase in Ca2+ binding in the pCa range (6.0-5.0) in which the single regulatory site of cardiac troponin C is titrated. This concentration of Dextran T-500 produced a reduction in fiber width equivalent to that produced by stretching fibers from sarcomere length 1.7 microns to sarcomere length 2.3 microns Osmotic compression of skinned rabbit psoas muscle fibers also enhanced Ca2+ sensitivity but there was no significant change in Ca(2+)-troponin C affinity. These data suggest that 1) an important component of length-dependent Ca2+ sensitivity in both cardiac and skeletal muscle is the change in interfilament spacing, and 2) in cardiac muscle a reduction in spacing, like increase in length, leads to a specific increase in Ca(2+)-troponin C affinity. Thus both filament overlap and filament separation contribute to the length dependence of Ca2+ sensitivity and Ca2+ binding in cardiac muscle.

The single lamprey neurofilament subunit (NF-180) lacks multiphosphorylation repeats and is expressed selectively in projection neurons.

The lamprey is considered the most primitive living vertebrate and its neurofilaments (NFs) are unique in being homopolymers of a single 180 kDa subunit (NF-180). Previous immunologic studies have suggested that the sidearm of NF-180 is highly phosphorylated selectively in the largest diameter axons. We report in this study the isolation and characterization of cDNA clones encoding the NF-180 lamprey protein. In situ hybridization with digoxigenin-labeled cRNA revealed NF-180 message exclusively in neurons with long axons, such as reticulospinal neurons and cranial motor neurons. The core of NF-180 was similar in structure to those of mammalian neurofilaments, but surprisingly, the carboxy sidearm lacked the multiphosphorylation repeats characteristic of higher vertebrate and invertebrate neurofilaments. Overall there was a paucity of potential phosphorylation sites in the NF-180 carboxy-terminus compared to NF-M and NF-H of mammals, fish and squid. This, along with the highly acidic nature of the NF-180 sidearm, makes it unlikely that phosphorylation of sidearm residues regulates interfilament spacing and axon diameter through global electrostatic repulsion of the carboxy-terminus away from the filament backbone. Furthermore, the expression of a single neurofilament subunit in the lamprey that is most similar to the NF-M of higher vertebrates suggests that all three mammalian neurofilament subunits evolved from a single NF-M-like precursor.

Regional modulation of neurofilament organization by myelination in normal axons

Previous studies in the hypomyelinating mouse mutant Trembler have suggested that demyelinating axons are smaller in caliber compared to normal axons, and that there are differences in the organization of axonal neurofilaments. In the normal PNS, however, the relationship between neurofilament organization and myelination has not been investigated extensively. In normal axons, only the initial segments, the nodes of Ranvier (approximately 1 micron), and the terminals are not covered by myelin. We took advantage of an unusual feature of the primary sensory neurons in the dorsal root ganglion, the relatively long nonmyelinated stem process (up to several hundred micrometers), to determine if the presence of myelination correlates with differences in cytoskeletal organization and neurofilament phosphorylation. Axonal caliber and neurofilament numbers were substantially greater in the myelinated internodes than in the stem process or nodes of Ranvier. Neurofilament spacing, assessed by measuring the nearest-neighbor neurofilament distance, was 25-50% less in the stem processes and nodes of Ranvier than in the myelinated internodes. In the myelinated internodes, neurofilaments had greater immunoreactivity for phosphorylated epitopes than those in the stem process. These findings indicate that interactions with Schwann cells modulate neurofilament phosphorylation within the ensheathed axonal segments, and that increased phosphorylation within myelinated internodes leads to greater interfilament spacing. Lastly, the myelinated internodes had three fold more neurofilaments, but the same number of microtubules. Both the increased neurofilament spacing and the increase in neurofilament numbers in myelinated internodes contribute to a greater axonal caliber in the myelinated internodes.

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

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

Ultrastructure of sarcoballs on the surface of skinned amphibian skeletal muscle fibres.

The formation of sarcoballs on the surface of skinned fibres from semitendinosus muscles of Xenopus laevis, and the sarcoplasmic reticulum content of the structures, have been studied using conventional electron microscopic techniques and immunoelectron microscopy. Examination of the fibres showed many membrane-bound blebs projecting from the surface in areas where vesicles of internal membranes (including sarcoplasmic reticulum, T-tubules and mitochondria) were clustered in interfilament spaces. The blebs varied in size from 1 micron to 150 microns and those with diameters > 10 microns are referred to as sarcoballs. Small blebs were often seen in close association with each other and might have fused during sarcoball formation. The interior of the sarcoball was filled with foam-like material made up of vesicles with diameters of 100 nm to 1.0 microns. The sarcoplasmic reticulum membrane content of the sarcoballs was evaluated using two monoclonal antibodies, one to the Ca2+ ATPase of the sarcoplasmic reticulum and the second to ryanodine receptor calcium release channels in the junctional-face membrane. The antibodies bound to some components of the surface and interior of the sarcoball, but not to mitochondrial-like structures and tubular vesicles. The results show that a large component of the sarcoball and its surface is derived from sarcoplasmic reticulum and suggest that mitochondria and T-tubules might also contribute membranes to the structures. Our hypothesis is that (a) blebs bud out from the surface of the skinned fibre following fusion of internal vesicles that are extruded along interfilament channels during unrestrained contractures, (b) blebs grow into sarcoballs by additional fusion of internal membrane vesicles and fusion of adjacent blebs, and (c) the sarcoball is a foam-like structure composed of bathing medium and membrane lipid (containing membrane proteins).

Creep characteristics of wire-drawn Cu-20%Nb

In this study the creep deformation and rupture characteristics of a wire-drawn Cu-20%Nb composite are investigated at 500°C and compared with the results obtained from pure copper. The results show that the creep rupture strength of Cu-20%Nb is considerably higher than that for pure copper. The rupture strength of Cu-20%Nb increases up to a certain draw ratio, in contrast to the room temperature strengthening. The stress exponents n are in the range 6.6–8.1 for Cu-20%Nb, in comparison with 2.75 in pure copper. The increase in the creep strength and the higher stress exponent values in the Cu-20%Nb are associated with the reduction in the power law creep damage mechanism. This is due to the constraint introduced on the matrix creep flow by the niobium phase rather than the development of high threshold stress values. While the increase in the length of niobium filaments and reduction in interfilament spacing with increasing draw ratio increase the constraint on the creep flow of the matrix, they also enhance the creep damage caused by the diffusion mechanisms because of the easy diffusion paths along the niobium filaments and the reduction in the matrix gain size.

Z-line/I-band and A-band lattices of intact frog sartorius muscle at altered interfilament spacing.

Muscle contraction has long been known to be affected by the osmolarity of the bathing solution. Part of this effect is caused by changes in interfilament spacing in the A-band. We have investigated the variation in spacing of the square lattice of thin filaments within and near the Z-line (the Z-line/I-band or Z-I lattice) in intact frog sartorius muscle over a wide range of osmolarities and compared it with the corresponding changes in the A-band lattice. Both lattices have a lower limit for compression and an upper limit for swelling. The spacing of the Z-I lattice is nearly proportional to that of the A-band, but shows a 2-3% variation at extreme shrinkage or swelling. In normal intact muscle, the osmotically-inactive volume of both lattices is between 20 and 30%. These in vivo measurements of lattice spacing differ significantly from those observed in electron micrographs. With moderate variations in osmolarity, lattice spacing and muscle fibre width show similar behaviour, but at extreme osmolarities, the lattice spacing changes less than the fibre width. An equatorial reflection was observed in intact muscle, previously identified in skinned muscle, which does not index on the A-band and which changes with osmolarity in a manner different from that observed for the A-band and Z-I lattices. This reflection may arise from changes in the ordering of the Z-I lattice or may involve components additional to the thick and thin filaments.

Flow through the feeding structures of suspension feeding zooplankton: a physical model approach

Simple multi-filament models of suspension feeding zooplankton feeding structures were constructed and the flow through and around the models was described. The fraction of water passing the filter depends on filament diameter, interfilament spacing and the number of filaments. Within a realistic range of Reynolds' numbers (Re) only a fraction of the head on water stream passed through the filters. The significance of morphological characteristics was also investigated for a biological more relevant 90° sweep. Spreading the filaments apart caused a significant increase in leakage compared with parallel filaments and, by introducing side branches the models acted as slightly leaky sieves. The leakage can change from 20-30% to almost 100% when Re increased from 0.1 to 1. Leakage depends on the orientation of the side branches indicating the importance of asymmetric drag during water processing at low Re. The observations are discussed in the context of the morphology and movements of feeding structures of ciliary feeding benthic larvae and of setal feeding copepods. The usefulness of physical models as a supplement to direct observations on zooplankton and mathematical models is emphasized.

Effect of mechanical deformation on Nb-Ti filament proximity-effect coupling at the edges of SSC cables

Magnetization as a function of transverse magnetic field and time was measured for short strands extracted from the centers and edges of five Nb-Ti Rutherford cables designed for use in Superconducting Super Collider dipole magnets. The multifilamentary strands all had 6- mu m-diameter filaments. Edge samples, which had severe mechanical deformation, showed small magnetic coupling losses at low fields, compared to no coupling losses for underformed center strands. The existence of sharp strand bends at cable edges decreases the interfilament spacing to the order of the coherence length in the normal matrix material, which increases the effective filament diameter and hysteresis loss at low fields. Microscopic studies of the cables' cross sections confirmed smaller interfilament separations in these samples. Flux creep measurements, represented by the time dependence of magnetization, showed little difference between edge and center samples.

Supercoiling of f-Actin filaments

In the X-ray diffraction pattern from oriented gels of actin-containing filaments sampling of layer lines indicating the development of a well-ordered pseudohexagonal lattice within the gels at interfilament spacings as large as 13 nm is observed. This value exceeds by 3 nm the largest estimate of an external diameter of pure f-actin. The development of layer line sampling is always accompanied by: (i) the appearance of strong forbidden meridional reflections on the 5.9- and 5.1-nm layer lines; (ii) a drastic intensification of the first (expected) 2.75-nm meridional reflection by a factor of about 4; (iii) the appearance of streaks, connecting near-meridional reflections on the 5.9-, 5.1-, and 37-nm layer lines; and (iv) a slight decrease in the number of subunits per turn of the basic f-actin helix. All these features strongly indicate that f-actin filaments are supercoiled and make regular local contacts between themselves, which may lead to periodic distortions of the mobile external domain in the actin subunits.

The Detection of Giardia muris and Giardia lamblia Cysts by Immunofluorescence in Animal Tissues and Fecal Samples Subjected to Cycles of Freezing and Thawing

The effects of freezing and thawing on the detection of selected Giardia spp. cysts were investigated using immunofluorescence, bright field microscopy, and low voltage scanning electron microscopy (SEM). Giardia muris cysts were obtained from either animal carcasses, fecal pellets, or isolated cyst preparations, whereas Giardia lamblia cysts were isolated from fecal samples. These samples were stained using an immunofluorescence technique after 1-3 freezing (-16 C) and thawing (20 C) cycles. Cysts were detected successfully by immunofluorescence in all samples. However, in those samples subjected to freeze-thawing, the cyst walls often became distorted and then were not detectable by bright field microscopy. Low voltage SEM demonstrated that the filaments in the distorted cyst wall underwent rearrangements of interfilament spacing. Quantitation of cyst recovery after freezing and thawing demonstrated that a substantial loss occurred after 1 cycle of alternating temperature when low concentrations of cysts were used, but not with high concentrations of cysts. Cyst recovery, after 3 freezing and thawing cycles, was dramatically lowered irrespective of the initial cyst concentration. These results demonstrated that immunofluorescence was an effective technique for the detection of Giardia spp. cysts in frozen samples and would suggest that freezing and thawing of fecal samples could prevent the detection of cysts when only bright field microscopy was employed.