Interfilament Spacing Research Articles

The effects of temperature, season, and nitric oxide on clearance rates in isolated gills of the heterodont clams Mercenaria mercenaria and Arctica islandica

Clearance of colloidal graphite (ml/min g dry weight) was measured in isolated gills treated with 10 −5 M 5-hydroxytryptamine (5HT) in the shallow water eurythermal clam Mercenaria mercenaria and the deeper water stenothermal clam Arctica islandica acclimated to 3 temperatures during the summer and winter. In Mercenaria there were no statistical differences in clearance rates in gills acclimated to 5, 10, and 22 °C during the summer and winter; thus the gills showed perfect temperature compensation (Q 10 = 1). Inhibition of nitric oxide synthesis with L-NAME significantly reduced clearance rates at all three temperatures during the winter but had no effect during the summer. Examination of gills treated with L-NAME and 5HT revealed that the lateral cilia beat at a significantly lower rate than gills treated with 5HT. In Arctica there was no temperature compensation – except at 5 and 10 °C during the winter – as evidenced by Q 10s of 4.0 (winter) and 4.5 (summer) for the temperature range of 0 to 10 °C. In addition, L-NAME had no effect upon clearance during either season. There were no significant differences in the activity of the lateral and frontal cilia, but the interfilament space and the diameters of the water tubes were greater in gills acclimated to 5 °C in comparison to 10 °C during the summer. Arctica gills clear at significantly higher rates than do those of Mercenaria. Examination of gills of both species at 10 °C revealed that the activities of the lateral and frontal cilia are significantly greater in Mercenaria in comparison to Arctica. In contrast the lateral cilia were longer and the interfilament space was larger in Arctica in comparison to Mercenaria.

Structural and functional aspects of the myosin essential light chain in cardiac muscle contraction

The myosin essential light chain (ELC) is a structural component of the actomyosin cross-bridge, but its function is poorly understood, especially the role of the cardiac specific N-terminal extension in modulating actomyosin interaction. Here, we generated transgenic (Tg) mice expressing the A57G (alanine to glycine) mutation in the cardiac ELC known to cause familial hypertrophic cardiomyopathy (FHC). The function of the ELC N-terminal extension was investigated with the Tg-Δ43 mouse model, whose myocardium expresses a truncated ELC. Low-angle X-ray diffraction studies on papillary muscle fibers in rigor revealed a decreased interfilament spacing (≈ 1.5 nm) and no alterations in cross-bridge mass distribution in Tg-A57G mice compared to Tg-WT, expressing the full-length nonmutated ELC. The truncation mutation showed a 1.3-fold increase in I(1,1)/I(1,0), indicating a shift of cross-bridge mass from the thick filament backbone toward the thin filaments. Mechanical studies demonstrated increased stiffness in Tg-A57G muscle fibers compared to Tg-WT or Tg-Δ43. The equilibrium constant for the cross-bridge force generation step was smallest in Tg-Δ43. These results support an important role for the N-terminal ELC extension in prepositioning the cross-bridge for optimal force production. Subtle changes in the ELC sequence were sufficient to alter cross-bridge properties and lead to pathological phenotypes.

Interactions between Planar Grafted Neurofilament Side-Arms

The side-arms of neurofilaments (NFs) have been proposed to be highly disordered, leading to an entropically and electrostatically based repulsion that modulates interfilament spacing. To characterize the behavior of two interacting polymer brushes in a system of this type, we performed molecular dynamics simulations of neurofilament brushes using a four bead reduced amino acid set coarse-grained model. In these simulations, we examined components of the neurofilament brush, NF-L, NF-M, and phosphorylated NF-H (NF-HP), individually. Each protein type was grafted to planar surfaces and simulations were performed for a range of separations of two apposed grafted surfaces. The calculated force-separation curves show the force increases as the reciprocal separation as predicted for polyelectrolyte brushes at high salt. All three systems can be overlapped on a single force-separation curve, which is not expected given the variation in amino acid sequence and charges on the polymers. Examination of structural properties shows scaling behavior in the average brush height, end-to-end distance, and the density interpenetration. Some of this scaling can be understood in terms of treating the NF proteins as effective polyelectrolytes, but some cannot suggesting a distinct polyampholyte behavior. Correlations are found between oppositely charged residues in opposite brushes. However, these correlations are weak in comparison to the strong correlations within each brush. In comparison with recent experimental data that observes condensed and expanded gel states, our results suggest that the condensed state structure involves significant interdigitation of the side-arms.

Thick-Filament Strain and Interfilament Spacing in Passive Muscle: Effect of Titin-Based Passive Tension

We studied the effect of titin-based passive tension on sarcomere structure by simultaneously measuring passive tension and low-angle x-ray diffraction patterns on passive fiber bundles from rabbit skinned psoas muscle. We used a stretch-hold-release protocol with measurement of x-ray diffraction patterns at various passive tension levels during the hold phase before and after passive stress relaxation. Measurements were performed in relaxing solution without and with dextran T-500 to compress the lattice toward physiological levels. The myofilament lattice spacing was measured in the A-band ( d 1,0) and Z-disk ( d Z) regions of the sarcomere. The axial spacing of the thick-filament backbone was determined from the sixth myosin meridional reflection (M6) and the equilibrium positions of myosin heads from the fourth myosin layer line peak position and the I 1,1/I 1,0 intensity ratio. Total passive tension was measured during the x-ray experiments, and a differential extraction technique was used to determine the relations between collagen- and titin-based passive tension and sarcomere length. Within the employed range of sarcomere lengths (∼2.2–3.4 μm), titin accounted for >80% of passive tension. X-ray results indicate that titin compresses both the A-band and Z-disk lattice spacing with viscoelastic behavior when fibers are swollen after skinning, and elastic behavior when the lattice is reduced with dextran. Titin also increases the axial thick-filament spacing, M6, in an elastic manner in both the presence and absence of dextran. No changes were detected in either I 1,1/I 1,0 or the position of peaks on the fourth myosin layer line during passive stress relaxation. Passive tension and M6 measurements were converted to thick-filament compliance, yielding a value of ∼85 m/N, which is several-fold larger than the thick-filament compliance determined by others during the tetanic tension plateau of activated intact muscle. This difference can be explained by the fact that thick filaments are more compliant at low tension (passive muscle) than at high tension (tetanic tension). The implications of our findings are discussed.

Tuning the Neurofilament Hydrogel Network - a Synchrotron X-Ray Scattering Study of Salt Dependent Response

Neurofilaments (NFs) are the cytoskeletal intermediate filament protein class expressed in neuronal cells and play a major a role in the maintenance and mechanical integrity of neuronal processes (i.e. the axon and dendrites). NFs assemble into flexible bottlebrushes from 3 different molecular weight subunits (NF-Low (NF-L), NF-Medium (NF-M), NF-High (NF-H)) with compositions that are specific for the axon and dendrites. The main variation in the subunits is the charge distribution of their unstructured C-termini sidearms. The sidearms are polyampholytes (i.e. containing both cationic and anionic amino acid residues), which enables interpenetration and interfilament attractions. We examine the strength and range of the electrostatic interfilament interactions by varying the salinity of the in vitro buffer. Reassembled (in vitro) binary system NF-hydrogels have revealed the different contributions of individual subunits to interfilament interactions and to network interfilament spacings [1-2]. This network tunability parallels with variable in vivo subunit expression in axons versus dendrites that results in variable network packing. We describe synchrotron x-ray scattering experiments that have allowed us to quantitatively study the changes in the microscopic structure of the NF gels as a function of salt and sidearm density. At high weight ratios of NF-M and NF-H, and as a function of increasing salt concentrations, NF gels exhibit an unexpectedly abrupt (most likely electrostatically driven) transition from a weakly oriented (nearly isotropic) low filament density gel with interfilament spacing d ∼ 1000A to a highly oriented liquid crystalline gel with high filament density and d ∼ 500A (NF-M) and ∼ 700A (NF-H).Funded by DOE DE-FG-02-06ER46314, NSF DMR-0803103, NIH R01-059288, HFSP[1] R. Beck, J. Deek, J.B. Jones, C.R. Safinya. Nature Materials. 9, 40 (2010)[2] J.B. Jones, C.R. Safinya. Biophysical Journal. 95, 823 (2008)

Conformational Dynamics of Neurofilament Side-Arms

The side-arms of neurofilaments (NFs) have been proposed to be highly disordered, leading to entropic repulsion that modulates interfilament spacing. To gain further insight into the dynamics and organization of the side-arms, we performed molecular dynamics simulations of neurofilament brushes using a coarse-grained model. The density profiles for three NF proteins, NF-L, NF-M, and phosphorylated NF-H (NF-HP), grafted to planar surfaces were calculated and examined as a function of component (salt, residues) and as a function of charge. Analysis of these profiles reveals that the NF with the shortest side arm, NF-L, is disproportionately long compared to the other NFs. The reason for difference is that NF-L is effectively a strong polyelectrolyte, while NF-M and NF-HP are effectively weaker polyelectrolytes. Further, we find cross-correlations between neurofilament side-arms within the brush, even for the NF-L polymers. These correlations occur because of strong attractions between the long sequence repeats of negative residues and the long postive residue repeats and impart a time average structure of the neurofilament brush that deviates from an ideal polymer in a theta solvent.

Differential roles of regulatory light chain and myosin binding protein-C phosphorylations in the modulation of cardiac force development

Phosphorylation of myosin regulatory light chain (RLC) by myosin light chain kinase (MLCK) and myosin binding protein-C (cMyBP-C) by protein kinase A (PKA) independently accelerate the kinetics of force development in ventricular myocardium. However, while MLCK treatment has been shown to increase the Ca(2+) sensitivity of force (pCa(50)), PKA treatment has been shown to decrease pCa(50), presumably due to cardiac troponin I phosphorylation. Further, MLCK treatment increases Ca(2+)-independent force and maximum Ca(2+)-activated force, whereas PKA treatment has no effect on either force. To investigate the structural basis underlying the kinase-specific differential effects on steady-state force, we used synchrotron low-angle X-ray diffraction to compare equatorial intensity ratios (I(1,1)/I(1,0)) to assess the proximity of myosin cross-bridge mass relative to actin and to compare lattice spacings (d(1,0)) to assess the inter-thick filament spacing in skinned myocardium following treatment with either MLCK or PKA. As we showed previously, PKA phosphorylation of cMyBP-C increases I(1,1)/I(1,0) and, as hypothesized, treatment with MLCK also increased I(1,1)/I(1,0), which can explain the accelerated rates of force development during activation. Importantly, interfilament spacing was reduced by 2 nm (3.5%) with MLCK treatment, but did not change with PKA treatment. Thus, RLC or cMyBP-C phosphorylation increases the proximity of cross-bridges to actin, but only RLC phosphorylation affects lattice spacing, which suggests that RLC and cMyBP-C modulate the kinetics of force development by similar structural mechanisms; however, the effect of RLC phosphorylation to increase the Ca(2+) sensitivity of force is mediated by a distinct mechanism, most probably involving changes in interfilament spacing.

Tuning of Neurofilament Hydrogel Network Features - a Synchrotron X-Ray Scattering Study of Salt Dependent Network Response

Neurofilaments (NFs) are cytoskeletal proteins expressed in neuronal cells, with a role in the maintenance and mechanical integrity of neuronal processes. NFs assemble as flexible cylinders from 3 protein subunits: NF-Low (NF-L), NF-Medium (NF-M), and NF-High (NF-H). The variable length and charge of the subunits sets the strength and range of the interactions, which are predominantly electrostatic. Reassembled (in vitro) binary system hydrogels have shown us the different contributions of individual subunits to interfilament interactions and thus to network characteristics [1,2]. We emulate cellular conditions by varying the salinity of the in vitro buffer: low salt conditions parallel higher inherent charge of the subunits, and high salt conditions parallel the lower inherent charge states of the subunits. The tunability of the network in vitro mirrors in vivo cellular control of the NF network via subunit phosphorylation, which may transition the network from a highly oriented rigid state to an isotropic gel with orientational plasticity. We describe synchrotron x-ray scattering experiments that have allowed us to quantitatively study the changes in the microscopic structure of the NF gels as a function of salt and sidearm density. At low NF-M and NF-H sidearm weight ratios, NF gels exhibit weak salt dependence. In contrast, at high weight ratios, and as a function of decreasing salt concentrations, NF gels exhibit an unexpectedly abrupt transition from highly oriented liquid crystalline gels with high filament density (α 1/d , d = interfilament spacing) to a weakly oriented (nearly isotropic) low filament density gel.Funded by DOE DE-FG-02-06ER46314, NSF DMR-0503347.[1] R. Beck, J. Deek, J.B. Jones, C.R. Safinya. Nature Materials, In Press[2] J.B. Jones, C.R. Safinya, Biophys. J. 95, 823 (2008)

Fabrication and evaluation of solid freeformed electromagnetic bandgap structures

Microwave electromagnetic bandgap (EBG) structures have potential applications in improving the radiating features of antennas and the transmission properties of waveguides. Extrusion freeforming is a rapid prototyping technique for assembling ceramic dielectric lattice structures directly from a computer design file. It is without heating, cooling or polymerization processes to contend with at the construction stage. Various limitations on overall build thickness prompted us to explore lamination by welding to produce larger three-dimensional quasi-crystals. Microwave transmission through normal and side incidence showed that the bandgap frequency was in the 90–110 GHz region in all directions, matching the design that was informed by computer modelling. In order to image the EBG internal crystal structure, micro-computed tomography was used for scanning and reconstruction. Finally, the effect of structural defects including roundness and sagging of filaments and deviation of inter-filament spacing caused by fabrication errors on bandgap frequency was investigated.

Structural Properties of Neurofilament Sidearms: Sequence-Based Modeling of Neurofilament Architecture

Neurofilaments (NFs) are essential cytoskeletal filaments that impart mechanical integrity to nerve cells. They are assembled from three distinct molecular mass proteins that bind to each other to form a 10-nm-diameter filamentous rod with sidearm extensions. The sidearms are considered to play a critical role in modulating interfilament spacing and axonal caliber. However, the precise mechanism by which NF protrusions regulate axonal diameter remains to be well understood. In particular, the role played by individual NF protrusions in specifying interfilament distances is yet to be established. To gain insight into the role of individual proteins, we investigated the structural organization of NF architecture under different phosphorylation conditions. To this end, a physically motivated sequence-based coarse-grain model of NF brush has been developed based on the three-dimensional architecture of NFs. The model incorporates the charge distribution of sidearms, including charges from the phosphorylation sites corresponding to Lys-Ser-Pro repeat motifs. The model also incorporates the proper grafting of the real NF sidearms based on the stoichiometry of the three subunits. The equilibrium structure of the NF brush is then investigated under different phosphorylation conditions. The phosphorylation of NF modifies the structural organization of sidearms. Upon phosphorylation, a dramatic change involving a transformation from a compact conformation to an extended conformation is found in the heavy NF (NF-H) protein. However, in spite of extensive phosphorylation sites present in the NF-H subunit, the tails of the medium NF subunit are found to be more extended than the NF-H sidearms. This supports the notion that medium NF protrusions are critical in regulating NF spacings and, hence, axonal caliber.

Direct Measurement of Inter-filament Forces in Neurofilament Networks: Synchrotron X-ray Diffraction Study under Osmotic Pressure

Neurofilaments (NFs) are the major protein constituents in neuronal processes (axons and dendrites) that impart mechanical stability and act as structural scaffolds. The filaments assemble from 3 different subunit proteins (NF-L, NF-M, NF-H) to form a 10 nm diameter flexible polymer with radiating unstructured sidearms. Recent work, showed that at high protein concentration, the NFs form a nematic hydrogel network with a well-defined interfilament spacing as can be measured by synchrotron small angle x-ray scattering (SAXS) [1]. The x-ray/phase behavior study showed the role the different subunit protein compositions play in the interfilament interaction. In order to directly elucidate the interfilament forces responsible for the mechanical properties of NFs hydrogel, we conducted a SAXS-osmotic pressure study, which yielded pressure-distance curves at different subunit compositions and monovalent salts. We show that filaments composed with NF-L and NF-M strongly attract each other through their polyampholyte sidearms, in particularly at high monovalent salt. However, filaments comprised of NF-L and NF-H, at high NF-H grafting density, show a distinctly different pressure-distance dependency, with much larger interfilament spacing and weaker salt dependence. Supported by DOE DE-FG-02-06ER46314, NIH GM-59288, NSF DMR-0503347, and the Human Frontier Science Program organization.[1] J.B. Jones, C.R. Safinya, Biophys. J. 95, 823 (2008).

Finite Element and Statistical Methods Applied to Actin Bundles and Networks under Load

Actin binding proteins (ABPs) organize F-actin into ensembles_generally bundles or networks_thereby affecting actin cytoskeletal function and dynamics. They also participate in binding F-actin to the cell membrane. Some ABPs, e.g. α-actinin, fascin, bind F-actin into parallel bundles whose inter-filament spacing, which depends upon the ABP length and distance between its actin-binding domains, may or may not allow F-actin to interact with other proteins, e.g. myosin. Other, generally longer, ABPs, e.g. filamins, bind F-actin into compliant, albeit tightly-entangled, orthogonal networks imbued with gel-like mechanical properties, as a result of the 2D-rotational flexibility of the V-shaped ABP hinges.As the actin structure in non-muscle cells is complex and dynamic, a couple of features have been made to build the 3D model: determination of the representative model space that contains enough number of actin filaments to model the typical actin-actin interactions over larger scales; identification of the average effect of actin crosslinking in the model space, including the possible links and number of actin filaments in the neighborhood with respect to actin length, length distribution, and number density; construction of the fundamental elements that repeat in the crosslinked structures and maintain the physical constraints of bundles and networks; application of Bell's model to back out the passive force and bond lifetime of actin structure under load.Myosin mini-filaments work together with actin filaments in non-muscle cells during cell migration and division. Instead of providing passive load bearing like ABPs, myosin filaments walk along F-actins to generate contraction forces. This model is being integrated with the presence of myosin mini-filaments and the relative interactions, into a larger model for the production and transmission of biochemically-mediated intracellular forces, and actin cytoskeleton dynamics in whole cells.

Effect of pH and Electrostatic Interactions on Myofilament Lattice Volume

Changes interfilament lattice spacing is a major determinant of force production in striated muscle with reductions in force generation being observed with both expansion and compression of the lattice spacing relative to the normal physiological values. Previous studies have concluded that lattice spacing depends complexly on the balance of outward repulsive forces and inward attractive and compressive forces between thick and thin filaments. Since lattice spacing has important implications on force generation, we examined the effects of alterations in filament charge, induced by changes in intracellular pH (by the rapid application and withdrawal of 30 mM NH4Cl), on lattice spacing in intact twitching cardiac trabeculae. Since we observed changes in sarcomere length in response to changes in intracellular pH and since changes in sarcomere length induce changes in lattice spacing as a result of the isovolumic volume behavior of intact sarcomeres, sarcomere length was maintained at a fixed value (∼2.2 μm) during the pH interventions. Lattice spacing increased (p < 0.05) following NH4Cl wash in (measured after 5 minutes) when the pH is estimated to increase to ∼7.8 (Balnave 2000 J. Physiol.) to 36.9 +/-0.2. Following the washout of NH4Cl (which is estimated to decrease pH to ∼6.5 (Swietach 2005 J. Physiol.)) lattice spacing increased to 37.6+/−0.4 elevated above (P < 0.05) the space observed at control pH. Since the isoelectric point for myofilaments is ∼5 (Naylor, Biophys J, 1985), our findings suggest that, in addition to electrostatic, van der Waals' and osmotic forces, other pH-sensitive forces are also critical determinants of the lattice spacing, possibly the M-protein, myomesin or other M-line proteins which are postulated to hold thick filaments together in the M-band of striated muscle.

Salt Dependence of Neurofilament Gel Phase Behavior - A Synchrotron X-ray Scattering Study

Neurofilaments (NFs) are cytoskeletal proteins expressed in neuronal cells, and are believed to play a role in the determination and maintenance of the neuronal cell shape and mechanical integrity. NFs self-assemble as flexible cylinders from 3 protein subunits: NF-Low (NF-L), NF-Medium (NF-M), and NF-High (NF-H). The three subunits are structurally conserved with the exception of their “tail” domains, composed of amino acid strands of increasing length and charge respectively. Screening of tail charges is achieved by varying the salinity of the in vitro buffer. At high concentrations, the filaments interact amongst themselves through their unstructured tails that branch out from the filament core and form a viscous gel. Polarized microscopy was used to map out phase diagrams of the resultant neurofilament hydrogels, and thus characterize salt dependent phase behavior. Reassembled separate networks of NF-L + NF-M and NF-L + NF-H show phase transitions from isotropic to nematic gel phases at distinct salt concentrations, thus reflecting the differences in the governing sidearm interactions [1].We will describe synchrotron x-ray scattering experiments that have allowed us to quantitatively study the microscopic structure of the NF gels: shifts in average interfilament spacing demonstrate the phase boundary between the isotropic and anisotropic NF-liquid crystal gel phases and how they are shifted as a result of varying the in vitro buffer salt concentrations. Funding provided by DOE DE-FG-02-06ER46314, NIH GM-59288, NSF DMR-0503347.[1] J.B. Jones, C.R. Safinya, Biophys. J. 95, 823 (2008).

Morphology of Neurofilament Protrusions: Sequence-Based Modeling of Neurofilament Brush

Neurofilaments are essential cytoskeletal filaments that impart mechanical integrity to nerve cells and regulate the cross-sectional area of axons. They are assembled from three distinct molecular weight proteins (NF-L, NF-M, NF-H) bound to each other laterally forming 10 nm diameter filamentous rods along with side-arm extensions. These protrusions contain an abundance of charged amino acid residues. The charged side-arms are considered to mediate the interactions between neighboring filaments, regulating interfilament spacing, and hence axonal caliber. The precise mechanism by which neurofilament protrusions regulate axonal diameter remains unsettled. In particular, the role of individual proteins has remained to be a matter of debate. Computer modeling is instrumental in demonstrating the details about the structural arrangement of individual protrusions. The present study employs Monte Carlo Simulations of neurofilament to reveal the role played by individual side-arms. The simulations are conducted under different phosphorylation state by making use of physically motivated 3D model of neurofilament brush. The model consists of a neurofilament backbone along with side-arm extensions that are distributed according to the stoichiometry of the three subunits. The side-arms are modeled at amino acid resolution with each amino acid represented by a hard sphere along with the corresponding charge valence. The phosphorylation states are specified through charges assigned to the serine amino acids of the Lys-Ser-Pro (KSP) repeat motifs of the side-arms. The equilibrium structure of the neurofilament brush has been studied via the model that maintained the proper charge distributions and grafting density of neurofilament side arms. It has been found that in spite of extensive phosphorylation sites present on NF-H, the tails of the medium sized neurofilament subunit (NF-M) is more elongated than NF-H tails. This suggests that NF-M protrusions are more critical in regulating neurofilament spacings and axonal caliber.

Effects of Sustained Length-Dependent Activation on In Situ Cross-Bridge Dynamics in Rat Hearts

The cellular basis of the length-dependent increases in contractile force in the beating heart has remained unclear. Our aim was to investigate whether length-dependent mediated increases in contractile force are correlated with myosin head proximity to actin filaments, and presumably the number of cross-bridges activated during a contraction. We therefore employed x-ray diffraction analyses of beat-to-beat contractions in spontaneously beating rat hearts under open-chest conditions simultaneous with recordings of left ventricle (LV) pressure-volume. Regional x-ray diffraction patterns were recorded from the anterior LV free wall under steady-state contractions and during acute volume loading (intravenous lactate Ringers infusion at 60 ml/h, <5 min duration) to determine the change in intensity ratio ( I 1,0/ I 1,1) and myosin interfilament spacing ( d 1,0). We found no significant change in end-diastolic (ED) intensity ratio, indicating that the proportion of myosin heads in proximity to actin was unchanged by fiber stretching. Intensity ratio decreased significantly more during the isovolumetric contraction phase during volume loading than under baseline contractions. A significant systolic increase in myosin head proximity to actin filaments correlated with the maximum rate of pressure increase. Hence, a reduction in interfilament spacing at end-diastole (∼0.5 nm) during stretch increased the proportion of cross-bridges activated. Furthermore, our recordings suggest that d 1,0 expansion was inversely related to LV volume but was restricted during contraction and sarcomere shortening to values smaller than the maximum during isovolumetric relaxation. Since ventricular volume, and presumably sarcomere length, was found to be directly related to interfilament spacing, these findings support a role for interfilament spacing in modulating cross-bridge formation and force developed before shortening.

When fibres go slack and cross bridges are free to run: a brilliant method to study kinetic properties of acto‐myosin interaction

When fibres go slack and cross bridges are free to run: a brilliant method to study kinetic properties of acto‐myosin interaction

Seasonal control of particle clearance by isolated gills from the clam Mercenaria mercenaria

Pieces of gill isolated from the clam Mercenaria mercenaria clear colloidal graphite from seawater, and the clearance rates are measurable. 5-Hydroxytryptamine (5-HT) had a biphasic effect on clearance rates: concentrations from 10(-6) to 10(-5) mol l(-1) 5-HT increased clearance, but higher concentrations reduced it. During the summer, the gills were less responsive to 5-HT: the threshold increased from 1 x 10(-6) to 5 x 10(-6) mol l(-1), and although the rate was still maximal at 10(-5) mol l(-1), it was significantly lower than the maximal rate in the winter. At 10(-5) mol l(-1) 5-HT, which maximized clearance, the lateral cilia were active, the interfilament space decreased and the diameter of the water tubes increased. Higher concentrations of 5-HT contracted the gill musculature, which inhibited the lateral cilia, decreased the interfilament space even more and decreased the diameter of the water tubes. The nitric oxide (NO) generator DEANO stimulated clearance in the winter but had no effect during the summer. L-NAME, an inhibitor of NO synthesis, diminished the effect of 5-HT during the winter but was ineffective during the summer. The diminished response to 5-HT of winter gills treated with L-NAME was statistically equal to the response of the gills to 5-HT during the summer. Dopamine (DA) inhibited clearance, and the gills were more sensitive to DA in winter than in summer. Microscopic examination of untreated gills revealed little or no lateral ciliary activity, and clearance was minimal. Thus, clearance rates of isolated gills behave in a manner consistent with the seasonality and pharmacology of the lateral cilia and branchial musculature.

Interfilament Spacing Is Preserved during Sarcomere Length Isometric Contractions in Rat Cardiac Trabeculae

It is generally assumed that the myofilament lattice in intact (i.e., nonskinned) striated muscle obeys constant volume. However, whether such is the case during the myocardial contraction is unknown. Accordingly, we measured interfilament spacing by x-ray diffraction in ultra-thin isolated rat right ventricular trabeculae during a short 10 ms shuttered exposure either just before electrical stimulation (diastole), or at the peak of the contraction (systole); sarcomere length (SL) was held constant throughout the contraction using an iterative feedback control system. SL was thus varied in a series of SL-clamped contractions; the relationship between SL and interfilament spacing was not different between diastole and systole within 1%; this was true also over a wide range of inotropic states induced by varied [Ca 2+] o. We conclude that the cardiac myofilament lattice maintains constant volume, and thus constant interfilament spacing, during contraction.

Electron Tomography of Swollen Rigor Fibers of Insect Flight Muscle Reveals a Short and Variably Angled S2 Domain

Subfragment 2 (S2), the segment that links the two myosin heads to the thick filament backbone, may serve as a swing-out adapter allowing crossbridge access to actin, as the elastic component of crossbridges and as part of a phosphorylation-regulated on-off switch for crossbridges in smooth muscle. Low-salt expansion increases interfilament spacing (from 52 nm to 67 nm) of rigor insect flight muscle fibers and exposes a tethering segment of S2 in many crossbridges. Docking an actoS1 atomic model into EM tomograms of swollen rigor fibers identifies in situ for the first time the location, length and angle assignable to a segment of S2. Correspondence analysis of 1831 38.7 nm crossbridge repeats grouped self-similar forms from which class averages could be computed. The full range of the variability in angles and lengths of exposed S2 was displayed by using class averages for atomic fittings of acto-S1, while S2 was modeled by fitting a length of coiled-coil to unaveraged individual repeats. This hybrid modeling shows that the average length of S2 tethers along the thick filament (except near the tapered ends) is ∼10 nm, or 16% of S2′s total length, with an angular range encompassing 90° axially and 120° azimuthally. The large range of S2 angles indicates that some rigor bridges produce positive force that must be balanced by others producing drag force. The short tethering segment clarifies constraints on the function of S2 in accommodating variable myosin head access to actin. We suggest that the short length of S2 may also favor intermolecular head−head interactions in IFM relaxed thick filaments.