Average Filament Length Research Articles

Functional morphology of gills and respiratory area of two active rheophilic fish species, Plagioscion squamosissimus and Prochilodus scrofa

Measurements of gill dimensions were carried out on two ecologically distinct active rheophilic teleost species, the curvina Plagioscion squamosissimus and the curimbatáProchilodus scrofa, and were analysed in relation to body mass according to the equation Y=aWb. The gill respiratory areas of P. squamosissimus and P. scrofa were large as expected for active fish and increased with increasing body mass (b=0.70 and 0.72, respectively) showing no significant difference between them. However, the large respiratory area of both species was realized in a different way revealing an adaptation to the plasticity of head components related to feeding habits. Consequently, significant differences were found between the number and average length of gill filaments and the bilateral area of secondary lamellae. The respiratory area of P. scrofa was due mainly to larger bilateral surface area of the secondary lamellae and its growth coefficient (b=0.51) that was significantly higher (P<0.05) than that found for P. squamosissimus (b= 0.36). The frequency of secondary lamellae mm−1 of filament was similar in both species (22 ± 2 on one side of gill filament). The dimensions of gill components and the respiratory area of these species suggest a complex interaction between head form, and feeding habits related to the functional morphology of the gills to meet the oxygen requirements of each species.

Capping protein terminates but does not initiate chemoattractant-induced actin assembly in Dictyostelium.

The first step in the directed movement of cells toward a chemotactic source involves the extension of pseudopods initiated by the focal nucleation and polymerization of actin at the leading edge of the cell. We have previously isolated a chemoattractant-regulated barbed-end capping activity from Dictyostelium that is uniquely associated with capping protein, also known as cap32/34. Although uncapping of barbed ends by capping protein has been proposed as a mechanism for the generation of free barbed ends after stimulation, in vitro and in situ analysis of the association of capping protein with the actin cytoskeleton after stimulation reveals that capping protein enters, but does not exit, the cytoskeleton during the initiation of actin polymerization. Increased association of capping protein with regions of the cell containing free barbed ends as visualized by exogenous rhodamine-labeled G-actin is also observed after stimulation. An approximate threefold increase in the number of filaments with free barbed ends is accompanied by increases in absolute filament number, whereas the average filament length remains constant. Therefore, a mechanism in which preexisting filaments are uncapped by capping protein, in response to stimulation leading to the generation of free barbed ends and filament elongation, is not supported. A model for actin assembly after stimulation, whereby free barbed ends are generated by either filament severing or de novo nucleation is proposed. In this model, exposure of free barbed ends results in actin assembly, followed by entry of free capping protein into the actin cytoskeleton, which acts to terminate, not initiate, the actin polymerization transient.

Actin isoform expression, cellular heterogeneity and contractile function in smooth muscle

Smooth muscles express four isoforms of actin: two smooth muscle specific and two cytoplasmic isoforms typically associated with the cytoskeleton of nonmuscle cells. The relative amounts of each isoform expressed and the total actin content vary with smooth muscle type, with development, in cell culture, pathologically, and potentially between cells within tissues. Our objective was to determine whether actin isoforms contribute to contractile diversity. Functional diversity may be the result of differences in the kinetics of the cross-bridge interaction with thin filaments consisting of different actin isoforms or of the fraction of cross-bridges developing force in series or in parallel resulting from thin filaments of different lengths. Our hypothesis was that functionally significant differences in actin isoform properties (i.e., myosin interactions or properties affecting thin filament lengths) would require isoform segregation into distinct populations of thin filaments within cellular domains (e.g., cytoskeletal and contractile) or in phenotypically different cells. We tested this hypothesis by determining the smooth muscle alpha- and gamma-actin and cytoplasmic beta-actin isoform composition of native thin filaments isolated from swine stomach using isoform-specific antibodies linked to colloidal gold beads with protein A (the cytoplasmic lambda-isoactin content was below the detection limit). The lengths of individual thin filaments were also estimated from electron micrographs. A statistically uniform population of thin filaments was observed consisting of randomly copolymerized isoactins in each filament with the same isoform proportions as the tissue. The average thin filament length was 1.35 +/- 0.06 (SEM) microns. These results, together with other studies, suggest that actin isoforms are functionally equivalent. The data imply that the high stress-generating and shortening capacities of smooth muscles are not primarily due to long thin filament to thick filament length ratios compared with striated muscles.

An experimental study of phytoplankton feeding in three tilapiine cichlids

Three species of tilapia (Oreochromis lidole, O. karongae and O. niloticus) were fed on four planktonic algal diets in the laboratory. The algal concentrations were measured over a 24‐h period and the ingestion rates calculated. In monocultures of large algae (average filament length=477 μm) O. niloticus had the highest ingestion rate, but on small algae (particle size <8 μm) O. lidole was the fastest, while O. karongae enhanced nanoplankton production. With algae of mixed sizes, O. niloticus was able to remove small algae quicker than the other fish species. Thus, the presence of larger algae can influence the impact of a planktivorous species on nanoplankton. O. lidole is likely to have the greatest impact on plankton communities heavily dominated by smaller algae.

Spatial and temporal variability in filament length of a toxic cyanobacterium (Anabaena affinis)

SUMMARY1. This study examines the distribution of Anabaena affinis filament lengths under natural conditions as a function of depth and season, and in the laboratory as a function of growth phase. Because Anabaena affinis is only toxic when consumed, both its filament length and position in the water column are important determinants of its potential impact on zooplankton populations.2. Star Lake (Norwich, Vermont, U.S.A.), a natural, eutrophic pond, remained thermally stratified throughout the Anabaena bloom. Filament number and length differed significantly with both sampling date and water depth. Most filaments occurred at 0.5 m, particularly at the height of the bloom. Throughout the entire water column average filament length decreased from approximately 0.53 mm in May to 0.14 mm in July. The shortest filaments occurred at the 2.5 m depth. Filament length distributions (combined for all depths) for 29 May, 12 June and 3 July, corresponding to the beginning, middle and end of the bloom, respectively, differed significantly among the three dates. These patterns most likely reflect variable growth conditions, both during the season and in the water column.3. In the laboratory, Anabaena filament length was affected by medium composition and growth phase. Filaments were significantly longer when grown in MBL than in ASM medium. Also, the average length of Anabaena filaments grown in MBL changed significantly as cultures aged; by day 13 filament length (2.01 ± 0.38 mm, mean ± SD) was twice that on day 0 (0.97 ± 0.71 mm). As cell concentration continued to increase, mean filament length gradually decreased.

Crowding-induced organization of cytoskeletal elements: II. Dissolution of spontaneously formed filament bundles by capping proteins.

Through calculations of molecular packing constraints in crowded solutions, we have previously shown that dispersions of filament forming proteins and soluble proteins can be unstable at physiological concentrations, such that tight bundles of filaments are formed spontaneously, in the absence of any accessory binding proteins. Here we consider the modulation of this phenomenon by capping proteins. The theory predicts that, by shortening the average filament length, capping alleviates the packing problem. As a result, the dispersed isotropic solution is stable over an expanded range of compositions.

Flexibility of F-actin in aqueous solution: a study on filaments of different average lengths

We have investigated the dynamics of F-actin with different average filament lengths in dilute and semidilute solution by polarized dynamic light scattering. The average degree of polymerization, (X), of F-actin was controlled by the actin-binding protein gelsolin. By variation of the molar ratio of actin/ gelsolin, we obtained a homologous series of filaments with 50 < (X) < 2000, the polydispersity of the system being characterized by an exponential distribution. Comparison of the electric field autocorrelation function decay profile in the dilute regime with model calculations for rigid and semiflexible rodlike polymers revealed that the microfilaments are rather stiff molecules. From measurements on the homologous series of different filament lengths the persistence length was determined to P=7.5+/-2.5 mu m. The contribution of internal bending motions to the correlation function was clearly discernible for longer filaments and at larger scattering vector, respectively. To characterize the concentration effect on the polarized dynamic structure factor, we studied solutions with (X) = 350 from 2.2 to 105 mu M actin. The transition from dilute to semidilute regime was detected at c(L)(3) approximate to 15. This is very similar to findings on other polymer systems with a narrower size distribution. While at low scattering angle the apparent diffusion coefficient is independent on concentration, we observed a decrease of D-app at theta=90 degrees. From this we concluded that the rotational motion of the rodlike molecules is restricted at higher C(L)(3). In addition, with increasing concentration a slow mode appeared in the experimental correlation functions. The amplitude decreased with increasing scattering vector. The nature of the slow relaxation process, also observed in other polymer systems, is still under debate.

Polarized and depolarized dynamic light scattering study on F-actin in solution: comparison with model calculations

The dynamics of F-actin of different average filament lengths are sudied by polarized and depolarized dynamic light scattering. The experimental data have been compared with model calculations for rigid and semiflexible macromolecules. The polarized DLS spectra follow the predictions for semiflexible rods with a persistence length of (7.5±2.5) μm. Became there is no theory available that includes flexibility in the calculation of the depolarized dynamic structure factor, to a first approximation we used the diffusion coefficients for semiflexible molecules in our rigid-rod model calculations

Formation of liquid crystals from actin filaments.

Actin is cross-linked by actin-binding proteins in the cytoplasm to form either isotropic or highly oriented anisotropic structures. The inherent orientation among actin filaments could influence whether an isotropic or highly oriented anisotropic structure is formed. A highly oriented state can arise spontaneously through the formation of liquid crystals as predicted by polymer theory. In this study, the ability of filamentous actin to form liquid crystalline domains was detected using the anisotropic component of scattered light and by observation of birefringence. As liquid crystalline domains formed, the intensity of the anisotropic component of scattered light increased, and birefringent macroscopic oriented domains were directly observed. The formation of liquid crystalline domains was dependent on the concentration of actin filaments and on the average filament length controlled by varying the ratio of gelsolin to actin monomers. The concentration of actin filaments required to form liquid crystalline domains increased moderately as the average length was decreased. At a fixed actin concentration, orientation among the filaments attained a maximum value at a ratio of actin to gelsolin in the range from 1500 to 2000 and decreased as the ratio was increased or decreased from this range. The results are not well explained by theoretical treatments for liquid crystal formation by monodisperse, charged worm-like chains. Differences from the theoretical predictions for formation of liquid crystals are most likely due to the polydisperse filament length of actin. This phenomenon may have important effects on the structural and rheological properties of the cytoplasm in living cells.

Vapor grown carbon fibers from pyrolysis of hydrocarbons: Modeling of filament growth and poisoning

A kinetic scheme has been proposed as a feasible model to explain the lengthening step in vapor grown carbon fibers from the pyrolysis of hydrocarbons. The two-parameter model assumes that the filaments lengthen by the reaction of a pyrolysis product with the growing filaments, and that the growth process ceases when the catalyst particle attached to the growing filament is poisoned by some reactive species in the gas phase. The model results show that the rate of filament growth is initially high and decreases monotonically. The final average filament length is found to depend only on the ratio of the rate of growth to the rate of poisoning. The model results can satisfactorily correlate the limited experimental data available in the open literature.

3D analysis of gelatin gel networks from transmission electron microscopy imaging

This paper presents a quantitative analysis of the structural parameters of gelatin gel networks derived from transmission electron micrographs. The network gel replicas were obtained by a quick-freezing, deep-etching and rotary replication method adapted to the study of fragile physical networks by Favard P. et al. (Biol. Cell 67 (1989) 201-207). Stereo electron micrographs were taken by tilting the replicas at increasing angles between – 35 to + 30○. A 3D reconstruction of the filamentous network was performed by stereoscopic and tomographic procedures. These procedures allowed us to measure the thicknesses of the replicas. The average length of filaments (triple helices) per unit volume and the average distance between filaments (mesh sizes of the networks) were derived for two polymer concentrations ($c=2~\%$ and $c=10~\%$). The results are in full agreement with optical rotation and small-angle neutron scattering measurements.

Motion of actin filaments in the presence of myosin heads and ATP

We measured, by fluorescence correlation spectroscopy, the motion of actin filaments in solution during hydrolysis of ATP by acto-heavy meromyosin (acto-HMM). The method relies on the fact that the intensity of fluorescence fluctuates as fluorescently labeled actin filaments enter and leave a small sample volume. The rapidity of these number fluctuations is characterized by the autocorrelation function, which decays to 0 in time that is related to the average velocity of translation of filaments. The time of decay of the autocorrelation function of bare actin filaments in solution was 10.59 +/- 0.85 s. Strongly bound (rigor) heads slowed down the diffusion. Direct observation of filaments under an optical microscope showed that addition of HMM did not change the average length or flexibility of actin filaments, suggesting that the decrease in diffusion was not due to a HMM-induced change in the shape of filaments. Rather, slowing down of translational motion was caused by an increase in the volume of the diffusing complex. Surprisingly, the addition of ATP to acto-HMM accelerated the motion of actin filaments. The acceleration was the greatest at the low molar ratios of HMM:actin. Direct observation of filaments under an optical microscope showed that in the presence of ATP the average length of filaments did not change and that the filaments became stiffer, suggesting that acceleration of diffusion was not due to an ATP-induced increase in flexibility of filaments. These results show that some of the energy of splitting of ATP is impaired to actin filaments and suggest that 0.06 +/- 0.02 of HMM interferes with the diffusion of actin filaments during hydrolysis of ATP.

Sea urchin egg villin: Identification of villin in a non-epithelial cell from an invertebrate species

Fertilization of sea urchin eggs results in the rapid polymerization of actin filaments and subsequent formation of a brush border-like cortical cytoskeleton. A 110 x 10(3) Mr (110K) actin binding protein has been purified from extracts of unfertilized Strongylocentrotus purpuratus eggs. Analysis of polymerization kinetics using fluorescence and viscometry assays demonstrated that 110K accelerated the nucleation phase of actin assembly only in the presence of elevated Ca2+. The Ca(2+)-mediated effects were correlated with a decrease in sedimentable polymer and a decrease in average filament length. Addition of Ca2+ to solutions of 110K and F-actin, polymerized in the presence of EGTA, resulted in a precipitous drop in viscosity and the decreased viscosity was fully reversible upon chelation of Ca2+. The Ca2+ threshold for 110K activation was in the 10(-6) to 10(-7) M range. Nucleated assembly experiments using Limulus sperm acrosomal processes demonstrated that egg 110K capped the barbed ends of actin filaments. In the absence of Ca2+, 110K organized actin filaments into bundles at pH values less than 7.4. Anti-egg 110K antibody crossreacted with chicken intestinal epithelial cell villin and anti-porcine villin headpiece monoclonal antibody crossreacted with 110K. Further, 110K possesses an approximately 10 x 10(3) Mr terminal polypeptide segment that is immunologically related to villin headpiece. These studies demonstrate that sea urchin egg 110K is functionally, immunologically and structurally related to villin, an actin binding protein expressed in specific epithelial tissues in vertebrates. Consequently, this finding provides insight into the potential mechanisms that might determine the genesis of the cortical brush border cytoarchitecture in sea urchin eggs and further sheds light on the evolution of the villin protein family.

Preparing well-oriented sols of straight bacterial flagellar filaments for X-ray fiber diffraction

Well-oriented sols of straight bacterial flagellar filaments have been obtained by preparing reconstituted flagellar filaments with an appropriate length distribution and choosing appropriate solvent conditions. An average filament length of 300 to 500 nm and the use of solvents with very low concentrations of salt has allowed us to prepare highly fluid sols that make flow orientation possible. X-ray fiber diffraction from these sols has shown distinct layer-line reflections to 3.5 Å resolution in the meridional direction. Layer-line intensities have been collected by the angular deconvolution method up to 5 Å resolution. The possibility of using a magnetic field to further improve the orientation has been explored and a solvent condition that makes flagellar sols sensitive to the magnetic field has been found. General applicability of the method to other systems is also discussed.

Kinetics of the interaction of a 41-kilodalton macrophage capping protein with actin: promotion of nucleation during prolongation of the lag period

A 41-kilodalton macrophage capping protein (MCP) has been isolated which is capable of forming complexes with actin monomers in addition to capping the barbed ends of actin filaments (Southwick & DiNubile, 1986). The protein is calcium activated in a fully reversible manner. Using kinetic assays, we determined a capping constant, defined here as a modified Kd, of 1 nM and a Kd of 3-4 microM for MCP-actin monomer complex formation. MCP weakly nucleates actin polymerization: more than 0.5 microM MCP is necessary to shorten the lag period, and 1 microM MCP at an actin/MCP ratio of 10 reduces the average length of actin filaments to about 200 molecules per filament. We determined that the actin nucleus that survives MCP inactivation contains a minimum number of five actin molecules. These experiments also make a point with respect to the interpretation of the prolongation of the lag period. We directly demonstrate that in the presence of an actin binding protein a prolongation of the lag period can be associated with increased nucleation, contrary to the usual interpretation in the literature that it indicates no or decreased nucleation by the actin binding protein.

Differential modulation of actin-severing activity of gelsolin by multiple isoforms of cultured rat cell tropomyosin: Potentiation of protective ability of tropomyosins by 83-kDa nonmuscle caldesmon

Abstract Multiple isoforms of tropomyosin (TM) of rat cultured cells show differential effects on actin-severing activity of gelsolin. Flow birefringence measurements have revealed that tropomyosin isoforms with high Mr values (high Mr TMs) partially protect actin filaments from fragmentation by gelsolin, while tropomyosins with low Mr values (low Mr TMs) have no significant protection even when the actin filaments have been fully saturated with low Mr TMs. We have also examined effect of nonmuscle caldesmon on the severing activity of gelsolin because 83-kDa nonmuscle caldesmon stimulates actin binding of rat cell TMs (Yamashiro-Matsumura, S., and Matsumura, F. (1988) J. Cell Biol. 106, 1973-1983). While nonmuscle caldesmon alone or low Mr TMs alone show no significant protection against fragmentation by gelsolin, the low Mr TMs coupled with 83-kDa protein are able to protect actin filaments. Further, high Mr TMs together with 83-kDa protein appear to block the severing activity completely. Electron microscopic analyses of length distribution of actin filaments have confirmed the results. The average length of control actin filaments is measured as 1.46 +/- microns, and gelsolin shortens the average length to 0.084 +/- 0.039 micron. Similar short average lengths are obtained when gelsolin severs actin complexed with low Mr TMs (0.080 +/- 0.045 micron) or with nonmuscle caldesmon (0.11 +/- 0.072 micron) while longer average length (0.22 +/- 0.18 micron) is measured in the presence of high Mr TMs. The simultaneous addition of nonmuscle caldesmon makes the average length considerably longer, i.e. 0.61 +/- 0.37 micron in the presence of low Mr TMs and 1.57 +/- 0.97 micron in the presence of high Mr TMs. Furthermore, the actin binding of gelsolin is strongly inhibited by co-addition of high Mr TMs and nonmuscle caldesmon. These results suggest that TM and gelsolin share the same binding site on actin molecules and that the differences in the actin affinities between TMs are related to their abilities of protection against gelsolin.

Substoichiometric concentrations of ATP-G-actin are required to anneal actin polymerized by calcium ions

At 3°C and pH 7.0, the addition of 40 nM ATP-G-actin to F-actin (12 μM as the monomer), polymerized in the presence of 4 mM CaCl 2, determines a substantial and rapid increase of the viscosity of the solution, which is accompanied by the incorporation of the ATP-G-actin added into the polymer. The hypothesis that the presence of ATP-actin at the filament end(s) promotes the annealing reaction is substantiated by the finding that, after the addition of ATP-G-actin, the average filament length is increased. This finding is relevant, not only because it provides evidences in favour of the existence of annealing but also because it shows that the concentration of ATP-G-actin influences the filaments length distribution through a mechanism different from the elongation reaction.

PH dependence of actin self-assembly

Fluorescence enhancement and fluorescence photobleaching recovery have been utilized to examine actin self-assembly over the pH range 6.6-8.0. The kinetics of assembly are faster and the critical concentrations are lower at lower pH. Filament diffusion coefficients are not a function of pH, indicating that average filament lengths are not pH dependent. Although critical actin concentrations are a sensitive function of the concentrations of various cations in the medium, the relative pH dependences of critical concentrations are similar for all combinations of cations employed. The pH dependence of actin self-assembly is sufficiently great that it should be taken into account when comparing data from different reports and when relating in vitro measurements to cytoplasmic mechanisms.

Heat-induced gelation of myosin filaments.

Various lengths of myosin filaments were prepared by changing the speed of lowering the ionic strength. Short filaments were formed by rapid dilution and the filament length became longer with decreasing the speed of lowering the ionic strength. The average length of myosin filaments ranged from 0.5 to 3.0 μm with varying the speed of dilution. Myosin filament suspensions were heated to form gels, and those gel strengths were evaluated as rigidity. The rigidity depended on the length of the filaments before heating. The longer the filaments, the higher the rigidities. The gels with higher rigidity had a fine strand-like network structure. In contrast, that showing lower rigidity had a coarse aggregated structure. When low protein concentrations of myosin filament suspensions were heated at 40°C, myosin filaments associated with increasing the incubation time. When heating was done at 60°C, aggregation of myosin filaments occurred in a short time. Electron microscopic observation suggests that the association and the following aggregation of myosin filaments per se cause formation of a whole network structure. Short myosin filaments began to aggregate randomly in an early stage of heating and they made clusters. But long filaments did not form a cluster; instead, some of them associated side-by-side to form bundles. These differences in the mode of aggregation among various lengths of myosin filaments seemed to be one of possible reasons for the different network structures observed in the gels.

The structure of myosin filaments and the properties of heat-induced gel in the presence and absence of C-protein.

Myosin filaments were prepared by either dialysis or rapid dilution to give a final condition of 0.1 m KC1 and pH 6.0, in the presence or absence of C-protein. The average length of myosin filaments prepared by dialysis was much longer than that by dilution whether C-protein is present or absent. C-protein reduced the diameters of both filaments but no significant effects on the filament length were observed. All of the myosin filament suspensions formed gels upon heating, but C-protein alone did not. The strengths of thermal gels of myosin filaments prepared by dialysis were much higher than those'by dilution. In addition, a fine strand-like network structure was formed in the gel from dialyzed myosin, but diluted myosin formed a rather coarse network structure. C- protein lowered the strengths of thermal gels of myosin filaments, especially that prepared by dialysis. These results suggest that the rheological properties of thermal gels of myosin filaments at low ionic strength depend on the structure of...