Role of plant growth substances in MS33-controlled stamen filament growth in Arabidopsis

The rapid growth of stamen filaments just before flower anthesis in Arabidopsis thaliana does not occur in the male sterile33 ( ms33 , formerly known as msZ ) mutant. ms33 filaments were approximately 40% shorter than the wild type (WT), and there was corresponding reduction in the epidermal cell length of filaments. This suggests that MS33 controls the final cell-elongation phase of filament growth. Both low temperatures and gibberellic acid (GA₃) restored filament and cell growth in intact ms33 flowers, but these treatments only had a small promotive effect on WT filaments. Decapitation experiments involving the removal of the anther had the opposite effect on WT and ms33 filaments; growth was inhibited in WT, but was increased in ms33 filaments. In young stamen primordia cultured in vitro, filament growth was less in WT, but more in ms33 , than in respective in vivo produced filaments. Plant growth substances (PGSs), GA₃ and indole-3-acetic acid (IAA) were promotive, zeatin had no effect, and abscisic acid (ABA) and ethrel inhibited filament growth in both intact and decapitated WT and ms33 filaments. Together these observations suggest that MS33 is activated immediately before anthesis and that the MS33 product either regulates temporal biosynthesis of gibberellins (GAs) and/or IAA or makes the filament tissue sensitive to these PGSs, which in turn trigger cell elongation and filament growth. The data also suggest that ms33 mutant anthers contain a relatively high ratio of growth inhibitors to promoters, which inhibits epidermal cell elongation and filament growth.     

Cdc42-induced actin filaments are protected from capping protein.

Each actin filament has a pointed and a barbed end, however, filament elongation occurs primarily at the barbed end. Capping proteins, by binding to the barbed end, can terminate this elongation. The rate of capping depends on the concentration of capping protein [1], and thus, if capping terminates elongation, the length of filaments should vary inversely with the concentration of capping protein. In cell extracts, such as those derived from neutrophils, new actin filaments can be nucleated by addition of GTPgammaS-activated Cdc42 (a small GTPase of the Rho family). To determine whether elongation of these filaments is terminated by capping, we manipulated the concentration of capping protein, the major calcium-independent capping protein in neutrophils, and observed the effects on filament lengths. Depletion of 70% of the capping protein from extracts increased the mean length of filaments elongated from spectrin-actin seeds (very short actin filaments with free barbed ends) but did not increase the mean length of filaments induced by Cdc42. Furthermore, doubling the concentration of capping protein in cell extracts by adding pure capping protein did not decrease the mean length of filaments induced by Cdc42. These results suggest that the barbed ends of Cdc42-induced filaments are protected from capping by capping protein.     

Direct demonstration of actin filament annealing in vitro

Direct electron microscopic examination confirms that short actin filaments rapidly anneal end-to-end in vitro, leading over time to an increase in filament length at steady state. During annealing of mixtures of native unlabeled filaments and glutaraldehyde-fixed filaments labeled with myosin subfragment-1, the structural polarity within heteropolymers is conserved absolutely. Annealing does not appear to require either ATP hydrolysis or the presence of exogenous actin monomers, suggesting that joining occurs through the direct association of filament ends. During recovery from sonication the initial rate of annealing is consistent with a second-order reaction involving the collision of two filament ends with an apparent annealing rate constant of 10(7) M-1s-1. This rapid phase lasts less than 10 s and is followed by a slow phase lasting minutes to hours. Annealing is calculated to contribute minimally to filament elongation during the initial stages of self-assembly. However, the rapid rate of annealing of sonicated fixed filaments observed in vitro suggests that it may be an efficient mechanism for repairing breaks in filaments and that annealing together with polymer-severing mechanisms may contribute significantly to the dynamics and function of actin filaments in vivo.     

THE RELATIONSHIP BETWEEN THE PROMOTION OF ELONGATION OF FERN PROTONEMATA BY LIGHT AND GROWTH SUBSTANCES

Germinating spores of the fern Onoclea sensibilis L. were grown in darkness, so that they developed as filaments (protonemata). Brief daily exposure of the filaments to red, far-red or blue light increased the rate of filament elongation. Filament elongation was also promoted by indoleacetic acid. When filament elongation was promoted with both indoleacetic acid and exposure to light, the growth promotions caused by red and far-red light were additive to auxin-induced growth. Blue light promoted elongation only at sub-optimal concentrations of auxin. Elongation induced by guanine was additive to red- and far-red-induced elongation. Gibberellic acid had no effect on elongation under any condition. Blue-light-induced elongation resembled auxin-induced elongation in its requirement for exogenous sucrose and sensitivity to inhibition by parachlorophenoxyisobutyric acid. Red and far-red light were active regardless of the presence or absence of sucrose and promoted elongation at a concentration of parachlorophenoxyisobutyric acid which completely inhibited blue-light-induced elongation.     

Nonlinear increase of elongation rate of actin filaments with actin monomer concentration

The nonlinear increase of the elongation rate of actin filaments above the critical monomer concentration was investigated by nucleated polymerization of actin. Significant deviations from linearity were observed when actin was polymerized in the presence of magnesium ions. When magnesium ions were replaced by potassium or calcium ions, no deviations from linearity could be detected. The nonlinearity was analyzed by two simple assembly mechanisms. In the first model, if the ATP hydrolysis by polymeric actin is approximately as fast as the incorporation of monomers into filaments, terminal subunits of lengthening filaments are expected to carry to some extent ADP. As ADP-containing subunits dissociate from the ends of actin filaments faster than ATP-containing subunits, the rate of elongation of actin filaments would be nonlinearly correlated with the monomer concentration. In the second model (conformational change model), actin monomers and filament subunits were assumed to occur in two conformations. The association and dissociation rates of actin molecules in the two conformations were thought to be different. The equilibrium distribution between the two conformations was assumed to be different for monomers and filament subunits. The ATP hydrolysis was thought to lag behind polymerization and conformational change. As under the experimental conditions the rate of ATP hydrolysis by polymeric actin was independent of the concentration of filament ends, the observed nonlinear increase of the rate of elongation with the monomer concentration above the critical monomer concentration was unlikely to be caused by ATP hydrolysis at the terminal subunits. The conformational change model turned out to be the simplest assembly mechanism by which all available experimental data could be explained.     

Nucleation limits the lengths of actin filaments assembled by formin

Formins stimulate actin polymerization by promoting both filament nucleation and elongation. Because nucleation and elongation draw upon a common pool of actin monomers, the rate at which each reaction proceeds influences the other. This interdependent mechanism determines the number of filaments assembled over the course of a polymerization reaction, as well as their equilibrium lengths. In this study, we used kinetic modeling and in vitro polymerization reactions to dissect the contributions of filament nucleation and elongation to the process of formin-mediated actin assembly. We found that the rates of nucleation and elongation evolve over the course of a polymerization reaction. The period over which each process occurs is a key determinant of the total number of filaments that are assembled, as well as their average lengths at equilibrium. Inclusion of formin in polymerization reactions speeds filament nucleation, thus increasing the number and shortening the lengths of filaments that are assembled over the course of the reaction. Modulation of the elongation rate produces modest changes in the equilibrium lengths of formin-bound filaments. However, the dependence of filament length on the elongation rate is limited by the number of filament ends generated via formin’s nucleation activity. Sustained elongation of small numbers of formin-bound filaments, therefore, requires inhibition of nucleation via monomer sequestration and a low concentration of activated formin. Our results underscore the mechanistic advantage for keeping formin’s nucleation efficiency relatively low in cells, where unregulated actin assembly would produce deleterious effects on cytoskeletal dynamics. Under these conditions, differences in the elongation rates mediated by formin isoforms are most likely to impact the kinetics of actin assembly.     

Quantitative analysis of the self-assembly strategies of intermediate filaments from tetrameric vimentin

In vitro assembly of intermediate filaments from tetrameric vimentin consists of a very rapid phase of tetramers laterally associating into unit-length filaments and a slow phase of filament elongation. We focus in this paper on a systematic quantitative investigation of two molecular models for filament assembly, recently proposed in (Kirmse et al. J. Biol. Chem. 282, 52 (2007), 18563-18572), through mathematical modeling, model fitting, and model validation. We analyze the quantitative contribution of each filament elongation strategy: with tetramers, with unit-length filaments, with longer filaments, or combinations thereof. In each case, we discuss the numerical fitting of the model with respect to one set of data, and its separate validation with respect to a second, different set of data. We introduce a high-resolution model for vimentin filament self-assembly, able to capture the detailed dynamics of filaments of arbitrary length. This provides much more predictive power for the model, in comparison to previous models where only the mean length of all filaments in the solution could be analyzed. We show how kinetic observations on low-resolution models can be extrapolated to the high-resolution model and used for lowering its complexity.     

PARAMETERS OF FILAMENT ELONGATION IN IPOMOEA NIL (CONVOLVULACEAE)

It has been thought for some time that morning glory filaments elongate in response to changes in concentrations of gibberellins (Murakami, 1973), but many other aspects of their growth have remained unstudied. In the present work, the interacting roles of gibberellin and ethylene in filament growth were examined. Filaments elongated ten-fold by epidermal cell elongation accompanied by ten-fold increases in fresh and dry weight. Applied gibberellins could stimulate filament growth in vitro, but gibberellin biosynthesis inhibitors had no effect. The putative gibberellin action inhibitor, ancymidol, reduced growth but the inhibition could be removed by blocking ethylene biosynthesis. Stimulators of the ethylene biosynthesis pathway and applied ethylene precursor (ACC) strongly inhibited filament elongation; ethylene biosynthesis inhibitors elicited as much growth as applied gibberellin. The filaments produced little ethylene at the time of the onset of growth. While the filaments produced ethylene rapidly before and after growth initiation, the closed flower bud had a relatively constant level of ethylene. It seems likely that in situ production of ethylene negatively influences filament growth.     

Hormonal influence on photocontrol of the protandry in the genus Helianthus

Under natural photoperiodic conditions protandry in hermaphrodite disc flowers of sunflower (Helianthus annuus L.) is determined by the different elongation rates of the style and filaments. The elongation of the filament and style starts simultaneously after the daily dark period, but the style growth rate is slower. When plants close to anthesis are exposed to continuous white light (WL) a loss of protandry occurs: the filaments do not grow far enough to extrude the anthers from the corolla. The histological analyses show that the number of filament epidermal cells remains unaltered after organ elongation and that cells respond to photoperiod only by cell expansion. Emasculation does not substantially inhibit filament cell expansion, whereas isolation of the filament or stamen from the corolla suggests that this organ could be the perception site of the filament growth stimulus. In vitro treatments with auxin (indole-3-acetic acid, IAA or alpha-naphthaleneacetic acid, NAA) reverses the inhibition of cell expansion caused by continuous WL, whereas gibberellic acid (GA(3)) at high concentrations reproduces the effect of continuous WL. Experiments carried out on various Helianthus spp. show that all these plants have evolved the same photo- and hormonal-control of the protandry. In experiments in which the light treatments were continued for 24 h, the auxins drastically reduced the inhibiting effect of red light (R) and dichromatic treatments FR (far red)+R, whereas GA(3) repressed filament extension regardless of light quality. As far as auxins are concerned, the response of sunflower filaments does not appear to be connected with the polar transport of the hormone. Moreover, the promoting effect of darkness is not mediated by an increase of endogenous free IAA in disc flowers. However, sunflower filaments manifested a similar temporal pattern of response to the light/dark cycle and to auxin.     

EMASCULATION EFFECTS ON THE STAMEN FILAMENT OF NIGELLA HTSPANICA AND THEIR PARTIAL REVERSAL BY GIBBERELLIC ACID

Anther removal from stamens whose filaments are 1–3 mm long restricts filament elongation to approximately 60% of the normal length. Removal of one-third to one-half of the anthers affects only the antherless filaments and does not appear to inhibit the growth of the other organs of the flower. Filament growth inhibition induced by anther removal involves both an inhibition of epidermal cell elongation along the length of the filament and also an inhibition of cell division. There is no evidence that the inhibition of filament growth is a response to damage caused by anther removal. Rather, it is suggested that anther removal severs a normal hormonal relationship existing between the anther and the developing filament. The application of gibberellic acid (GA₃) in lanolin paste stimulated the elongation of the antherless filaments to achieve an average of 87% of the filament length of adjacent intact stamens. The closer a filament is to having attained its final number of cells before anther removal, the closer does its length come to reaching the final length of filaments in intact stamens. The elongation of these antherless filaments with the application of GA₃ was accompanied by elongation of the epidermal cells of the filament to normal, or in some cases greater than normal, lengths. There is no evidence that GA₃ application affected the inhibition of epidermal cell devision induced by anther removal. The results of this study support the suggestion of Plack that emasculation-induced inhibition in the growth of floral organs and its reversal by GA₃ is a general phenomenon.     

A quantitative kinetic model for the in vitro assembly of intermediate filaments from tetrameric vimentin

In vitro assembly of intermediate filament proteins is a very rapid process. It starts without significant delay by lateral association of tetramer complexes into unit-length filaments (ULFs) after raising the ionic strength from low salt to physiological conditions (100 mM KCl). We employed electron and scanning force microscopy complemented by mathematical modeling to investigate the kinetics of in vitro assembly of human recombinant vimentin. From the average length distributions of the resulting filaments measured at increasing assembly times we simulated filament assembly and estimated specific reaction rate parameters. We modeled eight different potential pathways for vimentin filament elongation. Comparing the numerical with the experimental data we conclude that a two-step mechanism involving rapid formation of ULFs followed by ULF and filament annealing is the most robust scenario for vimentin assembly. These findings agree with the first two steps of the previously proposed three-step assembly model (Herrmann, H., and Aebi, U. (1998) Curr. Opin. Struct. Biol. 8, 177-185). In particular, our modeling clearly demonstrates that end-to-end annealing of ULFs and filaments is obligatory for forming long filaments, whereas tetramer addition to filament ends does not contribute significantly to filament elongation.     

Hydrostatic Pressure Shows That Lamellipodial Motility in Ascaris Sperm Requires Membrane-associated Major Sperm Protein Filament Nucleation and Elongation

Sperm from nematodes use a major sperm protein (MSP) cytoskeleton in place of an actin cytoskeleton to drive their ameboid locomotion. Motility is coupled to the assembly of MSP fibers near the leading edge of the pseudopod plasma membrane. This unique motility system has been reconstituted in vitro in cell-free extracts of sperm from Ascaris suum: inside-out vesicles derived from the plasma membrane trigger assembly of meshworks of MSP filaments, called fibers, that push the vesicle forward as they grow (Italiano, J.E., Jr., T.M. Roberts, M. Stewart, and C.A. Fontana. 1996. Cell. 84:105–114). We used changes in hydrostatic pressure within a microscope optical chamber to investigate the mechanism of assembly of the motile apparatus. The effects of pressure on the MSP cytoskeleton in vivo and in vitro were similar: pressures >50 atm slowed and >300 atm stopped fiber growth. We focused on the in vitro system to show that filament assembly occurs in the immediate vicinity of the vesicle. At 300 atm, fibers were stable, but vesicles often detached from the ends of fibers. When the pressure was dropped, normal fiber growth occurred from detached vesicles but the ends of fibers without vesicles did not grow. Below 300 atm, pressure modulates both the number of filaments assembled at the vesicle (proportional to fiber optical density and filament nucleation rate), and their rate of assembly (proportional to the rates of fiber growth and filament elongation). Thus, fiber growth is not simply because of the addition of subunits onto the ends of existing filaments, but rather is regulated by pressure-sensitive factors at or near the vesicle surface. Once a filament is incorporated into a fiber, its rates of addition and loss of subunits are very slow and disassembly occurs by pathways distinct from assembly. The effects of pressure on fiber assembly are sensitive to dilution of the extract but largely independent of MSP concentration, indicating that a cytosolic component other than MSP is required for vesicle-association filament nucleation and elongation. Based on these data we present a model for the mechanism of locomotion-associated MSP polymerization the principles of which may apply generally to the way cells assemble filaments locally to drive protrusion of the leading edge.     

ACID-INDUCED FILAMENT ELONGATION IN IPOMOEA NIL (CONVOLVULACEAE)

It has been known for some time that morning glory filaments elongate in response to increases in concentration of gibberellins (Murakami, 1973) and decreases in ethylene production (Koning and Raab, in press), but many other aspects of their growth have remained unstudied. In the present work, the possible role of gibberellin-stimulated proton efflux in filament growth was examined. Although applied gibberellins stimulated extensive filament growth in vitro and the pH of the incubating medium became acidified during growth, gibberellin also induced growth in media buffered at alkaline pH values. Acidic buffers alone elicited only a very small amount of growth. Fusicoccin, a potent stimulator of proton efflux, initially stimulated the rate of filament growth but elicited only a small increment of growth. In fact, continued presence of fusicoccin poisoned sustained gibberellin-induced growth. Vanadate ions, believed to inhibit proton efflux, had little effect upon gibberellin-induced growth except at extremely high concentrations. Based upon these results, it appears that the acid-induced component of growth stimulation by gibberellin is relatively minor in Ipomoea filaments. These results are quite different from those reported for filament elongation in Gaillardia (Koning, 1983a).     

Tropomodulin caps the pointed ends of actin filaments

Many proteins have been shown to cap the fast growing (barbed) ends of actin filaments, but none have been shown to block elongation and depolymerization at the slow growing (pointed) filament ends. Tropomodulin is a tropomyosin-binding protein originally isolated from red blood cells that has been localized by immunofluorescence staining to a site at or near the pointed ends of skeletal muscle thin filaments (Fowler, V. M., M. A., Sussman, P. G. Miller, B. E. Flucher, and M. P. Daniels. 1993. J. Cell Biol. 120: 411-420). Our experiments demonstrate that tropomodulin in conjunction with tropomyosin is a pointed end capping protein: it completely blocks both elongation and depolymerization at the pointed ends of tropomyosin-containing actin filaments in concentrations stoichiometric to the concentration of filament ends (Kd < or = 1 nM). In the absence of tropomyosin, tropomodulin acts as a "leaky" cap, partially inhibiting elongation and depolymerization at the pointed filament ends (Kd for inhibition of elongation = 0.1-0.4 microM). Thus, tropomodulin can bind directly to actin at the pointed filament end. Tropomodulin also doubles the critical concentration at the pointed ends of pure actin filaments without affecting either the rate of extent of polymerization at the barbed filament ends, indicating that tropomodulin does not sequester actin monomers. Our experiments provide direct biochemical evidence that tropomodulin binds to both the terminal tropomyosin and actin molecules at the pointed filament end, and is the long sought-after pointed end capping protein. We propose that tropomodulin plays a role in maintaining the narrow length distributions of the stable, tropomyosin-containing actin filaments in striated muscle and in red blood cells.     

THE ROLES OF AUXIN,ETHYLENE, AND ACID GROWTH IN FILAMENT ELONGATION IN GAILLARDIA GRANDIFLORA (ASTERACEAE)

Filament elongation and the role of auxin in this process in Gaillardia grandiflora was investigated. Filament elongation in vivo occurred just prior to anthesis and was accompanied by cell elongation and fresh weight increase. Filaments isolated and exposed to auxin in vitro grew more rapidly than controls and their growth was comparable to that of filaments in vivo. Furthermore, the natural auxin content of disc flowers (determined by double-standard isotope dilution analyses) increased just prior to anthesis and filament elongation. These results imply that auxin controls filament elongation. Applied ethylene slightly promoted filament elongation in vitro, and ethylene production of the flowers (determined by gas chromatography) slightly increased prior to filament growth. Fusicoccin and acidic buffers also stimulated elongation of isolated filaments. Thus, the role of auxin in controlling filament elongation in Gaillardia may involve stimulation of ethylene biosynthesis and acid growth.     

Ligand-dependent tau filament formation: implications for Alzheimer's disease progression.

The mechanism through which arachidonic acid induces the polymerization of tau protein into filaments under reducing conditions was characterized through a combination of fluorescence spectroscopy and electron microscopy. Results show that polymerization follows a ligand-mediated mechanism, where binding of arachidonic acid is an obligate step preceding tau-tau interaction. Homopolymerization begins with rapid (on the order of seconds) nucleation, followed by a slower elongation phase (on the order of hours). Although essentially all synthetic filaments have straight morphology at early time points, they interact with thioflavin-S and monoclonal antibody Alz50 much like authentic paired helical filaments, suggesting that the conformation of tau protein is similar in the two filament forms. Over a period of days, synthetic straight filaments gradually adopt paired helical morphology. These results define a novel pathway of tau filament formation under reducing conditions, where oxidation may contribute to final paired helical morphology, but is not a necessary prerequisite for efficient nucleation or elongation of tau filaments.     

Kinetic analysis of the growth rate of the flagellar hook in Salmonella typhimurium by the population balance method.

The growth rate of flagellar hooks in Salmonella typhimurium was analyzed by computer-aided simulation of the length distributions of mutant hooks of uncontrolled length (polyhooks). The wild-type hook has a relatively well-controlled length, with an average of 55 nm and a standard deviation of 6 nm. Mutations in the fliK gene give rise to polyhooks. A histogram of the lengths of polyhooks from a fliK mutant shows a peak at 55 nm with a long monotonic tail extending out to 1 microm. To analyze the growth rate, we employed the population balance method. Regression analysis showed that the histogram could fit a combination of two theoretical curves. In the first phase of growth, the hook starts with a very fast growth rate (40 nm/min), and then the rate exponentially slows until the length reaches 55 nm. In the second phase of growth, where the hook length is over 55 nm, the hook grows at a constant rate of 8 nm/min. Second mutations in either the fliK or flhB genes, as found in pseudorevertants from fliK mutants, give rise to polyhook filaments (phf). The ratio between the numbers of hooks with and without filament was 6:4. The calculated probability of filament attachment to polyhooks was low so that the proportion of hooks that start filament growth was only 2% per minute. The lengths of polyhooks with and without filaments were measured. A histogram of hook length in phf's was the same as that for polyhooks in single-site fliK mutants, against the expectation that the distribution would shift to a shorter average. The role of FliK in hook length control is discussed.     

Tropomyosin stabilizes the pointed end of actin filaments by slowing depolymerization

Tropomyosin is postulated to confer stability to actin filaments in nonmuscle cells. We have found that a nonmuscle tropomyosin isolated from the intestinal epithelium can directly stabilize actin filaments by slowing depolymerization from the pointed, or slow-growing, filament end. Kinetics of elongation and depolymerization from the pointed end were measured in fluorescence assays using pyrenylactin filaments capped at the barbed end by villin. The initial pointed end depolymerization rate in the presence of tropomyosin averaged 56% of the control rate. Elongation from the pointed filament end in the presence of tropomyosin occurred at a lower free G-actin concentration, although the on rate constant, kappa p+, was not greatly affected. Furthermore, in the presence of tropomyosin, the free G-actin concentration was lower at steady state. Therefore, nonmuscle tropomyosin stabilizes the pointed filament end by lowering the off rate constant, kappa p-.     

Filament ring formation in the dimorphic yeast Candida albicans

Stationary phase cultures of Candida albicans inoculated into fresh medium at 37 degrees C synchronously from buds at pH 4.5 and mycelia at pH 6.5. During bud formation, a filament ring forms just under the plasma membrane at the mother cell-bud junction at roughly the time of evagination. A filament ring also forms in mycelium-forming cells, but it appears later than in a budding cell and it is positioned along the elongating mycelium, on the average 2 microns from the mother cell-mycelium junction. Sections of filament rings in early and late budding cells and in mycelia appear similar. Each contains approximately 11 to 12 filaments equidistant from one another and closely associated with the plasma membrane. In both budding and mycelium-forming cells, the filament ring disappears when the primary septum grows inward. The close temporal and spatial association of the filament ring and the subsequent chitin-containing septum suggests a role for the filament ring in septum formation. In addition, a close temporal correlation is demonstrated between filament ring formation and the time at which cells become committed to bud formation at pH 4.5 and mycelium formation at pH 6.5. The temporal and spatial differences in filament ring formation between the two growth forms also suggest a simple model for the positioning of the filament ring.     

Tropomyosin inhibits the rate of actin polymerization by stabilizing actin filaments

Tropomyosin inhibition of the rate of spontaneous polymerization of actin is associated with binding of tropomyosin to actin filaments. Rate constants determined by using a direct electron microscopic assay of elongation showed that alpha alpha- and alpha beta-tropomyosin have a small or no effect on the rate of elongation at either end of the filaments. The most likely explanation for the inhibition of the rate of polymerization of actin in bulk samples is that tropomyosin reduces the number of filament ends by mechanical stabilization of the filaments.