The structure of the F-actin filament and the actin molecule.

A consensus view on the three-dimensional structure of the F-actin filament and the relative strength of the intersubunit contacts in the filament has been established from an atomic filament model and recent three-dimensional reconstructions from electron micrographs of F-actin filaments. Functional implications of recent structural and biochemical data indicating a rather dynamic filament structure are discussed.     

Tracer diffusion through F-actin: effect of filament length and cross-linking.

We have determined diffusion coefficients for small (50- to 70-nm diameter) fluorescein-thiocarbamoyl-labeled Ficoll tracers through F-actin as a function of filament length and cross-linking. fx45 was used to regulate filament length and avidin/biotinylated actin or ABP-280 was used to prepare cross-linked actin gels. We found that tracer diffusion was generally independent of filament length in agreement with theoretical predictions for diffusion through solutions of rods. However, in some experiments diffusion was slower through short (< or = 1.0 micron) filaments, although this result was not consistently reproducible. Measured diffusion coefficients through unregulated F-actin and filaments of lengths > 1.0 micron were more rapid than predicted by theory for tracer diffusion through rigid, random networks, which was consistent with some degree of actin bundling. Avidin-induced cross-linking of biotinylated F-actin did not affect diffusion through unregulated F-actin, but in cases where diffusion was slower through short filaments this cross-linking method resulted in enhanced tracer diffusion rates indistinguishable from unregulated F-actin. This finding, in conjunction with increased turbidity of 1.0-micron filaments upon avidin cross-linking, indicated that this cross-linking method induces F-actin bundling. By contrast, ABP-280 cross-linking retarded diffusion through unregulated F-actin and decreased turbidity. Tracer diffusion under these conditions was well approximated by the diffusion theory. Both cross-linking procedures resulted in gel formation as determined by falling ball viscometry. These results demonstrate that network microscopic geometry is dependent on the cross-linking method, although both methods markedly increase F-actin macroscopic viscosity.     

Coronin-1A stabilizes F-actin by bridging adjacent actin protomers and stapling opposite strands of the actin filament

Coronins are F-actin-binding proteins that are involved, in concert with Arp2/3, Aip1, and ADF/cofilin, in rearrangements of the actin cytoskeleton. An understanding of coronin function has been hampered by the absence of any structural data on its interaction with actin. Using electron microscopy and three-dimensional reconstruction, we show that coronin-1A binds to three protomers in F-actin simultaneously: it bridges subdomain 1 and subdomain 2 of two adjacent actin subunits along the same long-pitch strand, and it staples subdomain 1 and subdomain 4 of two actin protomers on different strands. Such a mode of binding explains how coronin can stabilize actin filaments in vitro. In addition, we show which residues of F-actin may participate in the interaction with coronin-1A. Human nebulin and Xin, as well as Salmonella invasion protein A, use a similar mechanism to stabilize actin filaments. We suggest that the stapling of subdomain 1 and subdomain 4 of two actin protomers on different strands is a common mechanism for F-actin stabilization utilized by many actin-binding proteins that have no homology.     

Actin filament attachments for sustained motility in vitro are maintained by filament bundling

We reconstructed cellular motility in vitro from individual proteins to investigate how actin filaments are organized at the leading edge. Using total internal reflection fluorescence microscopy of actin filaments, we tested how profilin, Arp2/3, and capping protein (CP) function together to propel thin glass nanofibers or beads coated with N-WASP WCA domains. Thin nanofibers produced wide comet tails that showed more structural variation in actin filament organization than did bead substrates. During sustained motility, physiological concentrations of Mg(2+) generated actin filament bundles that processively attached to the nanofiber. Reduction of total Mg(2+) abolished particle motility and actin attachment to the particle surface without affecting actin polymerization, Arp2/3 nucleation, or filament capping. Analysis of similar motility of microspheres showed that loss of filament bundling did not affect actin shell formation or symmetry breaking but eliminated sustained attachments between the comet tail and the particle surface. Addition of Mg(2+), Lys-Lys(2+), or fascin restored both comet tail attachment and sustained particle motility in low Mg(2+) buffers. TIRF microscopic analysis of filaments captured by WCA-coated beads in the absence of Arp2/3, profilin, and CP showed that filament bundling by polycation or fascin addition increased barbed end capture by WCA domains. We propose a model in which CP directs barbed ends toward the leading edge and polycation-induced filament bundling sustains processive barbed end attachment to the leading edge.     

Coincident filament distribution and histochemical localization of F-actin in the enterocytes of the larval dragonflyAeshna cyanea

Summary The fluorescence pattern of the enterocytes of larvalAeshna cyanea obtained by the incubation of cryosections with rhodaminephalloidin, a histochemical probe for F-actin, exactly coincides with the distribution of 6 nm filaments. These prevail as 1. core bundles in the microvilli 2. terminal web including the rootlets of the core bundles and and filaments associated with the adhesive junctions, 3. intercisternal arrays in the subapical stacks of smooth endoplasmic reticulum, and 4. cortical web underneath the basolateral plasma membranes.Abbreviations EDTA ethylenediaminetetraacetic acid - HMM heavy meromyosin - PBS phosphate-buffered saline - PIPES piperazine-N, N-bis (2-ethanesulfonic acid) - rER and sER rough and smooth endoplasmic reticulum     

Scinderin and cortical F-actin are components of the secretory machinery

Secretory vesicle exocytosis is the mechanism of release of neurotransmitters and neuropeptides. Secretory vesicles are localized in at least two morphologically and functionally distinct compartments: the reserve pool and the release-ready pool. Filamentous actin networks play an important role in this compartmentalization and in the trafficking of vesicles between these compartments. The cortical F-actin network constitutes a barrier (negative clamp) to the movement of secretory vesicles to release sites, and it must be locally disassembled to allow translocation of secretory vesicles in preparation for exocytosis. The disassembly of the cortical F-actin network is controlled by scinderin (a Ca(2+)-dependent F-actin severing protein) upon activation by Ca2+ entering the cells during stimulation. There are several factors that regulate scinderin activation (i.e., Ca2+ levels, phosphatidylinositol 4,5-bisphosphate (PIP2), etc.). The results suggest that scinderin and the cortical F-actin network are components of the secretory machinery.     

Protein bodies in corn endosperm are enclosed by and enmeshed in F-actin

Summary When corn endosperm is gently homogenized in a microfilament-stabilizing buffer and filtrates centrifuged at very low g forces, abundant protein bodies are present in the pellet. When stained with both rhodamine-phalloidin and fluorescein-S₁-myosin and viewed under epifluorescence, individual protein bodies appear to be enclosed in F-actin and groups of protein bodies appear to be enmeshed within a network of fine actin filaments. The yield of actinenclosed protein bodies is not affected by the addition of non-ionic detergents to the grinding medium, but it is severely decreased with the addition of cytoskeleton-disrupting agents including Tris-HCl, KCl, KI, and ammonium sulphate, and the interconnecting fine filaments are totally abolished. Heparin, which interacts with membrane-cytoskeleton complexes, causes apparent coagulation of the protein bodies. The evidence from fluorescent microscopy suggesting that F-actin surrounds and enmeshes the protein bodies is supported by electron microscopy, gel electrophoresis, and Western blot analysis.Abbreviations BSA bovine serum albumin - DTE dithioerythritol - EGTA ethylehe-glycol-bis(B-aminoethyl ether) N, N, N, N-tetraacetic acid - HEPES (N-[hydroxyethylpiperazine-N-[2-ethanesulfonic acid]) - HMM heavy meromyosin - MSB microfilament-stabilizing buffer - PMSF phenyl-methylsulfonyl fluoride - PTA phospho-tungstic acid - PTE polyoxyethylene-10-tridecyl ether - Tris Tris-(hydroxymethyl) aminomethaneM     

Pure F-actin networks are distorted and branched by steps in the critical-point drying method

Elucidation of the ultrastructural organization of actin networks is crucial for understanding the molecular mechanisms underlying actin-based motility. Results obtained from cytoskeletons and actin comets prepared by the critical-point procedure, followed by rotary shadowing, support recent models incorporating actin filament branching as a main feature of lamellipodia and pathogen propulsion. Since actin branches were not evident in earlier images obtained by negative staining, we explored how these differences arise. Accordingly, we have followed the structural fate of dense networks of pure actin filaments subjected to steps of the critical-point drying protocol. The filament networks have been visualized in parallel by both cryo-electron microscopy and negative staining. Our results demonstrate the selective creation of branches and other artificial structures in pure F-actin networks by the critical-point procedure and challenge the reliability of this method for preserving the detailed organization of actin assemblies that drive motility.     

Sucrose synthase oligomerization and F-actin association are regulated by sucrose concentration and phosphorylation

Sucrose synthase (SUS) is a key enzyme in plant metabolism, as it serves to cleave the photosynthetic end-product sucrose into UDP-glucose and fructose. SUS is generally assumed to be a tetrameric protein, but results in the present study suggest that SUS can form dimers as well as tetramers and that sucrose may be a regulatory factor for the oligomerization status of SUS. The oligomerization of SUS may also affect the cellular localization of the protein. We show that sucrose concentration modulates the ability of SUS1 to associate with F-actin in vitro and that calcium-dependent protein kinase-mediated phosphorylation of recombinant SUS1 at the Ser15 site is a negative regulator of its association with actin. Although high sucrose concentrations and hyperphosphorylation have been shown to promote SUS association with the plasma membrane, we show that the opposite is true for the SUS-actin association. We also show that SUS1 has a unique 28 residue coiled-coil domain that does not appear to play a role in oligomerization, but may prove to be significant in the future for interactions of SUS with other proteins. Collectively, these results highlight the multifaceted nature of SUS association with cellular structures.     

Intermediate filament structure: 2. Molecular interactions in the filament

Molecules of intermediate filament (IF) proteins contain a central rod domain in which the two constituent chains have a predominantly α-helical conformation and are coiled around one another to form segments of two-strand rope. Possible interactions between the two long segments, termed 1B and 2 were investigated by a technique successfully employed in studies of the modes of association of collagen molecules by Miller and coworkers. Prominent maxima were found in all of the six possible modes of association between the rod domain segments in individual IF proteins and certain maxima were found to be common to all IF. The surface lattice of the IF from α-keratin has been determined and possible bonding arrangements between the rod-domain segments are catalogued. A systematic search was carried out for combinations of interaction maxima which were consistent with the dimensions of the surface lattice. By the further application of stereochemical constraints, models for the topological arrangement of the rod-domain segments on the surface lattice were derived and these are illustrated and discussed.     

Molecular determination by electron microscopy of the actin filament end structure

In eukaryotic cells, actin filaments play various crucial roles by altering their spatial and temporal distributions in the cell. The distribution of actin filaments is regulated by the binding of end-binding proteins, including capping protein (CapZ in muscle), the Arp2/3 complex, gelsolin, formin and tropomodulin, to the end of the actin filament. In order to determine the nature of these regulations, structural elucidations of actin filament-end-binding protein complexes are crucially important. Here, we have developed new procedures on the basis of single-particle analysis to determine the structure of the end of actin filaments from electron micrographs. In these procedures, the polarity of the actin filament image, as well as the azimuth orientation and the axial position of each actin protomer within a short stretch near the filament end, were determined accurately. This improved both the stability and accuracy of the structural determination dramatically. We tested our procedures by reconstructing structures from simulated filament images, which were obtained from 24 model structures for the actin-CapZ complex. These model structures were generated by random docking of the atomic structure of CapZ to the barbed end of an atomic model of the actin filament. Of the 24 model structures, 23 were recovered correctly by the present procedures. We found that our analysis was robust against local aberrations of the helical twist near the end of the actin filament. Finally, the procedures were applied successfully to determine the structure of the actin-CapZ complex from real cryo-electron micrographs of the complex. This is the first method for elucidating the detailed 3D structures at the end of the actin filament.     

Insights into enzyme structure and dynamics elucidated by amide H/D exchange mass spectrometry

Conformational changes and protein dynamics play an important role in the catalytic efficiency of enzymes. Amide H/D exchange mass spectrometry (H/D exchange MS) is emerging as an efficient technique to study the local and global changes in protein structure and dynamics due to ligand binding, protein activation-inactivation by modification, and protein-protein interactions. By monitoring the selective exchange of hydrogen for deuterium along a peptide backbone, this sensitive technique probes protein motions and structural elements that may be relevant to allostery and function. In this report, several applications of H/D exchange MS are presented which demonstrate the unique capability of amide hydrogen/deuterium exchange mass spectrometry for examining dynamic and structural changes associated with enzyme catalysis.     

Kinetic analysis of F-actin depolymerization in polymorphonuclear leukocyte lysates indicates that chemoattractant stimulation increases actin filament number without altering the filament length distribution

The rate of filamentous actin (F-actin) depolymerization is proportional to the number of filaments depolarizing and changes in the rate are proportional to changes in filament number. To determine the number and length of actin filaments in polymorphonuclear leukocytes and the change in filament number and length that occurs during the increase in F-actin upon chemoattractant stimulation, the time course of cellular F-actin depolymerization in lysates of control and peptide-stimulated cells was examined. F-actin was quantified by the TRITC-labeled phalloidin staining of pelletable actin. Lysis in 1.2 M KCl and 10 microM DNase I minimized the effects of F-actin binding proteins and G-actin, respectively, on the kinetics of depolymerization. To determine filament number and length from a depolymerization time course, depolymerization kinetics must be limited by the actin monomer dissociation rate. Comparison of time courses of depolymerization in the presence (pointed ends free) or absence (barbed and pointed ends free) of cytochalasin suggested depolymerization occurred from both ends of the filament and that monomer dissociation was rate limiting. Control cells had 1.7 +/- 0.4 x 10(5) filaments with an average length of 0.29 +/- 0.09 microns. Chemo-attractant stimulation for 90 s at room temperature with 0.02 microM N-formylnorleucylleucylphenylalanine caused a twofold increase in F-actin and about a two-fold increase in the total number of actin filaments to 4.0 +/- 0.5 x 10(5) filaments with an average length of 0.27 +/- 0.07 microns. In both cases, most (approximately 80%) of the filaments were quite short (less than or equal to 0.18 micron). The length distributions of actin filaments in stimulated and control cells were similar.     

Helical structure of the human umbilical cord.

The helical structure of the human umbilical cord has been studied on 528 full-term cords from normal deliveries, 18 cords from aborted and developmentally normal fetuses with a CR length of 1.2-19.5 cm, 10 cords from monozygotic twins. In order to resolve the discrepancy in the nomenclature of the twist direction, a method has been suggested which takes into account the direction to which the fetus must have rotated to produce the twist. The incidence of the right helical pattern was 64.58%, of the left helical pattern 15.15%, and of the mixed patterns 17.43%; the twists were indeterminate in 1.89% and absent in 0.95% of the cords. Details of the mixed patterns are listed. The number of the uniform right twists ranged from 1 to 29 (7.5) and that of the left twists from 1 to 19 (6.7). There exists an exponential relation between the number of the twists and the ratio between length and thickness of the cords. The twists begin to appear during the early part of the 8th week, and their final number is possibly attained soon after the 9th week of development. In view of the absence of concordance in monozygotic twins, the helical nature of the cord is possibly controlled by factors which may be partly genetic and partly environmental.     

Effect of the length and effective diameter of F-actin on the filament orientation in liquid crystalline sols measured by x-ray fiber diffraction.

We examined factors that affect the filament orientation in F-actin sols to prepare highly well-oriented liquid crystalline sols suitable for x-ray fiber diffraction structure analysis. Filamentous particles such as F-actin spontaneously align with one another when concentrated above a certain threshold concentration. This alignment is attributed to the excluded volume effect of the particles. In trying to improve the orientation of F-actin sols, we focused on the excluded volume to see how it affects the alignment. The achievable orientation was sensitive to the ionic strength of the solvent; the filaments were better oriented at lower ionic strengths, where the effective diameter of the filament is relatively large. Sols of longer filaments were better oriented than those of shorter filaments at the same concentration, but the best achievable orientation was limited, probably because of the filament flexibility. The best strategy for making well-oriented F-actin sols is therefore to concentrate F-actin filaments of relatively short length (<1 micrometer) by slow centrifugation in a low-ionic-strength solvent (<30 mM).