Molecular dynamics study of interactions between polymorphic actin filaments and gelsolin segment-1

The assembly of protein actin into double-helical filaments promotes many eukaryotic cellular processes that are regulated by actin-binding proteins (ABPs). Actin filaments can adopt multiple conformations, known as structural polymorphism, which possibly influences the interaction between filaments and ABPs. Gelsolin is a Ca²⁺-regulated ABP that severs and caps actin filaments. Gelsolin binding modulates filament structure; however, it is not known how polymorphic actin filament structures influence an interaction of gelsolin S1 with the barbed-end of filament. Herein, we investigated how polymorphic structures of actin filaments affect the interactions near interfaces between the gelsolin segment 1 (S1) domain and the filament barbed-end. Using all-atom molecular dynamics simulations, we demonstrate that different tilted states of subunits modulate gelsolin S1 interactions with the barbed-end of polymorphic filaments. Hydrogen bonding and interaction energy at the filament-gelsolin S1 interface indicate distinct conformations of filament barbed ends, resulting in different interactions of gelsolin S1. This study demonstrates that filament's structural multiplicity plays important roles in the interactions of actin with ABPs.     

Identification and purification of actin from the subpellicular network of Toxoplasma gondii tachyzoites

Toxoplasma gondii infects cells through dynamic events dependent on actin. Although the presence of cortical actin has been widely suggested, visualisation and localisation of actin filaments has not been reported. The subpellicular cytoskeleton network is a recently described structure possibly involved in the dynamic events. Using non-ionic detergent extractions, the cortical cytoskeleton network was enriched and used for the isolation and identification of actin. Actin was detected by Western blots in extracts of cytoskeleton networks, and it was localised by gold staining in the network and in both the apical end and the posterior polar ring. Actin was isolated from subpellicular cytoskeleton extracts by binding to DNase I, and it polymerised in vitro as filaments that were gold-decorated by a monoclonal anti-actin antibody. Filaments bound the subfragment 1 of heavy meromyosin, although with atypical arrangements in comparison with the arrowheads observed in muscle actin filaments. Treatment with cytochalasin D and colchicine altered the structural organisation of the subpellicular network indicating the participation of actin filaments and microtubules in the maintenance of its structure. Actin filaments and microtubules, in the subpellicular network, participate reciprocally in the maintaining of the parasite's shape and the gliding motility.     

Conformation-specific antibodies reveal distinct actin structures in the nucleus and the cytoplasm

For many years the existence of actin in the nucleus has been doubted because of the lack of phalloidin staining as well as the failure to document nuclear actin filaments by electron microscopy. More recent findings reveal actin to be a component of chromatin remodeling complexes and of the machinery involved in RNA synthesis and transport. With distinct functions for nuclear actin emerging, the quest for its conformation and oligomeric/polymeric structure in the nucleus has resumed importance. We used chemically cross-linked 'lower dimer' (LD) to generate mouse monoclonal antibodies specific for different actin conformations. One of the resulting antibodies, termed 1C7, recognizes an epitope that is buried in the F-actin filament, but is surface-exposed in G-actin as well as in the LD. In immunofluorescence studies with different cell lines, 1C7 selectively reacts with non-filamentous actin in the cytoplasm. In addition, it detects a discrete form of actin in the nucleus, which is different from the nuclear actin revealed by the previously described 2G2 [Gonsior, S.M., Platz, S., Buchmeier, S., Scheer, U., Jockusch, B.M., Hinssen, H., 1999. J. Cell Sci. 112, 797]. Upon latrunculin-induced disassembly of the filamentous cytoskeleton in Rat2 fibroblasts, we observed a perinuclear accumulation of the 1C7-reactive actin conformation. In addition, latrunculin treatment led to the assembly of phalloidin-staining actin structures in chromatin-free regions of the nucleus in these cells. Our results indicate that distinct actin conformations and/or structures are present in the nucleus and the cytoplasm of different cell types and that their distribution varies in response to external signals.     

黄蝉和姜花生殖细胞内肌动蛋白微丝的定位

用荧光标记的鬼笔碱染色,对离体的黄蝉和姜花的生殖细胞内肌动蛋白微丝的分布进行了研究,结果证明两种植物的生殖细胞内部都存在一个微丝网络,黄蝉生殖细胞的比姜花的简单,微丝束较粗。但姜花生殖细胞的网络微丝束比黄蝉的更紧密地环绕着核。用免疫荧光技术在黄蝉生殖细胞的分裂前期和中期,可以观察到一些微丝束的存在,但在分裂后期和末期细胞内的肌动蛋白则变为颗粒状。     

Correlative immuno-electron-microscopic and immunofluorescent localization of actin in sensory and supporting cells of the inner ear by use of a low-temperature embedding resin

Summary The cochleas from chinchilla inner ears were processed in the cold through Lowicryl K4M, and cured by UV light. Thick (2 m) sections were reacted with primary antibodies raised against actin, and anti-actin antibodies localized by FITC epifluorescence. On thin sections from the same blocks anti-actin antibodies were localized ultrastructurally with secondary antibodies coupled to colloidal gold.In the hair cells, actin was present in the stereocilia and cuticular plate, regions where thin filaments were observed by electron microscopy. Colloidal gold was uniformly distributed over these regions and over the stereocilia rootlets demonstrating that actin was present in this region although previously in permeabilized cells, the rootlet was not decorated with myosin subfragment S-1. Actin was present in the pillar and Deiters supporting cells at the reticular lamina and at the basilar membrane, where a meshwork of thin filaments was seen by electron microscopy. Colloidal gold particles were also localized over the thin processes of the pillar and Deiters cells, and over the region of the Deiters cell which envelops the base of the outer hair cell. In these regions actin co-localized with microtubules along the entire length of the supporting cells.     

Structural interaction of cytoskeletal components

Three-dimensional cytoskeletal organization of detergent-treated epithelial African green monkey kidney cells (BSC-1) and chick embryo fibroblasts was studied in whole-mount preparations visualized in a high voltage electron microscope. Stereo images are generated at both low and high magnification to reveal both overall cytoskeletal morphology and details of the structural continuity of different filament types. By the use of an improved extraction procedure in combination with heavy meromyosin subfragment 1 decoration of actin filaments, several new features of filament organization are revealed that suggest that the cytoskeleton is a highly interconnected structural unit. In addition to actin filaments, intermediate filaments, and microtubules, a new class of filaments of 2- to 3-nm diameter and 30- to 300-nm length that do not bind heavy merymyosin is demonstrated. They form end-to-side contacts with other cytoskeletal filaments, thereby acting as linkers between various fibers, both like (e.g., actin- actin) and unlike (e.g., actin-intermediate filament, intermediate filament-microtubule). Their nature is unknown. In addition to 2- to 3-nm filaments, actin filaments are demonstrated to form end-to-side contacts with other filaments. Y-shaped actin filament “branches” are observed both in the cell periphery close to ruffles and in more central cell areas also populated by abundant intermediate filaments and microtubules. Arrowhead complexes formed by subfragment 1 decoration of actin filaments point towards the contact site. Actin filaments also form end-to-side contacts with microtubules and intermediate filaments. Careful inspection of numerous actin-microtubule contacts shows that microtubules frequently change their course at sites of contact. A variety of experimentally induced modifications of the frequency of actin-microtubule contacts can be shown to influence the course of microtubules. We conclude that bends in microtubules are imposed by structural interactions with other cytoskeletal elements. A structural and biochemical comparison of whole cells and cytoskeletons demonstrates that the former show a more inticate three-dimensional network and a more complex biochemical composition than the latter. An analysis of the time course of detergent extraction strongly suggests that the cytoskeleton forms a structural backbone with which a large number of proteins of the cytoplasmic ground substance associate in an ordered fashion to form the characteristic image of the “microtrabecular network” (J.J. Wolosewick and K.R. Porter. 1979. J. Cell Biol. 82: 114-139).     

Anti-actin antibodies generated against profilin:actin distinguish between non-filamentous and filamentous actin, and label cultured cells in a dotted pattern

Actin polymerization is a prominent feature of migrating cells, where it powers the protrusion of the leading edge. Many studies have characterized the well-ordered and dynamic arrangement of filamentous actin in this submembraneous space. However, less is known about the organization of unpolymerized actin. Previously, we reported on the use of covalently coupled profilin:actin to study actin dynamics and presented evidence that profilin-bound actin is a major source of actin for filament growth. To locate profilin:actin in the cell we have now used this non-dissociable complex for antibody generation, and obtained monospecific anti-actin and anti-profilin antibodies from two separate immunizations. Fluorescence microscopy revealed drastic differences in the staining pattern generated by the anti-actin antibody preparations. With one, distinct puncta appeared at the actin-rich leading edge and sometimes aligned with microtubules in the interior of the lamella, while the other displayed typical actin filament staining. Labelling experiments in vitro demonstrated failure of the first antibody to recognize filamentous actin and none of the two bound microtubules. The two anti-profilin antibodies purified in parallel generated a punctated pattern similar to that seen with the first anti-actin antibody. All antibody preparations labelled the nuclei.     

Crystallographic conformers of actin in a biologically active bundle of filaments

Actin carries out many of its cellular functions through its filamentous form; thus, understanding the detailed structure of actin filaments is an essential step in achieving a mechanistic understanding of actin function. The acrosomal bundle in the Limulus sperm has been shown to be a quasi-crystalline array with an asymmetric unit composed of a filament with 14 actin-scruin pairs. The bundle in its true discharge state penetrates the jelly coat of the egg. Our previous electron crystallographic reconstruction demonstrated that the actin filament cross-linked by scruin in this acrosomal bundle state deviates significantly from a perfect F-actin helix. In that study, the tertiary structure of each of the 14 actin protomers in the asymmetric unit of the bundle filament was assumed to be constant. In the current study, an actin filament atomic model in the acrosomal bundle has been refined by combining rigid-body docking with multiple actin crystal structures from the Protein Data Bank and constrained energy minimization. Our observation demonstrates that actin protomers adopt different tertiary conformations when they form an actin filament in the bundle. The scruin and bundle packing forces appear to influence the tertiary and quaternary conformations of actin in the filament of this biologically active bundle.     

Yeast actin patches are networks of branched actin filaments

Cortical actin patches are the most prominent actin structure in budding and fission yeast. Patches assemble, move, and disassemble rapidly. We investigated the mechanisms underlying patch actin assembly and motility by studying actin filament ultrastructure within a patch. Actin patches were partially purified from Saccharomyces cerevisiae and examined by negative-stain electron microscopy (EM). To identify patches in the EM, we correlated fluorescence and EM images of GFP-labeled patches. Patches contained a network of actin filaments with branches characteristic of Arp2/3 complex. An average patch contained 85 filaments. The average filament was only 50-nm (20 actin subunits) long, and the filament to branch ratio was 3:1. Patches lacking Sac6/fimbrin were unstable, and patches lacking capping protein were relatively normal. Our results are consistent with Arp2/3 complex-mediated actin polymerization driving yeast actin patch assembly and motility, as described by a variation of the dendritic nucleation model.     

Cytoskeleton and vesicle mobility in astrocytes

Exocytotic vesicles in astrocytes are increasingly viewed as essential in astrocyte-to-neuron communication in the brain. In neurons and excitable secretory cells, delivery of vesicles to the plasma membrane for exocytosis involves an interaction with the cytoskeleton, in particular microtubules and actin filaments. Whether cytoskeletal elements affect vesicle mobility in astrocytes is unknown. We labeled single vesicles with fluorescent atrial natriuretic peptide and monitored their mobility in rat astrocytes with depolymerized microtubules, actin, and intermediate filaments and in mouse astrocytes deficient in the intermediate filament proteins glial fibrillary acidic protein and vimentin. In astrocytes, as in neurons, microtubules participated in directional vesicle mobility, and actin filaments played an important role in this process. Depolymerization of intermediate filaments strongly affected vesicle trafficking and in their absence the fraction of vesicles with directional mobility was reduced.     

Actin and Arp2/3 localize at the centrosome of interphase cells

Although many actin binding proteins such as cortactin and the Arp2/3 activator WASH localize at the centrosome, the presence and conformation of actin at the centrosome has remained elusive. Here, we report the localization of actin at the centrosome in interphase but not in mitotic MDA-MB-231 cells. Centrosomal actin was detected with the anti-actin antibody 1C7 that recognizes antiparallel (“lower dimer”) actin dimers. In addition, we report the transient presence of the Arp2/3 complex at the pericentriolar matrix but not at the centrioles of interphase HEK 293T cells. Overexpression of an Arp2/3 component resulted in expansion of the pericentriolar matrix and selective accumulation of the Arp2/3 component in the pericentriolar matrix. Altogether, we hypothesize that the centrosome transiently recruits Arp2/3 to perform processes such as centrosome separation prior to mitotic entry, whereas the observed constitutive centrosomal actin staining in interphase cells reinforces the current model of actin-based centrosome reorientation toward the leading edge in migrating cells.     

Purification of cytoplasmic actin by affinity chromatography using the C-terminal half of gelsolin

A new rapid method of the cytoplasmic actin purification, not requiring the use of denaturants or high concentrations of salt, was developed, based on the affinity chromatography using the C-terminal half of gelsolin (G4-6), an actin filament severing and capping protein. When G4-6 expressed in Escherichia coli was added to the lysate of HeLa cells or insect cells infected with a baculovirus encoding the beta-actin gene, in the presence of Ca²⁺ and incubated overnight at 4 °C, actin and G4-6 were both detected in the supernatant. Following the addition of Ni-Sepharose beads to the mixture, only actin was eluted from the Ni-NTA column by a Ca²⁺-chelating solution. The functionality of the cytoplasmic actins thus purified was confirmed by measuring the rate of actin polymerization, the gliding velocity of actin filaments in an in vitro motility assay on myosin V-HMM, and the ability to activate the ATPase activity of myosin V-S1.     

Actin filaments in microridges of the oral mucosal epithelium in the carp Cyprinus carpio

Summary Actin filaments in the microridges on the surface of the fish oral mucosa taken from Cyprinus carpio were examined by electron microscopy after detergent extraction and decoration with myosin subfragment 1. After extraction with saponin, an irregular and densely packed meshwork of actin filaments was observed in the bases of the microridges, just lateral to the tight junctions with their fibrous undercoats. Actin filaments formed cores in the microridges and numerous linkages were seen between the filaments and the plasma membrane. Extraction with Triton X-100 and decoration with myosin subfragment 1 showed the ends of the actin filaments to be associated with the plasma membrane of the microridges, and in the bases of microridges the filament ends were anchored to intermediate filaments. Some actin filaments interconnected with the fibrous undercoats of the tight junctions. On the basis of these observations, the mechanism of the formation of microridges, including their pattern, is discussed.     

Characterization of filaments within the subacrosomal space of rat spermatids during spermiogenesis

Two types of filaments were observed within the subacrosomal space of rat spermatids. The first of these types was characterized as actin by demonstration of actin filament affinity for myosin S-1 subfragments. Actin filaments were noted in the subacrosomal space shortly after the acrosomal sac made contact with the nucleus. As the acrosome increased its surface area contact with the spermatid nucleus, the number of layers of subacrosomal filaments increased. Pre-treatment with detergent, which in addition to permeablizing cells to allow entry of S-1, also caused the acrosome to vesiculate and the subacrosomal space to widen. In such preparations filaments were more easily visualized and appeared to extend between the nuclear and acrosomal membranes, indicating, but not proving, attachment to these membranes. During spermatid clongation, the number of actin filaments in the subacrosomal space increased greatly, especially over the dorsal convex region of the spermatid head. The polarity of the majority of filaments was not ascertainable since filaments were tightly packed within the narrow subacrosomal space. In late spermiogenesis (steps 18 and 19), actin filaments were no longer detected within the subacrosomal space. A second and much thicker type of filamentous structure was observed in the subacrosomal space of spermatids at steps 14-17 of spermiogenesis. About 14 nm in diameter (10-15 nm measurement range depending on fixation protocol utilized), these filaments did not decorate with myosin S-1 subfragments and were found in subacrosomal regions not containing actin. Fourteen nanometer filaments were seen in parallel array along the ventral folded portion of the nuclear membrane and extended partially around the nucleus. Like actin filaments. 14 nm filaments were not seen in the subacrosomal space during late spermiogenesis.     

Chlamydia trachomatis Tarp Harbors Distinct G and F Actin Binding Domains That Bundle Actin Filaments

All species of Chlamydia undergo a unique developmental cycle that transitions between extracellular and intracellular environments and requires the capacity to invade new cells for dissemination. A chlamydial protein called Tarp has been shown to nucleate actin in vitro and is implicated in bacterial entry into human cells. Colocalization studies of ectopically expressed enhanced green fluorescent protein (EGFP)-Tarp indicate that actin filament recruitment is restricted to the C-terminal half of the effector protein. Actin filaments are presumably associated with Tarp via an actin binding alpha helix that is also required for actin nucleation in vitro, but this has not been investigated. Tarp orthologs from C. pneumoniae, C. muridarum, and C. caviae harbor between 1 and 4 actin binding domains located in the C-terminal half of the protein, but C. trachomatis serovar L2 has only one characterized domain. In this work, we examined the effects of domain-specific mutations on actin filament colocalization with EGFP-Tarp. We now demonstrate that actin filament colocalization with Tarp is dependent on two novel F-actin binding domains that endow the Tarp effector with actin-bundling activity. Furthermore, Tarp-mediated actin bundling did not require actin nucleation, as the ability to bundle actin filaments was observed in mutant Tarp proteins deficient in actin nucleation. These data shed molecular insight on the complex cytoskeletal rearrangements required for C. trachomatis entry into host cells.     

Cortical fine actin filaments in higher plant cells visualized by rhodamine-phalloidin after pretreatment with m-maleimidobenzoyl N-hydroxysuccinimide ester

Summary Actin filaments in cultured tobacco cells were stained by rhodamine-phalloidin after pretreatment with 100 M m-maleidobenzoyl N-hydroxysuccinimide ester (MBS) followed by formaldehyde fixation. The use of MBS prior to formaldehyde fixation enabled us to visualize fine, transversely arranged cortical actin filaments in a majority of interphase tobacco cells. It also enabled us to double-stain fine actin filaments and microtubules in the same cells. The pattern of actin filaments and that of microtubules in the cortical region of a single tobacco cell bore a close resemblance to each other. The method which employed MBS was found to be useful also in visualizing fine cortical actin filaments in inner epidermal cells of onion bulbs.Rhodamine-phalloidin seemed to induce the bundling of actin filaments both tobacco cells and in onion cells when it was applied to the cells which had not been subjected to fixation, indicating that the application of fluorescent-dye-labeled phallotoxins to unfixed cells involves the risk of observing artifically bundled actin filaments.     

Localization of actin filaments on mitotic apparatus in tobacco BY-2 cells

Actin filaments are among the major components of the cytoskeleton, and participate in various cellular dynamic processes. However, conflicting results had been obtained on the localization of actin filaments on the mitotic apparatus and their participation in the process of chromosome segregation. We demonstrated by using rhodamine-phalloidin staining, the localization of actin filaments on the mitotic spindles of tobacco BY-2 cells when the cells were treated with cytochalasin D. At prophase, several clear spots were observed at or near the kinetochores of the chromosomes. At anaphase, the actin filaments that appeared to be pulling chromosomes toward the division poles were demonstrated. However, as there was a slight possibility that these results might have been the artifacts of cytochalasin D treatment or the phalloidin staining, we analyzed the localization of actin filaments at the mitotic apparatus immunologically. We cloned a novel BY-2 -type actin cDNA and prepared a BY-2 actin antibody. The fluorescence of the anti-BY-2 actin antibody was clearly observed at the mitotic apparatus in both non-treated and cytochalasin D-treated BY-2 cells during mitosis. The facts that similar results were obtained in both actin staining with rhodamine-phalloidin and immunostaining with actin antibody strongly indicate the participation of actin in the organization of the spindle body or in the process of chromosome segregation. Furthermore, both filamentous actin and spindle bodies disappeared in the cells treated with propyzamide, which depolymerizes microtubules, supporting the notion that actin filaments are associated with microtubules organizing the spindle body.Hiroshi Yasuda and Katsuhiro Kanda contributed equally.     

Immunofluorescence evidence for cytoskeletal rearrangement accompanying pigment redistribution in goldfish xanthophores

Immunofluorescence and phase-contrast microscopic studies of goldfish xanthophores with aggregated or dispersed pigment show two unusual features. First, immunofluorescence studies with anti-actin show punctate structures instead of filaments. These punctate structures are unique for the xanthophores and are absent from both goldfish dermal non-pigment cells and a dedifferentiated cell line (GEM-81) derived from a goldfish xanthophore tumor. Comparison of immunofluorescence and phase-contrast microscopic images with electron microscopic images of thin sections and of Triton-insoluble cytoskeletons show that these punctate structures represent pterinosomes with radiating F-actin. The high local concentration of actin around the pterinosomes results in strong localized fluorescence such that, when the images have proper brightness for these structures, individual actin filaments elsewhere in the cell are too weak in their fluorescence to be visible in the micrographs. Second, whereas immunofluorescence images with anti-tubulin show typical patterns in xanthophores with either aggregated or dispersed pigment, namely, filaments radiating out from the microtubule organizing center, immunofluorescence images with anti-actin or with anti-intermediate filament proteins show different patterns in xanthophores with aggregated versus dispersed pigment. In cells with dispersed pigment, the punctate structures seen with anti-actin are relatively evenly distributed in the cytoplasm, and intermediate filaments appear usually as a dense perinuclear band and long filaments elsewhere in the cytoplasm. In cells with aggregated pigment, both intermediate filaments and pterinosomes with associated actin are largely excluded from the space occupied by the pigment aggregate, and the band of intermediate filaments surrounds not only the nucleus but also the pigment aggregate. The patterns of distribution of the different cytoskeleton components, together with previous results from this laboratory, indicate that formation of the pigment aggregate depends at least in part on the interaction between pigment organelles and microtubules. The possibility that intermediate filaments may play a role in the formation/stabilization of the pigment aggregate is discussed.     

Spatial distribution of filament elasticity determines the migratory behaviors of a cell

Any cellular response leading to morphological changes is highly tuned to balance the force generated from structural reorganization, provided by actin cytoskeleton. Actin filaments serve as the backbone of intracellular force, and transduce external mechanical signal via focal adhesion complex into the cell. During migration, cells not only undergo molecular changes but also rapid mechanical modulation. Here we focus on determining, the role of spatial distribution of mechanical changes of actin filaments in epithelial, mesenchymal, fibrotic and cancer cells with non-migration, directional migration, and non-directional migration behaviors using the atomic force microscopy. We found 1) non-migratory cells only generated one type of filament elasticity, 2) cells generating spatially distributed two types of filament elasticity showed directional migration, and 3) pathologic cells that autonomously generated two types of filament elasticity without spatial distribution were actively migrating non-directionally. The demonstration of spatial regulation of filament elasticity of different cell types at the nano-scale highlights the coupling of cytoskeletal function with physical characters at the sub-cellular level, and provides new research directions for migration related disease.     

Action of cytochalasin D on cytoskeletal networks

Extraction of SC-1 cells (African green monkey kidney) with the detergent Triton X-100 in combination with stereo high-voltage electron microscopy of whole mount preparations has been used as an approach to determine the mode of action of cytochalasin D on cells. The cytoskeleton of extracted BSC-1 cells consists of substrate-associated filament bundles (stress fibers) and a highly cross-linked network of four major filament types extending throughout the cell body; 10-nm filaments, actin microfilaments, microtubules, and 2- to 3-nm filaments. Actin filaments and 2- to 3-nm filaments form numerous end- to-side contacts with other cytoskeletal filaments. Cytochalasin D treatment severely disrupts network organization, increases the number of actin filament ends, and leads to the formation of filamentous aggregates or foci composed mainly of actin filaments. Metabolic inhibitors prevent filament redistribution, foci formation, and cell arborization, but not disorganization of the three-dimensional filament network. In cells first extracted and then treated with cytochalasin D, network organization is disrupted, and the number of free filament ends is increased. Supernates of preparations treated in this way contain both short actin filaments and network fragments (i.e., actin filaments in end-to-side contact with other actin filaments). It is proposed that the dramatic effects of cytochalasin D on cells result from both a direct interaction of the drug with the actin filament component of cytoskeletal networks and a secondary cellular response. The former leads to an immediate disruption of the ordered cytoskeletal network that appears to involve breaking of actin filaments, rather than inhibition of actin filament-filament interactions (i.e., disruption of end-to-side contacts). The latter engages network fragments in an energy-dependent (contractile) event that leads to the formation of filament foci.