The recently reported structural connectivity in F-actin between the DNase I binding loop on actin (residues 38-52) and the C-terminus region was investigated by fluorescence and proteolytic digestion methods. The binding of copper to Cys-374 on F- but not G-actin quenched the fluorescence of dansyl ethylenediamine (DED) attached to Gin-41 by more than 50%. The blocking of copper binding to DED-actin by N-ethylmaleimide labeling of Cys-374 on actin abolished the fluorescence quenching. The quenching of DED-actin fluorescence was restored in copolymers (1:9) of N-ethylmaleimide-DED-actin with unlabeled actin. The quenching of DED-actin fluorescence by copper was also abolished in copolymers (1:4) of DED-actin and N-ethylmaleimide-actin. These results show intermolecular coupling between loop 38-52 and the C-terminus in F-actin. Consistent with this, the rate of subtilisin cleavage of actin at loop 38-52 was increased by the bound copper by more than 10-fold in F-actin but not in G-actin. Neither acto-myosin subfragment-1 (S1) ATPase activity nor the tryptic digestion of G-actin and F-actin at the Lys-61 and Lys-69 sites were affected by the bound copper. These observations suggest that copper binding to Cys-374 does not induce extensive changes in actin structure and that the perturbation of loop 38-52 environment results from changes in the intermolecular contacts in F-actin. 相似文献
The organization of the actin cytoskeleton is impacted by the interplay between physical confinement, features of cross-linking proteins, and deformations of semiflexible actin filaments. Some cross-linking proteins preferentially bind filaments in parallel, although others bind more indiscriminately. However, a quantitative understanding of how the mode of binding influences the assembly of actin networks in confined environments is lacking. Here we employ coarse-grained computer simulations to study the dynamics and organization of semiflexible actin filaments in confined regions upon the addition of cross-linkers. We characterize how the emergent behavior is influenced by the system shape, the number and type of cross-linking proteins, and the length of filaments. Structures include isolated clusters of filaments, highly connected filament bundles, and networks of interconnected bundles and loops. Elongation of one dimension of the system promotes the formation of long bundles that align with the elongated axis. Dynamics are governed by rapid cross-linking into aggregates, followed by a slower change in their shape and connectivity. Cross-linking decreases the average bending energy of short or sparsely connected filaments by suppressing shape fluctuations. However, it increases the average bending energy in highly connected networks because filament bundles become deformed, and small numbers of filaments exhibit long-lived, highly unfavorable configurations. Indiscriminate cross-linking promotes the formation of high-energy configurations due to the increased likelihood of unfavorable, difficult-to-relax configurations at early times. Taken together, this work demonstrates physical mechanisms by which cross-linker binding and physical confinement impact the emergent behavior of actin networks, which is relevant both in cells and in synthetic environments. 相似文献
Changes in cell shape that occur in many cellular processes are thought to arise from polymerization of actin filaments near the cell membrane. End-to-end annealing of actin filaments is believed to play only a minor role in this process, as annealing in solution was shown to be a slow process, which is not typical for a bimolecular reaction, its rate constant decreasing over time, being inversely proportional to the filament length. Furthermore, in vitro studies on f-actin solutions were found to display an exponential steady-state length distribution. In the cell, many physiologically important parameters, such as mechanical strength or viscoelastic response are a direct function of the physical properties of the underlying actin cytoskeleton, such as actin filament length distribution and dynamics. How the underlying physical parameters of the actin cytoskeleton may be influenced by the cell surface or molecular crowding remains poorly understood. Using total internal reflection fluorescence (TIRF) microscopy we reinvestigated actin end-to-end annealing in vitro in a more realistic environment. We studied the process near a hydrophilic surface together with crowding agents, in order to mimic the physiological media near the cell membrane, which has substantial amounts of macromolecules present. We find that actin end-to-end annealing changes in three ways near a crowded hydrophilic surface as compared to solution. First the annealing rate becomes a factor of 20 faster than in solution. Second the rate of annealing becomes typical of a bimolecular reaction, shows no length dependence and is basically just a function of the square of the concentration of ends. Lastly the length distribution is Gaussian throughout the entire annealing process. This implicates that dynamic rearrangement of actin filaments by annealing near the leading edge of the cell, could change physical parameters like the mechanical response and contribute significantly to cell motility. 相似文献
Epithelial protein lost in neoplasm (EPLIN) is a cytoskeleton-associated protein encoded by a gene that is down-regulated in transformed cells. EPLIN increases the number and size of actin stress fibers and inhibits membrane ruffling induced by Rac. EPLIN has at least two actin binding sites. Purified recombinant EPLIN inhibits actin filament depolymerization and cross-links filaments in bundles. EPLIN does not affect the kinetics of spontaneous actin polymerization or elongation at the barbed end, but inhibits branching nucleation of actin filaments by Arp2/3 complex. Side binding activity may stabilize filaments and account for the inhibition of nucleation mediated by Arp2/3 complex. We propose that EPLIN promotes the formation of stable actin filament structures such as stress fibers at the expense of more dynamic actin filament structures such as membrane ruffles. Reduced expression of EPLIN may contribute to the motility of invasive tumor cells. 相似文献
We have measured the effects of cofilin on the conformation and dynamics of actin filaments labeled at Cys374 with erythrosin-iodoacetemide (ErIA), using time-resolved phosphorescence anisotropy (TPA). Cofilin quenches the phosphorescence intensity of actin-bound ErIA, indicating that binding changes the local environment of the probe. The cofilin concentration-dependence of the phosphorescence intensity is sigmoidal, consistent with cooperative actin filament binding. Model-independent analysis of the anisotropies indicates that cofilin increases the rates of the microsecond rotational motions of actin. In contrast to the reduction in phosphorescence intensity, the changes in the rates of rotational motions display non-nearest-neighbor cooperative interactions and saturate at substoichiometric cofilin binding densities. Detailed analysis of the TPA decays indicates that cofilin decreases the torsional rigidity (C) of actin, increasing the thermally driven root-mean-square torsional angle between adjacent filament subunits from approximately 4 degrees (C = 2.30 x 10(-27) Nm2 radian(-1)) to approximately 17 degrees (C = 0.13 x 10(-27) Nm2 radian(-1)) at 25 degrees C. We favor a mechanism in which cofilin binding shifts the equilibrium between thermal ErIA-actin filament conformers, and facilitates two distinct structural changes in actin. One is local in nature, which affects the structure of actin's C terminus and is likely to mediate nearest-neighbor cooperative binding and filament severing. The second is a change in the internal dynamics of actin, which displays non-nearest-neighbor cooperativity and increases the torsional flexibility of filaments. The long-range effects of cofilin on the torsional dynamics of actin may accelerate P(i) release from filaments and modulate interactions with other regulatory actin filament binding proteins. 相似文献
The polymerization-depolymerization dynamics of actin is a key process in a variety of cellular functions. Many spectroscopic studies have been performed in solution, but studies on single actin filaments have just begun. Here, we show that the time course of polymerization of individual filaments consists of a polymerization phase and a subsequent steady-state phase. During the steady-state phase, a treadmilling process of elongation at the barbed end and shortening at the pointed end occurs, in which both components of the process proceed at approximately the same rate. The time correlation of length fluctuation of the filaments in the steady-state phase showed that the polymerization-depolymerization dynamics follow a diffusion (stochastic) process, which cannot be explained by simple association and dissociation of monomers at both ends of the filaments. 相似文献
Despite the recognition that actin filaments are important for numerous cellular processes, and decades of investigation, the dynamics of in vitro actin filaments are still not completely understood. Here, we follow the time evolution of the length distribution of labeled actin reporter filaments in an unlabeled F-actin solution via fluorescence microscopy. Whereas treadmilling and diffusive length fluctuations cannot account for the observed dynamics, our results suggest that at low salt conditions, spontaneous fragmentation is crucial. 相似文献
Red blood cell (RBC) shape and deformability are supported by a planar network of short actin filament (F-actin) nodes (∼37 nm length, 15–18 subunits) interconnected by long spectrin strands at the inner surface of the plasma membrane. Spectrin-F-actin network structure underlies quantitative modeling of forces controlling RBC shape, membrane curvature, and deformation, yet the nanoscale organization and dynamics of the F-actin nodes in situ are not well understood. We examined F-actin distribution and dynamics in RBCs using fluorescent-phalloidin labeling of F-actin imaged by multiple microscopy modalities. Total internal reflection fluorescence and Zeiss Airyscan confocal microscopy demonstrate that F-actin is concentrated in multiple brightly stained F-actin foci ∼200–300 nm apart interspersed with dimmer F-actin staining regions. Single molecule stochastic optical reconstruction microscopy imaging of Alexa 647-phalloidin-labeled F-actin and computational analysis also indicates an irregular, nonrandom distribution of F-actin nodes. Treatment of RBCs with latrunculin A and cytochalasin D indicates that F-actin foci distribution depends on actin polymerization, while live cell imaging reveals dynamic local motions of F-actin foci, with lateral movements, appearance and disappearance. Regulation of F-actin node distribution and dynamics via actin assembly/disassembly pathways and/or via local extension and retraction of spectrin strands may provide a new mechanism to control spectrin-F-actin network connectivity, RBC shape, and membrane deformability. 相似文献
This review focuses on recent advances in the understanding of the organization and roles of actin filaments, and associated myosin motor proteins, in regulating the structure and function of the axon shaft. ‘Patches’ of actin filaments have emerged as a major type of actin filament organization in axons. In the distal axon, patches function as precursors to the formation of filopodia and branches. At the axon initial segment, patches locally capture membranous organelles and contribute to polarized trafficking. The trapping function of patches at the initial segment can be ascribed to interactions with myosin motors, and likely also applies to patches in the more distal axon. Finally, submembranous rings of actin filaments were recently described in axons, which form an actin‐spectrin cytoskeleton, likely contributing to the maintenance of axon integrity. Continued investigation into the roles of axonal actin filaments and myosins will shed light on fundamental aspects of the development, adult function and the repair of axons in the nervous system.
Mitotic spindles are microtubule-based structures responsible for chromosome partitioning during cell division. Although the roles of microtubules and microtubule-based motors in mitotic spindles are well established, whether or not actin filaments (F-actin) and F-actin-based motors (myosins) are required components of mitotic spindles has long been controversial. Based on the demonstration that myosin-10 (Myo10) is important for assembly of meiotic spindles, we assessed the role of this unconventional myosin, as well as F-actin, in mitotic spindles. We find that Myo10 localizes to mitotic spindle poles and is essential for proper spindle anchoring, normal spindle length, spindle pole integrity, and progression through metaphase. Furthermore, we show that F-actin localizes to mitotic spindles in dynamic cables that surround the spindle and extend between the spindle and the cortex. Remarkably, although proper anchoring depends on both F-actin and Myo10, the requirement for Myo10 in spindle pole integrity is F-actin independent, whereas F-actin and Myo10 actually play antagonistic roles in maintenance of spindle length. 相似文献
Salmonella bacteria cause more than three million deaths each year. They hijack cells and inject among other proteins SipA via a "molecular syringe" into the cell, which can tether actin subunits in opposing strands to form mechanically stabilized filaments which rapidly reshape the cells surface into extended ruffles, leading to bacterial internalization. Exactly how these ruffles form at a single filament level remains unknown. Our real time total internal fluorescence microscopy observations show that both bidirectional elongation of actin by SipA as well as end-to-end annealing of SipA-actin filaments are rapid processes. Complementary electron microscopy investigations demonstrate that crowding agents in vitro readily induce stiff bundles of SipA-actin filaments. Taken together these three effects, rapid SipA induced actin polymerization, filament annealing and bundle formation due to molecular crowding can explain how Salmonella invades cells at molecular level. 相似文献
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. 相似文献
Actin labeling at Cys(374) with tethramethylrhodamine derivatives (TMR-actin) has been widely used for direct observation of the in vitro filaments growth, branching, and treadmilling, as well as for the in vivo visualization of actin cytoskeleton. The advantage of TMR-actin is that it does not lock actin in filaments (as rhodamine-phalloidin does), possibly allowing for its use in investigating the dynamic assembly behavior of actin polymers. Although it is established that TMR-actin alone is polymerization incompetent, the impact of its copolymerization with unlabeled actin on filament structure and dynamics has not been tested yet. In this study, we show that TMR-actin perturbs the filaments structure when copolymerized with unlabeled actin; the resulting filaments are more fragile and shorter than the control filaments. Due to the increased severing of copolymer filaments, TMR-actin accelerates the polymerization of unlabeled actin in solution also at mole ratios lower than those used in most fluorescence microscopy experiments. The destabilizing and severing effect of TMR-actin is countered by filament stabilizing factors, phalloidin, S1, and tropomyosin. These results point to an analogy between the effects of TMR-actin and severing proteins on F-actin, and imply that TMR-actin may be inappropriate for investigations of actin filaments dynamics. 相似文献
In contrast to the actin filaments of muscle, which are stabilized by special proteins, actin filaments of the cytoskeleton are highly dynamic. In vitro observations at room temperature have led to the conclusion that the hydrolysis of ATP, which accompanies the polymerization of ATP-containing monomers, destabilizes the filaments of the actin skeleton. Many functions of this skeleton, such as signal transduction, the anchoring of cell adhesion complexes, and the transfer and generation of pulling forces, can obviously only be adequately performed by stable filaments. Here it is assumed that, at room temperature, the interaction of ADP-containing monomers is impaired by complexed water molecules that partly shield the binding surfaces. The possibility that, at higher temperatures, the interaction of the monomers is strong enough to prevent spontaneous filament depolymerization is explored. Using mechanical models that take into account binding forces and energies, the polymerization cycle expected under these conditions is described. It is shown that ATP serves primarily to prevent incorrect binding of the incoming monomer to the end of the filament ('adjusted fit'). In addition, it provides the free energy needed for disassembly of the expected stable filaments. 相似文献
Actin filaments and chloroplasts in guard cells play roles in stomatal function. However, detailed actin dynamics vary, and the roles that they play in chloroplast localization during stomatal movement remain to be determined. We examined the dynamics of actin filaments and chloroplast localization in transgenic tobacco expressing green fluorescent protein (GFP)-mouse talin in guard cells by time-lapse imaging. Actin filaments showed sliding, bundling and branching dynamics in moving guard cells. During stomatal movement, long filaments can be severed into small fragments, which can form longer filaments by end-joining activities. With chloroplast movement, actin filaments near chloroplasts showed severing and elongation activity in guard cells during stomatal movement. Cytochalasin B treatment abolished elongation, bundling and branching activities of actin filaments in guard cells, and these changes of actin filaments, and as a result, more chloroplasts were localized at the centre of guard cells. However, chloroplast turning to avoid high light, and sliding of actin fragments near the chloroplast, was unaffected following cytochalasin B treatment in guard cells. We suggest that the sliding dynamics of actin may play roles in chloroplast turning in guard cells. Our results indicate that the stochastic dynamics of actin filaments in guard cells regulate chloroplast localization during stomatal movement. 相似文献
It is essential to investigate the mechanical behaviour of cytoskeletal actin filaments in order to understand their critical role as mechanical components in various cellular functional activities. These actin filaments consisting of monomeric molecules function in the thermal fluctuations. Hence, it is important to understand their mechanical behaviour on the microscopic scale by comparing the stiffness based on thermal fluctuations with the one experimentally measured on the macroscopic scale. In this study, we perform a large-scale molecular dynamics (MD) simulation for a half-turn structure of an actin filament. We analyse its longitudinal and twisting Brownian motions in equilibrium and evaluated its apparent extensional and torsional stiffness on the nanosecond scale. Upon increasing the sampling-window durations for analysis, the apparent stiffness gradually decreases and exhibits a trend to converge to a value that is close to the experimental value. This suggests that by extrapolating the data obtained in the MD analysis, we can estimate the experimentally determined stiffness on the microsecond to millisecond scales. For shorter temporal scales, the apparent stiffness is larger than experimental values, indicating that fast, local motions of the molecular structure are dominant. To quantify the local structural changes within the filament on the nanosecond scale and investigate the molecular mechanisms, such as the binding of the actin-regulatory proteins to the filaments, it is preferable to analyse the mechanical behaviour on the nanometre and nanosecond scales using MD simulation. 相似文献
It has become accepted that deep-freeze-drying at or below -90 degrees C is necessary to preserve the structure of supramolecular assemblies such as actin filaments (AFs) for metal shadowing. This has kept the metal shadowing technique from widespread use in the study of proteins complexed with AFs because of the limited availability of the apparatus for deep-freeze-drying. I report here that adsorption to freshly cleaved mica, treatment with buffered uranyl acetate in glycerol solution, rinsing, and removal of liquid eliminate the need of freeze-drying to preserve the structure of AFs. This technique, in combination with metal shadowing, was applied to the study of AFs decorated with heavy meromyosin (HMM). It was observed that (1) when HMM molecules are associated with single AFs in the majority of cases only one head of each HMM molecule makes contact at the point furthest from the neck region; (2) binding of HMM causes bundling of AFs, probably by the two heads of each molecule binding different filaments; and (3) the binding of HMM to the bundled AFs appears to be more stable than that to a single AF. This method of specimen preparation requires no freeze-drying and is therefore easily applicable to other large protein complexes. 相似文献