The Role of Structural Dynamics of Actin in Class-Specific Myosin Motility

The structural dynamics of actin, including the tilting motion between the small and large domains, are essential for proper interactions with actin-binding proteins. Gly146 is situated at the hinge between the two domains, and we previously showed that a G146V mutation leads to severe motility defects in skeletal myosin but has no effect on motility of myosin V. The present study tested the hypothesis that G146V mutation impaired rotation between the two domains, leading to such functional defects. First, our study showed that depolymerization of G146V filaments was slower than that of wild-type filaments. This result is consistent with the distinction of structural states of G146V filaments from those of the wild type, considering the recent report that stabilization of actin filaments involves rotation of the two domains. Next, we measured intramolecular FRET efficiencies between two fluorophores in the two domains with or without skeletal muscle heavy meromyosin or the heavy meromyosin equivalent of myosin V in the presence of ATP. Single-molecule FRET measurements showed that the conformations of actin subunits of control and G146V actin filaments were different in the presence of skeletal muscle heavy meromyosin. This altered conformation of G146V subunits may lead to motility defects in myosin II. In contrast, distributions of FRET efficiencies of control and G146V subunits were similar in the presence of myosin V, consistent with the lack of motility defects in G146V actin with myosin V. The distribution of FRET efficiencies in the presence of myosin V was different from that in the presence of skeletal muscle heavy meromyosin, implying that the roles of actin conformation in myosin motility depend on the type of myosin.     

Structure and Dynamics of the Actin Filament

We used all-atom molecular dynamics simulations to investigate the structure and properties of the actin filament, starting with either the recent Oda model or the older Holmes model. Simulations of monomeric and polymerized actin show that polymerization changes the nucleotide-binding cleft, bringing together the Q137 side chain and bound ATP in a way that may enhance the ATP hydrolysis rate in the filament. Simulations with different bound nucleotides and conformations of the DNase I binding loop show that the persistence length of the filament depends only on loop conformation. Computational modeling reveals how bound phalloidin stiffens actin filaments and inhibits the release of γ-phosphate from ADP-P_i actin.     

Lineage Specificity of the Evolutionary Dynamics of the mtDNA D-Loop Region in Rodents

This paper reports an intraorder study on the D-loop-containing region of the mitochondrial DNA in rodents. A complete multialignment of this region is not feasible with the exception of some conserved regions. The comparative analysis of 25 complete rodent sequences from 23 species plus one lagomorph has revealed that only the central domain (CD), a conserved region of about 80 bp in the extended termination-associated sequences (ETAS) domain, adjacent to the CD, the ETAS1, and conserved sequence block (CSB) 1 blocks are present in all rodent species, whereas the presence of CSB2 and CSB3 is erratic within the order. We have also found a conserved region of 90 bp located between tRNAPro and ETAS1 present in fat dormouse, squirrel, guinea pig, and rabbit. Repeated sequences are present in both the ETAS and the CSB domain, but the repeats differ in length, copy number, and base composition in different species. The potential use of the D-loop for evolutionary studies has been investigated; the presence/absence of conserved blocks and/or repeated sequences cannot be used as a reliable phylogenetic marker, since in some cases they may be shared by distantly related organisms but not by close ones, while in other ones a relationship between tree topology and presence/absence of such motifs is observed. Better results can be obtained by the use of the CD, which, however, due to its reduced size, when used for tracing a phylogenetic tree, shows some nodes with low statistical support. Received: 26 February 2001 / Accepted: 6 June 2001     

Structural Polymorphism of Actin

Information on the structural polymorphism of a protein is essential to understand the mechanisms of how it functions at an atomic level. Numerous studies on actin have accumulated substantial amounts of information about its polymorphism, and there are over 200 published atomic structures of different forms of actin using crystallography, fiber diffraction, and electron microscopy. To characterize all the reported structures, we proposed simple parameters based on the discrete rigid bodies within the actin molecule and identified four conformation groups by cluster analysis: the F-form in naked F-actin, the C-form in cofilactin, the O-form in profilin–actin, and the G-form in the majority of actin-containing crystal structures. The G-form group included the most variations, but each conformational variation was convertible via a thermal fluctuation, whereas the F- and C-forms were not accessible from the G-form. The convertibility and accessibility of the structures were evaluated using molecular dynamics simulations. Information about conformational conversion among each group is useful for understanding the mechanisms of actin function.     

植物微丝骨架动态变化的调节

由球形肌动蛋白聚合而成的微丝骨架,又称肌动蛋白纤维,它在细胞运动、细胞形态建成以及物质运输等诸多生命活动中发挥重要作用。细胞内微丝的解聚和聚合动态特性是微丝骨架行使功能的重要基础,并受到如微丝结合蛋白、金属离子、小G蛋白等各种因素的严格控制。植物细胞微丝骨架的研究虽然晚于动物细胞,但也取得了飞速发展。本文对植物细胞内微丝骨架动态变化的作用机制及一些主要调节因子的最新研究进展做一介绍。     

Signaling, Actin Dynamics and Endocytosis

Endocytosis is an essential process for normal function of all living cells. Cells get nutrients, and control the surface-expressional level of proteins as well as membrane hemostats through the endocytosis. Endocytosis process is regulated in response to functional status of a particular cell. Signaling events and the endocytosis process go hand in hand to fulfill cellular functions. Although our understanding of the endocytosis process has grown rapidly during the last decade, little is known about how it is interconnected functionally with the signaling status of cells. During endocytosis, vesicles are formed from the plasma membrane through complex molecular machinery. The location where the vesicles are formed is rich in cortical actin cytoskeleton that supports the plasma membrane. To enter cells, vesicles have to diffuse through the cortical actin cytoskeleton. The actin cytoskeleton has a very dynamic structure and actively participates a wide variety of cellular functions. In addition to its central role in cytokinesis, cell shape, cell motility, and cell polarity, a connection between the endocytosis process and the actin cytoskeleton has been implicated in both yeast and mammalian system. In recent years the knowledge on how the actin cytoskeleton participates in the generation of coordinated cellular responses to external stimuli is grown rapidly. In this review, we focus on the potential roles of the actin cytoskeleton in regulating the endocytosis process in response to signaling events.     

Analysis of Actin FLAP Dynamics in the Leading Lamella

Background

The transport of labeled G-actin from the mid-lamella region to the leading edge in a highly motile malignant rat fibroblast line has been studied using fluorescence localization after photobleaching or FLAP, and the transit times recorded in these experiments were so fast that simple diffusion was deemed an insufficient explanation (see Zicha et al., Science, v. 300, pp. 142–145 [1]).

Methodology/Principal Findings

We re-examine the Zicha FLAP experiments using a two-phase reactive interpenetrating flow formalism to model the cytoplasm and the transport dynamics of bleached and unbleached actin. By allowing an improved treatment of effects related to the retrograde flow of the cytoskeleton and of the geometry and finite thickness of the lamella, this new analysis reveals a mechanism that can realistically explain the timing and the amplitude of all the FLAP signals observed in [1] without invoking special transport modalities.

Conclusions/Significance

We conclude that simple diffusion is sufficent to explain the observed transport rates, and that variations in the transport of labeled actin through the lamella are minor and not likely to be the cause of the observed physiological variations among different segments of the leading edge. We find that such variations in labeling can easily arise from differences and changes in the microscopic actin dynamics inside the edge compartment, and that the key dynamical parameter in this regard is the so-called “dilatation rate” (the velocity of cytoskeletal retrograde flow divided by a characteristic dimension of the edge compartment where rapid polymerization occurs). If our dilatation hypothesis is correct, the transient kinetics of bleached actin relocalization constitute a novel and very sensitive method for probing the cytoskeletal dynamics in leading edge micro-environments which are otherwise very difficult to directly interrogate.     

Regulation of Actin Dynamics in Pollen Tubes: Control of Actin Polymer Level

Actin cytoskeleton undergoes rapid reorganization in response to internal and external cues. How the dynamics of actin cytoskeleton are regulated, and how its dynamics relate to its function are fundamental questions in plant cell biology. The pollen tube is a well characterized actin-based cell morphogenesis in plants. One of the striking features of actin cytoskeleton characterized in the pollen tube is its surprisingly low level of actin polymer. This special phenomenon might relate to the function of actin cytoskeleton in pollen tubes. Understanding the molecular mechanism underlying this special phenomenon requires careful analysis of actin-binding proteins that modulate actin dynamics directly. Recent biochemical and biophysical analyses of several highly conserved plant actin-binding proteins reveal unusual and unexpected properties, which emphasizes the importance of carefully analyzing their action mechanism and cellular activity. In this review, we highlight an actin monomer sequestering protein, a barbed end capping protein and an F-actin severing and dynamizing protein in plant. We propose that these proteins function in harmony to regulate actin dynamics and maintain the low level of actin polymer in pollen tubes.     

Quantitative Analysis of Ezrin Turnover Dynamics in the Actin Cortex

Proteins of the ERM family (ezrin, moesin, radixin) play a fundamental role in tethering the membrane to the cellular actin cortex as well as regulating cortical organization and mechanics. Overexpression of dominant inactive forms of ezrin leads to fragilization of the membrane-cortex link and depletion of moesin results in softer cortices that disrupt spindle orientation during cytokinesis. Therefore, the kinetics of association of ERM proteins with the cortex likely influence the timescale of cortical signaling events and the dynamics of membrane interfacing to the cortex. However, little is known about ERM protein turnover at the membrane-cortex interface. Here, we examined cortical ezrin dynamics using fluorescence recovery after photobleaching experiments and single-molecule imaging. Using multiexponential fitting of fluorescence recovery curves, we showed that ezrin turnover resulted from three molecular mechanisms acting on very different timescales. The fastest turnover process was due to association/dissociation from the F-actin cortex, suggesting that ezrin acts as a link that leads to low friction between the membrane and the cortex. The second turnover process resulted from association/dissociation of ezrin from the membrane and the slowest turnover process resulted from the slow diffusion of ezrin in the plane of the membrane. In summary, ezrin-mediated membrane-cortex tethering resulted from long-lived interactions with the membrane via the FERM domain coupled with shorter-lived interactions with the cortex. The slow diffusion of membranous ezrin and its interaction partners relative to the cortex signified that signals emanating from membrane-associated ezrin may locally act to modulate cortical organization and contractility.     

Quantitative Analysis of Ezrin Turnover Dynamics in the Actin Cortex

Proteins of the ERM family (ezrin, moesin, radixin) play a fundamental role in tethering the membrane to the cellular actin cortex as well as regulating cortical organization and mechanics. Overexpression of dominant inactive forms of ezrin leads to fragilization of the membrane-cortex link and depletion of moesin results in softer cortices that disrupt spindle orientation during cytokinesis. Therefore, the kinetics of association of ERM proteins with the cortex likely influence the timescale of cortical signaling events and the dynamics of membrane interfacing to the cortex. However, little is known about ERM protein turnover at the membrane-cortex interface. Here, we examined cortical ezrin dynamics using fluorescence recovery after photobleaching experiments and single-molecule imaging. Using multiexponential fitting of fluorescence recovery curves, we showed that ezrin turnover resulted from three molecular mechanisms acting on very different timescales. The fastest turnover process was due to association/dissociation from the F-actin cortex, suggesting that ezrin acts as a link that leads to low friction between the membrane and the cortex. The second turnover process resulted from association/dissociation of ezrin from the membrane and the slowest turnover process resulted from the slow diffusion of ezrin in the plane of the membrane. In summary, ezrin-mediated membrane-cortex tethering resulted from long-lived interactions with the membrane via the FERM domain coupled with shorter-lived interactions with the cortex. The slow diffusion of membranous ezrin and its interaction partners relative to the cortex signified that signals emanating from membrane-associated ezrin may locally act to modulate cortical organization and contractility.     

Sensing Actin Dynamics through Adherens Junctions

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Actin Gene Family Dynamics in Cryptomonads and Red Algae

Here we present evidence for a complex evolutionary history of actin genes in red algae and cryptomonads, a group that acquired photosynthesis secondarily through the engulfment of a red algal endosymbiont. Four actin genes were found in the nuclear genome of the cryptomonad, Guillardia theta, and in the genome of the red alga, Galdieria sulphuraria, a member of the Cyanidiophytina. Phylogenetic analyses reveal that the both organisms possess two distinct sequence types, designated “type-1” and “type-2.” A weak but consistent phylogenetic affinity between the cryptomonad type-2 sequences and the type-2 sequences of G. sulphuraria and red algae belonging to the Rhodophytina was observed. This is consistent with the possibility that the cryptomonad type-2 sequences are derived from the red algal endosymbiont that gave rise to the cryptomonad nucleomorph and plastid. Red algae as a whole possess two very different actin sequence types, with G. sulphuraria being the only organism thus far known to possess both. The common ancestor of Rhodophytina and Cyanidiophytina may have had two actin genes, with differential loss explaining the distribution of these genes in modern-day groups. Our study provides new insight into the evolution and divergence of actin genes in cryptomonads and red algae, and in doing so underscores the challenges associated with heterogeneity in actin sequence evolution and ortholog/paralog detection.     

Role of ATP-Hydrolysis in the Dynamics of a Single Actin Filament

We study the stochastic dynamics of growth and shrinkage of single actin filaments taking into account insertion, removal, and ATP hydrolysis of subunits either according to the vectorial mechanism or to the random mechanism. In a previous work, we developed a model for a single actin or microtubule filament where hydrolysis occurred according to the vectorial mechanism: the filament could grow only from one end, and was in contact with a reservoir of monomers. Here we extend this approach in two ways—by including the dynamics of both ends and by comparing two possible mechanisms of ATP hydrolysis. Our emphasis is mainly on two possible limiting models for the mechanism of hydrolysis within a single filament, namely the vectorial or the random model. We propose a set of experiments to test the nature of the precise mechanism of hydrolysis within actin filaments.     

Structural Variations in Protein Superfamilies: Actin and Tubulin

Structures of homologous proteins are usually conserved during evolution, as are critical active site residues. This is the case for actin and tubulin, the two most important cytoskeleton proteins in eukaryotes. Actins and their related proteins (Arps) constitute a large superfamily whereas the tubulin family has fewer members. Unaligned sequences of these two protein families were analysed by searching for short groups of family-specific amino acid residues, that we call motifs, and by counting the number of residues from one motif to the next. For each sequence, the set of motif-to-motif residue counts forms a subfamily-specific pattern (landmark pattern) allowing actin and tubulin superfamily members to be identified and sorted into subfamilies. The differences between patterns of individual subfamilies are due to inserts and deletions (indels). Inserts appear to have arisen at an early stage in eukaryote evolution as suggested by the small but consistent kingdom-dependent differences found within many Arp subfamilies and in γ-tubulins. Inserts tend to be in surface loops where they can influence subfamily-specific function without disturbing the core structure of the protein. The relatively few indels found for tubulins have similar positions to established results, whereas we find many previously unreported indel positions and lengths for the metazoan Arps.     

Real-Time Dynamics of Emerging Actin Networks in Cell-Mimicking Compartments

Understanding the cytoskeletal functionality and its relation to other cellular components and properties is a prominent question in biophysics. The dynamics of actin cytoskeleton and its polymorphic nature are indispensable for the proper functioning of living cells. Actin bundles are involved in cell motility, environmental exploration, intracellular transport and mechanical stability. Though the viscoelastic properties of actin-based structures have been extensively probed, the underlying microstructure dynamics, especially their disassembly, is not fully understood. In this article, we explore the rich dynamics and emergent properties exhibited by actin bundles within flow-free confinements using a microfluidic set-up and epifluorescence microscopy. After forming entangled actin filaments within cell-sized quasi two-dimensional confinements, we induce their bundling using three different fundamental mechanisms: counterion condensation, depletion interactions and specific protein-protein interactions. Intriguingly, long actin filaments form emerging networks of actin bundles via percolation leading to remarkable properties such as stress generation and spindle-like intermediate structures. Simultaneous sharing of filaments in different links of the network is an important parameter, as short filaments do not form networks but segregated clusters of bundles instead. We encounter a hierarchical process of bundling and its subsequent disassembly. Additionally, our study suggests that such percolated networks are likely to exist within living cells in a dynamic fashion. These observations render a perspective about differential cytoskeletal responses towards numerous stimuli.     

Spontaneous Structural Changes in Actin Regulate G-F Transformation

Transformations between G- (monomeric) and F-actin (polymeric) are important in cellular behaviors such as migration, cytokinesis, and morphing. In order to understand these transitions, we combined single-molecule Förster resonance energy transfer with total internal reflection fluorescence microscopy to examine conformational changes of individual actin protomers. We found that the protomers can take different conformational states and that the transition interval is in the range of hundreds of seconds. The distribution of these states was dependent on the environment, suggesting that actin undergoes spontaneous structural changes that accommodate itself to polymerization.