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.     

The Role of Actin Cytoskeletal Tension in Oscillatory Fluid Flow Induced Osteogenesis

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Formation and Dynamics of Waves in a Cortical Model of Cholinergic Modulation

Acetylcholine (ACh) is a regulator of neural excitability and one of the neurochemical substrates of sleep. Amongst the cellular effects induced by cholinergic modulation are a reduction in spike-frequency adaptation (SFA) and a shift in the phase response curve (PRC). We demonstrate in a biophysical model how changes in neural excitability and network structure interact to create three distinct functional regimes: localized asynchronous, traveling asynchronous, and traveling synchronous. Our results qualitatively match those observed experimentally. Cortical activity during slow wave sleep (SWS) differs from that during REM sleep or waking states. During SWS there are traveling patterns of activity in the cortex; in other states stationary patterns occur. Our model is a network composed of Hodgkin-Huxley type neurons with a M-current regulated by ACh. Regulation of ACh level can account for dynamical changes between functional regimes. Reduction of the magnitude of this current recreates the reduction in SFA the shift from a type 2 to a type 1 PRC observed in the presence of ACh. When SFA is minimal (in waking or REM sleep state, high ACh) patterns of activity are localized and easily pinned by network inhomogeneities. When SFA is present (decreasing ACh), traveling waves of activity naturally arise. A further decrease in ACh leads to a high degree of synchrony within traveling waves. We also show that the level of ACh determines how sensitive network activity is to synaptic heterogeneity. These regimes may have a profound functional significance as stationary patterns may play a role in the proper encoding of external input as memory and traveling waves could lead to synaptic regularization, giving unique insights into the role and significance of ACh in determining patterns of cortical activity and functional differences arising from the patterns.     

The Architecture of Traveling Actin Waves Revealed by Cryo-Electron Tomography

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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。     

The Cytoskeletal Proteins Actin and Tubulin in the Spermatozoa of Four Decapod Crabs (Crustacea,Decapoda)

The mature spermatozoa of four species of European decapod crabs (Clibanarius erythropus, Maja squinado, Cancer pagurusand Potamon fluviatile)have been investigated using indirect immuno-fluorescence techniques for the presence of the cytoskeletal proteins actin and tubulin. Indirect immunofluorescence labelling with monoclonal anti-actin antibody and three different monoclonal anti-tubulin antibodies indicate that actin is present in the spermatozoa of all four species, but tubulins are restricted to the two species with microtubular arms, Clibanariusand Maja.The pattern of actin fluorescence varies between the spermatozoa of the four species, with Majaand Cancershowing intense fluorescence in the acrosome vesicle and in elements of the sperm cell involved in the acrosome reaction. The spermatozoon of each species is described ultrastructurally using transmission electron microscopy and correlations made between observed patterns of fluorescence and the cellular components described. No obvious filamentous actin (F-actin) is visible in the electron micrographs of the spermatozoa of any of the species. In most cases the fluorescence is sufficiently specific to indicate in which region of the mature sperm cell the actin and tubulin occurs. Actin is acrosomal in Maja, Cancerand Potamonbut appears to be cytoplasmic in Clibanarius, while the tubulins appear only to be present in the cytoplasm of Clibanarius, Majaand Cancer.     

A Three-Dimensional Computer Simulation Model Reveals the Mechanisms for Self-Organization of Plant Cortical Microtubules into Oblique Arrays

The noncentrosomal cortical microtubules (CMTs) of plant cells self-organize into a parallel three-dimensional (3D) array that is oriented transverse to the cell elongation axis in wild-type plants and is oblique in some of the mutants that show twisted growth. To study the mechanisms of CMT array organization, we developed a 3D computer simulation model based on experimentally observed properties of CMTs. Our computer model accurately mimics transverse array organization and other fundamental properties of CMTs observed in rapidly elongating wild-type cells as well as the defective CMT phenotypes observed in the Arabidopsis mor1-1 and fra2 mutants. We found that CMT interactions, boundary conditions, and the bundling cutoff angle impact the rate and extent of CMT organization, whereas branch-form CMT nucleation did not significantly impact the rate of CMT organization but was necessary to generate polarity during CMT organization. We also found that the dynamic instability parameters from twisted growth mutants were not sufficient to generate oblique CMT arrays. Instead, we found that parameters regulating branch-form CMT nucleation and boundary conditions at the end walls are important for forming oblique CMT arrays. Together, our computer model provides new mechanistic insights into how plant CMTs self-organize into specific 3D arrangements.     

The Yeast V159N Actin Mutant Reveals Roles for Actin Dynamics In Vivo

Actin with a Val 159 to Asn mutation (V159N) forms actin filaments that depolymerize slowly because of a failure to undergo a conformational change after inorganic phosphate release. Here we demonstrate that expression of this actin results in reduced actin dynamics in vivo, and we make use of this property to study the roles of rapid actin filament turnover. Yeast strains expressing the V159N mutant (act1-159) as their only source of actin have larger cortical actin patches and more actin cables than wild-type yeast. Rapid actin dynamics are not essential for cortical actin patch motility or establishment of cell polarity. However, fluid phase endocytosis is defective in act1-159 strains. act1-159 is synthetically lethal with cofilin and profilin mutants, supporting the conclusion that mutations in all of these genes impair the polymerization/ depolymerization cycle. In contrast, act1-159 partially suppresses the temperature sensitivity of a tropomyosin mutant, and the loss of cytoplasmic cables seen in fimbrin, Mdm20p, and tropomyosin null mutants, suggesting filament stabilizing functions for these actin-binding proteins. Analysis of the cables in these double-mutant cells supports a role for fimbrin in organizing cytoplasmic cables and for Mdm20p and tropomyosin in excluding cofilin from the cables.     

Role of Cyclic Nucleotide-Dependent Actin Cytoskeletal Dynamics: [Ca2+]i and Force Suppression in Forskolin-Pretreated Porcine Coronary Arteries

Initiation of force generation during vascular smooth muscle contraction involves a rise in intracellular calcium ([Ca²⁺]_i) and phosphorylation of myosin light chains (MLC). However, reversal of these two processes alone does not account for the force inhibition that occurs during relaxation or inhibition of contraction, implicating that other mechanisms, such as actin cytoskeletal rearrangement, play a role in the suppression of force. In this study, we hypothesize that forskolin-induced force suppression is dependent upon changes in actin cytoskeletal dynamics. To focus on the actin cytoskeletal changes, a physiological model was developed in which forskolin treatment of intact porcine coronary arteries (PCA) prior to treatment with a contractile agonist resulted in complete suppression of force. Pretreatment of PCA with forskolin suppressed histamine-induced force generation but did not abolish [Ca²⁺]_i rise or MLC phosphorylation. Additionally, forskolin pretreatment reduced filamentous actin in histamine-treated tissues, and prevented histamine-induced changes in the phosphorylation of the actin-regulatory proteins HSP20, VASP, cofilin, and paxillin. Taken together, these results suggest that forskolin-induced complete force suppression is dependent upon the actin cytoskeletal regulation initiated by the phosphorylation changes of the actin regulatory proteins and not on the MLC dephosphorylation. This model of complete force suppression can be employed to further elucidate the mechanisms responsible for smooth muscle tone, and may offer cues to pathological situations, such as hypertension and vasospasm.     

Scinderin,a Ca2+-Dependent Actin Filament Severing Protein that Controls Cortical Actin Network Dynamics During Secretion

Secretory vesicles are localized in specific compartments within neurosecretory cells. These are different pools in which vesicles are in various states of releasability. The transit of vesicles between compartments is controlled and regulated by Ca²⁺, scinderin and the cortical F-actin network. Cortical F-actin disassembly is produced by the filament severing activity of scinderin. This Ca²⁺-dependent activity of scinderin together with its Ca²⁺-independent actin nucleating activity, control cortical F-actin dynamics during the secretory cycle. A good understanding of the interaction of actin with scinderin and of the role of this protein in secretion has been provided by the analysis of the molecular structure of scinderin together with the use of recombinant proteins corresponding to its different domains.     

Three-Dimensional Self-Gravito-Acoustic Solitary Waves in a Degenerate Quantum Plasma System

Plasma Physics Reports - Three-dimensional self-gravito-acoustic solitary waves (SGASWs) in a general (but realistic) self-gravitating degenerate quantum plasma media consisting of heavy...     

A Correlative Analysis of Actin Filament Assembly, Structure, and Dynamics

The effect of the type of metal ion (i.e., Ca²⁺, Mg²⁺, or none) bound to the high-affinity divalent cation binding site (HAS) of actin on filament assembly, structure, and dynamics was investigated in the absence and presence of the mushroom toxin phalloidin. In agreement with earlier reports, we found the polymerization reaction of G-actin into F-actin filaments to be tightly controlled by the type of divalent cation residing in its HAS. Moreover, novel polymerization data are presented indicating that LD, a dimer unproductive by itself, does incorporate into growing F-actin filaments. This observation suggests that during actin filament formation, in addition to the obligatory nucleation– condensation pathway involving UD, a productive filament dimer, a facultative, LD-based pathway is implicated whose abundance strongly depends on the exact polymerization conditions chosen. The “ragged” and “branched” filaments observed during the early stages of assembly represent a hallmark of LD incorporation and might be key to producing an actin meshwork capable of rapidly assembling and disassembling in highly motile cells. Hence, LD incorporation into growing actin filaments might provide an additional level of regulation of actin cytoskeleton dynamics. Regarding the structure and mechanical properties of the F-actin filament at steady state, no significant correlation with the divalent cation residing in its HAS was found. However, compared to native filaments, phalloidin-stabilized filaments were stiffer and yielded subtle but significant structural changes. Together, our data indicate that whereas the G-actin conformation is tightly controlled by the divalent cation in its HAS, the F-actin conformation appears more robust than this variation. Hence, we conclude that the structure and dynamics of the Mg–F-actin moiety within the thin filament are not significantly modulated by the cyclic Ca²⁺ release as it occurs in muscle contraction to regulate the actomyosin interaction via troponin.     

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.     

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.