Actin Filament Bundles Research Articles

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 Mg2+ generated actin filament bundles that processively attached to the nanofiber. Reduction of total Mg2+ 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 Mg2+, Lys-Lys2+, or fascin restored both comet tail attachment and sustained particle motility in low Mg2+ 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.

Ezrin regulates microvillus morphogenesis by promoting distinct activities of Eps8 proteins

The mechanisms that regulate actin filament polymerization resulting in the morphogenesis of the brush border microvilli in epithelial cells remain unknown. Eps8, the prototype of a family of proteins capable of capping and bundling actin filaments, has been shown to bundle the microvillar actin filaments. We report that Eps8L1a, a member of the Eps8 family and a novel ezrin-interacting partner, controls microvillus length through its capping activity. Depletion of Eps8L1a leads to the formation of long microvilli, whereas its overexpression has the opposite effect. We demonstrate that ezrin differentially modulates the actin-capping and -bundling activities of Eps8 and Eps8L1a during microvillus assembly. Coexpression of ezrin with Eps8 promotes the formation of membrane ruffles and tufts of microvilli, whereas expression of ezrin and Eps8L1a induces the clustering of actin-containing structures at the cell surface. These distinct morphological changes are neither observed when a mutant of ezrin defective in its binding to Eps8/Eps8L1a is coexpressed with Eps8 or Eps8L1a nor observed when ezrin is expressed with mutants of Eps8 or Eps8L1a defective in the actin-bundling or -capping activities, respectively. Our data show a synergistic effect of ezrin and Eps8 proteins in the assembly and organization of actin microvillar filaments.

Reprogramming cell shape with laser nano-patterning

Cell shape in vitro can be directed by geometrically defined micropatterned adhesion substrates. However conventional methods are limited by the fixed micropattern design, which cannot recapitulate the dynamic changes of the cell microenvironment. Here, we manipulate the shape of living cells in real time by using a tightly focused pulsed laser to introduce additional geometrically defined adhesion sites. The sub-micrometer resolution of the laser patterning allowed us to identify the critical distances between cell adhesion sites required for cell shape extension and contraction. This easy-to-handle method allows the precise control of specific actin-based structures that regulate cell architecture. Actin filament bundles or branched meshworks were induced, displaced or removed in response to specific dynamic modifications of the cell adhesion pattern. Isotropic branched actin meshworks could be forced to assemble new stress fibers locally and polarised in response to specific geometrical cues.

Bundling actin filaments from membranes: some novel players.

Progress in live-cell imaging of the cytoskeleton has significantly extended our knowledge about the organization and dynamics of actin filaments near the plasma membrane of plant cells. Noticeably, two populations of filamentous structures can be distinguished. On the one hand, fine actin filaments which exhibit an extremely dynamic behavior basically characterized by fast polymerization and prolific severing events, a process referred to as actin stochastic dynamics. On the other hand, thick actin bundles which are composed of several filaments and which are comparatively more stable although they constantly remodel as well. There is evidence that the actin cytoskeleton plays critical roles in trafficking and signaling at both the cell cortex and organelle periphery but the exact contribution of actin bundles remains unclear. A common view is that actin bundles provide the long-distance tracks used by myosin motors to deliver their cargo to growing regions and accordingly play a particularly important role in cell polarization. However, several studies support that actin bundles are more than simple passive highways and display multiple and dynamic roles in the regulation of many processes, such as cell elongation, polar auxin transport, stomatal and chloroplast movement, and defense against pathogens. The list of identified plant actin-bundling proteins is ever expanding, supporting that plant cells shape structurally and functionally different actin bundles. Here I review the most recently characterized actin-bundling proteins, with a particular focus on those potentially relevant to membrane trafficking and/or signaling.

Single Molecule Movement of Full-Length Myosin X

Myosin X is an unconventional myosin that is critical for cargo transportation to filopodia, and is associated with the mechanism underlying filopodia formation and extension. We previously reported that myosin X-HMM is a processive motor with large ∼34 nm step size. Our myosin X-HMM dominantly shows the large step size on both single actin filament and fascin-actin bundle although a minor ∼18 nm step size can be seen on fascin-actin bundle [Sun et al. (2010) Nat. Struct. Mol. Biol., 17, 485-491]. On the other hands, Rock and his colleagues recently reported that their myosin X-HMM only showed ∼18 nm step size [Nagy et al. (2010) J. Biol. Chem., 285, 26608-26617; Kapitein et al. (2010) Biophys. J., 99, 2143-2152]. Thus, the actual step size of myosin X remains to be elucidated. To address the question of innate step size of myosin X molecules, we produced the full-length myosin X construct with a leucine zipper motif at the C-terminal end (M10FullLZ) to ensure the dimer formation. We first compared the localization of M10FullLZ with the wild type myosin X full-length (M10Full) in COS7 cells, and confirmed that M10FullLZ localized at the tip of filopodia, which is similar to M10Full. We next expressed M10FullLZ in Sf9 cells and purified for motility assay. The M10FullLZ could move actin filaments in PIP3 dependent manner by in vitro multi-molecule motility assay, consistent with the result of M10Full. We then analyzed the single molecular step size of M10FullLZ using a total internal reflection fluorescence microscope. The mean step size of M10FullLZ was the large ∼34 nm on single actin filaments. This result suggests that the innate step size of myosin X is ∼34 nm. The step size of M10FullLZ on actin bundles in filopodia is now underway.

Filamin A controls matrix metalloprotease activity and regulates cell invasion in human fibrosarcoma cells.

Filamins are an important family of actin-binding proteins that, in addition to bundling actin filaments, link cell surface adhesion proteins, signaling receptors and channels to the actin cytoskeleton, and serve as scaffolds for an array of intracellular signaling proteins. Filamins are known to regulate the actin cytoskeleton, act as mechanosensors that modulate tissue responses to matrix density, control cell motility and inhibit activation of integrin adhesion receptors. In this study, we extend the repertoire of filamin activities to include control of extracellular matrix (ECM) degradation. We show that knockdown of filamin increases matrix metalloproteinase (MMP) activity and induces MMP2 activation, enhancing the ability of cells to remodel the ECM and increasing their invasive potential, without significantly altering two-dimensional random cell migration. We further show that within filamin A, the actin-binding domain is necessary, but not sufficient, to suppress the ECM degradation seen in filamin-A-knockdown cells and that dimerization and integrin binding are not required. Filamin mutations are associated with neuronal migration disorders and a range of congenital malformations characterized by skeletal dysplasia and various combinations of cardiac, craniofacial and intestinal anomalies. Furthermore, in breast cancers loss of filamin A has been correlated with increased metastatic potential. Our data suggest that effects on ECM remodeling and cell invasion should be considered when attempting to provide cellular explanations for the physiological and pathological effects of altered filamin expression or filamin mutations.

Arabidopsis VILLIN2 and VILLIN3 are required for the generation of thick actin filament bundles and for directional organ growth.

In plant cells, actin filament bundles serve as tracks for myosin-dependent organelle movement and play a role in the organization of the cytoplasm. Although virtually all plant cells contain actin filament bundles, the role of the different actin-bundling proteins remains largely unknown. In this study, we investigated the role of the actin-bundling protein villin in Arabidopsis (Arabidopsis thaliana). We used Arabidopsis T-DNA insertion lines to generate a double mutant in which VILLIN2 (VLN2) and VLN3 transcripts are truncated. Leaves, stems, siliques, and roots of vln2 vln3 double mutant plants are twisted, which is caused by local differences in cell length. Microscopy analysis of the actin cytoskeleton showed that in these double mutant plants, thin actin filament bundles are more abundant while thick actin filament bundles are virtually absent. In contrast to full-length VLN3, truncated VLN3 lacking the headpiece region does not rescue the phenotype of the vln2 vln3 double mutant. Our results show that villin is involved in the generation of thick actin filament bundles in several cell types and suggest that these bundles are involved in the regulation of coordinated cell expansion.

Motility and Segregation of Hsp104-Associated Protein Aggregates in Budding Yeast

During yeast cell division, aggregates of damaged proteins are segregated asymmetrically between the bud and the mother. It is thought that protein aggregates are cleared from the bud via actin cable-based retrograde transport toward the mother and that Bni1p formin regulates this transport. Here, we examined the dynamics of Hsp104-associated protein aggregates by video microscopy, particle tracking, and image correlation analysis. We show that protein aggregates undergo random walk without directional bias. Clearance of heat-induced aggregates from the bud does not depend on formin proteins but occurs mostly through dissolution via Hsp104p chaperon. Aggregates formed naturally in aged cells also exhibit random walk but do not dissolve during observation. Although our data do not disagree with a role for actin or cell polarity in aggregate segregation, modeling suggests that their asymmetric inheritance can be a predictable outcome of aggregates' slow diffusion and the geometry of yeast cells.

Adjudin disrupts spermatogenesis via the action of some unlikely partners

Adjudin, 1-(2,4-dichlorobenzyl)-1H-indazole-3-carbohydrazide (formerly called AF-2364), is a potent analog of lonidamine [1-(2,4-dichlorobenzyl)-1H-indazole-3-carboxylic acid] known to disrupt germ cell adhesion, most notably elongating and elongated spermatids, in the seminiferous epithelium of adult rat testes and thus, leads to infertility in rats. Since the population of spermatogonia and spermatogonial stem cells (SSCs) in the seminiferous tubules is not significantly reduced by the treatment of rats with adjudin, adjudin-induced infertility is highly reversible, which enables reinitiation of spermatogenesis and germ cell re-population of the voided seminiferous epithelium. Furthermore, adjudin appears to exert its effects at the testis-specific atypical adherens junction (AJ) type known as ectoplasmic specialization (ES), most notably the apical ES at the Sertoli cell-spermatid interface. Thus, the hypothalamic-pituitary-gonadal axis is not unaffected and systemic side-effects are minimal. This also makes adjudin a potential candidate for male contraceptive development. Herein, we critically evaluate recent findings in the field and provide an updated model regarding the mechanism underlying adjudin-induced apical ES disruption. In short, adjudin targets actin filament bundles at the apical ES, the hallmark ultrastructure of this testis-specific junction type not found in any other epithelia/endothelia in mammals, by suppressing the expression of Eps8 (epidermal growth factor receptor pathway substrate 8), an actin capping protein that also plays a role in actin bundling, so that actin filament bundles can no longer be maintained at the apical ES. This is concomitant with a mis-localization of Arp3 (actin-related protein 3, a component of the Arp2/3 complex that induces actin nucleation/branching) recruited by drebrin E, causing "unwanted" actin branching, further destabilizing actin filament bundles at the apical ES. Additionally, adjudin blocks the expression of PAR6 (partitioning defective protein 6) and 14-3-3 (also known as PAR5) considerably at the apical ES, disrupting the homeostasis of endocytic vesicle-mediated protein trafficking, which in turn leads to an increase in protein endocytosis. The net result of these changes destabilizes cell adhesion and induces degeneration of the apical ES, causing premature release of spermatids, mimicking spermiation.

High expression of Lifeact in Arabidopsis thaliana reduces dynamic reorganization of actin filaments but does not affect plant development

Lifeact is a novel probe that labels actin filaments in a wide range of organisms. We compared the localization and reorganization of Lifeact:Venus-labeled actin filaments in Arabidopsis root hairs and root epidermal cells of lines that express different levels of Lifeact: Venus with that of actin filaments labeled with GFP:FABD2, a commonly used probe in plants. Unlike GFP:FABD2, Lifeact:Venus labeled the highly dynamic fine F-actin in the subapical region of tip-growing root hairs. Lifeact:Venus expression at varying levels was not observed to affect plant development. However, at expression levels comparable to those of GFP:FABD2 in a well-characterized marker line, Lifeact:Venus reduced reorganization rates of bundles of actin filaments in root epidermal cells. Reorganization rates of cytoplasmic strands, which reflect the reorganization of the actin cytoskeleton, were also reduced in these lines. Moreover, in the same line, Lifeact:Venus-decorated actin filaments were more resistant to depolymerization by latrunculin B than those in an equivalent GFP:FABD2-expressing line. In lines where Lifeact: Venus is expressed at lower levels, these effects are less prominent or even absent. We conclude that Lifeact: Venus reduces remodeling of the actin cytoskeleton in Arabidopsis in a concentration-dependent manner. Since this reduction occurs at expression levels that do not cause defects in plant development, selection of normally growing plants is not sufficient to determine optimal Lifeact expression levels. When correct expression levels of Lifeact have been determined, it is a valuable probe that labels dynamic populations of actin filaments such as fine F-actin, better than FABD2 does.

BioEssays 10/2011

BioEssaysVolume 33, Issue 10 Cover PictureFree Access BioEssays 10/2011 First published: 15 September 2011 https://doi.org/10.1002/bies.201190052AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Abstract ‘Tug of war’ between cells: Cell-cell junction development depends on the forces applied. The cover image shows adherens junctions (magenta) with actin filaments (green) in an NRK fibroblast sheet. This type of adherens junction has an actin filament bundle oriented perpendicularly to the plasma membrane. Because actin bundles on both sides of the adherens junction are oriented along a straight line, it is obvious that both actin bundles are pulling each other and that adherens junctions are responsible for transmitting forces between cells. Recent studies reported that structure/function of adherens junctions/cadherin-mediated cell adhesions changes according to the forces applied to them. In this issue (pages 732–736) Shigenobu Yonemura gives an overview of mechanotransduction at adherens junctions and its possible molecular mechanism. The importance of α-catenin in the cadherin-catenin complex in a force-balancing mechanism for morphogenesis in multicellular organisms is highlighted. Cover by S. Yonemura Volume33, Issue10October 2011 RelatedInformation

Golgi Body Motility in the Plant Cell Cortex Correlates with Actin Cytoskeleton Organization

The actin cytoskeleton is involved in the transport and positioning of Golgi bodies, but the actin-based processes that determine the positioning and motility behavior of Golgi bodies are not well understood. In this work, we have studied the relationship between Golgi body motility behavior and actin organization in intercalary growing root epidermal cells during different developmental stages. We show that in these cells two distinct actin configurations are present, depending on the developmental stage. In small cells of the early root elongation zone, fine filamentous actin (F-actin) occupies the whole cell, including the cortex. In larger cells in the late elongation zone that have almost completed cell elongation, actin filament bundles are interspersed with areas containing this fine F-actin and areas without F-actin. Golgi bodies in areas with the fine F-actin exhibit a non-directional, wiggling type of motility. Golgi bodies in areas containing actin filament bundles move up to 7 μm s⁻¹. Since the motility of Golgi bodies changes when they enter an area with a different actin configuration, we conclude that the type of movement depends on the actin organization and not on the individual organelle. Our results show that the positioning of Golgi bodies depends on the local actin organization.

Barriers to Diabetes Prevention and Control Among American Indians

North Carolina's American Indian population experiences a disproportionate diabetes burden, in terms of both a high prevalence of the disease and excess diabetes-related death and disability. Concerted efforts need to be made to provide culturally appropriate and easily accessible education, health care, and health-promoting resources in these vulnerable communities.

Measuring Forces between Two Single Actin Filaments during Bundle Formation

Bundles of filamentous actin are dominant cytoskeletal structures, which play a crucial role in various cellular processes. As yet quantifying the fundamental interaction between two individual actin filaments forming the smallest possible bundle has not been realized. Applying holographic optical tweezers integrated with a microfluidic platform, we were able to measure the forces between two actin filaments during bundle formation. Quantitative analysis yields forces up to 0.2 pN depending on the concentration of bundling agents.

Mechanism of Actin Filament Bundling by Fascin

Fascin is the main actin filament bundling protein in filopodia. Because of the important role filopodia play in cell migration, fascin is emerging as a major target for cancer drug discovery. However, an understanding of the mechanism of bundle formation by fascin is critically lacking. Fascin consists of four β-trefoil domains. Here, we show that fascin contains two major actin-binding sites, coinciding with regions of high sequence conservation in β-trefoil domains 1 and 3. The site in β-trefoil-1 is located near the binding site of the fascin inhibitor macroketone and comprises residue Ser-39, whose phosphorylation by protein kinase C down-regulates actin bundling and formation of filopodia. The site in β-trefoil-3 is related by pseudo-2-fold symmetry to that in β-trefoil-1. The two sites are ∼5 nm apart, resulting in a distance between actin filaments in the bundle of ∼8.1 nm. Residue mutations in both sites disrupt bundle formation in vitro as assessed by co-sedimentation with actin and electron microscopy and severely impair formation of filopodia in cells as determined by rescue experiments in fascin-depleted cells. Mutations of other areas of the fascin surface also affect actin bundling and formation of filopodia albeit to a lesser extent, suggesting that, in addition to the two major actin-binding sites, fascin makes secondary contacts with other filaments in the bundle. In a high resolution crystal structure of fascin, molecules of glycerol and polyethylene glycol are bound in pockets located within the two major actin-binding sites. These molecules could guide the rational design of new anticancer fascin inhibitors.

Mechanics of Intracellular Stress Fibers: A Short Review

The mechanical response of living cells against external stimuli depends largely on the physical and biochemical properties of an intracellular structure called cytoskeleton. Three main kinds of filamentous protein structures, namely, microtubules, intermediate filaments, and actin thin filaments comprise the cytoskeleton. In the case of cells cultured on an adhesive substrate, the development of actin-based cytoskeleton, especially linearly bundled actin filaments called stress fibers (SFs), is well established. SFs are formed close to the cell membrane with one or both of their ends associated with the transmembrane structure called the focal adhesion (FA). In this article, we review some fundamental facts about SFs in live cells starting from the early description of the bundled filaments as observed under optical and electron microscopies. The morphology, chemical components, and biological functions of SFs, and recent work on the mechanical nature of SFs are reviewed.

Mechanisms of neuronal growth cone guidance: An historical perspective

At the distal most aspect of motile extending axons and dendrites lies the growth cone, a hand like macroorganelle of membrane bound cytoskeleton, packed with receptors, adhesion molecules, molecular motors, and an army of regulatory and signaling proteins. Splayed out along the substratum in vitro, the growth cone resembles an open hand with bundles of filamentous actin, barbed ends outstretched, as if fingers extending from a central domain of dynamic microtubule plus ends. The growth cone acts first as a sensory platform, analyzing the environment ahead for the presence of guidance cues, secondly as a mechanical dynamo establishing focal contact with the extracellular matrix to drive processive forward outgrowth, and thirdly as a forward biochemical command center where signals are interrogated to inform turning, extension, retraction, or branching. During his career, Paul Letourneau has made major contributions to our understanding of how growth cones respond to their environment. Here, we will summarize some of these major advances in their historical context. Letourneau's contributions have provided insights into cytoskeletal organization, growth cone dynamics, and signaling pathways. His recent work has described some important molecules and molecular mechanisms involved in growth cone turning. Although much remains to be understood about this important and intriguing structure, Letourneau's contributions have provided us with "growth cone guidance."

Actin Filament Bundling by Fimbrin Is Important for Endocytosis, Cytokinesis, and Polarization in Fission Yeast

Through the coordinated action of diverse actin-binding proteins, cells simultaneously assemble actin filaments with distinct architectures and dynamics to drive different processes. Actin filament cross-linking proteins organize filaments into higher order networks, although the requirement of cross-linking activity in cells has largely been assumed rather than directly tested. Fission yeast Schizosaccharomyces pombe assembles actin into three discrete structures: endocytic actin patches, polarizing actin cables, and the cytokinetic contractile ring. The fission yeast filament cross-linker fimbrin Fim1 primarily localizes to Arp2/3 complex-nucleated branched filaments of the actin patch and by a lesser amount to bundles of linear antiparallel filaments in the contractile ring. It is unclear whether Fim1 associates with bundles of parallel filaments in actin cables. We previously discovered that a principal role of Fim1 is to control localization of tropomyosin Cdc8, thereby facilitating cofilin-mediated filament turnover. Therefore, we hypothesized that the bundling ability of Fim1 is dispensable for actin patches but is important for the contractile ring and possibly actin cables. By directly visualizing actin filament assembly using total internal reflection fluorescence microscopy, we determined that Fim1 bundles filaments in both parallel and antiparallel orientations and efficiently bundles Arp2/3 complex-branched filaments in the absence but not the presence of actin capping protein. Examination of cells exclusively expressing a truncated version of Fim1 that can bind but not bundle actin filaments revealed that bundling activity of Fim1 is in fact important for all three actin structures. Therefore, fimbrin Fim1 has diverse roles as both a filament "gatekeeper" and as a filament cross-linker.

RefilinB (FAM101B) targets FilaminA to organize perinuclear actin networks and regulates nuclear shape

The intracellular localization and shape of the nucleus plays a central role in cellular and developmental processes. In fibroblasts, nuclear movement and shape are controlled by a specific perinuclear actin network made of contractile actin filament bundles called transmembrane actin-associated nuclear (TAN) lines that form a structure called the actin cap. The identification of regulatory proteins associated with this specific actin cytoskeletal dynamic is a priority for understanding actin-based changes in nuclear shape and position in normal and pathological situations. Here, we first identify a unique family of actin regulators, the refilin proteins (RefilinA and RefilinB), that stabilize specifically perinuclear actin filament bundles. We next identify the actin-binding filamin A (FLNA) protein as the downstream effector of refilins. Refilins act as molecular switches to convert FLNA from an actin branching protein into one that bundles. In NIH 3T3 fibroblasts, the RefilinB/FLNA complex organizes the perinuclear actin filament bundles forming the actin cap. Finally, we demonstrate that in epithelial normal murine mammary gland (NmuMG) cells, the RefilinB/FLNA complex controls formation of a new perinuclear actin network that accompanies nuclear shape changes during the epithelial-mesenchymal transition (EMT). Our studies open perspectives for further functional analyses of this unique actin-based network and shed light on FLNA function during development and in human syndromes associated with FLNA mutations.

The Plant-Specific Actin Binding Protein SCAB1 Stabilizes Actin Filaments and Regulates Stomatal Movement inArabidopsis

Microfilament dynamics play a critical role in regulating stomatal movement; however, the molecular mechanism underlying this process is not well understood. We report here the identification and characterization of STOMATAL CLOSURE-RELATED ACTIN BINDING PROTEIN1 (SCAB1), an Arabidopsis thaliana actin binding protein. Plants lacking SCAB1 were hypersensitive to drought stress and exhibited reduced abscisic acid-, H(2)O(2)-, and CaCl(2)-regulated stomatal movement. In vitro and in vivo analyses revealed that SCAB1 binds, stabilizes, and bundles actin filaments. SCAB1 shares sequence similarity only with plant proteins and contains a previously undiscovered actin binding domain. During stomatal closure, actin filaments switched from a radial orientation in open stomata to a longitudinal orientation in closed stomata. This switch took longer in scab1 plants than in wild-type plants and was correlated with the delay in stomatal closure seen in scab1 mutants in response to drought stress. Our results suggest that SCAB1 is required for the precise regulation of actin filament reorganization during stomatal closure.