Actin Filament Arrays Research Articles

Direct observation of cortactin protecting Arp2/3-actin filament branch junctions from GMF-mediated destabilization

How cells tightly control the formation and turnover of branched actin filament arrays to drive cell motility, endocytosis, and other cellular processes is still not well understood. Here, we investigated the mechanistic relationship between two binding partners of the Arp2/3 complex, glia maturation factor (GMF) and cortactin. Individually, GMF and cortactin have opposite effects on the stability of actin filament branches, but it is unknown how they work in concert with each other to govern branch turnover. Using TIRF microscopy, we observe that GMF’s branch destabilizing activities are potently blocked by cortactin (IC50 = 1.3 nM) and that this inhibition requires direct interactions of cortactin with Arp2/3 complex. The simplest model that would explain these results is competition for binding Arp2/3 complex. However, we find that cortactin and GMF do not compete for free Arp2/3 complex in solution. Further, we use single molecule analysis to show that cortactin’s on-rate (3 ×107 s−1 M−1) and off-rate (0.03 s−1) at branch junctions are minimally affected by excess GMF. Together, these results show that cortactin binds with high affinity to branch junctions, where it blocks the destabilizing effects of GMF, possibly by a mechanism that is allosteric in nature. In addition, the affinities we measure for cortactin at actin filament branch junctions (Kd = 0.9 nM) and filament sides (Kd = 206 nM) are approximately 20-fold stronger than previously reported. These observations contribute to an emerging view of molecular complexity in how Arp2/3 complex is regulated through the integration of multiple inputs.

G-actin diffusion is insufficient to achieve F-actin assembly in fast-treadmilling protrusions

To generate forces that drive migration of a eukaryotic cell, arrays of actin filaments (F-actin) are assembled at the cell’s leading membrane edge. To maintain cell propulsion and respond to dynamic external cues, actin filaments must be disassembled to regenerate the actin monomers (G-actin), and transport of G-actin from sites of disassembly back to the leading edge completes the treadmilling cycle and limits the flux of F-actin assembly. Whether or not molecular diffusion is sufficient for G-actin transport has been a long-standing topic of debate, in part because the dynamic nature of cell motility and migration hinders the estimation of transport parameters. In this work, we applied an experimental system in which cells adopt an approximately constant and symmetrical shape; they cannot migrate but exhibit fast, steady treadmilling in the thin region protruding from the cell. Using fluorescence recovery after photobleaching, we quantified the relative concentrations and corresponding fluxes of F- and G-actin in this system. In conjunction with mathematical modeling, constrained by measured features of each region of interest, this approach revealed that diffusion alone cannot account for the transport of G-actin to the leading edge. Although G-actin diffusion and vectorial transport might vary with position in the protruding region, good agreement with the fluorescence recovery after photobleaching measurements was achieved by a model with constant G-actin diffusivity ∼2 μm2/s and anterograde G-actin velocity less than 1 μm/s.

Microvilli, myosin contractility and apical actin organization are regulated locally though ezrin bound gaps

The structure of microvilli at the apical surface of epithelial cells consists of a core bundle of parallel actin filaments enveloped by the plasma membrane. Just beneath these structures exists a dense array of interwoven actin filaments where the core bundles are anchored. To create this finely ordered structure, links are produced between both actin-to-actin and actin-to-plasma-membrane interactions. These links are achieved by multiple myosin classes (myosin classes 1, 2, 7, and 9), actin bundling proteins (e.g.

Mechanobiology of muscle and myofibril morphogenesis.

Muscles generate forces for animal locomotion. The contractile apparatus of muscles is the sarcomere, a highly regular array of large actin and myosin filaments linked by gigantic titin springs. During muscle development many sarcomeres assemble in series into long periodic myofibrils that mechanically connect the attached skeleton elements. Thus, ATP-driven myosin forces can power movement of the skeleton. Here we review muscle and myofibril morphogenesis, with a particular focus on their mechanobiology. We describe recent progress on the molecular structure of sarcomeres and their mechanical connections to the skeleton. We discuss current models predicting how tension coordinates the assembly of key sarcomeric components to periodic myofibrils that then further mature during development. This requires transcriptional feedback mechanisms that may help to coordinate myofibril assembly and maturation states with the transcriptional program. To fuel the varying energy demands of muscles we also discuss the close mechanical interactions of myofibrils with mitochondria and nuclei to optimally support powerful or enduring muscle fibers.

Computational toolbox for ultrastructural quantitative analysis of filament networks in cryo-ET data

A precise quantitative description of the ultrastructural characteristics underlying biological mechanisms is often key to their understanding. This is particularly true for dynamic extra- and intracellular filamentous assemblies, playing a role in cell motility, cell integrity, cytokinesis, tissue formation and maintenance. For example, genetic manipulation or modulation of actin regulatory proteins frequently manifests in changes of the morphology, dynamics, and ultrastructural architecture of actin filament-rich cell peripheral structures, such as lamellipodia or filopodia. However, the observed ultrastructural effects often remain subtle and require sufficiently large datasets for appropriate quantitative analysis. The acquisition of such large datasets has been enabled by recent advances in high-throughput cryo-electron tomography (cryo-ET) methods. This also necessitates the development of complementary approaches to maximize the extraction of relevant biological information. We have developed a computational toolbox for the semi-automatic quantification of segmented and vectorized filamentous networks from pre-processed cryo-electron tomograms, facilitating the analysis and cross-comparison of multiple experimental conditions. GUI-based components simplify the processing of data and allow users to obtain a large number of ultrastructural parameters describing filamentous assemblies. We demonstrate the feasibility of this workflow by analyzing cryo-ET data of untreated and chemically perturbed branched actin filament networks and that of parallel actin filament arrays. In principle, the computational toolbox presented here is applicable for data analysis comprising any type of filaments in regular (i.e. parallel) or random arrangement. We show that it can ease the identification of key differences between experimental groups and facilitate the in-depth analysis of ultrastructural data in a time-efficient manner.

Filament-guided filament assembly provides structural memory of filament alignment during cytokinesis.

During cytokinesis, animal cells rapidly remodel the equatorial cortex to build an aligned array of actin filaments called the contractile ring. Local reorientation of filaments by active equatorial compression is thought to underlie the emergence of filament alignment during ring assembly. Here, combining single molecule analysis and modeling in one-cell C.elegans embryos, we show that filaments turnover is far too fast for reorientation of individual filaments by equatorial compression to explain the observed alignment, even if favorably oriented filaments are selectively stabilized. By tracking single formin/CYK-1::GFP particles to monitor local filament assembly, we identify a mechanism that we call filament-guided filament assembly (FGFA), in which existing filaments serve as templates to orient the growth of new filaments. FGFA sharply increases the effective lifetime of filament orientation, providing structural memory that allows cells to build highly aligned filament arrays in response to equatorial compression, despite rapid turnover of individual filaments.

Coactosin Promotes F-Actin Protrusion in Growth Cones Under Cofilin-Related Signaling Pathway.

During brain development, axon outgrowth and its subsequent pathfinding are reliant on a highly motile growth cone located at the tip of the axon. Actin polymerization that is regulated by actin-depolymerizing factors homology (ADF-H) domain-containing family drives the formation of lamellipodia and filopodia at the leading edge of growth cones for axon guidance. However, the precise localization and function of ADF-H domain-containing proteins involved in axon extension and retraction remain unclear. We have previously shown that transcripts and proteins of coactosin-like protein 1 (COTL1), an ADF-H domain-containing protein, are observed in neurites and axons in chick embryos. Coactosin overexpression analysis revealed that this protein was localized to axonal growth cones and involved in axon extension in the midbrain. We further examined the specific distribution of coactosin and cofilin within the growth cone using superresolution microscopy, structured illumination microscopy, which overcomes the optical diffraction limitation and is suitable to the analysis of cellular dynamic movements. We found that coactosin was tightly associated with F-actin bundles at the growth cones and that coactosin overexpression promoted the expansion of lamellipodia and extension of growth cones. Coactosin knockdown in oculomotor neurons resulted in an increase in the levels of the inactive, phosphorylated form of cofilin and dysregulation of actin polymerization and axonal elongation, which suggests that coactosin promoted axonal growth in a cofilin-dependent manner. Indeed, the application of a dominant-negative form of LIMK1, a downstream effector of GTPases, reversed the effect of coactosin knockdown on axonal growth by enhancing cofilin activity. Combined, our results indicate that coactosin functions promote the assembly of protrusive actin filament arrays at the leading edge for growth cone motility.

Actin Cell Cortex: Structure and Molecular Organization.

The actin cytoskeleton consists of structurally and biochemically different actin filament arrays. Among them, the actin cortex is thought to have key roles in cell mechanics, but remains a poorly characterized part of the actin cytoskeleton. The cell cortex is typically defined as a thin layer of actin meshwork that uniformly underlies the plasma membrane of the entire cell. However, this definition applies only to specific cases. In general, the cortex structure and subcellular distribution vary significantly across cell types and physiological states of the cell. In this review, I focus on our current knowledge of the structure and molecular composition of the cell cortex.

Atypical Myosin Tunes Dendrite Arbor Subdivision

Dendrite arbor pattern determines the functional characteristics of a neuron. It is founded on primary branch structure, defined through cell intrinsic and transcription-factor-encoded mechanisms. Developing arbors have extensive acentrosomal microtubule dynamics, and here, we report an unexpected role for the atypical actin motor Myo6 in creating primary branch structure by specifying the position, polarity,and targeting of these events. We carried out invivo time-lapse imaging of Drosophila adult sensory neuron differentiation, integrating machine-learning-based quantification of arbor patterning with molecular-level tracking of cytoskeletal remodeling. This revealed that Myo6 and the transcription factor Knot regulate transient surges of microtubule polymerization at dendrite tips; they drive retrograde extension of an actin filament array that specifies anterograde microtubule polymerization and guides these microtubules to subdivide the tip into multiple branches. Primary branches delineate functional compartments; this tunable branching mechanism is key to define and diversify dendrite arbor compartmentalization.

Mitochondrial dynamics and segregation during the asymmetric division of Arabidopsis zygotes

The zygote is the first cell of a multicellular organism. In most angiosperms, the zygote divides asymmetrically to produce an embryo-precursor apical cell and a supporting basal cell. Zygotic division should properly segregate symbiotic organelles, because they cannot be synthesized de novo. In this study, we revealed the real-time dynamics of the principle source of ATP biogenesis, mitochondria, in Arabidopsis thaliana zygotes using live-cell observations and image quantifications. In the zygote, the mitochondria formed the extended structure associated with the longitudinal array of actin filaments (F-actins) and were polarly distributed along the apical-basal axis. The mitochondria were then temporally fragmented during zygotic division, and the resulting apical cells inherited mitochondria at higher concentration compared to the basal cells. Further observation of postembryonic organs showed that these mitochondrial behaviours are characteristic of the zygote. Overall, our results showed that the zygote has spatiotemporal regulation that unequally distributes the mitochondria.

Three-dimensional structure of the basketweave Z-band in midshipman fish sonic muscle

Striated muscle enables movement in all animals by the contraction of myriads of sarcomeres joined end to end by the Z-bands. The contraction is due to tension generated in each sarcomere between overlapping arrays of actin and myosin filaments. At the Z-band, actin filaments from adjoining sarcomeres overlap and are cross-linked in a regular pattern mainly by the protein α-actinin. The Z-band is dynamic, reflected by the 2 regular patterns seen in transverse section electron micrographs; the so-called small-square and basketweave forms. Although these forms are attributed, respectively, to relaxed and actively contracting muscles, the basketweave form occurs in certain relaxed muscles as in the muscle studied here. We used electron tomography and subtomogram averaging to derive the 3D structure of the Z-band in the swimbladder sonic muscle of type I male plainfin midshipman fish (Porichthys notatus), into which we docked the crystallographic structures of actin and α-actinin. The α-actinin links run diagonally between connected pairs of antiparallel actin filaments and are oriented at an angle of about 25° away from the actin filament axes. The slightly curved and flattened structure of the α-actinin rod has a distinct fit into the map. The Z-band model provides a detailed understanding of the role of α-actinin in transmitting tension between actin filaments in adjoining sarcomeres.

Ultrastructure and dynamics of the actin-myosin II cytoskeleton during mitochondrial fission.

Mitochondrial fission involves preconstriction of the organelle followed by scission by dynamin-related protein Drp1. Preconstriction is facilitated by actin and nonmuscle myosin II through a mechanism that remains unclear, mostly, due to unknown cytoskeleton ultrastructure at mitochondrial constrictions. Here, using platinum replica electron microscopy, we show that mitochondria in cells are embedded into an interstitial cytoskeletal network containing abundant unbranched actin filaments. Both spontaneous and induced mitochondrial constrictions typically associate with a criss-cross array of long actin filaments that comprise a part of this interstitial network. Nonmuscle myosin II is found adjacent to mitochondria without specific enrichments at the constriction sites. During ionomycin-induced mitochondrial fission, F-actin clouds colocalize with mitochondrial constriction sites, whereas myosin II clouds mostly fluctuate nearby. We propose that myosin II promotes mitochondrial constriction by inducing stochastic deformations of the interstitial actin network, which applies pressure onto mitochondrial surface, thus initiating curvature-sensing mechanisms that complete mitochondrial constriction.

Polar vacuolar distribution is essential for accurate asymmetric division of Arabidopsis zygotes

In most flowering plants, the asymmetric cell division of the zygote is the initial step in establishing the apical-basal axis of the mature plant. The zygote is polarized, possessing the nucleus at the apical tip and large vacuoles at the basal end. Despite their known polar localization, whether the positioning of the vacuoles and the nucleus is coordinated and what the role of the vacuole is in the asymmetric zygotic division remain elusive. In the present study, we utilized a live-cell imaging system to visualize the dynamics of vacuoles during the entire process of zygote polarization in Arabidopsis Image analysis revealed that the vacuoles formed tubular strands around the apically migrating nucleus. They gradually accumulated at the basal region and filled the space, resulting in asymmetric distribution in the mature zygote. To assess the role of vacuoles in the zygote, we screened various vacuole mutants and identified that shoot gravitropism2 (sgr2), in which the vacuolar structural change was impaired, failed to form tubular vacuoles and to polarly distribute the vacuole. In sgr2, large vacuoles occupied the apical tip and thus nuclear migration was blocked, resulting in a more symmetric zygotic division. We further observed that tubular vacuole formation and asymmetric vacuolar distribution both depended on the longitudinal array of actin filaments. Overall, our results show that vacuolar dynamics is crucial not only for the polar distribution along actin filaments but also for adequate nuclear positioning, and consequently zygote-division asymmetry.

A transcriptomics resource reveals a transcriptional transition during ordered sarcomere morphogenesis in flight muscle.

Muscles organise pseudo-crystalline arrays of actin, myosin and titin filaments to build force-producing sarcomeres. To study sarcomerogenesis, we have generated a transcriptomics resource of developing Drosophila flight muscles and identified 40 distinct expression profile clusters. Strikingly, most sarcomeric components group in two clusters, which are strongly induced after all myofibrils have been assembled, indicating a transcriptional transition during myofibrillogenesis. Following myofibril assembly, many short sarcomeres are added to each myofibril. Subsequently, all sarcomeres mature, reaching 1.5 µm diameter and 3.2 µm length and acquiring stretch-sensitivity. The efficient induction of the transcriptional transition during myofibrillogenesis, including the transcriptional boost of sarcomeric components, requires in part the transcriptional regulator Spalt major. As a consequence of Spalt knock-down, sarcomere maturation is defective and fibers fail to gain stretch-sensitivity. Together, this defines an ordered sarcomere morphogenesis process under precise transcriptional control - a concept that may also apply to vertebrate muscle or heart development.

Palladin Compensates for the Arp2/3 Complex and Supports Actin Structures during Listeria Infections.

ABSTRACTPalladin is an important component of motile actin-rich structures and nucleates branched actin filament arrays in vitro. Here we examine the role of palladin during Listeria monocytogenes infections in order to tease out novel functions of palladin. We show that palladin is co-opted by L. monocytogenes during its cellular entry and intracellular motility. Depletion of palladin resulted in shorter and misshapen comet tails, and when actin- or VASP-binding mutants of palladin were overexpressed in cells, comet tails disintegrated or became thinner. Comet tail thinning resulted in parallel actin bundles within the structures. To determine whether palladin could compensate for the Arp2/3 complex, we overexpressed palladin in cells treated with the Arp2/3 inhibitor CK-666. In treated cells, bacterial motility could be initiated and maintained when levels of palladin were increased. To confirm these findings, we utilized a cell line depleted of multiple Arp2/3 complex subunits. Within these cells, L. monocytogenes failed to generate comet tails. When palladin was overexpressed in this Arp2/3 functionally null cell line, the ability of L. monocytogenes to generate comet tails was restored. Using purified protein components, we demonstrate that L. monocytogenes actin clouds and comet tails can be generated (in a cell-free system) by palladin in the absence of the Arp2/3 complex. Collectively, our results demonstrate that palladin can functionally replace the Arp2/3 complex during bacterial actin-based motility.

Rice Morphology Determinant-Mediated Actin Filament Organization Contributes to Pollen Tube Growth.

For successful fertilization in angiosperms, rapid tip growth in pollen tubes delivers the male gamete into the ovules. The actin-binding protein-mediated organization of the actin cytoskeleton within the pollen tube plays a crucial role in this polarized process. However, the mechanism underlying the polarity of the actin filament (F-actin) array and behaviors in pollen tube growth remain largely unknown. Here, we demonstrate that an actin-organizing protein, Rice Morphology Determinant (RMD), a type II formin from rice (Oryza sativa), controls pollen tube growth by modulating the polarity and distribution of the F-actin array. The rice rmd mutant exhibits abnormal pollen tube growth and a decreased germination rate of the pollen grain in vitro and in vivo. The rmd pollen tubes display a disorganized F-actin pattern with disrupted apical actin density and shank longitudinal cable direction/arrangement, indicating the novel role of RMD in F-actin polarity during tip growth. Consistent with this role, RMD localizes at the tip of the rice pollen tube, which is essential for pollen tube growth and polarity as well as F-actin organization. Furthermore, the direction and characteristics of the RMD-guided F-actin array positively regulate the deposition of cell wall components and the pattern and velocity of cytoplasmic streaming during rice pollen tube growth. Collectively, our results suggest that RMD is essential for the spatial regulation of pollen tube growth via modulating F-actin organization and array orientation in rice. This work provides insights into tip-focused cell growth and polarity.

Thick Filament Protein Network, Functions, and Disease Association.

Sarcomeres consist of highly ordered arrays of thick myosin and thin actin filaments along with accessory proteins. Thick filaments occupy the center of sarcomeres where they partially overlap with thin filaments. The sliding of thick filaments past thin filaments is a highly regulated process that occurs in an ATP-dependent manner driving muscle contraction. In addition to myosin that makes up the backbone of the thick filament, four other proteins which are intimately bound to the thick filament, myosin binding protein-C, titin, myomesin, and obscurin play important structural and regulatory roles. Consistent with this, mutations in the respective genes have been associated with idiopathic and congenital forms of skeletal and cardiac myopathies. In this review, we aim to summarize our current knowledge on the molecular structure, subcellular localization, interacting partners, function, modulation via posttranslational modifications, and disease involvement of these five major proteins that comprise the thick filament of striated muscle cells. © 2018 American Physiological Society. Compr Physiol 8:631-709, 2018.

Mechanism of Ultra-Fast Actin-Myosin Sliding Producing Cytoplasmic Streaming in Giant Algal Cell, Studied Using the Centrifuge Microscope

In giant intermodal cells of green algae Chara collaria, cytoplasmic streaming is produced by ATP-dependent sliding between myosin heads extending from amorphous cytoplasmic organelles and actin filament arrays (actin cables) fixed on chloroplast rows. The velocity of cytoplasmic streaming is many times faster than the maximum myofilament sliding in skeletal muscle. In this article, we compared steady-state force-velocity (P-V) relations between cytoplasmic myosin and skeletal and cardiac muscle myosins using the centrifuge microscope, in which myosincoated latex beads were made to slide along the actin cables under various centrifugal forces. In contrast with the hyperbolic P-V relation of actin-myosin sliding in skeletal and cardiac myosins, the P-V relation of cytoplasmic myosin versus actin cable sliding was a straight line, indicating a very large duty ratio and a very small rate of chemomechanical energy conversion. Possible mechanisms of the ultra-fast actin-myosin sliding are discussed.Highlights• The velocity of cytoplasmic streaming, caused by ATP-dependent sliding between cytoplasmic myosin and actin cables in giant algal cells is many times faster than ATP-dependent actin-myosin sliding in skeletal and cardiac muscles.• The mechanism of ultra-fast actin-myosin sliding was studied using the centrifuge microscope, in which beads coated with cytoplasmic myosin were made to slide along actin cables under various centrifugal forces serving as loads against cytoplasmic myosin versus actin cable sliding.• Unlike the hyperbolic force-velocity (P-V) relation of skeletal and cardiac muscle actin-myosin sliding, the P-V relation of cytoplasmic actin myosin sliding was a straight line irrespective of the force generated by cytoplasmic myosin.• These results indicate a very large duty ratio and a very small efficiency of chemo-mechanical energy conversion in cytoplasmic actin-myosin sliding.

The Actin Cytoskeleton: Functional Arrays for Cytoplasmic Organization and Cell Shape Control.

Functionally distinct actin filament arrays cluster organelles and define cellular scale flow patterns for secretion.

Auditory Hair Cell Stereocilia: Balancing Stability to Gain Sensitivity?

Auditory Hair Cell Stereocilia: Balancing Stability to Gain Sensitivity?