Toxoplasma gondii F-actin forms an extensive filamentous network required for material exchange and parasite maturation.

PubMed

Periz, Javier; Whitelaw, Jamie; Harding, Clare; Gras, Simon; Del Rosario Minina, Mario Igor; Latorre-Barragan, Fernanda; Lemgruber, Leandro; Reimer, Madita Alice; Insall, Robert; Heaslip, Aoife; Meissner, Markus

2017-03-21

Apicomplexan actin is important during the parasite's life cycle. Its polymerization kinetics are unusual, permitting only short, unstable F-actin filaments. It has not been possible to study actin in vivo and so its physiological roles have remained obscure, leading to models distinct from conventional actin behaviour. Here a modified version of the commercially available actin-chromobody was tested as a novel tool for visualising F-actin dynamics in Toxoplasma gondii. Cb labels filamentous actin structures within the parasite cytosol and labels an extensive F-actin network that connects parasites within the parasitophorous vacuole and allows vesicles to be exchanged between parasites. In the absence of actin, parasites lack a residual body and inter-parasite connections and grow in an asynchronous and disorganized manner. Collectively, these data identify new roles for actin in the intracellular phase of the parasites lytic cycle and provide a robust new tool for imaging parasitic F-actin dynamics.

Single-Molecule Studies of Actin Assembly and Disassembly Factors

PubMed Central

Smith, Benjamin A.; Gelles, Jeff; Goode, Bruce L.

2014-01-01

The actin cytoskeleton is very dynamic and highly regulated by multiple associated proteins in vivo. Understanding how this system of proteins functions in the processes of actin network assembly and disassembly requires methods to dissect the mechanisms of activity of individual factors and of multiple factors acting in concert. The advent of single-filament and single-molecule fluorescence imaging methods has provided a powerful new approach to discovering actin-regulatory activities and obtaining direct, quantitative insights into the pathways of molecular interactions that regulate actin network architecture and dynamics. Here we describe techniques for acquisition and analysis of single-molecule data, applied to the novel challenges of studying the filament assembly and disassembly activities of actin-associated proteins in vitro. We discuss the advantages of single-molecule analysis in directly visualizing the order of molecular events, measuring the kinetic rates of filament binding and dissociation, and studying the coordination among multiple factors. The methods described here complement traditional biochemical approaches in elucidating actin-regulatory mechanisms in reconstituted filamentous networks. PMID:24630103

Memory Dynamics in Cross-linked Actin Networks

NASA Astrophysics Data System (ADS)

Scheff, Danielle; Majumdar, Sayantan; Gardel, Margaret

Cells demonstrate the remarkable ability to adapt to mechanical stimuli through rearrangement of the actin cytoskeleton, a cross-linked network of actin filaments. In addition to its importance in cell biology, understanding this mechanical response provides strategies for creation of novel materials. A recent study has demonstrated that applied stress can encode mechanical memory in these networks through changes in network geometry, which gives rise to anisotropic shear response. Under later shear, the network is stiffer in the direction of the previously applied stress. However, the dynamics behind the encoding of this memory are unknown. To address this question, we explore the effect of varying either the rigidity of the cross-linkers or the length of actin filament on the time scales required for both memory encoding and over which it later decays. While previous experiments saw only a long-lived memory, initial results suggest another mechanism where memories relax relatively quickly. Overall, our study is crucial for understanding the process by which an external stress can impact network arrangement and thus the dynamics of memory formation.

Fascin- and α-Actinin-Bundled Networks Contain Intrinsic Structural Features that Drive Protein Sorting.

PubMed

Winkelman, Jonathan D; Suarez, Cristian; Hocky, Glen M; Harker, Alyssa J; Morganthaler, Alisha N; Christensen, Jenna R; Voth, Gregory A; Bartles, James R; Kovar, David R

2016-10-24

Cells assemble and maintain functionally distinct actin cytoskeleton networks with various actin filament organizations and dynamics through the coordinated action of different sets of actin-binding proteins. The biochemical and functional properties of diverse actin-binding proteins, both alone and in combination, have been increasingly well studied. Conversely, how different sets of actin-binding proteins properly sort to distinct actin filament networks in the first place is not nearly as well understood. Actin-binding protein sorting is critical for the self-organization of diverse dynamic actin cytoskeleton networks within a common cytoplasm. Using in vitro reconstitution techniques including biomimetic assays and single-molecule multi-color total internal reflection fluorescence microscopy, we discovered that sorting of the prominent actin-bundling proteins fascin and α-actinin to distinct networks is an intrinsic behavior, free of complicated cellular signaling cascades. When mixed, fascin and α-actinin mutually exclude each other by promoting their own recruitment and inhibiting recruitment of the other, resulting in the formation of distinct fascin- or α-actinin-bundled domains. Subdiffraction-resolution light microscopy and negative-staining electron microscopy revealed that fascin domains are densely packed, whereas α-actinin domains consist of widely spaced parallel actin filaments. Importantly, other actin-binding proteins such as fimbrin and espin show high specificity between these two bundle types within the same reaction. Here we directly observe that fascin and α-actinin intrinsically segregate to discrete bundled domains that are specifically recognized by other actin-binding proteins. Copyright © 2016 Elsevier Ltd. All rights reserved.

A structural study of F-actin - filamin networks

NASA Astrophysics Data System (ADS)

Ahrens-Braunstein, Ashley; Nguyen, Lam; Hirst, Linda

2010-03-01

The cell's ability to move and contract is attributed to the semi-flexible filamentous protein, F -actin, one of the three filaments in the cytoskeleton. Actin bundling can be formed by a cross-linking actin binding protein (ABP) filamin. By examining filamin's cross-linking abilities at different concentrations and molar ratios, we can study the flexibility, structure and multiple network formations created when cross-linking F-actin with this protein. We have studied the phase diagram of this protein system using fluorescence microscopy, analyzing the network structures observed in the context of a coarse grained molecular dynamics simulation carried out by our group.

The Stationary-Phase Cells of Saccharomyces cerevisiae Display Dynamic Actin Filaments Required for Processes Extending Chronological Life Span.

PubMed

Vasicova, Pavla; Lejskova, Renata; Malcova, Ivana; Hasek, Jiri

2015-11-01

Stationary-growth-phase Saccharomyces cerevisiae yeast cultures consist of nondividing cells that undergo chronological aging. For their successful survival, the turnover of proteins and organelles, ensured by autophagy and the activation of mitochondria, is performed. Some of these processes are engaged in by the actin cytoskeleton. In S. cerevisiae stationary-phase cells, F actin has been shown to form static aggregates named actin bodies, subsequently cited to be markers of quiescence. Our in vivo analyses revealed that stationary-phase cultures contain cells with dynamic actin filaments, besides the cells with static actin bodies. The cells with dynamic actin displayed active endocytosis and autophagy and well-developed mitochondrial networks. Even more, stationary-phase cell cultures grown under calorie restriction predominantly contained cells with actin cables, confirming that the presence of actin cables is linked to successful adaptation to stationary phase. Cells with actin bodies were inactive in endocytosis and autophagy and displayed aberrations in mitochondrial networks. Notably, cells of the respiratory activity-deficient cox4Δ strain displayed the same mitochondrial aberrations and actin bodies only. Additionally, our results indicate that mitochondrial dysfunction precedes the formation of actin bodies and the appearance of actin bodies corresponds to decreased cell fitness. We conclude that the F-actin status reflects the extent of damage that arises from exponential growth. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

The Stationary-Phase Cells of Saccharomyces cerevisiae Display Dynamic Actin Filaments Required for Processes Extending Chronological Life Span

PubMed Central

Lejskova, Renata; Malcova, Ivana

2015-01-01

Stationary-growth-phase Saccharomyces cerevisiae yeast cultures consist of nondividing cells that undergo chronological aging. For their successful survival, the turnover of proteins and organelles, ensured by autophagy and the activation of mitochondria, is performed. Some of these processes are engaged in by the actin cytoskeleton. In S. cerevisiae stationary-phase cells, F actin has been shown to form static aggregates named actin bodies, subsequently cited to be markers of quiescence. Our in vivo analyses revealed that stationary-phase cultures contain cells with dynamic actin filaments, besides the cells with static actin bodies. The cells with dynamic actin displayed active endocytosis and autophagy and well-developed mitochondrial networks. Even more, stationary-phase cell cultures grown under calorie restriction predominantly contained cells with actin cables, confirming that the presence of actin cables is linked to successful adaptation to stationary phase. Cells with actin bodies were inactive in endocytosis and autophagy and displayed aberrations in mitochondrial networks. Notably, cells of the respiratory activity-deficient cox4Δ strain displayed the same mitochondrial aberrations and actin bodies only. Additionally, our results indicate that mitochondrial dysfunction precedes the formation of actin bodies and the appearance of actin bodies corresponds to decreased cell fitness. We conclude that the F-actin status reflects the extent of damage that arises from exponential growth. PMID:26351139

Actin cable dynamics and Rho/Rock orchestrate a polarized cytoskeletal architecture in the early steps of assembling a stratified epithelium.

PubMed

Vaezi, Alec; Bauer, Christoph; Vasioukhin, Valeri; Fuchs, Elaine

2002-09-01

To enable stratification and barrier function, the epidermis must permit self-renewal while maintaining adhesive connections. By generating K14-GFP-actin mice to monitor actin dynamics in cultured primary keratinocytes, we uncovered a role for the actin cytoskeleton in establishing cellular organization. During epidermal sheet formation, a polarized network of nascent intercellular junctions and radial actin cables assemble in the apical plane of the monolayer. These actin fibers anchor to a central actin-myosin network, creating a tension-based plane of cytoskeleton across the apical surface of the sheet. Movement of the sheet surface relative to its base expands the zone of intercellular overlap, catalyzing new sites for nascent intercellular junctions. This polarized cytoskeleton is dependent upon alpha-catenin, Rho, and Rock, and its regulation may be important for wound healing and/or stratification, where coordinated tissue movements are involved.

A master equation approach to actin polymerization applied to endocytosis in yeast.

PubMed

Wang, Xinxin; Carlsson, Anders E

2017-12-01

We present a Master Equation approach to calculating polymerization dynamics and force generation by branched actin networks at membranes. The method treats the time evolution of the F-actin distribution in three dimensions, with branching included as a directional spreading term. It is validated by comparison with stochastic simulations of force generation by actin polymerization at obstacles coated with actin "nucleation promoting factors" (NPFs). The method is then used to treat the dynamics of actin polymerization and force generation during endocytosis in yeast, using a model in which NPFs form a ring around the endocytic site, centered by a spot of molecules attaching the actin network strongly to the membrane. We find that a spontaneous actin filament nucleation mechanism is required for adequate forces to drive the process, that partial inhibition of branching and polymerization lead to different characteristic responses, and that a limited range of polymerization-rate values provide effective invagination and obtain correct predictions for the effects of mutations in the active regions of the NPFs.

A master equation approach to actin polymerization applied to endocytosis in yeast

PubMed Central

Wang, Xinxin

2017-01-01

We present a Master Equation approach to calculating polymerization dynamics and force generation by branched actin networks at membranes. The method treats the time evolution of the F-actin distribution in three dimensions, with branching included as a directional spreading term. It is validated by comparison with stochastic simulations of force generation by actin polymerization at obstacles coated with actin “nucleation promoting factors” (NPFs). The method is then used to treat the dynamics of actin polymerization and force generation during endocytosis in yeast, using a model in which NPFs form a ring around the endocytic site, centered by a spot of molecules attaching the actin network strongly to the membrane. We find that a spontaneous actin filament nucleation mechanism is required for adequate forces to drive the process, that partial inhibition of branching and polymerization lead to different characteristic responses, and that a limited range of polymerization-rate values provide effective invagination and obtain correct predictions for the effects of mutations in the active regions of the NPFs. PMID:29240771

Arp2/3 complex–dependent actin networks constrain myosin II function in driving retrograde actin flow

PubMed Central

Yang, Qing; Zhang, Xiao-Feng; Pollard, Thomas D.

2012-01-01

The Arp2/3 complex nucleates actin filaments to generate networks at the leading edge of motile cells. Nonmuscle myosin II produces contractile forces involved in driving actin network translocation. We inhibited the Arp2/3 complex and/or myosin II with small molecules to investigate their respective functions in neuronal growth cone actin dynamics. Inhibition of the Arp2/3 complex with CK666 reduced barbed end actin assembly site density at the leading edge, disrupted actin veils, and resulted in veil retraction. Strikingly, retrograde actin flow rates increased with Arp2/3 complex inhibition; however, when myosin II activity was blocked, Arp2/3 complex inhibition now resulted in slowing of retrograde actin flow and veils no longer retracted. Retrograde flow rate increases induced by Arp2/3 complex inhibition were independent of Rho kinase activity. These results provide evidence that, although the Arp2/3 complex and myosin II are spatially segregated, actin networks assembled by the Arp2/3 complex can restrict myosin II–dependent contractility with consequent effects on growth cone motility. PMID:22711700

Actin Interacting Protein1 and Actin Depolymerizing Factor Drive Rapid Actin Dynamics in Physcomitrella patens[W

PubMed Central

Augustine, Robert C.; Pattavina, Kelli A.; Tüzel, Erkan; Vidali, Luis; Bezanilla, Magdalena

2011-01-01

The remodeling of actin networks is required for a variety of cellular processes in eukaryotes. In plants, several actin binding proteins have been implicated in remodeling cortical actin filaments (F-actin). However, the extent to which these proteins support F-actin dynamics in planta has not been tested. Using reverse genetics, complementation analyses, and cell biological approaches, we assessed the in vivo function of two actin turnover proteins: actin interacting protein1 (AIP1) and actin depolymerizing factor (ADF). We report that AIP1 is a single-copy gene in the moss Physcomitrella patens. AIP1 knockout plants are viable but have reduced expansion of tip-growing cells. AIP1 is diffusely cytosolic and functions in a common genetic pathway with ADF to promote tip growth. Specifically, ADF can partially compensate for loss of AIP1, and AIP1 requires ADF for function. Consistent with a role in actin remodeling, AIP1 knockout lines accumulate F-actin bundles, have fewer dynamic ends, and have reduced severing frequency. Importantly, we demonstrate that AIP1 promotes and ADF is essential for cortical F-actin dynamics. PMID:22003077

The spatial response of nonlinear strain propagation in response to actively driven microspheres through entangled actin networks

NASA Astrophysics Data System (ADS)

Falzone, Tobias; Blair, Savanna; Robertson-Anderson, Rae

2015-03-01

The semiflexible biopolymer actin, a ubiquitous component of nearly all biological organisms, plays an important role in many mechanically-driven processes such as muscle contraction, cancer invasion and cell motility. As such, entangled actin networks, which possess unique and complex viscoelastic properties, have been the subject of much theoretical and experimental work. However, due to this viscoelastic complexity, much is still unknown regarding the correlation of the applied stress on actin networks to the induced filament strain at the molecular and micro scale. Here, we use simultaneous optical trapping and fluorescence microscopy to characterize the link between applied microscopic forces and strain propagation as a function of strain rate and concentration. Specifically, we track fiduciary markers on entangled actin filaments before, during and after actively driving embedded microspheres through the network. These measurements provide much needed insight into the molecular-level dynamics connecting stress and strain in semiflexible polymer networks.

Investigating sub-spine actin dynamics in rat hippocampal neurons with super-resolution optical imaging.

PubMed

Tatavarty, Vedakumar; Kim, Eun-Ji; Rodionov, Vladimir; Yu, Ji

2009-11-09

Morphological changes in dendritic spines represent an important mechanism for synaptic plasticity which is postulated to underlie the vital cognitive phenomena of learning and memory. These morphological changes are driven by the dynamic actin cytoskeleton that is present in dendritic spines. The study of actin dynamics in these spines traditionally has been hindered by the small size of the spine. In this study, we utilize a photo-activation localization microscopy (PALM)-based single-molecule tracking technique to analyze F-actin movements with approximately 30-nm resolution in cultured hippocampal neurons. We were able to observe the kinematic (physical motion of actin filaments, i.e., retrograde flow) and kinetic (F-actin turn-over) dynamics of F-actin at the single-filament level in dendritic spines. We found that F-actin in dendritic spines exhibits highly heterogeneous kinematic dynamics at the individual filament level, with simultaneous actin flows in both retrograde and anterograde directions. At the ensemble level, movements of filaments integrate into a net retrograde flow of approximately 138 nm/min. These results suggest a weakly polarized F-actin network that consists of mostly short filaments in dendritic spines.

The F-Actin Binding Protein Cortactin Regulates the Dynamics of the Exocytotic Fusion Pore through its SH3 Domain

PubMed Central

González-Jamett, Arlek M.; Guerra, María J.; Olivares, María J.; Haro-Acuña, Valentina; Baéz-Matus, Ximena; Vásquez-Navarrete, Jacqueline; Momboisse, Fanny; Martinez-Quiles, Narcisa; Cárdenas, Ana M.

2017-01-01

Upon cell stimulation, the network of cortical actin filaments is rearranged to facilitate the neurosecretory process. This actin rearrangement includes both disruption of the preexisting actin network and de novo actin polymerization. However, the mechanism by which a Ca2+ signal elicits the formation of new actin filaments remains uncertain. Cortactin, an actin-binding protein that promotes actin polymerization in synergy with the nucleation promoting factor N-WASP, could play a key role in this mechanism. We addressed this hypothesis by analyzing de novo actin polymerization and exocytosis in bovine adrenal chromaffin cells expressing different cortactin or N-WASP domains, or cortactin mutants that fail to interact with proline-rich domain (PRD)-containing proteins, including N-WASP, or to be phosphorylated by Ca2+-dependent kinases, such as ERK1/2 and Src. Our results show that the activation of nicotinic receptors in chromaffin cells promotes cortactin translocation to the cell cortex, where it colocalizes with actin filaments. We further found that, in association with PRD-containing proteins, cortactin contributes to the Ca2+-dependent formation of F-actin, and regulates fusion pore dynamics and the number of exocytotic events induced by activation of nicotinic receptors. However, whereas the actions of cortactin on the fusion pore dynamics seems to depend on the availability of monomeric actin and its phosphorylation by ERK1/2 and Src kinases, cortactin regulates the extent of exocytosis by a mechanism independent of actin polymerization. Together our findings point out a role for cortactin as a critical modulator of actin filament formation and exocytosis in neuroendocrine cells. PMID:28522963

Altered Actin Centripetal Retrograde Flow in Physically Restricted Immunological Synapses

PubMed Central

Yu, Cheng-han; Wu, Hung-Jen; Kaizuka, Yoshihisa; Vale, Ronald D.; Groves, Jay T.

2010-01-01

Antigen recognition by T cells involves large scale spatial reorganization of numerous receptor, adhesion, and costimulatory proteins within the T cell-antigen presenting cell (APC) junction. The resulting patterns can be distinctive, and are collectively known as the immunological synapse. Dynamical assembly of cytoskeletal network is believed to play an important role in driving these assembly processes. In one experimental strategy, the APC is replaced with a synthetic supported membrane. An advantage of this configuration is that solid structures patterned onto the underlying substrate can guide immunological synapse assembly into altered patterns. Here, we use mobile anti-CD3ε on the spatial-partitioned supported bilayer to ligate and trigger T cell receptor (TCR) in live Jurkat T cells. Simultaneous tracking of both TCR clusters and GFP-actin speckles reveals their dynamic association and individual flow patterns. Actin retrograde flow directs the inward transport of TCR clusters. Flow-based particle tracking algorithms allow us to investigate the velocity distribution of actin flow field across the whole synapse, and centripetal velocity of actin flow decreases as it moves toward the center of synapse. Localized actin flow analysis reveals that, while there is no influence on actin motion from substrate patterns directly, velocity differences of actin are observed over physically trapped TCR clusters. Actin flow regains its velocity immediately after passing through confined TCR clusters. These observations are consistent with a dynamic and dissipative coupling between TCR clusters and viscoelastic actin network. PMID:20686692

Mechanics of composite actin networks: in vitro and cellular perspectives

NASA Astrophysics Data System (ADS)

Upadhyaya, Arpita

2014-03-01

Actin filaments and associated actin binding proteins play an essential role in governing the mechanical properties of eukaryotic cells. Even though cells have multiple actin binding proteins (ABPs) that exist simultaneously to maintain the structural and mechanical integrity of the cellular cytoskeleton, how these proteins work together to determine the properties of actin networks is not well understood. The ABP, palladin, is essential for the integrity of cell morphology and movement during development. Palladin coexists with alpha-actinin in stress fibers and focal adhesions and binds to both actin and alpha-actinin. To obtain insight into how mutually interacting actin crosslinking proteins modulate the properties of actin networks, we have characterized the micro-structure and mechanics of actin networks crosslinked with palladin and alpha-actinin. Our studies on composite networks of alpha-actinin/palladin/actin show that palladin and alpha-actinin synergistically determine network viscoelasticity. We have further examined the role of palladin in cellular force generation and mechanosensing. Traction force microscopy revealed that TAFs are sensitive to substrate stiffness as they generate larger forces on substrates of increased stiffness. Contrary to expectations, knocking down palladin increased the forces generated by cells, and also inhibited the ability to sense substrate stiffness for very stiff gels. This was accompanied by significant differences in the actin organization and adhesion dynamics of palladin knock down cells. Perturbation experiments also suggest altered myosin activity in palladin KD cells. Our results suggest that the actin crosslinkers such as palladin and myosin motors coordinate for optimal cell function and to prevent aberrant behavior as in cancer metastasis.

PI(3,5)P2 controls endosomal branched actin dynamics by regulating cortactin–actin interactions

PubMed Central

Hong, Nan Hyung; Qi, Aidong

2015-01-01

Branched actin critically contributes to membrane trafficking by regulating membrane curvature, dynamics, fission, and transport. However, how actin dynamics are controlled at membranes is poorly understood. Here, we identify the branched actin regulator cortactin as a direct binding partner of phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) and demonstrate that their interaction promotes turnover of late endosomal actin. In vitro biochemical studies indicated that cortactin binds PI(3,5)P2 via its actin filament-binding region. Furthermore, PI(3,5)P2 competed with actin filaments for binding to cortactin, thereby antagonizing cortactin activity. These findings suggest that PI(3,5)P2 formation on endosomes may remove cortactin from endosome-associated branched actin. Indeed, inhibition of PI(3,5)P2 production led to cortactin accumulation and actin stabilization on Rab7+ endosomes. Conversely, inhibition of Arp2/3 complex activity greatly reduced cortactin localization to late endosomes. Knockdown of cortactin reversed PI(3,5)P2-inhibitor–induced actin accumulation and stabilization on endosomes. These data suggest a model in which PI(3,5)P2 binding removes cortactin from late endosomal branched actin networks and thereby promotes net actin turnover. PMID:26323691

A disassembly-driven mechanism explains F-actin-mediated chromosome transport in starfish oocytes

PubMed Central

Bun, Philippe; Dmitrieff, Serge; Belmonte, Julio M

2018-01-01

While contraction of sarcomeric actomyosin assemblies is well understood, this is not the case for disordered networks of actin filaments (F-actin) driving diverse essential processes in animal cells. For example, at the onset of meiosis in starfish oocytes a contractile F-actin network forms in the nuclear region transporting embedded chromosomes to the assembling microtubule spindle. Here, we addressed the mechanism driving contraction of this 3D disordered F-actin network by comparing quantitative observations to computational models. We analyzed 3D chromosome trajectories and imaged filament dynamics to monitor network behavior under various physical and chemical perturbations. We found no evidence of myosin activity driving network contractility. Instead, our observations are well explained by models based on a disassembly-driven contractile mechanism. We reconstitute this disassembly-based contractile system in silico revealing a simple architecture that robustly drives chromosome transport to prevent aneuploidy in the large oocyte, a prerequisite for normal embryonic development. PMID:29350616

Drosophila growth cones: a genetically tractable platform for the analysis of axonal growth dynamics.

PubMed

Sánchez-Soriano, Natalia; Gonçalves-Pimentel, Catarina; Beaven, Robin; Haessler, Ulrike; Ofner-Ziegenfuss, Lisa; Ballestrem, Christoph; Prokop, Andreas

2010-01-01

The formation of neuronal networks, during development and regeneration, requires outgrowth of axons along reproducible paths toward their appropriate postsynaptic target cells. Axonal extension occurs at growth cones (GCs) at the tips of axons. GC advance and navigation requires the activity of their cytoskeletal networks, comprising filamentous actin (F-actin) in lamellipodia and filopodia as well as dynamic microtubules (MTs) emanating from bundles of the axonal core. The molecular mechanisms governing these two cytoskeletal networks, their cross-talk, and their response to extracellular signaling cues are only partially understood, hindering our conceptual understanding of how regulated changes in GC behavior are controlled. Here, we introduce Drosophila GCs as a suitable model to address these mechanisms. Morphological and cytoskeletal readouts of Drosophila GCs are similar to those of other models, including mammals, as demonstrated here for MT and F-actin dynamics, axonal growth rates, filopodial structure and motility, organizational principles of MT networks, and subcellular marker localization. Therefore, we expect fundamental insights gained in Drosophila to be translatable into vertebrate biology. The advantage of the Drosophila model over others is its enormous amenability to combinatorial genetics as a powerful strategy to address the complexity of regulatory networks governing axonal growth. Thus, using pharmacological and genetic manipulations, we demonstrate a role of the actin cytoskeleton in a specific form of MT organization (loop formation), known to regulate GC pausing behavior. We demonstrate these events to be mediated by the actin-MT linking factor Short stop, thus identifying an essential molecular player in this context.

ACF7 regulates cytoskeletal-focal adhesion dynamics and migration and has ATPase activity.

PubMed

Wu, Xiaoyang; Kodama, Atsuko; Fuchs, Elaine

2008-10-03

Coordinated interactions between microtubule (MT) and actin cytoskeletons are involved in many polarized cellular processes. Spectraplakins are enormous (>500 kDa) proteins able to bind both MTs and actin filaments (F-actin) directly. To elucidate the physiological significance and functions of mammalian spectraplakin ACF7, we've conditionally targeted it in skin epidermis. Intriguingly, ACF7 deficiency compromises the targeting of microtubules along F-actin to focal adhesions (FAs), stabilizes FA-actin networks, and impairs epidermal migration. Exploring underlying mechanisms, we show that ACF7's binding domains for F-actin, MTs, and MT plus-end proteins are not sufficient to rescue the defects in FA-cytoskeletal dynamics and migration functions of ACF7 null keratinocytes. We've uncovered an intrinsic actin-regulated ATPase domain in ACF7 and demonstrate that it is both functional and essential for these roles. Our findings provide insight into the functions of this important cytoskeletal crosslinking protein in regulating dynamic interactions between MTs and F-actin to sustain directional cell movement.

Resemblance of actin-binding protein/actin gels to covalently crosslinked networks

NASA Astrophysics Data System (ADS)

Janmey, Paul A.; Hvidt, Søren; Lamb, Jennifer; Stossel, Thomas P.

1990-05-01

THE maintainance of the shape of cells is often due to their surface elasticity, which arises mainly from an actin-rich cytoplasmic cortex1,2. On locomotion, phagocytosis or fission, however, these cells become partially fluid-like. The finding of proteins that can bind to actin and control the assembly of, or crosslink, actin filaments, and of intracellular messages that regulate the activities of some of these actin-binding proteins, indicates that such 'gel sol' transformations result from the rearrangement of cortical actin-rich networks3. Alternatively, on the basis of a study of the mechanical properties of mixtures of actin filaments and an Acanthamoeba actin-binding protein, α-actinin, it has been proposed that these transformations can be accounted for by rapid exchange of crosslinks between actin filaments4: the cortical network would be solid when the deformation rate is greater than the rate of crosslink exchange, but would deform or 'creep' when deformation is slow enough to permit crosslinker molecules to rearrange. Here we report, however, that mixtures of actin filaments and actin-binding protein (ABP), an actin crosslinking protein of many higher eukaryotes, form gels Theologically equivalent to covalently crosslinked networks. These gels do not creep in response to applied stress on a time scale compatible with most cell-surface movements. These findings support a more complex and controlled mechanism underlying the dynamic mechanical properties of cortical cytoplasm, and can explain why cells do not collapse under the constant shear forces that often exist in tissues.

Lifeact-mEGFP Reveals a Dynamic Apical F-Actin Network in Tip Growing Plant Cells

PubMed Central

Hepler, Peter K.; Bezanilla, Magdalena

2009-01-01

Background Actin is essential for tip growth in plants. However, imaging actin in live plant cells has heretofore presented challenges. In previous studies, fluorescent probes derived from actin-binding proteins often alter growth, cause actin bundling and fail to resolve actin microfilaments. Methodology/Principal Findings In this report we use Lifeact-mEGFP, an actin probe that does not affect the dynamics of actin, to visualize actin in the moss Physcomitrella patens and pollen tubes from Lilium formosanum and Nicotiana tobaccum. Lifeact-mEGFP robustly labels actin microfilaments, particularly in the apex, in both moss protonemata and pollen tubes. Lifeact-mEGFP also labels filamentous actin structures in other moss cell types, including cells of the gametophore. Conclusions/Significance Lifeact-mEGFP, when expressed at optimal levels does not alter moss protonemal or pollen tube growth. We suggest that Lifeact-mEGFP represents an exciting new versatile probe for further studies of actin's role in tip growing plant cells. PMID:19478943

Actin-filament disassembly: it takes two to shrink them fast.

PubMed

Winterhoff, Moritz; Faix, Jan

2015-06-01

Actin-filament disassembly is indispensable for replenishing the pool of polymerizable actin and allows continuous dynamic remodelling of the actin cytoskeleton. A new study now reveals that ADF/cofilin preferentially dismantles branched networks and provides new insights into the collaborative work of ADF/cofilin and Aip1 on filament disassembly at the molecular level. Copyright © 2015 Elsevier Ltd. All rights reserved.

Mena/VASP and αII-Spectrin complexes regulate cytoplasmic actin networks in cardiomyocytes and protect from conduction abnormalities and dilated cardiomyopathy

PubMed Central

2013-01-01

Background In the heart, cytoplasmic actin networks are thought to have important roles in mechanical support, myofibrillogenesis, and ion channel function. However, subcellular localization of cytoplasmic actin isoforms and proteins involved in the modulation of the cytoplasmic actin networks are elusive. Mena and VASP are important regulators of actin dynamics. Due to the lethal phenotype of mice with combined deficiency in Mena and VASP, however, distinct cardiac roles of the proteins remain speculative. In the present study, we analyzed the physiological functions of Mena and VASP in the heart and also investigated the role of the proteins in the organization of cytoplasmic actin networks. Results We generated a mouse model, which simultaneously lacks Mena and VASP in the heart. Mena/VASP double-deficiency induced dilated cardiomyopathy and conduction abnormalities. In wild-type mice, Mena and VASP specifically interacted with a distinct αII-Spectrin splice variant (SH3i), which is in cardiomyocytes exclusively localized at Z- and intercalated discs. At Z- and intercalated discs, Mena and β-actin localized to the edges of the sarcomeres, where the thin filaments are anchored. In Mena/VASP double-deficient mice, β-actin networks were disrupted and the integrity of Z- and intercalated discs was markedly impaired. Conclusions Together, our data suggest that Mena, VASP, and αII-Spectrin assemble cardiac multi-protein complexes, which regulate cytoplasmic actin networks. Conversely, Mena/VASP deficiency results in disrupted β-actin assembly, Z- and intercalated disc malformation, and induces dilated cardiomyopathy and conduction abnormalities. PMID:23937664

Mena/VASP and αII-Spectrin complexes regulate cytoplasmic actin networks in cardiomyocytes and protect from conduction abnormalities and dilated cardiomyopathy.

PubMed

Benz, Peter M; Merkel, Carla J; Offner, Kristin; Abeßer, Marco; Ullrich, Melanie; Fischer, Tobias; Bayer, Barbara; Wagner, Helga; Gambaryan, Stepan; Ursitti, Jeanine A; Adham, Ibrahim M; Linke, Wolfgang A; Feller, Stephan M; Fleming, Ingrid; Renné, Thomas; Frantz, Stefan; Unger, Andreas; Schuh, Kai

2013-08-12

In the heart, cytoplasmic actin networks are thought to have important roles in mechanical support, myofibrillogenesis, and ion channel function. However, subcellular localization of cytoplasmic actin isoforms and proteins involved in the modulation of the cytoplasmic actin networks are elusive. Mena and VASP are important regulators of actin dynamics. Due to the lethal phenotype of mice with combined deficiency in Mena and VASP, however, distinct cardiac roles of the proteins remain speculative. In the present study, we analyzed the physiological functions of Mena and VASP in the heart and also investigated the role of the proteins in the organization of cytoplasmic actin networks. We generated a mouse model, which simultaneously lacks Mena and VASP in the heart. Mena/VASP double-deficiency induced dilated cardiomyopathy and conduction abnormalities. In wild-type mice, Mena and VASP specifically interacted with a distinct αII-Spectrin splice variant (SH3i), which is in cardiomyocytes exclusively localized at Z- and intercalated discs. At Z- and intercalated discs, Mena and β-actin localized to the edges of the sarcomeres, where the thin filaments are anchored. In Mena/VASP double-deficient mice, β-actin networks were disrupted and the integrity of Z- and intercalated discs was markedly impaired. Together, our data suggest that Mena, VASP, and αII-Spectrin assemble cardiac multi-protein complexes, which regulate cytoplasmic actin networks. Conversely, Mena/VASP deficiency results in disrupted β-actin assembly, Z- and intercalated disc malformation, and induces dilated cardiomyopathy and conduction abnormalities.

Active turnover regulates pattern formation and stress transmission in disordered acto-myosin networks

NASA Astrophysics Data System (ADS)

McCall, Patrick; Stam, Samantha; Kovar, David; Gardel, Margaret

The shape and mechanics of animal cells are controlled by a dynamic, thin network of semiflexible actin filaments and myosin-II motor proteins called the actomyosin cortex. Motor-generated stresses in the cortex drive changes in cell shape during cell division and morphogenesis, while dynamic turnover of actin filaments dissipates stress. The relative effects that force generation, force dissipation, and disassembly and reassembly of material have on motion in these networks are unknown. We find that cross-linked actin networks in vitro contract under myosin-generated stresses, resulting in partial filament disassembly, the formation of asters, and clustering of myosin motors. We observe a rapid restoration of uniform polymer density in the presence of the assembly factors which catalyze network turnover through elongation of severed actin filaments. When severing is accelerated further by the addition of a severing protein, network contraction and motor clustering are dramatically suppressed. We test the relative effects of material regeneration and force transmission using image analysis, and conclude that the dominant mechanism for this effect is relatively short-lived stresses that do not propagate over considerable distance or push network deformation into the nonlinear contractile regime we have previously characterized. Our results present a framework to understand cytoskeletal active matter that are influenced by a complex interplay between stress generation, network reorganization, and polymer turnover.

The CAMSAP3-ACF7 Complex Couples Noncentrosomal Microtubules with Actin Filaments to Coordinate Their Dynamics.

PubMed

Ning, Wenxiu; Yu, Yanan; Xu, Honglin; Liu, Xiaofei; Wang, Daiwei; Wang, Jing; Wang, Yingchun; Meng, Wenxiang

2016-10-10

For adaptation to complex cellular functions, dynamic cytoskeletal networks are required. There are two major components of the cytoskeleton, microtubules and actin filaments, which form an intricate network maintaining an exquisite cooperation to build the physical basis for their cellular function. However, little is known about the molecular mechanism underlying their synergism. Here, we show that in Caco2 epithelial cells, noncentrosomal microtubules crosstalk with F-actin through their minus ends and contribute to the regulation of focal adhesion size and cell migration. We demonstrate that ACF7, a member of the spectraplakin family of cytoskeletal crosslinking proteins, interacts with Nezha (also called CAMSAP3) at the minus ends of noncentrosomal microtubules and anchors them to actin filaments. Those noncentrosomal microtubules cooperate with actin filaments through retrograde flow to keep their length and orientation perpendicular to the cell edge as well as regulate focal adhesion size and cell migration. Copyright © 2016 Elsevier Inc. All rights reserved.

Competing dynamic phases of active polymer networks

NASA Astrophysics Data System (ADS)

Freedman, Simon; Banerjee, Shiladitya; Dinner, Aaron R.

Recent experiments on in-vitro reconstituted assemblies of F-actin, myosin-II motors, and cross-linking proteins show that tuning local network properties can changes the fundamental biomechanical behavior of the system. For example, by varying cross-linker density and actin bundle rigidity, one can switch between contractile networks useful for reshaping cells, polarity sorted networks ideal for directed molecular transport, and frustrated networks with robust structural properties. To efficiently investigate the dynamic phases of actomyosin networks, we developed a coarse grained non-equilibrium molecular dynamics simulation of model semiflexible filaments, molecular motors, and cross-linkers with phenomenologically defined interactions. The simulation's accuracy was verified by benchmarking the mechanical properties of its individual components and collective behavior against experimental results at the molecular and network scales. By adjusting the model's parameters, we can reproduce the qualitative phases observed in experiment and predict the protein characteristics where phase crossovers could occur in collective network dynamics. Our model provides a framework for understanding cells' multiple uses of actomyosin networks and their applicability in materials research. Supported by the Department of Defense (DoD) through the National Defense Science & Engineering Graduate Fellowship (NDSEG) Program.

From Cytoskeleton to Gene Expression: Actin in the Nucleus.

PubMed

Viita, Tiina; Vartiainen, Maria K

2017-01-01

Although most people still associate actin mainly with the cytoskeleton, several lines of evidence, with the earliest studies dating back to decades ago, have emphasized the importance of actin also inside the cell nucleus. Actin has been linked to many gene expression processes from gene activation to chromatin remodeling, but also to maintenance of genomic integrity and intranuclear movement of chromosomes and chromosomal loci. Recent advances in visualizing different forms and dynamic properties of nuclear actin have clearly advanced our understanding of the basic concepts by which actin operates in the nucleus. In this chapter we address the different breakthroughs in nuclear actin studies, as well as discuss the regulation nuclear actin and the importance of nuclear actin dynamics in relation to its different nuclear functions. Our aim is to highlight the fact that actin should be considered as an essential component of the cell nucleus, and its nuclear actions should be taken into account also in experiments on cytoplasmic actin networks.

Isotropic actomyosin dynamics promote organization of the apical cell cortex in epithelial cells.

PubMed

Klingner, Christoph; Cherian, Anoop V; Fels, Johannes; Diesinger, Philipp M; Aufschnaiter, Roland; Maghelli, Nicola; Keil, Thomas; Beck, Gisela; Tolić-Nørrelykke, Iva M; Bathe, Mark; Wedlich-Soldner, Roland

2014-10-13

Although cortical actin plays an important role in cellular mechanics and morphogenesis, there is surprisingly little information on cortex organization at the apical surface of cells. In this paper, we characterize organization and dynamics of microvilli (MV) and a previously unappreciated actomyosin network at the apical surface of Madin-Darby canine kidney cells. In contrast to short and static MV in confluent cells, the apical surfaces of nonconfluent epithelial cells (ECs) form highly dynamic protrusions, which are often oriented along the plane of the membrane. These dynamic MV exhibit complex and spatially correlated reorganization, which is dependent on myosin II activity. Surprisingly, myosin II is organized into an extensive network of filaments spanning the entire apical membrane in nonconfluent ECs. Dynamic MV, myosin filaments, and their associated actin filaments form an interconnected, prestressed network. Interestingly, this network regulates lateral mobility of apical membrane probes such as integrins or epidermal growth factor receptors, suggesting that coordinated actomyosin dynamics contributes to apical cell membrane organization. © 2014 Klingner et al.

Load Adaptation of Lamellipodial Actin Networks.

PubMed

Mueller, Jan; Szep, Gregory; Nemethova, Maria; de Vries, Ingrid; Lieber, Arnon D; Winkler, Christoph; Kruse, Karsten; Small, J Victor; Schmeiser, Christian; Keren, Kinneret; Hauschild, Robert; Sixt, Michael

2017-09-21

Actin filaments polymerizing against membranes power endocytosis, vesicular traffic, and cell motility. In vitro reconstitution studies suggest that the structure and the dynamics of actin networks respond to mechanical forces. We demonstrate that lamellipodial actin of migrating cells responds to mechanical load when membrane tension is modulated. In a steady state, migrating cell filaments assume the canonical dendritic geometry, defined by Arp2/3-generated 70° branch points. Increased tension triggers a dense network with a broadened range of angles, whereas decreased tension causes a shift to a sparse configuration dominated by filaments growing perpendicularly to the plasma membrane. We show that these responses emerge from the geometry of branched actin: when load per filament decreases, elongation speed increases and perpendicular filaments gradually outcompete others because they polymerize the shortest distance to the membrane, where they are protected from capping. This network-intrinsic geometrical adaptation mechanism tunes protrusive force in response to mechanical load. Copyright © 2017 Elsevier Inc. All rights reserved.

Decidable and undecidable arithmetic functions in actin filament networks

NASA Astrophysics Data System (ADS)

Schumann, Andrew

2018-01-01

The plasmodium of Physarum polycephalum is very sensitive to its environment, and reacts to stimuli with appropriate motions. Both the sensory and motor stages of these reactions are explained by hydrodynamic processes, based on fluid dynamics, with the participation of actin filament networks. This paper is devoted to actin filament networks as a computational medium. The point is that actin filaments, with contributions from many other proteins like myosin, are sensitive to extracellular stimuli (attractants as well as repellents), and appear and disappear at different places in the cell to change aspects of the cell structure—e.g. its shape. By assembling and disassembling actin filaments, some unicellular organisms, like Amoeba proteus, can move in response to various stimuli. As a result, these organisms can be considered a simple reversible logic gate—extracellular signals being its inputs and motions its outputs. In this way, we can implement various logic gates on amoeboid behaviours. These networks can embody arithmetic functions within p-adic valued logic. Furthermore, within these networks we can define the so-called diagonalization for deducing undecidable arithmetic functions.

Continuum mechanical model for cross-linked actin networks with contractile bundles

NASA Astrophysics Data System (ADS)

Ferreira, J. P. S.; Parente, M. P. L.; Natal Jorge, R. M.

2018-01-01

In the context of a mechanical approach to cell biology, there is a close relationship between cellular function and mechanical properties. In recent years, an increasing amount of attention has been given to the coupling between biochemical and mechanical signals by means of constitutive models. In particular, on the active contractility of the actin cytoskeleton. Given the importance of the actin contraction on the physiological functions, this study propose a constitutive model to describe how the filamentous network controls its mechanics actively. Embedded in a soft isotropic ground substance, the network behaves as a viscous mechanical continuum, comprised of isotropically distributed cross-linked actin filaments and actomyosin bundles. Trough virtual rheometry experiments, the present model relates the dynamics of the myosin motors with the network stiffness, which is to a large extent governed by the time-scale of the applied deformations/forces.

Actin-induced dimerization of palladin promotes actin-bundling

PubMed Central

Vattepu, Ravi; Yadav, Rahul; Beck, Moriah R

2015-01-01

A subset of actin binding proteins is able to form crosslinks between two or more actin filaments, thus producing structures of parallel or networked bundles. These actin crosslinking proteins interact with actin through either bivalent binding or dimerization. We recently identified two binding sites within the actin binding domain of palladin, an actin crosslinking protein that plays an important role in normal cell adhesion and motility during wound healing and embryonic development. In this study, we show that actin induces dimerization of palladin. Furthermore, the extent of dimerization reflects earlier comparisons of actin binding and bundling between different domains of palladin. On the basis of these results we hypothesized that actin binding may promote a conformational change that results in dimerization of palladin, which in turn may drive the crosslinking of actin filaments. The proximal distance between two actin binding sites on crosslinking proteins determines the ultrastructural properties of the filament network, therefore we also explored interdomain interactions using a combination of chemical crosslinking experiments and actin cosedimentation assays. Limited proteolysis data reveals that palladin is less susceptible to enzyme digestion after actin binding. Our results suggest that domain movements in palladin are necessary for interactions with actin and are induced by interactions with actin filaments. Accordingly, we put forth a model linking the structural changes to functional dynamics. PMID:25307943

Control of actin-based motility through localized actin binding

PubMed Central

Banigan, Edward J.; Lee, Kun-Chun; Liu, Andrea J.

2014-01-01

A wide variety of cell biological and biomimetic systems use actin polymerization to drive motility. It has been suggested that an object such as a bacterium can propel itself by self-assembling a high concentration of actin behind it if it is repelled by actin. However, it is also known that it is essential for the moving object to bind actin. Therefore, a key question is how the actin tail can propel an object when it both binds and repels the object. We present a physically consistent Brownian dynamics model for actin-based motility that includes the minimal components of the dendritic nucleation model and allows for both attractive and repulsive interactions between actin and a moveable disk. We find that the concentration gradient of filamentous actin generated by polymerization is sufficient to propel the object, even with moderately strong binding interactions. Additionally, actin binding can act as a biophysical cap, and may directly control motility through modulation of network growth. Overall, this mechanism is robust in that it can drive motility against a load up to a stall pressure that depends on the Young’s modulus of the actin network and can explain several aspects of actin-based motility. PMID:24225232

Quantitative Analysis of Statics and Dynamics of Actin Cables in Fission Yeast

NASA Astrophysics Data System (ADS)

Yusuf, Eddy; Wu, Jian-Qiu; Vavylonis, Dimitrios

2010-03-01

The assembly of actin and tubulin proteins into long filaments and bundles, i.e. closely-packed filaments, underlies important cellular processes such as cell motility, intracellular transport, and cell division. Recent theoretical and experimental work has addressed the nonequilibrium dynamics of single microtubules within live cells [1]. Actin filaments usually form dense networks that prevents microscopic imaging of individual filaments or bundles. Here, we studied actin dynamics using fission yeast that has low-density actin cytoskeleton consisting of actin cables (actin bundles aligned along the long axis of the cell) and ``actin patches.'' Yeast cells expressing GFP-CHD were imaged by 3D confocal microscopy. Stretching open active contours [2] were used to segment and track individual actin cables. We analyzed their curvature distribution, the tangent correlation, and the temporal bending amplitude fluctuations. We contrast our findings to equilibrium fluctuating semiflexible polymers and to microtubules in cells. We calculate the important time and length scales for the actin cables. We also discuss our findings within the broad context of understanding actin assembly in cells. [1] C. P. Brangwynne et. al., Phys. Rev. Lett. 100, 118104 (2008) [2] H. Li et. al., Proc. of the IEEE Int'l Symposium on Biomedical Imaging: From Nano to Macro, ISBI'09

The yeast actin cytoskeleton.

PubMed

Mishra, Mithilesh; Huang, Junqi; Balasubramanian, Mohan K

2014-03-01

The actin cytoskeleton is a complex network of dynamic polymers, which plays an important role in various fundamental cellular processes, including maintenance of cell shape, polarity, cell division, cell migration, endocytosis, vesicular trafficking, and mechanosensation. Precise spatiotemporal assembly and disassembly of actin structures is regulated by the coordinated activity of about 100 highly conserved accessory proteins, which nucleate, elongate, cross-link, and sever actin filaments. Both in vivo studies in a wide range of organisms from yeast to metazoans and in vitro studies of purified proteins have helped shape the current understanding of actin dynamics and function. Molecular genetics, genome-wide functional analysis, sophisticated real-time imaging, and ultrastructural studies in concert with biochemical analysis have made yeast an attractive model to understand the actin cytoskeleton, its molecular dynamics, and physiological function. Studies of the yeast actin cytoskeleton have contributed substantially in defining the universal mechanism regulating actin assembly and disassembly in eukaryotes. Here, we review some of the important insights generated by the study of actin cytoskeleton in two important yeast models the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Cofilin1-dependent actin dynamics control DRP1-mediated mitochondrial fission

PubMed Central

Rehklau, Katharina; Hoffmann, Lena; Gurniak, Christine B; Ott, Martin; Witke, Walter; Scorrano, Luca; Culmsee, Carsten; Rust, Marco B

2017-01-01

Mitochondria form highly dynamic networks in which organelles constantly fuse and divide. The relevance of mitochondrial dynamics is evident from its implication in various human pathologies, including cancer or neurodegenerative, endocrine and cardiovascular diseases. Dynamin-related protein 1 (DRP1) is a key regulator of mitochondrial fission that oligomerizes at the mitochondrial outer membrane and hydrolyzes GTP to drive mitochondrial fragmentation. Previous studies demonstrated that DRP1 recruitment and mitochondrial fission is promoted by actin polymerization at the mitochondrial surface, controlled by the actin regulatory proteins inverted formin 2 (INF2) and Spire1C. These studies suggested the requirement of additional actin regulatory activities to control DRP1-mediated mitochondrial fission. Here we show that the actin-depolymerizing protein cofilin1, but not its close homolog actin-depolymerizing factor (ADF), is required to maintain mitochondrial morphology. Deletion of cofilin1 caused mitochondrial DRP1 accumulation and fragmentation, without altering mitochondrial function or other organelles’ morphology. Mitochondrial morphology in cofilin1-deficient cells was restored upon (i) re-expression of wild-type cofilin1 or a constitutively active mutant, but not of an actin-binding-deficient mutant, (ii) pharmacological destabilization of actin filaments and (iii) genetic depletion of DRP1. Our work unraveled a novel function for cofilin1-dependent actin dynamics in mitochondrial fission, and identified cofilin1 as a negative regulator of mitochondrial DRP1 activity. We conclude that cofilin1 is required for local actin dynamics at mitochondria, where it may balance INF2/Spire1C-induced actin polymerization. PMID:28981113

Percolation mechanism drives actin gels to the critically connected state

NASA Astrophysics Data System (ADS)

Lee, Chiu Fan; Pruessner, Gunnar

2016-05-01

Cell motility and tissue morphogenesis depend crucially on the dynamic remodeling of actomyosin networks. An actomyosin network consists of an actin polymer network connected by cross-linker proteins and motor protein myosins that generate internal stresses on the network. A recent discovery shows that for a range of experimental parameters, actomyosin networks contract to clusters with a power-law size distribution [J. Alvarado, Nat. Phys. 9, 591 (2013), 10.1038/nphys2715]. Here, we argue that actomyosin networks can exhibit a robust critical signature without fine-tuning because the dynamics of the system can be mapped onto a modified version of percolation with trapping (PT), which is known to show critical behavior belonging to the static percolation universality class without the need for fine-tuning of a control parameter. We further employ our PT model to generate experimentally testable predictions.

Nephrin Regulates Lamellipodia Formation by Assembling a Protein Complex That Includes Ship2, Filamin and Lamellipodin

PubMed Central

Venkatareddy, Madhusudan; Cook, Leslie; Abuarquob, Kamal; Verma, Rakesh; Garg, Puneet

2011-01-01

Actin dynamics has emerged at the forefront of podocyte biology. Slit diaphragm junctional adhesion protein Nephrin is necessary for development of the podocyte morphology and transduces phosphorylation-dependent signals that regulate cytoskeletal dynamics. The present study extends our understanding of Nephrin function by showing in cultured podocytes that Nephrin activation induced actin dynamics is necessary for lamellipodia formation. Upon activation Nephrin recruits and regulates a protein complex that includes Ship2 (SH2 domain containing 5′ inositol phosphatase), Filamin and Lamellipodin, proteins important in regulation of actin and focal adhesion dynamics, as well as lamellipodia formation. Using the previously described CD16-Nephrin clustering system, Nephrin ligation or activation resulted in phosphorylation of the actin crosslinking protein Filamin in a p21 activated kinase dependent manner. Nephrin activation in cell culture results in formation of lamellipodia, a process that requires specialized actin dynamics at the leading edge of the cell along with focal adhesion turnover. In the CD16-Nephrin clustering model, Nephrin ligation resulted in abnormal morphology of actin tails in human podocytes when Ship2, Filamin or Lamellipodin were individually knocked down. We also observed decreased lamellipodia formation and cell migration in these knock down cells. These data provide evidence that Nephrin not only initiates actin polymerization but also assembles a protein complex that is necessary to regulate the architecture of the generated actin filament network and focal adhesion dynamics. PMID:22194892

Rheology of Membrane-Attached Minimal Actin Cortices.

PubMed

Nöding, Helen; Schön, Markus; Reinermann, Corinna; Dörrer, Nils; Kürschner, Aileen; Geil, Burkhard; Mey, Ingo; Heussinger, Claus; Janshoff, Andreas; Steinem, Claudia

2018-04-26

The actin cortex is a thin cross-linked network attached to the plasma membrane, which is responsible for the cell's shape during migration, division, and growth. In a reductionist approach, we created a minimal actin cortex (MAC) attached to a lipid membrane to correlate the filamentous actin architecture with its viscoelastic properties. The system is composed of a supported 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine bilayer doped with the receptor lipid phosphatidylinositol(4,5)-bisphosphate (PtdIns(4,5)P 2 ) to which a constitutively active mutant of ezrin, which is a direct membrane-cytoskeleton linker, is bound. The formation of the MAC on the supported lipid bilayer is analyzed as a function of increasing PtdIns(4,5)P 2 /ezrin pinning points, revealing an increase in the intersections between actin filaments, that is, the node density of the MAC. Bead tracking microrheology on the membrane-attached actin network provides information about its viscoelastic properties. The results show that ezrin serves as a dynamic cross-linker for the actin cortex attached to the lipid bilayer and that the stiffness of the network is influenced by the pinning point density, relating the plateau storage modulus G 0 to the node density of the MAC.

Branched actin networks push against each other at adherens junctions to maintain cell-cell adhesion.

PubMed

Efimova, Nadia; Svitkina, Tatyana M

2018-05-07

Adherens junctions (AJs) are mechanosensitive cadherin-based intercellular adhesions that interact with the actin cytoskeleton and carry most of the mechanical load at cell-cell junctions. Both Arp2/3 complex-dependent actin polymerization generating pushing force and nonmuscle myosin II (NMII)-dependent contraction producing pulling force are necessary for AJ morphogenesis. Which actin system directly interacts with AJs is unknown. Using platinum replica electron microscopy of endothelial cells, we show that vascular endothelial (VE)-cadherin colocalizes with Arp2/3 complex-positive actin networks at different AJ types and is positioned at the interface between two oppositely oriented branched networks from adjacent cells. In contrast, actin-NMII bundles are located more distally from the VE-cadherin-rich zone. After Arp2/3 complex inhibition, linear AJs split, leaving gaps between cells with detergent-insoluble VE-cadherin transiently associated with the gap edges. After NMII inhibition, VE-cadherin is lost from gap edges. We propose that the actin cytoskeleton at AJs acts as a dynamic push-pull system, wherein pushing forces maintain extracellular VE-cadherin transinteraction and pulling forces stabilize intracellular adhesion complexes. © 2018 Efimova and Svitkina.

Prestressed F-actin networks cross-linked by hinged filamins replicate mechanical properties of cells

NASA Astrophysics Data System (ADS)

Gardel, M. L.; Nakamura, F.; Hartwig, J. H.; Crocker, J. C.; Stossel, T. P.; Weitz, D. A.

2006-02-01

We show that actin filaments, shortened to physiological lengths by gelsolin and cross-linked with recombinant human filamins (FLNs), exhibit dynamic elastic properties similar to those reported for live cells. To achieve elasticity values of comparable magnitude to those of cells, the in vitro network must be subjected to external prestress, which directly controls network elasticity. A molecular requirement for the strain-related behavior at physiological conditionsis a flexible hinge found in FLNa and some FLNb molecules. Basic physical properties of the in vitro filamin-F-actin network replicate the essential mechanical properties of living cells. This physical behavior could accommodate passive deformation and internal organelle trafficking at low strains yet resist externally or internally generated high shear forces. cytoskeleton | cell mechanics | nonlinear rheology

Model for adhesion clutch explains biphasic relationship between actin flow and traction at the cell leading edge

PubMed Central

Craig, Erin M.; Stricker, Jonathan; Gardel, Margaret L.; Mogilner, Alex

2015-01-01

Cell motility relies on the continuous reorganization of a dynamic actin-myosin-adhesion network at the leading edge of the cell, in order to generate protrusion at the leading edge and traction between the cell and its external environment. We analyze experimentally measured spatial distributions of actin flow, traction force, myosin density, and adhesion density in control and pharmacologically perturbed epithelial cells in order to develop a mechanical model of the actin-adhesion-myosin self-organization at the leading edge. A model in which the F-actin network is treated as a viscous gel, and adhesion clutch engagement is strengthened by myosin but weakened by actin flow, can explain the measured molecular distributions and correctly predict the spatial distributions of the actin flow and traction stress. We test the model by comparing its predictions with measurements of the actin flow and traction stress in cells with fast and slow actin polymerization rates. The model predicts how the location of the lamellipodium-lamellum boundary depends on the actin viscosity and adhesion strength. The model further predicts that the location of the lamellipodium-lamellum boundary is not very sensitive to the level of myosin contraction. PMID:25969948

Recruitment Kinetics of Tropomyosin Tpm3.1 to Actin Filament Bundles in the Cytoskeleton Is Independent of Actin Filament Kinetics.

PubMed

Appaduray, Mark A; Masedunskas, Andrius; Bryce, Nicole S; Lucas, Christine A; Warren, Sean C; Timpson, Paul; Stear, Jeffrey H; Gunning, Peter W; Hardeman, Edna C

2016-01-01

The actin cytoskeleton is a dynamic network of filaments that is involved in virtually every cellular process. Most actin filaments in metazoa exist as a co-polymer of actin and tropomyosin (Tpm) and the function of an actin filament is primarily defined by the specific Tpm isoform associated with it. However, there is little information on the interdependence of these co-polymers during filament assembly and disassembly. We addressed this by investigating the recovery kinetics of fluorescently tagged isoform Tpm3.1 into actin filament bundles using FRAP analysis in cell culture and in vivo in rats using intracellular intravital microscopy, in the presence or absence of the actin-targeting drug jasplakinolide. The mobile fraction of Tpm3.1 is between 50% and 70% depending on whether the tag is at the C- or N-terminus and whether the analysis is in vivo or in cultured cells. We find that the continuous dynamic exchange of Tpm3.1 is not significantly impacted by jasplakinolide, unlike tagged actin. We conclude that tagged Tpm3.1 may be able to undergo exchange in actin filament bundles largely independent of the assembly and turnover of actin.

Jmy regulates oligodendrocyte differentiation via modulation of actin cytoskeleton dynamics.

PubMed

Azevedo, Maria M; Domingues, Helena S; Cordelières, Fabrice P; Sampaio, Paula; Seixas, Ana I; Relvas, João B

2018-05-06

During central nervous system development, oligodendrocytes form structurally and functionally distinct actin-rich protrusions that contact and wrap around axons to assemble myelin sheaths. Establishment of axonal contact is a limiting step in myelination that relies on the oligodendrocyte's ability to locally coordinate cytoskeletal rearrangements with myelin production, under the control of a transcriptional differentiation program. The molecules that provide fine-tuning of actin dynamics during oligodendrocyte differentiation and axon ensheathment remain largely unidentified. We performed transcriptomics analysis of soma and protrusion fractions from rat brain oligodendrocyte progenitors and found a subcellular enrichment of mRNAs in newly-formed protrusions. Approximately 30% of protrusion-enriched transcripts encode proteins related to cytoskeleton dynamics, including the junction mediating and regulatory protein Jmy, a multifunctional regulator of actin polymerization. Here, we show that expression of Jmy is upregulated during myelination and is required for the assembly of actin filaments and protrusion formation during oligodendrocyte differentiation. Quantitative morphodynamics analysis of live oligodendrocytes showed that differentiation is driven by a stereotypical actin network-dependent "cellular shaping" program. Disruption of actin dynamics via knockdown of Jmy leads to a program fail resulting in oligodendrocytes that do not acquire an arborized morphology and are less efficient in contacting neurites and forming myelin wraps in co-cultures with neurons. Our findings provide new mechanistic insight into the relationship between cell shape dynamics and differentiation in development. © 2018 Wiley Periodicals, Inc.

Liquid droplets of cross-linked actin filaments

NASA Astrophysics Data System (ADS)

Weirich, Kimberly; Banerjee, Shiladitya; Dasbiswas, Kinjal; Vaikuntanathan, Suriyanarayan; Gardel, Margaret

Soft materials constructed from biomolecules self-assemble into a myriad of structures that work in concert to support cell physiology. One critical soft material is the actin cytoskeleton, a viscoelastic gel composed of cross-linked actin filaments. Although actin networks are primarily known for their elastic properties, which are crucial to regulating cell mechanics, the viscous behavior has been theorized to enable shape changes and flows. We experimentally demonstrate a fluid phase of cross-linked actin, where cross-linker condenses dilute short actin filaments into spindle-shaped droplets, or tactoids. Tactoids have shape dynamics consistent with a continuum model of liquid crystal droplets. The cross-linker, which acts as a long range attractive interaction, analogous to molecular cohesion, controls the tactoid shape and dynamics, which reports on the liquid's interfacial tension and viscosity. We investigate how the cross-linker properties and filament length influence the liquid properties. These results demonstrate a novel mechanism to control organization of the actin cytoskeleton and provide insight into design principles for complex, macromolecular liquid phases.

The myosin passenger protein Smy1 controls actin cable structure and dynamics by acting as a formin damper

PubMed Central

Chesarone-Cataldo, Melissa; Guérin, Christophe; Yu, Jerry H.; Wedlich-Soldner, Roland; Blanchoin, Laurent; Goode, Bruce L.

2011-01-01

Summary Formins are a conserved family of proteins with robust effects in promoting actin nucleation and elongation. However, the mechanisms restraining formin activities in cells to generate actin networks with particular dynamics and architectures are not well understood. In S. cerevisiae, formins assemble actin cables, which serve as tracks for myosin-dependent intracellular transport. Here, we show that the kinesin-like myosin passenger-protein Smy1 interacts with the FH2 domain of the formin Bnr1 to decrease rates of actin filament elongation, which is distinct from the formin displacement activity of Bud14. In vivo analysis of smy1Δ mutants demonstrates that this ‘damper’ mechanism is critical for maintaining proper actin cable architecture, dynamics, and function. We directly observe Smy1–3GFP being transported by myosin V and transiently pausing at the neck in a manner dependent on Bnr1. These observations suggest that Smy1 is part of a negative feedback mechanism that detects cable length and prevents overgrowth. PMID:21839918

In silico reconstitution of Listeria propulsion exhibits nano-saltation.

PubMed

Alberts, Jonathan B; Odell, Garrett M

2004-12-01

To understand how the actin-polymerization-mediated movements in cells emerge from myriad individual protein-protein interactions, we developed a computational model of Listeria monocytogenes propulsion that explicitly simulates a large number of monomer-scale biochemical and mechanical interactions. The literature on actin networks and L. monocytogenes motility provides the foundation for a realistic mathematical/computer simulation, because most of the key rate constants governing actin network dynamics have been measured. We use a cluster of 80 Linux processors and our own suite of simulation and analysis software to characterize salient features of bacterial motion. Our "in silico reconstitution" produces qualitatively realistic bacterial motion with regard to speed and persistence of motion and actin tail morphology. The model also produces smaller scale emergent behavior; we demonstrate how the observed nano-saltatory motion of L. monocytogenes,in which runs punctuate pauses, can emerge from a cooperative binding and breaking of attachments between actin filaments and the bacterium. We describe our modeling methodology in detail, as it is likely to be useful for understanding any subcellular system in which the dynamics of many simple interactions lead to complex emergent behavior, e.g., lamellipodia and filopodia extension, cellular organization, and cytokinesis.

Probing GFP-actin diffusion in living cells using fluorescence correlation spectroscopy.

PubMed

Engelke, Hanna; Heinrich, Doris; Rädler, Joachim O

2010-12-22

The cytoskeleton of eukaryotic cells is continuously remodeled by polymerization and depolymerization of actin. Consequently, the relative content of polymerized filamentous actin (F-actin) and monomeric globular actin (G-actin) is subject to temporal and spatial fluctuations. Since fluorescence correlation spectroscopy (FCS) can measure the diffusion of fluorescently labeled actin it seems likely that FCS allows us to determine the dynamics and hence indirectly the structural properties of the cytoskeleton components with high spatial resolution. To this end we investigate the FCS signal of GFP-actin in living Dictyostelium discoideum cells and explore the inherent spatial and temporal signatures of the actin cytoskeleton. Using the free green fluorescent protein (GFP) as a reference, we find that actin diffusion inside cells is dominated by G-actin and slower than diffusion in diluted cell extract. The FCS signal in the dense cortical F-actin network near the cell membrane is probed using the cytoskeleton protein LIM and is found to be slower than cytosolic G-actin diffusion. Furthermore, we show that polymerization of the cytoskeleton induced by Jasplakinolide leads to a substantial decrease of G-actin diffusion. Pronounced fluctuations in the distribution of the FCS correlation curves can be induced by latrunculin, which is known to induce actin waves. Our work suggests that the FCS signal of GFP-actin in combination with scanning or spatial correlation techniques yield valuable information about the local dynamics and concomitant cytoskeletal properties.

Gel-sol transition of the cytoplasm and its regulation

NASA Astrophysics Data System (ADS)

Janmey, Paul A.

1991-05-01

The cytoplasm of motile cells contains a dynamic system of filamentous protein polymers that endow the cell with elasticity permitting it to maintain its shape in the presence of mechanical forces encountered in vivo. Part of this cytoskeleton is composed of filaments of polymerized actin. Remodeling of this network is required for cell motility and cytoplasmic restructuring, and the reversible polymerization of actin per se has been suggested to cause morphologic changes such as cell ruffling and pseudopd extension. Changes in the degree of polymerization of acting and in the association of actin filaments into supramolecular structures are often associated with cell activation. Such activation is initiated by extracellular signals that bind to receptors which are often coupled by G-proteins to the production of intracellular second messangers. Cytoplasmic gel-sol transitions therefore can occur by formation and dissolution of actin networks, mediated by a variety of actin-binding proteins which are regulated by intracellular signalling molecules such as Ca2+ and polyphosphoinositides. The effects of three actin binding proteins: profilin, gelsolin and ABP (Tilamin) on the polymerization of actin and the viscoelasticity of the resulting networks measured in vitro suggest possible roles of these proteins in vivo. In particular, gelsolin, which activated by Ca2+ to sever and cap actin filaments, and released from filament ends by PIP2, appears to be a likely candidate for regulation of gel-sol transitions in response to cell activation. Recent results demonstrate that the hydrolysis of ATP that occurs following actin polymerization also influences the structure of the resulting filament. In addition being regulated by acting-binding proteins, the viscoelasticity of actin networks is also affected by the presence of the other two classes of cytoplasmic protein polymers, microtubules and intermediate filaments.

Biochemical and bioinformatic analysis of the MYO19 motor domain

PubMed Central

Adikes, Rebecca C.; Unrath, William C.; Yengo, Christopher M.; Quintero, Omar A.

2014-01-01

Mitochondrial dynamics are dependent on both the microtubule and actin cytoskeletal systems. Evidence for the involvement of myosin motors has been described in many systems, and until recently a candidate mitochondrial transport motor had not been described in vertebrates. Myosin-XIX (MYO19) was predicted to represent a novel class of myosin and had previously been shown to bind to mitochondria and increase mitochondrial network dynamics when ectopically expressed. Our analyses comparing ∼40 MYO19 orthologs to ∼2000 other myosin motor domain sequences identified instances of homology well-conserved within class XIX myosins that were not found in other myosin classes, suggesting MYO19-specific mechanochemistry. Steady-state biochemical analyses of the MYO19 motor domain indicate that Homo sapiens MYO19 is a functional motor. Insect cell-expressed constructs bound calmodulin as a light chain at the predicted stoichiometry and displayed actin-activated ATPase activity. MYO19 constructs demonstrated high actin affinity in the presence of ATP in actin-cosedimentation assays, and translocated actin filaments in gliding assays. Expression of GFP-MYO19 containing a mutation impairing ATPase activity did not enhance mitochondrial network dynamics, as occurs with wild-type MYO19, indicating that myosin motor activity is required for mitochondrial motility. The measured biochemical properties of MYO19 suggest it is a high-duty ratio motor that could serve to transport mitochondria or anchor mitochondria, depending upon the cellular microenvironment. PMID:23568824

Transient Binding and Viscous Dissipation in Semi-flexible Polymer Networks

NASA Astrophysics Data System (ADS)

Lieleg, Oliver; Claessens, Mireille; Bausch, Andreas

2008-03-01

Nature specifically chooses from a myriad of actin binding proteins (ABPs) to tailor the cytoskeletal microstructure. Herein, cells rely on the dynamics of the cytoskeleton as its structural and mechanical adaptability is crucial to allow for dynamic processes. A molecular understanding of such biological complexity calls for an in vitro system with well-defined structural rearrangements and cross-linker dynamics to elucidate the physical origin of the unique viscoelastic properties of cells. As we present here, the frequency-dependent viscoelastic response of cross-linked in vitro actin networks is determined by the binding kinetics of cross-linking molecules. Independent from the particular network structure, the viscous dissipation (loss modulus) exhibits a pronounced minimum in an intermediate frequency which is dominated by elasticity. We show that in this frequency regime the molecular origin of the viscoelastic response is given by the non-static nature of actin/ABP bonds as they are subjugated to chemical on/off kinetics. The time scale of the resulting stress release is set by the lifetime distribution of the cross-linking molecule and therefore can be tuned independently from other relaxation mechanisms. We speculate that unbinding of distinct cross-links might be the molecular mechanism employed by cells for mechanosensing.

Analysis of microtubule growth dynamics arising from altered actin network structure and contractility in breast tumor cells

NASA Astrophysics Data System (ADS)

Ory, Eleanor C.; Bhandary, Lekhana; E Boggs, Amanda; Chakrabarti, Kristi R.; Parker, Joshua; Losert, Wolfgang; Martin, Stuart S.

2017-04-01

The periphery of epithelial cells is shaped by opposing cytoskeletal physical forces generated predominately by two dynamic force generating systems—growing microtubule ends push against the boundary from the cell center, and the actin cortex contracts the attached plasma membrane. Here we investigate how changes to the structure and dynamics of the actin cortex alter the dynamics of microtubules. Current drugs target actin polymerization and contraction to reduce cell division and invasiveness; however, the impacts on microtubule dynamics remain incompletely understood. Using human MCF-7 breast tumor cells expressing GFP-tagged microtubule end-binding-protein-1 (EB1) and coexpression of cytoplasmic fluorescent protein mCherry, we map the trajectories of growing microtubule ends and cytoplasmic boundary respectively. Based on EB1 tracks and cytoplasmic boundary outlines, we calculate the speed, distance from cytoplasmic boundary, and straightness of microtubule growth. Actin depolymerization with Latrunculin-A reduces EB1 growth speed as well as allows the trajectories to extend beyond the cytoplasmic boundary. Blebbistatin, a direct myosin-II inhibitor, reduced EB1 speed and yielded less straight EB1 trajectories. Inhibiting signaling upstream of myosin-II contractility via the Rho-kinase inhibitor, Y-27632, altered EB1 dynamics differently from Blebbistatin. These results indicate that reduced actin cortex integrity can induce distinct alterations in microtubule dynamics. Given recent findings that tumor stem cell characteristics are increased by drugs which reduce actin contractility or stabilize microtubules, it remains important to clearly define how cytoskeletal drugs alter the interactions between these two filament systems in tumor cells.

Overview of Single-Molecule Speckle (SiMS) Microscopy and Its Electroporation-Based Version with Efficient Labeling and Improved Spatiotemporal Resolution.

PubMed

Yamashiro, Sawako; Watanabe, Naoki

2017-07-06

Live-cell single-molecule imaging was introduced more than a decade ago, and has provided critical information on remodeling of the actin cytoskeleton, the motion of plasma membrane proteins, and dynamics of molecular motor proteins. Actin remodeling has been the best target for this approach because actin and its associated proteins stop diffusing when assembled, allowing visualization of single-molecules of fluorescently-labeled proteins in a state specific manner. The approach based on this simple principle is called Single-Molecule Speckle (SiMS) microscopy. For instance, spatiotemporal regulation of actin polymerization and lifetime distribution of actin filaments can be monitored directly by tracking actin SiMS. In combination with fluorescently labeled probes of various actin regulators, SiMS microscopy has contributed to clarifying the processes underlying recycling, motion and remodeling of the live-cell actin network. Recently, we introduced an electroporation-based method called eSiMS microscopy, with high efficiency, easiness and improved spatiotemporal precision. In this review, we describe the application of live-cell single-molecule imaging to cellular actin dynamics and discuss the advantages of eSiMS microscopy over previous SiMS microscopy.

Diffusion, capture and recycling of SCAR/WAVE and Arp2/3 complexes observed in cells by single-molecule imaging.

PubMed

Millius, Arthur; Watanabe, Naoki; Weiner, Orion D

2012-03-01

The SCAR/WAVE complex drives lamellipodium formation by enhancing actin nucleation by the Arp2/3 complex. Phosphoinositides and Rac activate the SCAR/WAVE complex, but how SCAR/WAVE and Arp2/3 complexes converge at sites of nucleation is unknown. We analyzed the single-molecule dynamics of WAVE2 and p40 (subunits of the SCAR/WAVE and Arp2/3 complexes, respectively) in XTC cells. We observed lateral diffusion of both proteins and captured the transition of p40 from diffusion to network incorporation. These results suggest that a diffusive 2D search facilitates binding of the Arp2/3 complex to actin filaments necessary for nucleation. After nucleation, the Arp2/3 complex integrates into the actin network and undergoes retrograde flow, which results in its broad distribution throughout the lamellipodium. By contrast, the SCAR/WAVE complex is more restricted to the cell periphery. However, with single-molecule imaging, we also observed WAVE2 molecules undergoing retrograde motion. WAVE2 and p40 have nearly identical speeds, lifetimes and sites of network incorporation. Inhibition of actin retrograde flow does not prevent WAVE2 association and disassociation with the membrane but does inhibit WAVE2 removal from the actin cortex. Our results suggest that membrane binding and diffusion expedites the recruitment of nucleation factors to a nucleation site independent of actin assembly, but after network incorporation, ongoing actin polymerization facilitates recycling of SCAR/WAVE and Arp2/3 complexes.

Diffusion, capture and recycling of SCAR/WAVE and Arp2/3 complexes observed in cells by single-molecule imaging

PubMed Central

Millius, Arthur; Watanabe, Naoki; Weiner, Orion D.

2012-01-01

The SCAR/WAVE complex drives lamellipodium formation by enhancing actin nucleation by the Arp2/3 complex. Phosphoinositides and Rac activate the SCAR/WAVE complex, but how SCAR/WAVE and Arp2/3 complexes converge at sites of nucleation is unknown. We analyzed the single-molecule dynamics of WAVE2 and p40 (subunits of the SCAR/WAVE and Arp2/3 complexes, respectively) in XTC cells. We observed lateral diffusion of both proteins and captured the transition of p40 from diffusion to network incorporation. These results suggest that a diffusive 2D search facilitates binding of the Arp2/3 complex to actin filaments necessary for nucleation. After nucleation, the Arp2/3 complex integrates into the actin network and undergoes retrograde flow, which results in its broad distribution throughout the lamellipodium. By contrast, the SCAR/WAVE complex is more restricted to the cell periphery. However, with single-molecule imaging, we also observed WAVE2 molecules undergoing retrograde motion. WAVE2 and p40 have nearly identical speeds, lifetimes and sites of network incorporation. Inhibition of actin retrograde flow does not prevent WAVE2 association and disassociation with the membrane but does inhibit WAVE2 removal from the actin cortex. Our results suggest that membrane binding and diffusion expedites the recruitment of nucleation factors to a nucleation site independent of actin assembly, but after network incorporation, ongoing actin polymerization facilitates recycling of SCAR/WAVE and Arp2/3 complexes. PMID:22349699

Force-velocity relation for actin-polymerization-driven motility from Brownian dynamics simulations.

PubMed

Lee, Kun-Chun; Liu, Andrea J

2009-09-02

We report numerical simulation results for the force-velocity relation for actin-polymerization-driven motility. We use Brownian dynamics to solve a physically consistent formulation of the dendritic nucleation model with semiflexible filaments that self-assemble and push a disk. We find that at small loads, the disk speed is independent of load, whereas at high loads, the speed decreases and vanishes at a characteristic stall pressure. Our results demonstrate that at small loads, the velocity is controlled by the reaction rates, whereas at high loads the stall pressure is determined by the mechanical properties of the branched actin network. The behavior is consistent with experiments and with our recently proposed self-diffusiophoretic mechanism for actin-polymerization-driven motility. New in vitro experiments to measure the force-velocity relation are proposed.

Comparative Dynamics of Retrograde Actin Flow and Focal Adhesions: Formation of Nascent Adhesions Triggers Transition from Fast to Slow Flow

PubMed Central

Alexandrova, Antonina Y.; Arnold, Katya; Schaub, Sébastien; Vasiliev, Jury M.; Meister, Jean-Jacques; Bershadsky, Alexander D.; Verkhovsky, Alexander B.

2008-01-01

Dynamic actin network at the leading edge of the cell is linked to the extracellular matrix through focal adhesions (FAs), and at the same time it undergoes retrograde flow with different dynamics in two distinct zones: the lamellipodium (peripheral zone of fast flow), and the lamellum (zone of slow flow located between the lamellipodium and the cell body). Cell migration involves expansion of both the lamellipodium and the lamellum, as well as formation of new FAs, but it is largely unknown how the position of the boundary between the two flow zones is defined, and how FAs and actin flow mutually influence each other. We investigated dynamic relationship between focal adhesions and the boundary between the two flow zones in spreading cells. Nascent FAs first appeared in the lamellipodium. Within seconds after the formation of new FAs, the rate of actin flow decreased locally, and the lamellipodium/lamellum boundary advanced towards the new FAs. Blocking fast actin flow with cytochalasin D resulted in rapid dissolution of nascent FAs. In the absence of FAs (spreading on poly-L-lysine-coated surfaces) retrograde flow was uniform and the velocity transition was not observed. We conclude that formation of FAs depends on actin dynamics, and in its turn, affects the dynamics of actin flow by triggering transition from fast to slow flow. Extension of the cell edge thus proceeds through a cycle of lamellipodium protrusion, formation of new FAs, advance of the lamellum, and protrusion of the lamellipodium from the new base. PMID:18800171

An epidermal plakin that integrates actin and microtubule networks at cellular junctions.

PubMed

Karakesisoglou, I; Yang, Y; Fuchs, E

2000-04-03

Plakins are cytoskeletal linker proteins initially thought to interact exclusively with intermediate filaments (IFs), but recently were found to associate additionally with actin and microtubule networks. Here, we report on ACF7, a mammalian orthologue of the Drosophila kakapo plakin genetically involved in epidermal-muscle adhesion and neuromuscular junctions. While ACF7/kakapo is divergent from other plakins in its IF-binding domain, it has at least one actin (K(d) = 0.35 microM) and one microtubule (K(d) approximately 6 microM) binding domain. Similar to its fly counterpart, ACF7 is expressed in the epidermis. In well spread epidermal keratinocytes, ACF7 discontinuously decorates the cytoskeleton at the cell periphery, including microtubules (MTs) and actin filaments (AFs) that are aligned in parallel converging at focal contacts. Upon calcium induction of intercellular adhesion, ACF7 and the cytoskeleton reorganize at cell-cell borders but with different kinetics from adherens junctions and desmosomes. Treatments with cytoskeletal depolymerizing drugs reveal that ACF7's cytoskeletal association is dependent upon the microtubule network, but ACF7 also appears to stabilize actin at sites where microtubules and microfilaments meet. We posit that ACF7 may function in microtubule dynamics to facilitate actin-microtubule interactions at the cell periphery and to couple the microtubule network to cellular junctions. These attributes provide a clear explanation for the kakapo mutant phenotype in flies.

Advances in the mechanical modeling of filamentous actin and its cross-linked networks on multiple scales.

PubMed

Unterberger, Michael J; Holzapfel, Gerhard A

2014-11-01

The protein actin is a part of the cytoskeleton and, therefore, responsible for the mechanical properties of the cells. Starting with the single molecule up to the final structure, actin creates a hierarchical structure of several levels exhibiting a remarkable behavior. The hierarchy spans several length scales and limitations in computational power; therefore, there is a call for different mechanical modeling approaches for the different scales. On the molecular level, we may consider each atom in molecular dynamics simulations. Actin forms filaments by combining the molecules into a double helix. In a model, we replace molecular subdomains using coarse-graining methods, allowing the investigation of larger systems of several atoms. These models on the nanoscale inform continuum mechanical models of large filaments, which are based on worm-like chain models for polymers. Assemblies of actin filaments are connected with cross-linker proteins. Models with discrete filaments, so-called Mikado models, allow us to investigate the dependence of the properties of networks on the parameters of the constituents. Microstructurally motivated continuum models of the networks provide insights into larger systems containing cross-linked actin networks. Modeling of such systems helps to gain insight into the processes on such small scales. On the other hand, they call for verification and hence trigger the improvement of established experiments and the development of new methods.

Actin dynamics in Amoeba proteus motility.

PubMed

Pomorski, P; Krzemiński, P; Wasik, A; Wierzbicka, K; Barańska, J; Kłopocka, W

2007-01-01

We studied the distribution of the endogenous Arp2/3 complex in Amoeba proteus and visualised the ratio of filamentous (F-actin) to total actin in living cells. The presented results show that in the highly motile Amoeba proteus, Arp2/3 complex-dependent actin polymerisation is involved in the formation of the branching network of the contractile layer, adhesive structures, and perinuclear cytoskeleton. The aggregation of the Arp2/3 complex in the cortical network, with the exception of the uroid and advancing fronts, and the spatial orientation of microfilaments at the leading edge suggest that actin polymerisation in this area is not sufficient to provide the driving force for membrane displacement. The examined proteins were enriched in the pinocytotic pseudopodia and the perinuclear cytoskeleton in pinocytotic amoebae. In migrating amoebae, the course of changes in F-actin concentration corresponded with the distribution of tension in the cell cortex. The maximum level of F-actin in migrating amoebae was observed in the middle-posterior region and in the front of retracting pseudopodia. Arp2/3 complex-dependent actin polymerisation did not seem to influence F-actin concentration. The strongly condensed state of the microfilament system could be attributed to strong isometric contraction of the cortical layer accompanied by its retraction from distal cell regions. Isotonic contraction was limited to the uroid.

Alpha-actinin binding kinetics modulate cellular dynamics and force generation

PubMed Central

Ehrlicher, Allen J.; Krishnan, Ramaswamy; Guo, Ming; Bidan, Cécile M.; Weitz, David A.; Pollak, Martin R.

2015-01-01

The actin cytoskeleton is a key element of cell structure and movement whose properties are determined by a host of accessory proteins. Actin cross-linking proteins create a connected network from individual actin filaments, and though the mechanical effects of cross-linker binding affinity on actin networks have been investigated in reconstituted systems, their impact on cellular forces is unknown. Here we show that the binding affinity of the actin cross-linker α-actinin 4 (ACTN4) in cells modulates cytoplasmic mobility, cellular movement, and traction forces. Using fluorescence recovery after photobleaching, we show that an ACTN4 mutation that causes human kidney disease roughly triples the wild-type binding affinity of ACTN4 to F-actin in cells, increasing the dissociation time from 29 ± 13 to 86 ± 29 s. This increased affinity creates a less dynamic cytoplasm, as demonstrated by reduced intracellular microsphere movement, and an approximate halving of cell speed. Surprisingly, these less motile cells generate larger forces. Using traction force microscopy, we show that increased binding affinity of ACTN4 increases the average contractile stress (from 1.8 ± 0.7 to 4.7 ± 0.5 kPa), and the average strain energy (0.4 ± 0.2 to 2.1 ± 0.4 pJ). We speculate that these changes may be explained by an increased solid-like nature of the cytoskeleton, where myosin activity is more partitioned into tension and less is dissipated through filament sliding. These findings demonstrate the impact of cross-linker point mutations on cell dynamics and forces, and suggest mechanisms by which such physical defects lead to human disease. PMID:25918384

The role of actin networks in cellular mechanosensing

NASA Astrophysics Data System (ADS)

Azatov, Mikheil

Physical processes play an important role in many biological phenomena, such as wound healing, organ development, and tumor metastasis. During these processes, cells constantly interact with and adapt to their environment by exerting forces to mechanically probe the features of their surroundings and generating appropriate biochemical responses. The mechanisms underlying how cells sense the physical properties of their environment are not well understood. In this thesis, I present my studies to investigate cellular responses to the stiffness and topography of the environment. In order to sense the physical properties of their environment, cells dynamically reorganize the structure of their actin cytoskeleton, a dynamic network of biopolymers, altering the shape and spatial distribution of protein assemblies. Several observations suggest that proteins that crosslink actin filaments may play an important role in cellular mechanosensitivity. Palladin is an actin-crosslinking protein that is found in the lamellar actin network, stress fibers and focal adhesions, cellular structures that are critical for mechanosensing of the physical environment. By virtue of its close interactions with these structures in the cell, palladin may play an important role in cell mechanics. However, the role of actin crosslinkers in general, and palladin in particular, in cellular force generation and mechanosensing is not well known. I have investigated the role of palladin in regulating the plasticity of the actin cytoskeleton and cellular force generation in response to alterations in substrate stiffness. I have shown that the expression levels of palladin modulate the forces exerted by cells and their ability to sense substrate stiffness. Perturbation experiments also suggest that palladin levels in cells altered myosin motor activity. These results suggest that the actin crosslinkers, such as palladin, and myosin motors coordinate for optimal cell function and to prevent aberrant behavior as in cancer metastasis. In addition to stiffness, the local geometry or topography of the surface has been shown to modulate the movement, morphology, and cytoskeletal organization of cells. However, the effect of topography on fluctuations of intracellular structures, which arise from motor driven activity on a viscoelastic actin network are not known. I have used nanofabricated substrates with parallel ridges to show that the cell shape, the actin cytoskeleton and focal adhesions all align along the direction of the ridges, exhibiting a biphasic dependence on the spacing between ridges. I further demonstrated that palladin bands along actin stress fibers undergo a complex diffusive motion with velocities aligned along the direction of ridges. These results provide insight into the mechanisms of cellular mechanosensing of the environment, suggesting a complex interplay between the actin cytoskeleton and cellular adhesions in coordinating cellular response to surface topography. Overall, this work has advanced our understanding of mechanisms that govern cellular responses to their physical environment.

The heel and toe of the cell’s foot: A multifaceted approach for understanding the structure and dynamics of focal adhesions

PubMed Central

Wolfenson, Haguy; Henis, Yoav I.; Geiger, Benjamin; Bershadsky, Alexander D.

2010-01-01

Focal adhesions (FAs) are large clusters of transmembrane receptors of the integrin family and a multitude of associated cytoplasmic “plaque” proteins, which connect the extracellular matrix-bound receptors with the actin cytoskeleton. The formation of nearly stationary focal adhesions defines a boundary between dense and highly dynamic actin network in lamellipodium and the sparser and more diverse cytoskeletal organization in the lamella proper, creating a template for the organization of entire actin network. The major “mechanical” and “sensory” functions of FAs, namely, the nucleation and regulation of the contractile, myosin-II-containing, stress fibers and the mechanosensing of external surfaces depend, to a major extent, on the dynamics of molecular components within FAs. A central element in FA regulation concerns the positive feedback loop, based on the most intriguing feature of FAs, namely, their dependence on mechanical tension developing by the growing stress fibers. FAs grow in response to such tension, and rapidly disassemble upon its relaxation. In this article we address the mechanistic relationships between the process of FA development, maturation and dissociation and the dynamic molecular events, which take place in different regions of the FA, primarily in the distal end of this structure (the “toe”) and the proximal “heel”, and discuss the central role of local mechanical forces in orchestrating the complex interplay between FAs and the actin system. PMID:19598236

Spontaneous actin dynamics in contractile rings

NASA Astrophysics Data System (ADS)

Kruse, Karsten; Wollrab, Viktoria; Thiagarajan, Raghavan; Wald, Anne; Riveline, Daniel

Networks of polymerizing actin filaments are known to be capable to self-organize into a variety of structures. For example, spontaneous actin polymerization waves have been observed in living cells in a number of circumstances, notably, in crawling neutrophils and slime molds. During later stages of cell division, they can also spontaneously form a contractile ring that will eventually cleave the cell into two daughter cells. We present a framework for describing networks of polymerizing actin filaments, where assembly is regulated by various proteins. It can also include the effects of molecular motors. We show that the molecular processes driven by these proteins can generate various structures that have been observed in contractile rings of fission yeast and mammalian cells. We discuss a possible functional role of each of these patterns. The work was supported by Agence Nationale de la Recherche, France, (ANR-10-LABX-0030-INRT) and by Deutsche Forschungsgemeinschaft through SFB1027.

Inter-Cellular Forces Orchestrate Contact Inhibition of Locomotion

PubMed Central

Davis, John R.; Luchici, Andrei; Mosis, Fuad; Thackery, James; Salazar, Jesus A.; Mao, Yanlan; Dunn, Graham A.; Betz, Timo; Miodownik, Mark; Stramer, Brian M.

2015-01-01

Summary Contact inhibition of locomotion (CIL) is a multifaceted process that causes many cell types to repel each other upon collision. During development, this seemingly uncoordinated reaction is a critical driver of cellular dispersion within embryonic tissues. Here, we show that Drosophila hemocytes require a precisely orchestrated CIL response for their developmental dispersal. Hemocyte collision and subsequent repulsion involves a stereotyped sequence of kinematic stages that are modulated by global changes in cytoskeletal dynamics. Tracking actin retrograde flow within hemocytes in vivo reveals synchronous reorganization of colliding actin networks through engagement of an inter-cellular adhesion. This inter-cellular actin-clutch leads to a subsequent build-up in lamellar tension, triggering the development of a transient stress fiber, which orchestrates cellular repulsion. Our findings reveal that the physical coupling of the flowing actin networks during CIL acts as a mechanotransducer, allowing cells to haptically sense each other and coordinate their behaviors. PMID:25799385

Structure and Dynamics of an Arp2/3 Complex-independent Component of the Lamellipodial Actin Network

PubMed Central

Henson, John H.; Cheung, David; Fried, Christopher A.; Shuster, Charles B.; McClellan, Mary K.; Voss, Meagen K.; Sheridan, John T.; Oldenbourg, Rudolf

2010-01-01

Sea urchin coelomocytes contain an unusually broad lamellipodial region and have served as a useful model experimental system for studying the process of actin-based retrograde/centripetal flow. In the current study the small molecule drug 2,3-butanedione monoxime (BDM) was employed as a means of delocalizing the Arp2/3 complex from the cell edge in an effort to investigate the Arp2/3 complex-independent aspects of retrograde flow. Digitally-enhanced phase contrast, fluorescence and polarization light microscopy, along with rotary shadow TEM methods demonstrated that BDM treatment resulted in the centripetal displacement of the Arp2/3 complex and the associated dendritic lamellipodial (LP) actin network from the cell edge. In its wake there remained an array of elongate actin filaments organized into concave arcs that displayed retrograde flow at approximately one quarter the normal rate. Actin polymerization inhibitor experiments indicated that these arcs were generated by polymerization at the cell edge, while active myosin-based contraction in BDM treated cells was demonstrated by localization with anti-phospho-MRLC antibody, the retraction of the cytoskeleton in the presence of BDM, and the response of the BDM arcs to laser-based severing. The results suggest that BDM treatment reveals an Arp2/3 complex-independent actin structure in coelomocytes consisting of elongate filaments integrated into the LP network and that these filaments represent a potential connection between the LP network and the central cytoskeleton. PMID:19530177

The actin-microtubule cross-linking activity of Drosophila Short stop is regulated by intramolecular inhibition

PubMed Central

Applewhite, Derek A.; Grode, Kyle D.; Duncan, Mara C.; Rogers, Stephen L.

2013-01-01

Actin and microtubule dynamics must be precisely coordinated during cell migration, mitosis, and morphogenesis—much of this coordination is mediated by proteins that physically bridge the two cytoskeletal networks. We have investigated the regulation of the Drosophila actin-microtubule cross-linker Short stop (Shot), a member of the spectraplakin family. Our data suggest that Shot's cytoskeletal cross-linking activity is regulated by an intramolecular inhibitory mechanism. In its inactive conformation, Shot adopts a “closed” conformation through interactions between its NH2-terminal actin-binding domain and COOH-terminal EF-hand-GAS2 domain. This inactive conformation is targeted to the growing microtubule plus end by EB1. On activation, Shot binds along the microtubule through its COOH-terminal GAS2 domain and binds to actin with its NH2-terminal tandem CH domains. We propose that this mechanism allows Shot to rapidly cross-link dynamic microtubules in response to localized activating signals at the cell cortex. PMID:23885120

Multiscale Modelling for investigating single molecule effects on the mechanics of actin filaments

NASA Astrophysics Data System (ADS)

A, Deriu Marco; C, Bidone Tamara; Laura, Carbone; Cristina, Bignardi; M, Montevecchi Franco; Umberto, Morbiducci

2011-12-01

This work presents a preliminary multiscale computational investigation of the effects of nucleotides and cations on the mechanics of actin filaments (F-actin). At the molecular level, Molecular Dynamics (MD) simulations are employed to characterize the rearrangements of the actin monomers (G-actin) in terms of secondary structures evolution in physiological conditions. At the mesoscale level, a coarse grain (CG) procedure is adopted where each monomer is represented by means of Elastic Network Modeling (ENM) technique. At the macroscale level, actin filaments up to hundreds of nanometers are assumed as isotropic and elastic beams and characterized via Rotation Translation Block (RTB) analysis. F-actin bound to adenosine triphosphate (ATP) shows a persistence length around 5 μm, while actin filaments bound to adenosine diphosphate (ADP) have a persistence length of about 3 μm. With magnesium bound to the high affinity binding site of G-actin, the persistence length of F-actin decreases to about 2 μm only in the ADP-bound form of the filament, while the same ion has no effects, in terms of stiffness variation, on the ATP-bound form of F-actin. The molecular mechanisms behind these changes in flexibility are herein elucidated. Thus, this study allows to analyze how the local binding of cations and nucleotides on G-actin induce molecular rearrangements that transmit to the overall F-actin, characterizing shifts of mechanical properties, that can be related with physiological and pathological cellular phenomena, as cell migration and spreading. Further, this study provides the basis for upcoming investigating of network and cellular remodelling at higher length scales.

Effects of polymerization and nucleotide identity on the conformational dynamics of the bacterial actin homolog MreB

PubMed Central

Colavin, Alexandre; Hsin, Jen; Huang, Kerwyn Casey

2014-01-01

The assembly of protein filaments drives many cellular processes, from nucleoid segregation, growth, and division in single cells to muscle contraction in animals. In eukaryotes, shape and motility are regulated through cycles of polymerization and depolymerization of actin cytoskeletal networks. In bacteria, the actin homolog MreB forms filaments that coordinate the cell-wall synthesis machinery to regulate rod-shaped growth and contribute to cellular stiffness through unknown mechanisms. Like actin, MreB is an ATPase and requires ATP to polymerize, and polymerization promotes nucleotide hydrolysis. However, it is unclear whether other similarities exist between MreB and actin because the two proteins share low sequence identity and have distinct cellular roles. Here, we use all-atom molecular dynamics simulations to reveal surprising parallels between MreB and actin structural dynamics. We observe that MreB exhibits actin-like polymerization-dependent structural changes, wherein polymerization induces flattening of MreB subunits, which restructures the nucleotide-binding pocket to favor hydrolysis. MreB filaments exhibited nucleotide-dependent intersubunit bending, with hydrolyzed polymers favoring a straighter conformation. We use steered simulations to demonstrate a coupling between intersubunit bending and the degree of flattening of each subunit, suggesting cooperative bending along a filament. Taken together, our results provide molecular-scale insight into the diversity of structural states of MreB and the relationships among polymerization, hydrolysis, and filament properties, which may be applicable to other members of the broad actin family. PMID:24550504

Effects of polymerization and nucleotide identity on the conformational dynamics of the bacterial actin homolog MreB.

PubMed

Colavin, Alexandre; Hsin, Jen; Huang, Kerwyn Casey

2014-03-04

The assembly of protein filaments drives many cellular processes, from nucleoid segregation, growth, and division in single cells to muscle contraction in animals. In eukaryotes, shape and motility are regulated through cycles of polymerization and depolymerization of actin cytoskeletal networks. In bacteria, the actin homolog MreB forms filaments that coordinate the cell-wall synthesis machinery to regulate rod-shaped growth and contribute to cellular stiffness through unknown mechanisms. Like actin, MreB is an ATPase and requires ATP to polymerize, and polymerization promotes nucleotide hydrolysis. However, it is unclear whether other similarities exist between MreB and actin because the two proteins share low sequence identity and have distinct cellular roles. Here, we use all-atom molecular dynamics simulations to reveal surprising parallels between MreB and actin structural dynamics. We observe that MreB exhibits actin-like polymerization-dependent structural changes, wherein polymerization induces flattening of MreB subunits, which restructures the nucleotide-binding pocket to favor hydrolysis. MreB filaments exhibited nucleotide-dependent intersubunit bending, with hydrolyzed polymers favoring a straighter conformation. We use steered simulations to demonstrate a coupling between intersubunit bending and the degree of flattening of each subunit, suggesting cooperative bending along a filament. Taken together, our results provide molecular-scale insight into the diversity of structural states of MreB and the relationships among polymerization, hydrolysis, and filament properties, which may be applicable to other members of the broad actin family.

RhoA regulates actin network dynamics during apical surface emergence in multiciliated epithelial cells

PubMed Central

Sedzinski, Jakub; Hannezo, Edouard; Tu, Fan; Biro, Maté

2017-01-01

ABSTRACT Homeostatic replacement of epithelial cells from basal precursors is a multistep process involving progenitor cell specification, radial intercalation and, finally, apical surface emergence. Recent data demonstrate that actin-based pushing under the control of the formin protein Fmn1 drives apical emergence in nascent multiciliated epithelial cells (MCCs), but little else is known about this actin network or the control of Fmn1. Here, we explore the role of the small GTPase RhoA in MCC apical emergence. Disruption of RhoA function reduced the rate of apical surface expansion and decreased the final size of the apical domain. Analysis of cell shapes suggests that RhoA alters the balance of forces exerted on the MCC apical surface. Finally, quantitative time-lapse imaging and fluorescence recovery after photobleaching studies argue that RhoA works in concert with Fmn1 to control assembly of the specialized apical actin network in MCCs. These data provide new molecular insights into epithelial apical surface assembly and could also shed light on mechanisms of apical lumen formation. PMID:28089989

RhoA regulates actin network dynamics during apical surface emergence in multiciliated epithelial cells.

PubMed

Sedzinski, Jakub; Hannezo, Edouard; Tu, Fan; Biro, Maté; Wallingford, John B

2017-01-15

Homeostatic replacement of epithelial cells from basal precursors is a multistep process involving progenitor cell specification, radial intercalation and, finally, apical surface emergence. Recent data demonstrate that actin-based pushing under the control of the formin protein Fmn1 drives apical emergence in nascent multiciliated epithelial cells (MCCs), but little else is known about this actin network or the control of Fmn1. Here, we explore the role of the small GTPase RhoA in MCC apical emergence. Disruption of RhoA function reduced the rate of apical surface expansion and decreased the final size of the apical domain. Analysis of cell shapes suggests that RhoA alters the balance of forces exerted on the MCC apical surface. Finally, quantitative time-lapse imaging and fluorescence recovery after photobleaching studies argue that RhoA works in concert with Fmn1 to control assembly of the specialized apical actin network in MCCs. These data provide new molecular insights into epithelial apical surface assembly and could also shed light on mechanisms of apical lumen formation. © 2017. Published by The Company of Biologists Ltd.

Toward the Structure of Dynamic Membrane-Anchored Actin Networks

PubMed Central

Weber, Igor

2007-01-01

In the cortex of a motile cell, membrane-anchored actin filaments assemble into structures of varying shape and function. Filopodia are distinguished by a core of bundled actin filaments within finger-like extensions of the membrane. In a recent paper by Medalia et al1 cryo-electron tomography has been used to reconstruct, from filopodia of Dictyostelium cells, the 3-dimensional organization of actin filaments in connection with the plasma membrane. A special arrangement of short filaments converging toward the filopod's tip has been called a “terminal cone”. In this region force is applied for protrusion of the membrane. Here we discuss actin organization in the filopodia of Dictyostelium in the light of current views on forces that are generated by polymerizing actin filaments, and on the resistance of membranes against deformation that counteracts these forces. PMID:19262130

The coordinating role of IQGAP1 in the regulation of local, endosome-specific actin networks

PubMed Central

Samson, Edward B.; Tsao, David S.; Zimak, Jan; McLaughlin, R. Tyler; Trenton, Nicholaus J.; Mace, Emily M.; Orange, Jordan S.; Schweikhard, Volker

2017-01-01

ABSTRACT IQGAP1 is a large, multi-domain scaffold that helps orchestrate cell signaling and cytoskeletal mechanics by controlling interactions among a spectrum of receptors, signaling intermediates, and cytoskeletal proteins. While this coordination is known to impact cell morphology, motility, cell adhesion, and vesicular traffic, among other functions, the spatiotemporal properties and regulatory mechanisms of IQGAP1 have not been fully resolved. Herein, we describe a series of super-resolution and live-cell imaging analyses that identified a role for IQGAP1 in the regulation of an actin cytoskeletal shell surrounding a novel membranous compartment that localizes selectively to the basal cortex of polarized epithelial cells (MCF-10A). We also show that IQGAP1 appears to both stabilize the actin coating and constrain its growth. Loss of compartmental IQGAP1 initiates a disassembly mechanism involving rapid and unconstrained actin polymerization around the compartment and dispersal of its vesicle contents. Together, these findings suggest IQGAP1 achieves this control by harnessing both stabilizing and antagonistic interactions with actin. They also demonstrate the utility of these compartments for image-based investigations of the spatial and temporal dynamics of IQGAP1 within endosome-specific actin networks. PMID:28455356

Organization and function of the actin cytoskeleton in developing root cells.

PubMed

Blancaflor, Elison B; Wang, Yuh-Shuh; Motes, Christy M

2006-01-01

The actin cytoskeleton is a highly dynamic structure, which mediates various cellular functions in large part through accessory proteins that tilt the balance between monomeric G-actin and filamentous actin (F-actin) or by facilitating interactions between actin and the plasma membrane, microtubules, and other organelles. Roots have become an attractive model to study actin in plant development because of their simple anatomy and accessibility of some root cell types such as root hairs for microscopic analyses. Roots also exhibit a remarkable developmental plasticity and possess a delicate sensory system that is easily manipulated, so that one can design experiments addressing a range of important biological questions. Many facets of root development can be regulated by the diverse actin network found in the various root developmental regions. Various molecules impinge on this actin scaffold to define how a particular root cell type grows or responds to a specific environmental signal. Although advances in genomics are leading the way toward elucidating actin function in roots, more significant strides will be realized when such tools are combined with improved methodologies for accurately depicting how actin is organized in plant cells.

Live cell imaging of actin dynamics in dexamethasone-treated porcine trabecular meshwork cells.

PubMed

Fujimoto, Tomokazu; Inoue, Toshihiro; Inoue-Mochita, Miyuki; Tanihara, Hidenobu

2016-04-01

The regulation of the actin cytoskeleton in trabecular meshwork (TM) cells is important for controlling outflow of the aqueous humor. In some reports, dexamethasone (DEX) increased the aqueous humor outflow resistance and induced unusual actin structures, such as cross-linked actin networks (CLAN), in TM cells. However, the functions and dynamics of CLAN in TM cells are not completely known, partly because actin stress fibers have been observed only in fixed cells. We conducted live-cell imaging of the actin dynamics in TM cells with or without DEX treatment. An actin-green fluorescent protein (GFP) fusion construct with a modified insect virus was transfected into porcine TM cells. Time-lapse imaging of live TM cells treated with 25 μM Y-27632 and 100 nM DEX was performed using an inverted fluorescence microscope. Fluorescent images were recorded every 15 s for 30 min after Y-27632 treatment or every 30 min for 72 h after DEX treatment. The GFP-actin was expressed in 22.7 ± 10.9% of the transfected TM cells. In live TM cells, many actin stress fibers were observed before the Y-27632 treatment. Y-27632 changed the cell shape and decreased stress fibers in a time-dependent manner. In fixed cells, CLAN-like structures were seen in 26.5 ± 1.7% of the actin-GFP expressed PTM cells treated with DEX for 72 h. In live imaging, there was 28% CLAN-like structure formation at 72 h after DEX treatment, and the lifetime of CLAN-like structures increased after DEX treatment. The DEX-treated cells with CLAN-like structures showed less migration than DEX-treated cells without CLAN-like structures. Furthermore, the control cells (without DEX treatment) with CLAN-like structures also showed less migration than the control cells without CLAN-like structures. These results suggested that CLAN-like structure formation was correlated with cell migration in TM cells. Live cell imaging of the actin cytoskeleton provides valuable information on the actin dynamics in TM cells. Copyright © 2016 Elsevier Ltd. All rights reserved.

Actin cytoskeleton of chemotactic amoebae operates close to the onset of oscillations

PubMed Central

Westendorf, Christian; Negrete, Jose; Bae, Albert J.; Sandmann, Rabea; Bodenschatz, Eberhard; Beta, Carsten

2013-01-01

The rapid reorganization of the actin cytoskeleton in response to external stimuli is an essential property of many motile eukaryotic cells. Here, we report evidence that the actin machinery of chemotactic Dictyostelium cells operates close to an oscillatory instability. When averaging the actin response of many cells to a short pulse of the chemoattractant cAMP, we observed a transient accumulation of cortical actin reminiscent of a damped oscillation. At the single-cell level, however, the response dynamics ranged from short, strongly damped responses to slowly decaying, weakly damped oscillations. Furthermore, in a small subpopulation, we observed self-sustained oscillations in the cortical F-actin concentration. To substantiate that an oscillatory mechanism governs the actin dynamics in these cells, we systematically exposed a large number of cells to periodic pulse trains of different frequencies. Our results indicate a resonance peak at a stimulation period of around 20 s. We propose a delayed feedback model that explains our experimental findings based on a time-delay in the regulatory network of the actin system. To test the model, we performed stimulation experiments with cells that express GFP-tagged fusion proteins of Coronin and actin-interacting protein 1, as well as knockout mutants that lack Coronin and actin-interacting protein 1. These actin-binding proteins enhance the disassembly of actin filaments and thus allow us to estimate the delay time in the regulatory feedback loop. Based on this independent estimate, our model predicts an intrinsic period of 20 s, which agrees with the resonance observed in our periodic stimulation experiments. PMID:23431176

Aggregatibacter actinomycetemcomitans lipopolysaccharide affects human gingival fibroblast cytoskeletal organization.

PubMed

Gutiérrez-Venegas, Gloria; Contreras-Marmolejo, Luis Arturo; Román-Alvárez, Patricia; Barajas-Torres, Carolina

2008-04-01

The cytoskeleton is a dynamic structure that plays a key role in maintaining cell morphology and function. This study investigates the effect of bacterial wall lipopolysaccharide (LPS), a strong inflammatory agent, on the dynamics and organization of actin, tubulin, vimentin, and vinculin proteins in human gingival fibroblasts (HGF). A time-dependent study showed a noticeable change in actin architecture after 1.5 h of incubation with LPS (1 microg/ml) with the formation of orthogonal fibers and further accumulation of actin filament at the cell periphery by 24 h. When 0.01-10 microg/ml of LPS was added to human gingival fibroblast cultures, cells acquired a round, flat shape and gradually developed cytoplasmic ruffling. Lipopolysaccharides extracted from Aggregatibacter actinomycetemcomitans periodontopathogenic bacteria promoted alterations in F-actin stress fibres of human gingival cells. Normally, human gingival cells have F-actin fibres that are organized in linear distribution throughout the cells, extending along the cell's length. LPS-treated cells exhibited changes in cytoskeletal protein organization, and F-actin was reorganized by the formation of bundles underneath and parallel to the cell membrane. We also found the reorganization of the vimentin network into vimentin bundling after 1.5 h of treatment. HGF cells exhibited diffuse and granular gamma-tubulin stain. There was no change in LPS-treated HGF. However, vinculin plaques distributed in the cell body diminished after LPS treatment. We conclude that the dynamic and structured organization of cytoskeletal filaments and actin assembly in human gingival fibroblasts is altered by LPS treatment and is accompanied by a decrease in F-actin pools.

Cortactin Branches Out: Roles in Regulating Protrusive Actin Dynamics

PubMed Central

Ammer, Amanda Gatesman; Weed, Scott A.

2008-01-01

Since its discovery in the early 1990’s, cortactin has emerged as a key signaling protein in many cellular processes, including cell adhesion, migration, endocytosis, and tumor invasion. While the list of cellular functions influenced by cortactin grows, the ability of cortactin to interact with and alter the cortical actin network is central to its role in regulating these processes. Recently, several advances have been made in our understanding of the interaction between actin and cortactin, providing insight into how these two proteins work together to provide a framework for normal and altered cellular function. This review examines how regulation of cortactin through post-translational modifications and interactions with multiple binding partners elicits changes in cortical actin cytoskeletal organization, impacting the regulation and formation of actin-rich motility structures. PMID:18615630

Actin dynamics at focal adhesions: a common endpoint and putative therapeutic target for proteinuric kidney diseases.

PubMed

Sever, Sanja; Schiffer, Mario

2018-06-01

Proteinuria encompasses diverse causes including both genetic diseases and acquired forms such as diabetic and hypertensive nephropathy. The basis of proteinuria is a disturbance in size selectivity of the glomerular filtration barrier, which largely depends on the podocyte: a terminally differentiated epithelial cell type covering the outer surface of the glomerulus. Compromised podocyte structure is one of the earliest signs of glomerular injury. The phenotype of diverse animal models and podocyte cell culture firmly established the essential role of the actin cytoskeleton in maintaining functional podocyte structure. Podocyte foot processes, actin-based membrane extensions, contain 2 molecularly distinct "hubs" that control actin dynamics: a slit diaphragm and focal adhesions. Although loss of foot processes encompasses disassembly of slit diaphragm multiprotein complexes, as long as cells are attached to the glomerular basement membrane, focal adhesions will be the sites in which stress due to filtration flow is counteracted by forces generated by the actin network in foot processes. Numerous studies within last 20 years have identified actin binding and regulatory proteins as well as integrins as essential components of signaling and actin dynamics at focal adhesions in podocytes, suggesting that some of them may become novel, druggable targets for proteinuric kidney diseases. Here we review evidence supporting the idea that current treatments for chronic kidney diseases beneficially and directly target the podocyte actin cytoskeleton associated with focal adhesions and suggest that therapeutic reagents that target the focal adhesion-regulated actin cytoskeleton in foot processes have potential to modernize treatments for chronic kidney diseases. Copyright © 2018 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

A QUICK Screen for Lrrk2 Interaction Partners – Leucine-rich Repeat Kinase 2 is Involved in Actin Cytoskeleton Dynamics*

PubMed Central

Meixner, Andrea; Boldt, Karsten; Van Troys, Marleen; Askenazi, Manor; Gloeckner, Christian J.; Bauer, Matthias; Marto, Jarrod A.; Ampe, Christophe; Kinkl, Norbert; Ueffing, Marius

2011-01-01

Mutations in human leucine-rich repeat kinase 2 (Lrrk2), a protein of yet unknown function, are linked to Parkinson's disease caused by degeneration of midbrain dopaminergic neurons. The protein comprises several domains including a GTPase and a kinase domain both affected by several pathogenic mutations. To elucidate the molecular interaction network of endogenous Lrrk2 under stoichiometric constraints, we applied QUICK (quantitative immunoprecipitation combined with knockdown) in NIH3T3 cells. The identified interactome reveals actin isoforms as well as actin-associated proteins involved in actin filament assembly, organization, rearrangement, and maintenance, suggesting that the biological function of Lrrk2 is linked to cytoskeletal dynamics. In fact, we demonstrate Lrrk2 de novo binding to F-actin and its ability to modulate its assembly in vitro. When tested in intact cells, knockdown of Lrrk2 causes morphological alterations in NIH3T3 cells. In developing dopaminergic midbrain primary neurons, Lrrk2 knockdown results in shortened neurite processes, indicating a physiological role of Lrrk2 in cytoskeletal organization and dynamics of dopaminergic neurons. Hence, our results demonstrate that molecular interactions as well as the physiological function of Lrrk2 are closely related to the organization of the actin-based cytoskeleton, a crucial feature of neuronal development and neuron function. PMID:20876399

Distinct cytoskeleton populations and extensive crosstalk control Ciona notochord tubulogenesis.

PubMed

Dong, Bo; Deng, Wei; Jiang, Di

2011-04-01

Cell elongation is a fundamental process that allows cells and tissues to adopt new shapes and functions. During notochord tubulogenesis in the ascidian Ciona intestinalis, a dramatic elongation of individual cells takes place that lengthens the notochord and, consequently, the entire embryo. We find a novel dynamic actin- and non-muscle myosin II-containing constriction midway along the anteroposterior aspect of each notochord cell during this process. Both actin polymerization and myosin II activity are required for the constriction and cell elongation. Discontinuous localization of myosin II in the constriction indicates that the actomyosin network produces local contractions along the circumference. This reveals basal constriction by the actomyosin network as a novel mechanism for cell elongation. Following elongation, the notochord cells undergo a mesenchymal-epithelial transition and form two apical domains at opposite ends. Extracellular lumens then form at the apical surfaces. We show that cortical actin and Ciona ezrin/radixin/moesin (ERM) are essential for lumen formation and that a polarized network of microtubules, which contributes to lumen development, forms in an actin-dependent manner at the apical cortex. Later in notochord tubulogenesis, when notochord cells initiate a bi-directional crawling movement on the notochordal sheath, the microtubule network rotates 90° and becomes organized as parallel bundles extending towards the leading edges of tractive lamellipodia. This process is required for the correct organization of actin-based protrusions and subsequent lumen coalescence. In summary, we establish the contribution of the actomyosin and microtubule networks to notochord tubulogenesis and reveal extensive crosstalk and regulation between these two cytoskeleton components.

Inter-cellular forces orchestrate contact inhibition of locomotion.

PubMed

Davis, John R; Luchici, Andrei; Mosis, Fuad; Thackery, James; Salazar, Jesus A; Mao, Yanlan; Dunn, Graham A; Betz, Timo; Miodownik, Mark; Stramer, Brian M

2015-04-09

Contact inhibition of locomotion (CIL) is a multifaceted process that causes many cell types to repel each other upon collision. During development, this seemingly uncoordinated reaction is a critical driver of cellular dispersion within embryonic tissues. Here, we show that Drosophila hemocytes require a precisely orchestrated CIL response for their developmental dispersal. Hemocyte collision and subsequent repulsion involves a stereotyped sequence of kinematic stages that are modulated by global changes in cytoskeletal dynamics. Tracking actin retrograde flow within hemocytes in vivo reveals synchronous reorganization of colliding actin networks through engagement of an inter-cellular adhesion. This inter-cellular actin-clutch leads to a subsequent build-up in lamellar tension, triggering the development of a transient stress fiber, which orchestrates cellular repulsion. Our findings reveal that the physical coupling of the flowing actin networks during CIL acts as a mechanotransducer, allowing cells to haptically sense each other and coordinate their behaviors. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

There is more than one way to model an elephant. Experiment-driven modeling of the actin cytoskeleton.

PubMed

Ditlev, Jonathon A; Mayer, Bruce J; Loew, Leslie M

2013-02-05

Mathematical modeling has established its value for investigating the interplay of biochemical and mechanical mechanisms underlying actin-based motility. Because of the complex nature of actin dynamics and its regulation, many of these models are phenomenological or conceptual, providing a general understanding of the physics at play. But the wealth of carefully measured kinetic data on the interactions of many of the players in actin biochemistry cries out for the creation of more detailed and accurate models that could permit investigators to dissect interdependent roles of individual molecular components. Moreover, no human mind can assimilate all of the mechanisms underlying complex protein networks; so an additional benefit of a detailed kinetic model is that the numerous binding proteins, signaling mechanisms, and biochemical reactions can be computationally organized in a fully explicit, accessible, visualizable, and reusable structure. In this review, we will focus on how comprehensive and adaptable modeling allows investigators to explain experimental observations and develop testable hypotheses on the intracellular dynamics of the actin cytoskeleton. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

There is More Than One Way to Model an Elephant. Experiment-Driven Modeling of the Actin Cytoskeleton

PubMed Central

Ditlev, Jonathon A.; Mayer, Bruce J.; Loew, Leslie M.

2013-01-01

Mathematical modeling has established its value for investigating the interplay of biochemical and mechanical mechanisms underlying actin-based motility. Because of the complex nature of actin dynamics and its regulation, many of these models are phenomenological or conceptual, providing a general understanding of the physics at play. But the wealth of carefully measured kinetic data on the interactions of many of the players in actin biochemistry cries out for the creation of more detailed and accurate models that could permit investigators to dissect interdependent roles of individual molecular components. Moreover, no human mind can assimilate all of the mechanisms underlying complex protein networks; so an additional benefit of a detailed kinetic model is that the numerous binding proteins, signaling mechanisms, and biochemical reactions can be computationally organized in a fully explicit, accessible, visualizable, and reusable structure. In this review, we will focus on how comprehensive and adaptable modeling allows investigators to explain experimental observations and develop testable hypotheses on the intracellular dynamics of the actin cytoskeleton. PMID:23442903

Mesoscopic model for filament orientation in growing actin networks: the role of obstacle geometry

NASA Astrophysics Data System (ADS)

Weichsel, Julian; Schwarz, Ulrich S.

2013-03-01

Propulsion by growing actin networks is a universal mechanism used in many different biological systems, ranging from the sheet-like lamellipodium of crawling animal cells to the actin comet tails induced by certain bacteria and viruses in order to move within their host cells. Although the core molecular machinery for actin network growth is well preserved in all of these cases, the geometry of the propelled obstacle varies considerably. During recent years, filament orientation distribution has emerged as an important observable characterizing the structure and dynamical state of the growing network. Here we derive several continuum equations for the orientation distribution of filaments growing behind stiff obstacles of various shapes and validate the predicted steady state orientation patterns by stochastic computer simulations based on discrete filaments. We use an ordinary differential equation approach to demonstrate that for flat obstacles of finite size, two fundamentally different orientation patterns peaked at either ±35° or +70°/0°/ - 70° exhibit mutually exclusive stability, in agreement with earlier results for flat obstacles of very large lateral extension. We calculate and validate phase diagrams as a function of model parameters and show how this approach can be extended to obstacles with piecewise straight contours. For curved obstacles, we arrive at a partial differential equation in the continuum limit, which again is in good agreement with the computer simulations. In all cases, we can identify the same two fundamentally different orientation patterns, but only within an appropriate reference frame, which is adjusted to the local orientation of the obstacle contour. Our results suggest that two fundamentally different network architectures compete with each other in growing actin networks, irrespective of obstacle geometry, and clarify how simulated and electron tomography data have to be analyzed for non-flat obstacle geometries.

Consequences of Molecular-Scale Non-Equilibrium Activity on the Dynamics and Mechanics of Self-Assembled Actin-Based Structures and Materials

NASA Astrophysics Data System (ADS)

Marshall Mccall, Patrick

Living cells are hierarchically self-organized forms of active soft matter: molecules on the nanometer scale form functional structures and organelles on the micron scale, which then compose cells on the scale of 10s of microns. While the biological functions of intracellular organelles are defined by the composition and properties of the structures themselves, how those bulk properties emerge from the properties and interactions of individual molecules remains poorly understood. Actin, a globular protein which self-assembles into dynamic semi-flexible polymers, is the basic structural material of cells and the major component of many functional organelles. In this thesis, I have used purified actin as a model system to explore the interplay between molecular-scale dynamics and organelle-scale functionality, with particular focus on the role of molecular-scale non-equilibrium activity. One of the most canonical forms of molecular-scale non-equilibrium activity is that of mechanoenzymes, also called motor proteins. These proteins utilized the free energy liberated by hydrolysis of ATP to perform mechanical work, thereby introducing non-equilibrium "active" stresses on the molecular scale. Combining experiments with mathematical modeling, we demonstrate in this thesis that non-equilibrium motor activity is sufficient to drive self-organization and pattern formation of the multimeric actin-binding motor protein Myosin II on 1D reconstituted actomyosin bundles. Like myosin, actin is itself an ATPase. However, nono-equilibrium ATP hydrolysis on actin is known to regulate the stability and assembly kinetics of actin filaments rather than generate active stresses per se. At the level of single actin filaments, the inhomogeneous nucleotide composition generated along the filament length by hydrolysis directs binding of regulatory proteins like cofilin, which mediate filament disassembly and thereby accelerate actin filament turnover. The concequences of this non-equilibrium turnover on the steady-state properties of collections of filaments remained unclear. Here, I reconstituted tunable, non-equilibrium actin turnover dynamics in entangled solutions of actin filaments as a model of the actin cortex of living cells. We found that this non-equilibrium turnover decouples solution mechanics from microstructure, enabling structurally indistinguishable materials to behave effectively as either viscous fluids or elastic gels. Additionally, we employed computer simulations to identify the dynamical regime in which actin turnover controls the effective viscosity of 2D cross-linked actin networks in the presence of motors. Additionally, I examine in this thesis the localization and self-assembly of actin filaments in condensed liquid phases called polyelectrolyte coacervates as a model membrane-less organelle. We find that concentration of actin through spontaneous partitioning preferentially to the coacervate phase accelerates the assembly of filaments. These filaments then localize to the coacervate-bulk interface, generating particles with visco-elastic shells surrounding liquid cores. In this case, the properties of the condensed phase enable regulation of actin assembly dynamics.

Actin dynamics, architecture, and mechanics in cell motility.

PubMed

Blanchoin, Laurent; Boujemaa-Paterski, Rajaa; Sykes, Cécile; Plastino, Julie

2014-01-01

Tight coupling between biochemical and mechanical properties of the actin cytoskeleton drives a large range of cellular processes including polarity establishment, morphogenesis, and motility. This is possible because actin filaments are semi-flexible polymers that, in conjunction with the molecular motor myosin, can act as biological active springs or "dashpots" (in laymen's terms, shock absorbers or fluidizers) able to exert or resist against force in a cellular environment. To modulate their mechanical properties, actin filaments can organize into a variety of architectures generating a diversity of cellular organizations including branched or crosslinked networks in the lamellipodium, parallel bundles in filopodia, and antiparallel structures in contractile fibers. In this review we describe the feedback loop between biochemical and mechanical properties of actin organization at the molecular level in vitro, then we integrate this knowledge into our current understanding of cellular actin organization and its physiological roles.

Filopodial retraction force is generated by cortical actin dynamics and controlled by reversible tethering at the tip

PubMed Central

Bornschlögl, Thomas; Romero, Stéphane; Vestergaard, Christian L.; Joanny, Jean-François; Van Nhieu, Guy Tran; Bassereau, Patricia

2013-01-01

Filopodia are dynamic, finger-like plasma membrane protrusions that sense the mechanical and chemical surroundings of the cell. Here, we show in epithelial cells that the dynamics of filopodial extension and retraction are determined by the difference between the actin polymerization rate at the tip and the retrograde flow at the base of the filopodium. Adhesion of a bead to the filopodial tip locally reduces actin polymerization and leads to retraction via retrograde flow, reminiscent of a process used by pathogens to invade cells. Using optical tweezers, we show that filopodial retraction occurs at a constant speed against counteracting forces up to 50 pN. Our measurements point toward retrograde flow in the cortex together with frictional coupling between the filopodial and cortical actin networks as the main retraction-force generator for filopodia. The force exerted by filopodial retraction, however, is limited by the connection between filopodial actin filaments and the membrane at the tip. Upon mechanical rupture of the tip connection, filopodia exert a passive retraction force of 15 pN via their plasma membrane. Transient reconnection at the tip allows filopodia to continuously probe their surroundings in a load-and-fail manner within a well-defined force range. PMID:24198333

Mechanical Detection of a Long-Range Actin Network Emanating from a Biomimetic Cortex

PubMed Central

Bussonnier, Matthias; Carvalho, Kevin; Lemière, Joël; Joanny, Jean-François; Sykes, Cécile; Betz, Timo

2014-01-01

Actin is ubiquitous globular protein that polymerizes into filaments and forms networks that participate in the force generation of eukaryotic cells. Such forces are used for cell motility, cytokinesis, and tissue remodeling. Among those actin networks, we focus on the actin cortex, a dense branched network beneath the plasma membrane that is of particular importance for the mechanical properties of the cell. Here we reproduce the cellular cortex by activating actin filament growth on a solid surface. We unveil the existence of a sparse actin network that emanates from the surface and extends over a distance that is at least 10 times larger than the cortex itself. We call this sparse actin network the “actin cloud” and characterize its mechanical properties with optical tweezers. We show, both experimentally and theoretically, that the actin cloud is mechanically relevant and that it should be taken into account because it can sustain forces as high as several picoNewtons (pN). In particular, it is known that in plant cells, actin networks similar to the actin cloud have a role in positioning the nucleus; in large oocytes, they play a role in driving chromosome movement. Recent evidence shows that such networks even prevent granule condensation in large cells. PMID:25140420

A spatiotemporal characterization method for the dynamic cytoskeleton.

PubMed

Alhussein, Ghada; Shanti, Aya; Farhat, Ilyas A H; Timraz, Sara B H; Alwahab, Noaf S A; Pearson, Yanthe E; Martin, Matthew N; Christoforou, Nicolas; Teo, Jeremy C M

2016-05-01

The significant gap between quantitative and qualitative understanding of cytoskeletal function is a pressing problem; microscopy and labeling techniques have improved qualitative investigations of localized cytoskeleton behavior, whereas quantitative analyses of whole cell cytoskeleton networks remain challenging. Here we present a method that accurately quantifies cytoskeleton dynamics. Our approach digitally subdivides cytoskeleton images using interrogation windows, within which box-counting is used to infer a fractal dimension (Df ) to characterize spatial arrangement, and gray value intensity (GVI) to determine actin density. A partitioning algorithm further obtains cytoskeleton characteristics from the perinuclear, cytosolic, and periphery cellular regions. We validated our measurement approach on Cytochalasin-treated cells using transgenically modified dermal fibroblast cells expressing fluorescent actin cytoskeletons. This method differentiates between normal and chemically disrupted actin networks, and quantifies rates of cytoskeletal degradation. Furthermore, GVI distributions were found to be inversely proportional to Df , having several biophysical implications for cytoskeleton formation/degradation. We additionally demonstrated detection sensitivity of differences in Df and GVI for cells seeded on substrates with varying degrees of stiffness, and coated with different attachment proteins. This general approach can be further implemented to gain insights on dynamic growth, disruption, and structure of the cytoskeleton (and other complex biological morphology) due to biological, chemical, or physical stimuli. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

A spatiotemporal characterization method for the dynamic cytoskeleton

PubMed Central

Alhussein, Ghada; Shanti, Aya; Farhat, Ilyas A. H.; Timraz, Sara B. H.; Alwahab, Noaf S. A.; Pearson, Yanthe E.; Martin, Matthew N.; Christoforou, Nicolas

2016-01-01

The significant gap between quantitative and qualitative understanding of cytoskeletal function is a pressing problem; microscopy and labeling techniques have improved qualitative investigations of localized cytoskeleton behavior, whereas quantitative analyses of whole cell cytoskeleton networks remain challenging. Here we present a method that accurately quantifies cytoskeleton dynamics. Our approach digitally subdivides cytoskeleton images using interrogation windows, within which box‐counting is used to infer a fractal dimension (D f) to characterize spatial arrangement, and gray value intensity (GVI) to determine actin density. A partitioning algorithm further obtains cytoskeleton characteristics from the perinuclear, cytosolic, and periphery cellular regions. We validated our measurement approach on Cytochalasin‐treated cells using transgenically modified dermal fibroblast cells expressing fluorescent actin cytoskeletons. This method differentiates between normal and chemically disrupted actin networks, and quantifies rates of cytoskeletal degradation. Furthermore, GVI distributions were found to be inversely proportional to D f, having several biophysical implications for cytoskeleton formation/degradation. We additionally demonstrated detection sensitivity of differences in D f and GVI for cells seeded on substrates with varying degrees of stiffness, and coated with different attachment proteins. This general approach can be further implemented to gain insights on dynamic growth, disruption, and structure of the cytoskeleton (and other complex biological morphology) due to biological, chemical, or physical stimuli. © 2016 The Authors. Cytoskeleton Published by Wiley Periodicals, Inc. PMID:27015595

In vitro studies of actin filament and network dynamics

PubMed Central

Mullins, R Dyche; Hansen, Scott D

2013-01-01

Now that many genomes have been sequenced, a central concern of cell biology is to understand how the proteins they encode work together to create living matter. In vitro studies form an essential part of this program because understanding cellular functions of biological molecules often requires isolating them and reconstituting their activities. In particular, many elements of the actin cytoskeleton were first discovered by biochemical methods and their cellular functions deduced from in vitro experiments. We highlight recent advances that have come from in vitro studies, beginning with studies of actin filaments, and ending with multi-component reconstitutions of complex actin-based processes, including force-generation and cell spreading. We describe both scientific results and the technical innovations that made them possible. PMID:23267766

Stress-enhanced gelation: a dynamic nonlinearity of elasticity.

PubMed

Yao, Norman Y; Broedersz, Chase P; Depken, Martin; Becker, Daniel J; Pollak, Martin R; Mackintosh, Frederick C; Weitz, David A

2013-01-04

A hallmark of biopolymer networks is their sensitivity to stress, reflected by pronounced nonlinear elastic stiffening. Here, we demonstrate a distinct dynamical nonlinearity in biopolymer networks consisting of filamentous actin cross-linked by α-actinin-4. Applied stress delays the onset of relaxation and flow, markedly enhancing gelation and extending the regime of solidlike behavior to much lower frequencies. We show that this macroscopic network response can be accounted for at the single molecule level by the increased binding affinity of the cross-linker under load, characteristic of catch-bond-like behavior.

Scaling of F-actin network rheology to probe single filament elasticity and dynamics.

PubMed

Gardel, M L; Shin, J H; MacKintosh, F C; Mahadevan, L; Matsudaira, P A; Weitz, D A

2004-10-29

The linear and nonlinear viscoelastic response of networks of cross-linked and bundled cytoskeletal filaments demonstrates remarkable scaling with both frequency and applied prestress, which helps elucidate the origins of the viscoelasticity. The frequency dependence of the shear modulus reflects the underlying single-filament relaxation dynamics for 0.1-10 rad/sec. Moreover, the nonlinear strain stiffening of such networks exhibits a universal form as a function of prestress; this is quantitatively explained by the full force-extension relation of single semiflexible filaments.

Tropomodulins: pointed-end capping proteins that regulate actin filament architecture in diverse cell types

PubMed Central

Yamashiro, Sawako; Gokhin, David S.; Kimura, Sumiko; Nowak, Roberta B.; Fowler, Velia M.

2012-01-01

Tropomodulins are a family of four proteins (Tmods 1–4) that cap the pointed ends of actin filaments in actin cytoskeletal structures in a developmentally regulated and tissue-specific manner. Unique among capping proteins, Tmods also bind tropomyosins (TMs), which greatly enhance the actin filament pointed-end capping activity of Tmods. Tmods are defined by a tropomyosin (TM)-regulated/Pointed-End Actin Capping (TM-Cap) domain in their unstructured N-terminal portion, followed by a compact, folded Leucine-Rich Repeat/Pointed-End Actin Capping (LRR-Cap) domain. By inhibiting actin monomer association and dissociation from pointed ends, Tmods regulate regulate actin dynamics and turnover, stabilizing actin filament lengths and cytoskeletal architecture. In this review, we summarize the genes, structural features, molecular and biochemical properties, actin regulatory mechanisms, expression patterns, and cell and tissue functions of Tmods. By understanding Tmods’ functions in the context of their molecular structure, actin regulation, binding partners, and related variants (leiomodins 1–3), we can draw broad conclusions that can explain the diverse morphological and functional phenotypes that arise from Tmod perturbation experiments in vitro and in vivo. Tmod-based stabilization and organization of intracellular actin filament networks provide key insights into how the emergent properties of the actin cytoskeleton drive tissue morphogenesis and physiology. PMID:22488942

Cell migration without a lamellipodium: translation of actin dynamics into cell movement mediated by tropomyosin.

PubMed

Gupton, Stephanie L; Anderson, Karen L; Kole, Thomas P; Fischer, Robert S; Ponti, Aaron; Hitchcock-DeGregori, Sarah E; Danuser, Gaudenz; Fowler, Velia M; Wirtz, Denis; Hanein, Dorit; Waterman-Storer, Clare M

2005-02-14

The actin cytoskeleton is locally regulated for functional specializations for cell motility. Using quantitative fluorescent speckle microscopy (qFSM) of migrating epithelial cells, we previously defined two distinct F-actin networks based on their F-actin-binding proteins and distinct patterns of F-actin turnover and movement. The lamellipodium consists of a treadmilling F-actin array with rapid polymerization-dependent retrograde flow and contains high concentrations of Arp2/3 and ADF/cofilin, whereas the lamella exhibits spatially random punctae of F-actin assembly and disassembly with slow myosin-mediated retrograde flow and contains myosin II and tropomyosin (TM). In this paper, we microinjected skeletal muscle alphaTM into epithelial cells, and using qFSM, electron microscopy, and immunolocalization show that this inhibits functional lamellipodium formation. Cells with inhibited lamellipodia exhibit persistent leading edge protrusion and rapid cell migration. Inhibition of endogenous long TM isoforms alters protrusion persistence. Thus, cells can migrate with inhibited lamellipodia, and we suggest that TM is a major regulator of F-actin functional specialization in migrating cells.

Relating microstructure to rheology of a bundled and cross-linked F-actin network in vitro

NASA Astrophysics Data System (ADS)

Shin, J. H.; Gardel, M. L.; Mahadevan, L.; Matsudaira, P.; Weitz, D. A.

2004-06-01

The organization of individual actin filaments into higher-order structures is controlled by actin-binding proteins (ABPs). Although the biological significance of the ABPs is well documented, little is known about how bundling and cross-linking quantitatively affect the microstructure and mechanical properties of actin networks. Here we quantify the effect of the ABP scruin on actin networks by using imaging techniques, cosedimentation assays, multiparticle tracking, and bulk rheology. We show how the structure of the actin network is modified as the scruin concentration is varied, and we correlate these structural changes to variations in the resultant network elasticity.

Triptolide disrupts the actin-based Sertoli-germ cells adherens junctions by inhibiting Rho GTPases expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Xiang; Zhao, Fang

Triptolide (TP), derived from the medicinal plant Triterygium wilfordii Hook. f. (TWHF), is a diterpene triepoxide with variety biological and pharmacological activities. However, TP has been restricted in clinical application due to its narrow therapeutic window especially in reproductive system. During spermatogenesis, Sertoli cell cytoskeleton plays an essential role in facilitating germ cell movement and cell-cell actin-based adherens junctions (AJ). At Sertoli cell-spermatid interface, the anchoring device is a kind of AJ, known as ectoplasmic specializations (ES). In this study, we demonstrate that β-actin, an important component of cytoskeleton, has been significantly down-regulated after TP treatment. TP can inhibit themore » expression of Rho GTPase such as, RhoA, RhoB, Cdc42 and Rac1. Downstream of Rho GTPase, Rho-associated protein kinase (ROCKs) gene expressions were also suppressed by TP. F-actin immunofluorescence proved that TP disrupts Sertoli cells cytoskeleton network. As a result of β-actin down-regulation, TP treatment increased expression of testin, which indicating ES has been disassembled. In summary, this report illustrates that TP induces cytoskeleton dysfunction and disrupts cell-cell adherens junctions via inhibition of Rho GTPases. - Highlights: • Triptolide induced the disruption of Sertoli-germ cell adherens junction. • Rho GTPases expression and actin dynamics have been suppressed by triptolide. • Actin-based adherens junction is a potential antifertility target of triptolide. • Rho-Rock is involved in the regulation of actin dynamics.« less

Noisy Oscillations in the Actin Cytoskeleton of Chemotactic Amoeba.

PubMed

Negrete, Jose; Pumir, Alain; Hsu, Hsin-Fang; Westendorf, Christian; Tarantola, Marco; Beta, Carsten; Bodenschatz, Eberhard

2016-09-30

Biological systems with their complex biochemical networks are known to be intrinsically noisy. Here we investigate the dynamics of actin polymerization of amoeboid cells, which are close to the onset of oscillations. We show that the large phenotypic variability in the polymerization dynamics can be accurately captured by a generic nonlinear oscillator model in the presence of noise. We determine the relative role of the noise with a single dimensionless, experimentally accessible parameter, thus providing a quantitative description of the variability in a population of cells. Our approach, which rests on a generic description of a system close to a Hopf bifurcation and includes the effect of noise, can characterize the dynamics of a large class of noisy systems close to an oscillatory instability.

Noisy Oscillations in the Actin Cytoskeleton of Chemotactic Amoeba

NASA Astrophysics Data System (ADS)

Negrete, Jose; Pumir, Alain; Hsu, Hsin-Fang; Westendorf, Christian; Tarantola, Marco; Beta, Carsten; Bodenschatz, Eberhard

2016-09-01

Biological systems with their complex biochemical networks are known to be intrinsically noisy. Here we investigate the dynamics of actin polymerization of amoeboid cells, which are close to the onset of oscillations. We show that the large phenotypic variability in the polymerization dynamics can be accurately captured by a generic nonlinear oscillator model in the presence of noise. We determine the relative role of the noise with a single dimensionless, experimentally accessible parameter, thus providing a quantitative description of the variability in a population of cells. Our approach, which rests on a generic description of a system close to a Hopf bifurcation and includes the effect of noise, can characterize the dynamics of a large class of noisy systems close to an oscillatory instability.

A Mechanistic Model of the Actin Cycle

PubMed Central

Bindschadler, M.; Osborn, E. A.; Dewey, C. F.; McGrath, J. L.

2004-01-01

We have derived a broad, deterministic model of the steady-state actin cycle that includes its major regulatory mechanisms. Ours is the first model to solve the complete nucleotide profile within filaments, a feature that determines the dynamics and geometry of actin networks at the leading edges of motile cells, and one that has challenged investigators developing models to interpret steady-state experiments. We arrived at the nucleotide profile through analytic and numerical approaches that completely agree. Our model reproduces behaviors seen in numerous experiments with purified proteins, but allows a detailed inspection of the concentrations and fluxes that might exist in these experiments. These inspections provide new insight into the mechanisms that determine the rate of actin filament treadmilling. Specifically, we find that mechanisms for enhancing Pi release from the ADP·Pi intermediate on filaments, for increasing the off rate of ADP-bound subunits at pointed ends, and the multiple, simultaneous functions of profilin, make unique and essential contributions to increased treadmilling. In combination, these mechanisms have a theoretical capacity to increase treadmilling to levels limited only by the amount of available actin. This limitation arises because as the cycle becomes more dynamic, it tends toward the unpolymerized state. PMID:15111391

The Effect of Crosslinking on the Microscale Stress Response and Molecular Deformations in Actin Networks

NASA Astrophysics Data System (ADS)

Gurmessa, Bekele; Fitzpatrick, Robert; Valdivia, Jonathon; Anderson, Rae M. R.

Actin, the most abundant protein in eukaryotic cells, is a semi-flexible biopolymer in the cytoskeleton that plays a crucial structural and mechanical role in cell stability, motion and replication, as well as muscle contraction. Most of these mechanically driven structural changes in cells stem from the complex viscoelastic nature of entangled actin networks and the presence of a myriad of proteins that cross-link actin filaments. Despite their importance, the mechanical response of actin networks is not yet well understood, particularly at the molecular level. Here, we use optical trapping - coupled with fluorescence microscopy - to characterize the microscale stress response and induced filament deformations in entangled and cross-linked actin networks subject to localized mechanical perturbations. In particular, we actively drive a microsphere 10 microns through an entangled or cross- linked actin network at a constant speed and measure the resistive force that the deformed actin filaments exert on the bead during and following strain. We simultaneously visualize and track individual sparsely-labeled actin filaments to directly link force response to molecular deformations, and map the propagation of the initially localized perturbation field throughout the rest of the network (~100 um). By varying the concentration of actin and cross-linkers we directly determine the role of crosslinking and entanglements on the length and time scales of stress propagation, molecular deformation and relaxation mechanisms in actin networks.

Myosin-II sets the optimal response time scale of chemotactic amoeba

NASA Astrophysics Data System (ADS)

Hsu, Hsin-Fang; Westendorf, Christian; Tarantola, Marco; Bodenschatz, Eberhard; Beta, Carsten

2014-03-01

The response dynamics of the actin cytoskeleton to external chemical stimuli plays a fundamental role in numerous cellular functions. One of the key players that governs the dynamics of the actin network is the motor protein myosin-II. Here we investigate the role of myosin-II in the response of the actin system to external stimuli. We used a microfluidic device in combination with a photoactivatable chemoattractant to apply stimuli to individual cells with high temporal resolution. We directly compare the actin dynamics in Dictyostelium discodelium wild type (WT) cells to a knockout mutant that is deficient in myosin-II (MNL). Similar to the WT a small population of MNL cells showed self-sustained oscillations even in absence of external stimuli. The actin response of MNL cells to a short pulse of chemoattractant resembles WT during the first 15 sec but is significantly delayed afterward. The amplitude of the dominant peak in the power spectrum from the response time series of MNL cells to periodic stimuli with varying period showed a clear resonance peak at a forcing period of 36 sec, which is significantly delayed as compared to the resonance at 20 sec found for the WT. This shift indicates an important role of myosin-II in setting the response time scale of motile amoeba. Institute of Physics und Astronomy, University of Potsdam, Karl-Liebknecht-Str. 24/25, 14476 Potsdam, Germany.

Physics of Cellular Movements

NASA Astrophysics Data System (ADS)

Sackmann, Erich; Keber, Felix; Heinrich, Doris

2010-04-01

The survival of cells depends on perpetual active motions, including (a) bending excitations of the soft cell envelopes, (b) the bidirectional transport of materials and organelles between the cell center and the periphery, and (c) the ongoing restructuring of the intracellular macromolecular scaffolds mediating global cell changes associated with cell adhesion locomotion and phagocytosis. Central questions addressed are the following: How can this bustling motion of extremely complex soft structures be characterized and measured? What are the major driving forces? Further topics include (a) the active dynamic control of global shape changes by the interactive coupling of the aster-like soft scaffold of microtubules and the network of actin filaments associated with the cell envelope (the actin cortex) and (b) the generation of propulsion forces by solitary actin gelation waves propagating within the actin cortex.

Pure F-actin networks are distorted and branched by steps in the critical-point drying method.

PubMed

Resch, Guenter P; Goldie, Kenneth N; Hoenger, Andreas; Small, J Victor

2002-03-01

Elucidation of the ultrastructural organization of actin networks is crucial for understanding the molecular mechanisms underlying actin-based motility. Results obtained from cytoskeletons and actin comets prepared by the critical-point procedure, followed by rotary shadowing, support recent models incorporating actin filament branching as a main feature of lamellipodia and pathogen propulsion. Since actin branches were not evident in earlier images obtained by negative staining, we explored how these differences arise. Accordingly, we have followed the structural fate of dense networks of pure actin filaments subjected to steps of the critical-point drying protocol. The filament networks have been visualized in parallel by both cryo-electron microscopy and negative staining. Our results demonstrate the selective creation of branches and other artificial structures in pure F-actin networks by the critical-point procedure and challenge the reliability of this method for preserving the detailed organization of actin assemblies that drive motility. (c) 2002 Elsevier Science (USA).

Mechanical coupling between transsynaptic N-cadherin adhesions and actin flow stabilizes dendritic spines

PubMed Central

Chazeau, Anaël; Garcia, Mikael; Czöndör, Katalin; Perrais, David; Tessier, Béatrice; Giannone, Grégory; Thoumine, Olivier

2015-01-01

The morphology of neuronal dendritic spines is a critical indicator of synaptic function. It is regulated by several factors, including the intracellular actin/myosin cytoskeleton and transcellular N-cadherin adhesions. To examine the mechanical relationship between these molecular components, we performed quantitative live-imaging experiments in primary hippocampal neurons. We found that actin turnover and structural motility were lower in dendritic spines than in immature filopodia and increased upon expression of a nonadhesive N-cadherin mutant, resulting in an inverse relationship between spine motility and actin enrichment. Furthermore, the pharmacological stimulation of myosin II induced the rearward motion of actin structures in spines, showing that myosin II exerts tension on the actin network. Strikingly, the formation of stable, spine-like structures enriched in actin was induced at contacts between dendritic filopodia and N-cadherin–coated beads or micropatterns. Finally, computer simulations of actin dynamics mimicked various experimental conditions, pointing to the actin flow rate as an important parameter controlling actin enrichment in dendritic spines. Together these data demonstrate that a clutch-like mechanism between N-cadherin adhesions and the actin flow underlies the stabilization of dendritic filopodia into mature spines, a mechanism that may have important implications in synapse initiation, maturation, and plasticity in the developing brain. PMID:25568337

Filamentous actin organization in the unfertilized sea urchin egg cortex.

PubMed

Henson, J H; Begg, D A

1988-06-01

We have investigated the organization of filamentous actin in the cortex of unfertilized eggs of the sea urchins Strongylocentrotus purpuratus and Lytechinus variegatus. Rhodamine phalloidin and anti-actin immunofluorescent staining of isolated cortices reveal a punctate pattern of fluorescent sources. Comparison of this pattern with SEM images of microvillar morphology and distribution indicates that filamentous actin in the cortex is predominantly localized in the microvilli. Thin-section TEM and quick-freeze deep-etch ultrastructure of isolated cortices demonstrates that this microvillar-associated actin is in a novel organizational state composed of very short filaments arranged in a tight network and that these filament networks form mounds that extend beyond the plane of the plasma membrane. Actin filaments within the networks do not exhibit free ends and make end-on attachments with the membrane only within the region of the evaginating microvilli. Myosin S-1 dissociable crosslinks, 2-3 nm in diameter, are observed between network filaments and between network filaments and the membrane. A second population of long, individual actin filaments is observed in close lateral association with the plasma membrane and frequently complexes with the microvillar actin networks. The filamentous actin of the unfertilized egg cortex may participate in establishing the mechanical properties of the egg surface and may function in nucleating the assembly of cortical actin following fertilization.

Boundaries steer the contraction of active gels

NASA Astrophysics Data System (ADS)

Schuppler, Matthias; Keber, Felix C.; Kröger, Martin; Bausch, Andreas R.

2016-10-01

Cells set up contractile actin arrays to drive various shape changes and to exert forces to their environment. To understand their assembly process, we present here a reconstituted contractile system, comprising F-actin and myosin II filaments, where we can control the local activation of myosin by light. By stimulating different symmetries, we show that the force balancing at the boundaries determine the shape changes as well as the dynamics of the global contraction. Spatially anisotropic attachment of initially isotropic networks leads to a self-organization of highly aligned contractile fibres, being reminiscent of the order formation in muscles or stress fibres. The observed shape changes and dynamics are fully recovered by a minimal physical model.

Characterization of actin filament deformation in response to actively driven microspheres propagated through entangled actin networks

NASA Astrophysics Data System (ADS)

Falzone, Tobias; Blair, Savanna; Robertson-Anderson, Rae

2014-03-01

The semi-flexible biopolymer actin is a ubiquitous component of nearly all biological organisms, playing an important role in many biological processes such as cell structure and motility, cancer invasion and metastasis, muscle contraction, and cell signaling. Concentrated actin networks possess unique viscoelastic properties that have been the subject of much theoretical and experimental work. However, much is still unknown regarding the correlation of the applied stress on the network to the induced filament strain at the molecular level. Here, we use dual optical traps alongside fluorescence microscopy to carry out active microrheology measurements that link mechanical stress to structural response at the micron scale. Specifically, we actively drive microspheres through entangled actin networks while simultaneously measuring the force the surrounding filaments exert on the sphere and visualizing the deformation and subsequent relaxation of fluorescent labeled filaments within the network. These measurements, which provide much needed insight into the link between stress and strain in actin networks, are critical for clarifying our theoretical understanding of the complex viscoelastic behavior exhibited in actin networks.

Dissection of molecular assembly dynamics by tracking orientation and position of single molecules in live cells

PubMed Central

McQuilken, Molly; La Riviere, Patrick J.; Occhipinti, Patricia; Verma, Amitabh; Oldenbourg, Rudolf; Gladfelter, Amy S.; Tani, Tomomi

2016-01-01

Regulation of order, such as orientation and conformation, drives the function of most molecular assemblies in living cells but remains difficult to measure accurately through space and time. We built an instantaneous fluorescence polarization microscope, which simultaneously images position and orientation of fluorophores in living cells with single-molecule sensitivity and a time resolution of 100 ms. We developed image acquisition and analysis methods to track single particles that interact with higher-order assemblies of molecules. We tracked the fluctuations in position and orientation of molecules from the level of an ensemble of fluorophores down to single fluorophores. We tested our system in vitro using fluorescently labeled DNA and F-actin, in which the ensemble orientation of polarized fluorescence is known. We then tracked the orientation of sparsely labeled F-actin network at the leading edge of migrating human keratinocytes, revealing the anisotropic distribution of actin filaments relative to the local retrograde flow of the F-actin network. Additionally, we analyzed the position and orientation of septin-GFP molecules incorporated in septin bundles in growing hyphae of a filamentous fungus. Our data indicate that septin-GFP molecules undergo positional fluctuations within ∼350 nm of the binding site and angular fluctuations within ∼30° of the central orientation of the bundle. By reporting position and orientation of molecules while they form dynamic higher-order structures, our approach can provide insights into how micrometer-scale ordered assemblies emerge from nanoscale molecules in living cells. PMID:27679846

Molecular Mechanics of the α-Actinin Rod Domain: Bending, Torsional, and Extensional Behavior

PubMed Central

Golji, Javad; Collins, Robert; Mofrad, Mohammad R. K.

2009-01-01

α-Actinin is an actin crosslinking molecule that can serve as a scaffold and maintain dynamic actin filament networks. As a crosslinker in the stressed cytoskeleton, α-actinin can retain conformation, function, and strength. α-Actinin has an actin binding domain and a calmodulin homology domain separated by a long rod domain. Using molecular dynamics and normal mode analysis, we suggest that the α-actinin rod domain has flexible terminal regions which can twist and extend under mechanical stress, yet has a highly rigid interior region stabilized by aromatic packing within each spectrin repeat, by electrostatic interactions between the spectrin repeats, and by strong salt bridges between its two anti-parallel monomers. By exploring the natural vibrations of the α-actinin rod domain and by conducting bending molecular dynamics simulations we also predict that bending of the rod domain is possible with minimal force. We introduce computational methods for analyzing the torsional strain of molecules using rotating constraints. Molecular dynamics extension of the α-actinin rod is also performed, demonstrating transduction of the unfolding forces across salt bridges to the associated monomer of the α-actinin rod domain. PMID:19436721

NHERF1 regulates actin cytoskeleton organization through modulation of α-actinin-4 stability.

PubMed

Sun, Licui; Zheng, Junfang; Wang, Qiqi; Song, Ran; Liu, Hua; Meng, Ran; Tao, Tao; Si, Yang; Jiang, Wenguo; He, Junqi

2016-02-01

The actin cytoskeleton is composed of a highly dynamic network of filamentous proteins, yet the molecular mechanism that regulates its organization and remodeling remains elusive. In this study, Na(+)/H(+) exchanger regulatory factor (NHERF)-1 loss-of-function and gain-of-function experiments reveal that polymerized actin cytoskeleton (F-actin) in HeLa cells is disorganized by NHERF1, whereas actin protein expression levels exhibit no detectable change. To elucidate the molecular mechanism underlying actin cytoskeleton disorganization by NHERF1, a combined 2-dimensional electrophoresis-matrix-assisted laser desorption/ionization-time of flight mass spectrometry approach was used to screen for proteins regulated by NHERF1 in HeLa cells. α-Actinin-4, an actin cross-linking protein, was identified. Glutathione S-transferase pull-down and coimmunoprecipitation studies showed the α-actinin-4 carboxyl-terminal region specifically interacted with the NHERF1 postsynaptic density 95/disc-large/zona occludens-1 domain. The NHERF1/α-actinin-4 interaction increased α-actinin-4 ubiquitination and decreased its expression levels, resulting in actin cytoskeleton disassembly. Our study identified α-actinin-4 as a novel NHERF1 interaction partner and provided new insights into the regulatory mechanism of the actin cytoskeleton by NHERF1. © FASEB.

Two Functionally Distinct Sources of Actin Monomers Supply the Leading Edge of Lamellipodia

PubMed Central

Vitriol, Eric A.; McMillen, Laura M.; Kapustina, Maryna; Gomez, Shawn M.; Vavylonis, Dimitrios; Zheng, James Q.

2015-01-01

Summary Lamellipodia, the sheet-like protrusions of motile cells, consist of networks of actin filaments (F-actin) regulated by the ordered assembly from and disassembly into actin monomers (G-actin). Traditionally, G-actin is thought to exist as a homogeneous pool. Here, we show that there are two functionally and molecularly distinct sources of G-actin that supply lamellipodial actin networks. G-actin originating from the cytosolic pool requires the monomer binding protein thymosin β4 (Tβ4) for optimal leading edge localization, is targeted to formins, and is responsible for creating an elevated G/F-actin ratio that promotes membrane protrusion. The second source of G-actin comes from recycled lamellipodia F-actin. Recycling occurs independently of Tβ4 and appears to regulate lamellipodia homeostasis. Tβ4-bound G-actin specifically localizes to the leading edge because it doesn’t interact with Arp2/3-mediated polymerization sites found throughout the lamellipodia. These findings demonstrate that actin networks can be constructed from multiple sources of monomers with discrete spatiotemporal functions. PMID:25865895

Bidirectional Interplay between Vimentin Intermediate Filaments and Contractile Actin Stress Fibers.

PubMed

Jiu, Yaming; Lehtimäki, Jaakko; Tojkander, Sari; Cheng, Fang; Jäälinoja, Harri; Liu, Xiaonan; Varjosalo, Markku; Eriksson, John E; Lappalainen, Pekka

2015-06-16

The actin cytoskeleton and cytoplasmic intermediate filaments contribute to cell migration and morphogenesis, but the interplay between these two central cytoskeletal elements has remained elusive. Here, we find that specific actin stress fiber structures, transverse arcs, interact with vimentin intermediate filaments and promote their retrograde flow. Consequently, myosin-II-containing arcs are important for perinuclear localization of the vimentin network in cells. The vimentin network reciprocally restricts retrograde movement of arcs and hence controls the width of flat lamellum at the leading edge of the cell. Depletion of plectin recapitulates the vimentin organization phenotype of arc-deficient cells without affecting the integrity of vimentin filaments or stress fibers, demonstrating that this cytoskeletal cross-linker is required for productive interactions between vimentin and arcs. Collectively, our results reveal that plectin-mediated interplay between contractile actomyosin arcs and vimentin intermediate filaments controls the localization and dynamics of these two cytoskeletal systems and is consequently important for cell morphogenesis. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Anomalous yet Brownian.

PubMed

Wang, Bo; Anthony, Stephen M; Bae, Sung Chul; Granick, Steve

2009-09-08

We describe experiments using single-particle tracking in which mean-square displacement is simply proportional to time (Fickian), yet the distribution of displacement probability is not Gaussian as should be expected of a classical random walk but, instead, is decidedly exponential for large displacements, the decay length of the exponential being proportional to the square root of time. The first example is when colloidal beads diffuse along linear phospholipid bilayer tubes whose radius is the same as that of the beads. The second is when beads diffuse through entangled F-actin networks, bead radius being less than one-fifth of the actin network mesh size. We explore the relevance to dynamic heterogeneity in trajectory space, which has been extensively discussed regarding glassy systems. Data for the second system might suggest activated diffusion between pores in the entangled F-actin networks, in the same spirit as activated diffusion and exponential tails observed in glassy systems. But the first system shows exceptionally rapid diffusion, nearly as rapid as for identical colloids in free suspension, yet still displaying an exponential probability distribution as in the second system. Thus, although the exponential tail is reminiscent of glassy systems, in fact, these dynamics are exceptionally rapid. We also compare with particle trajectories that are at first subdiffusive but Fickian at the longest measurement times, finding that displacement probability distributions fall onto the same master curve in both regimes. The need is emphasized for experiments, theory, and computer simulation to allow definitive interpretation of this simple and clean exponential probability distribution.

Mesoscopic model of actin-based propulsion.

PubMed

Zhu, Jie; Mogilner, Alex

2012-01-01

Two theoretical models dominate current understanding of actin-based propulsion: microscopic polymerization ratchet model predicts that growing and writhing actin filaments generate forces and movements, while macroscopic elastic propulsion model suggests that deformation and stress of growing actin gel are responsible for the propulsion. We examine both experimentally and computationally the 2D movement of ellipsoidal beads propelled by actin tails and show that neither of the two models can explain the observed bistability of the orientation of the beads. To explain the data, we develop a 2D hybrid mesoscopic model by reconciling these two models such that individual actin filaments undergoing nucleation, elongation, attachment, detachment and capping are embedded into the boundary of a node-spring viscoelastic network representing the macroscopic actin gel. Stochastic simulations of this 'in silico' actin network show that the combined effects of the macroscopic elastic deformation and microscopic ratchets can explain the observed bistable orientation of the actin-propelled ellipsoidal beads. To test the theory further, we analyze observed distribution of the curvatures of the trajectories and show that the hybrid model's predictions fit the data. Finally, we demonstrate that the model can explain both concave-up and concave-down force-velocity relations for growing actin networks depending on the characteristic time scale and network recoil. To summarize, we propose that both microscopic polymerization ratchets and macroscopic stresses of the deformable actin network are responsible for the force and movement generation.

WASp-dependent actin cytoskeleton stability at the dendritic cell immunological synapse is required for extensive, functional T cell contacts.

PubMed

Malinova, Dessislava; Fritzsche, Marco; Nowosad, Carla R; Armer, Hannah; Munro, Peter M G; Blundell, Michael P; Charras, Guillaume; Tolar, Pavel; Bouma, Gerben; Thrasher, Adrian J

2016-05-01

The immunological synapse is a highly structured and molecularly dynamic interface between communicating immune cells. Although the immunological synapse promotes T cell activation by dendritic cells, the specific organization of the immunological synapse on the dendritic cell side in response to T cell engagement is largely unknown. In this study, confocal and electron microscopy techniques were used to investigate the role of dendritic cell actin regulation in immunological synapse formation, stabilization, and function. In the dendritic cell-restricted absence of the Wiskott-Aldrich syndrome protein, an important regulator of the actin cytoskeleton in hematopoietic cells, the immunological synapse contact with T cells occupied a significantly reduced surface area. At a molecular level, the actin network localized to the immunological synapse exhibited reduced stability, in particular, of the actin-related protein-2/3-dependent, short-filament network. This was associated with decreased polarization of dendritic cell-associated ICAM-1 and MHC class II, which was partially dependent on Wiskott-Aldrich syndrome protein phosphorylation. With the use of supported planar lipid bilayers incorporating anti-ICAM-1 and anti-MHC class II antibodies, the dendritic cell actin cytoskeleton organized into recognizable synaptic structures but interestingly, formed Wiskott-Aldrich syndrome protein-dependent podosomes within this area. These findings demonstrate that intrinsic dendritic cell cytoskeletal remodeling is a key regulatory component of normal immunological synapse formation, likely through consolidation of adhesive interaction and modulation of immunological synapse stability. © The Author(s).

Identifying the architecture of a supracellular actomyosin network that induces tissue folding

NASA Astrophysics Data System (ADS)

Yevick, Hannah; Stoop, Norbert; Dunkel, Jorn; Martin, Adam

During embryonic development, the establishment of correct tissue form ensures proper tissue function. Yet, how the thousands of cells within a tissue coordinate force production to sculpt tissue shape is poorly understood. One important tissue shape change is tissue folding where a cell sheet bends to form a closed tube. Drosophila (fruit fly) embryos undergo such a folding event, called ventral furrow formation. The ventral furrow is associated with a supracellular network of actin and myosin, where actin-myosin fibers assemble and connect between cells. It is not known how this tissue-wide network grows and connects over time, how reproducible it is between embryos, and what determines its architecture. Here, we used topological feature analysis to quantitatively and dynamically map the connections and architecture of this supracellular network across hundreds of cells in the folding tissue. We identified the importance of the cell unit in setting up the tissue-scale architecture of the network. Our mathematical framework allows us to explore stereotypic properties of the myosin network such that we can investigate the reproducibility of mechanical connections for a morphogenetic process. NIH F32.

Elastic Coupling of Nascent apCAM Adhesions to Flowing Actin Networks

PubMed Central

Mejean, Cecile O.; Schaefer, Andrew W.; Buck, Kenneth B.; Kress, Holger; Shundrovsky, Alla; Merrill, Jason W.; Dufresne, Eric R.; Forscher, Paul

2013-01-01

Adhesions are multi-molecular complexes that transmit forces generated by a cell’s acto-myosin networks to external substrates. While the physical properties of some of the individual components of adhesions have been carefully characterized, the mechanics of the coupling between the cytoskeleton and the adhesion site as a whole are just beginning to be revealed. We characterized the mechanics of nascent adhesions mediated by the immunoglobulin-family cell adhesion molecule apCAM, which is known to interact with actin filaments. Using simultaneous visualization of actin flow and quantification of forces transmitted to apCAM-coated beads restrained with an optical trap, we found that adhesions are dynamic structures capable of transmitting a wide range of forces. For forces in the picoNewton scale, the nascent adhesions’ mechanical properties are dominated by an elastic structure which can be reversibly deformed by up to 1 µm. Large reversible deformations rule out an interface between substrate and cytoskeleton that is dominated by a number of stiff molecular springs in parallel, and favor a compliant cross-linked network. Such a compliant structure may increase the lifetime of a nascent adhesion, facilitating signaling and reinforcement. PMID:24039928

Force feedback controls motor activity and mechanical properties of self-assembling branched actin networks

PubMed Central

Bieling, Peter; Li, Tai-De; Weichsel, Julian; McGorty, Ryan; Jreij, Pamela; Huang, Bo; Fletcher, Daniel A.; Mullins, R. Dyche

2016-01-01

Branched actin networks–created by the Arp2/3 complex, capping protein, and a nucleation promoting factor– generate and transmit forces required for many cellular processes, but their response to force is poorly understood. To address this, we assembled branched actin networks in vitro from purified components and used simultaneous fluorescence and atomic force microscopy to quantify their molecular composition and material properties under various forces. Remarkably, mechanical loading of these self-assembling materials increases their density, power, and efficiency. Microscopically, increased density reflects increased filament number and altered geometry, but no change in average length. Macroscopically, increased density enhances network stiffness and resistance to mechanical failure beyond those of isotropic actin networks. These effects endow branched actin networks with memory of their mechanical history that shapes their material properties and motor activity. This work reveals intrinsic force feedback mechanisms by which mechanical resistance makes self-assembling actin networks stiffer, stronger, and more powerful. PMID:26771487

Nonequilibrium stabilization of an RNA/protein droplet emulsion by nuclear actin

NASA Astrophysics Data System (ADS)

Brangwynne, Clifford

2013-03-01

Actin plays a structural role in the cytoplasm. However, actin takes on new functions and structures in the nucleus that are poorly understood. The nuclei of the large oocytes of the frog X. laevisspecifically accumulate actin to reach high concentrations; however, it remains unclear if this actin polymerizes into a network, and what, if any, structural role such an actin network might play. Here, we use microrheological and confocal imaging techniques to probe the local architecture and mechanics of the nucleus. Our data show that actin forms a weak network that spatially organizes the nucleus by kinetically stabilizing embedded liquid-like RNA/protein bodies which are important for cell growth. In actin-disrupted nuclei this RNA/protein droplet emulsion is destabilized leading to homotypic coalescence into single large droplets. Our data provide intriguing new insights into why large cell nuclei require an actin-based structural scaffold.

Actin organization and endocytic trafficking are controlled by a network linking NIMA-related kinases to the CDC-42-SID-3/ACK1 pathway

PubMed Central

Bernazzani, Sarina M.

2018-01-01

Molting is an essential process in the nematode Caenorhabditis elegans during which the epidermal apical extracellular matrix, termed the cuticle, is detached and replaced at each larval stage. The conserved NIMA-related kinases NEKL-2/NEK8/NEK9 and NEKL-3/NEK6/NEK7, together with their ankyrin repeat partners, MLT-2/ANKS6, MLT-3/ANKS3, and MLT-4/INVS, are essential for normal molting. In nekl and mlt mutants, the old larval cuticle fails to be completely shed, leading to entrapment and growth arrest. To better understand the molecular and cellular functions of NEKLs during molting, we isolated genetic suppressors of nekl molting-defective mutants. Using two independent approaches, we identified CDC-42, a conserved Rho-family GTPase, and its effector protein kinase, SID-3/ACK1. Notably, CDC42 and ACK1 regulate actin dynamics in mammals, and actin reorganization within the worm epidermis has been proposed to be important for the molting process. Inhibition of NEKL–MLT activities led to strong defects in the distribution of actin and failure to form molting-specific apical actin bundles. Importantly, this phenotype was reverted following cdc-42 or sid-3 inhibition. In addition, repression of CDC-42 or SID-3 also suppressed nekl-associated defects in trafficking, a process that requires actin assembly and disassembly. Expression analyses indicated that components of the NEKL–MLT network colocalize with both actin and CDC-42 in specific regions of the epidermis. Moreover, NEKL–MLT components were required for the normal subcellular localization of CDC-42 in the epidermis as well as wild-type levels of CDC-42 activation. Taken together, our findings indicate that the NEKL–MLT network regulates actin through CDC-42 and its effector SID-3. Interestingly, we also observed that downregulation of CDC-42 in a wild-type background leads to molting defects, suggesting that there is a fine balance between NEKL–MLT and CDC-42–SID-3 activities in the epidermis. PMID:29608564

Actin organization and endocytic trafficking are controlled by a network linking NIMA-related kinases to the CDC-42-SID-3/ACK1 pathway.

PubMed

Lažetić, Vladimir; Joseph, Braveen B; Bernazzani, Sarina M; Fay, David S

2018-04-01

Molting is an essential process in the nematode Caenorhabditis elegans during which the epidermal apical extracellular matrix, termed the cuticle, is detached and replaced at each larval stage. The conserved NIMA-related kinases NEKL-2/NEK8/NEK9 and NEKL-3/NEK6/NEK7, together with their ankyrin repeat partners, MLT-2/ANKS6, MLT-3/ANKS3, and MLT-4/INVS, are essential for normal molting. In nekl and mlt mutants, the old larval cuticle fails to be completely shed, leading to entrapment and growth arrest. To better understand the molecular and cellular functions of NEKLs during molting, we isolated genetic suppressors of nekl molting-defective mutants. Using two independent approaches, we identified CDC-42, a conserved Rho-family GTPase, and its effector protein kinase, SID-3/ACK1. Notably, CDC42 and ACK1 regulate actin dynamics in mammals, and actin reorganization within the worm epidermis has been proposed to be important for the molting process. Inhibition of NEKL-MLT activities led to strong defects in the distribution of actin and failure to form molting-specific apical actin bundles. Importantly, this phenotype was reverted following cdc-42 or sid-3 inhibition. In addition, repression of CDC-42 or SID-3 also suppressed nekl-associated defects in trafficking, a process that requires actin assembly and disassembly. Expression analyses indicated that components of the NEKL-MLT network colocalize with both actin and CDC-42 in specific regions of the epidermis. Moreover, NEKL-MLT components were required for the normal subcellular localization of CDC-42 in the epidermis as well as wild-type levels of CDC-42 activation. Taken together, our findings indicate that the NEKL-MLT network regulates actin through CDC-42 and its effector SID-3. Interestingly, we also observed that downregulation of CDC-42 in a wild-type background leads to molting defects, suggesting that there is a fine balance between NEKL-MLT and CDC-42-SID-3 activities in the epidermis.

Actin retrograde flow and actomyosin II arc contraction drive receptor cluster dynamics at the immunological synapse in Jurkat T cells

PubMed Central

Yi, Jason; Wu, Xufeng S.; Crites, Travis; Hammer, John A.

2012-01-01

Actin retrograde flow and actomyosin II contraction have both been implicated in the inward movement of T cell receptor (TCR) microclusters and immunological synapse formation, but no study has integrated and quantified their relative contributions. Using Jurkat T cells expressing fluorescent myosin IIA heavy chain and F-tractin—a novel reporter for F-actin—we now provide direct evidence that the distal supramolecular activation cluster (dSMAC) and peripheral supramolecular activation cluster (pSMAC) correspond to lamellipodial (LP) and lamellar (LM) actin networks, respectively, as hypothesized previously. Our images reveal concentric and contracting actomyosin II arcs/rings at the LM/pSMAC. Moreover, the speeds of centripetally moving TCR microclusters correspond very closely to the rates of actin retrograde flow in the LP/dSMAC and actomyosin II arc contraction in the LM/pSMAC. Using cytochalasin D and jasplakinolide to selectively inhibit actin retrograde flow in the LP/dSMAC and blebbistatin to selectively inhibit actomyosin II arc contraction in the LM/pSMAC, we demonstrate that both forces are required for centripetal TCR microcluster transport. Finally, we show that leukocyte function–associated antigen 1 clusters accumulate over time at the inner aspect of the LM/pSMAC and that this accumulation depends on actomyosin II contraction. Thus actin retrograde flow and actomyosin II arc contraction coordinately drive receptor cluster dynamics at the immunological synapse. PMID:22219382

Actin protofilament orientation in deformation of the erythrocyte membrane skeleton.

PubMed Central

Picart, C; Dalhaimer, P; Discher, D E

2000-01-01

The red cell's spectrin-actin network is known to sustain local states of shear, dilation, and condensation, and yet the short actin filaments are found to maintain membrane-tangent and near-random azimuthal orientations. When calibrated with polarization results for single actin filaments, imaging of micropipette-deformed red cell ghosts has allowed an assessment of actin orientations and possible reorientations in the network. At the hemispherical cap of the aspirated projection, where the network can be dilated severalfold, filaments have the same membrane-tangent orientation as on a relatively unstrained portion of membrane. Likewise, over the length of the network projection pulled into the micropipette, where the network is strongly sheared in axial extension and circumferential contraction, actin maintains its tangent orientation and is only very weakly aligned with network extension. Similar results are found for the integral membrane protein Band 3. Allowing for thermal fluctuations, we deduce a bound for the effective coupling constant, alpha, between network shear and azimuthal orientation of the protofilament. The finding that alpha must be about an order of magnitude or more below its tight-coupling value illustrates how nanostructural kinematics can decouple from more macroscopic responses. Monte Carlo simulations of spectrin-actin networks at approximately 10-nm resolution further support this conclusion and substantiate an image of protofilaments as elements of a high-temperature spin glass. PMID:11106606

Actin filament bundling by fimbrin is important for endocytosis, cytokinesis, and polarization in fission yeast.

PubMed

Skau, Colleen T; Courson, David S; Bestul, Andrew J; Winkelman, Jonathan D; Rock, Ronald S; Sirotkin, Vladimir; Kovar, David R

2011-07-29

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.

Feedback Interactions of Polymerized Actin with the Cell Membrane: Waves, Pulses, and Oscillations

NASA Astrophysics Data System (ADS)

Carlsson, Anders

Polymerized filaments of the protein actin have crucial functions in cell migration, and in bending the cell membrane to drive endocytosis or the formation of protrusions. The nucleation and polymerization of actin filaments are controlled by upstream agents in the cell membrane, including nucleation-promoting factors (NPFs) that activate the Arp2/3 complex to form new branches on pre-existing filaments. But polymerized actin (F-actin) also feeds back on the assembly of NPFs. We explore the effects of the resulting feedback loop of F-actin and NPFs on two phenomena: actin pulses that drive endocytosis in yeast, and actin waves traveling along the membrane of several cell types. In our model of endocytosis in yeast, the actin network is grown explicitly in three dimensions, exerts a negative feedback interaction on localized patch of NPFs in the membrane, and bends the membrane by exerting a distribution of forces. This model explains observed actin and NPF pulse dynamics, and the effects of several interventions including i) NPF mutations, ii) inhibition of actin polymerization, and iii) deletion of a protein that allows F-actin to bend the cell membrane. The model predicts that mutation of the active region of an NPF will enhance the accumulation of that NPF, and we confirm this prediction by quantitative fluorescence microscopy. For actin waves, we treat a similar model, with NPFs distributed over a larger region of the cell membrane. This model naturally generates actin waves, and predicts a transition from wave behavior to spatially localized oscillations when NPFs are confined to a small region. We also predict a transition from waves to static polarization as the negative-feedback coupling between F-actin and the NPFs is reduced. Supported by NIGMS Grant R01 GM107667.

Dynamics of Actin Cables in Polarized Growth of the Filamentous Fungus Aspergillus nidulans

PubMed Central

Bergs, Anna; Ishitsuka, Yuji; Evangelinos, Minoas; Nienhaus, G. U.; Takeshita, Norio

2016-01-01

Highly polarized growth of filamentous fungi requires a continuous supply of proteins and lipids to the hyphal tip. This transport is managed by vesicle trafficking via the actin and microtubule cytoskeletons and their associated motor proteins. Particularly, actin cables originating from the hyphal tip are essential for hyphal growth. Although, specific marker proteins have been developed to visualize actin cables in filamentous fungi, the exact organization and dynamics of actin cables has remained elusive. Here, we observed actin cables using tropomyosin (TpmA) and Lifeact fused to fluorescent proteins in living Aspergillus nidulans hyphae and studied the dynamics and regulation. GFP tagged TpmA visualized dynamic actin cables formed from the hyphal tip with cycles of elongation and shrinkage. The elongation and shrinkage rates of actin cables were similar and approximately 0.6 μm/s. Comparison of actin markers revealed that high concentrations of Lifeact reduced actin dynamics. Simultaneous visualization of actin cables and microtubules suggests temporally and spatially coordinated polymerization and depolymerization between the two cytoskeletons. Our results provide new insights into the molecular mechanism of ordered polarized growth regulated by actin cables and microtubules. PMID:27242709

Hierarchical self-assembly of actin in micro-confinements using microfluidics

PubMed Central

Deshpande, Siddharth; Pfohl, Thomas

2012-01-01

We present a straightforward microfluidics system to achieve step-by-step reaction sequences in a diffusion-controlled manner in quasi two-dimensional micro-confinements. We demonstrate the hierarchical self-organization of actin (actin monomers—entangled networks of filaments—networks of bundles) in a reversible fashion by tuning the Mg2+ ion concentration in the system. We show that actin can form networks of bundles in the presence of Mg2+ without any cross-linking proteins. The properties of these networks are influenced by the confinement geometry. In square microchambers we predominantly find rectangular networks, whereas triangular meshes are predominantly found in circular chambers. PMID:24032070

Coupling of the hydration water dynamics and the internal dynamics of actin detected by quasielastic neutron scattering

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fujiwara, Satoru, E-mail: fujiwara.satoru@jaea.go.jp; Plazanet, Marie; Oda, Toshiro

2013-02-15

Highlights: ► Quasielastic neutron scattering spectra of F-actin and G-actin were measured. ► Analysis of the samples in D{sub 2}O and H{sub 2}O provided the spectra of hydration water. ► The first layer hydration water around F-actin is less mobile than around G-actin. ► This difference in hydration water is in concert with the internal dynamics of actin. ► Water outside the first layer behaves bulk-like but influenced by the first layer. -- Abstract: In order to characterize dynamics of water molecules around F-actin and G-actin, quasielastic neutron scattering experiments were performed on powder samples of F-actin and G-actin, hydratedmore » either with D{sub 2}O or H{sub 2}O, at hydration ratios of 0.4 and 1.0. By combined analysis of the quasielastic neutron scattering spectra, the parameter values characterizing the dynamics of the water molecules in the first hydration layer and those of the water molecules outside of the first layer were obtained. The translational diffusion coefficients (D{sub T}) of the hydration water in the first layer were found to be 1.2 × 10{sup −5} cm{sup 2}/s and 1.7 × 10{sup −5} cm{sup 2}/s for F-actin and G-actin, respectively, while that for bulk water was 2.8 × 10{sup −5} cm{sup 2}/s. The residence times were 6.6 ps and 5.0 ps for F-actin and G-actin, respectively, while that for bulk water was 0.62 ps. These differences between F-actin and G-actin, indicating that the hydration water around G-actin is more mobile than that around F-actin, are in concert with the results of the internal dynamics of F-actin and G-actin, showing that G-actin fluctuates more rapidly than F-actin. This implies that the dynamics of the hydration water is coupled to the internal dynamics of the actin molecules. The D{sub T} values of the water molecules outside of the first hydration layer were found to be similar to that of bulk water though the residence times are strongly affected by the first hydration layer. This supports the recent observation on intracellular water that shows bulk-like behavior.« less

Dynamic multiprotein assemblies shape the spatial structure of cell signaling.

PubMed

Nussinov, Ruth; Jang, Hyunbum

2014-01-01

Cell signaling underlies critical cellular decisions. Coordination, efficiency as well as fail-safe mechanisms are key elements. How the cell ensures that these hallmarks are at play are important questions. Cell signaling is often viewed as taking place through discrete and cross-talking pathways; oftentimes these are modularized to emphasize distinct functions. While simple, convenient and clear, such models largely neglect the spatial structure of cell signaling; they also convey inter-modular (or inter-protein) spatial separation that may not exist. Here our thesis is that cell signaling is shaped by a network of multiprotein assemblies. While pre-organized, the assemblies and network are loose and dynamic. They contain transiently-associated multiprotein complexes which are often mediated by scaffolding proteins. They are also typically anchored in the membrane, and their continuum may span the cell. IQGAP1 scaffolding protein which binds proteins including Raf, calmodulin, Mek, Erk, actin, and tens more, with actin shaping B-cell (and likely other) membrane-anchored nanoclusters and allosterically polymerizing in dynamic cytoskeleton formation, and Raf anchoring in the membrane along with Ras, provides a striking example. The multivalent network of dynamic proteins and lipids, with specific interactions forming and breaking, can be viewed as endowing gel-like properties. Collectively, this reasons that efficient, productive and reliable cell signaling takes place primarily through transient, preorganized and cooperative protein-protein interactions spanning the cell rather than stochastic, diffusion-controlled processes. Copyright © 2014 Elsevier Ltd. All rights reserved.

αE-catenin regulates actin dynamics independently of cadherin-mediated cell–cell adhesion

PubMed Central

Benjamin, Jacqueline M.; Kwiatkowski, Adam V.; Yang, Changsong; Korobova, Farida; Pokutta, Sabine; Svitkina, Tatyana

2010-01-01

αE-catenin binds the cell–cell adhesion complex of E-cadherin and β-catenin (β-cat) and regulates filamentous actin (F-actin) dynamics. In vitro, binding of αE-catenin to the E-cadherin–β-cat complex lowers αE-catenin affinity for F-actin, and αE-catenin alone can bind F-actin and inhibit Arp2/3 complex–mediated actin polymerization. In cells, to test whether αE-catenin regulates actin dynamics independently of the cadherin complex, the cytosolic αE-catenin pool was sequestered to mitochondria without affecting overall levels of αE-catenin or the cadherin–catenin complex. Sequestering cytosolic αE-catenin to mitochondria alters lamellipodia architecture and increases membrane dynamics and cell migration without affecting cell–cell adhesion. In contrast, sequestration of cytosolic αE-catenin to the plasma membrane reduces membrane dynamics. These results demonstrate that the cytosolic pool of αE-catenin regulates actin dynamics independently of cell–cell adhesion. PMID:20404114

Cations Modulate Actin Bundle Mechanics, Assembly Dynamics, and Structure.

PubMed

Castaneda, Nicholas; Zheng, Tianyu; Rivera-Jacquez, Hector J; Lee, Hyun-Ju; Hyun, Jaekyung; Balaeff, Alexander; Huo, Qun; Kang, Hyeran

2018-04-12

Actin bundles are key factors in the mechanical support and dynamic reorganization of the cytoskeleton. High concentrations of multivalent counterions promote bundle formation through electrostatic attraction between actin filaments that are negatively charged polyelectrolytes. In this study, we evaluate how physiologically relevant divalent cations affect the mechanical, dynamic, and structural properties of actin bundles. Using a combination of total internal reflection fluorescence microscopy, transmission electron microscopy, and dynamic light scattering, we demonstrate that divalent cations modulate bundle stiffness, length distribution, and lateral growth. Molecular dynamics simulations of an all-atom model of the actin bundle reveal specific actin residues coordinate cation-binding sites that promote the bundle formation. Our work suggests that specific cation interactions may play a fundamental role in the assembly, structure, and mechanical properties of actin bundles.

Concentration profiles of actin-binding molecules in lamellipodia

NASA Astrophysics Data System (ADS)

Falcke, Martin

2016-04-01

Motile cells form lamellipodia in the direction of motion, which are flat membrane protrusions containing an actin filament network. The network flows rearward relative to the leading edge of the lamellipodium due to actin polymerization at the front. Thus, actin binding molecules are subject to transport towards the rear of the cell in the bound state and diffuse freely in the unbound state. We analyze this reaction-diffusion-advection process with respect to the concentration profiles of these species and provide an analytic approximation for them. Network flow may cause a depletion zone of actin binding molecules close to the leading edge. The existence of such zone depends on the free molecule concentration in the cell body, on the ratio of the diffusion length to the distance bound molecules travel rearward with the flow before dissociating, and the ratio of the diffusion length to the width of the region with network flow and actin binding. Our calculations suggest the existence of depletion zones for the F-actin cross-linkers filamin and α-actinin in fish keratocytes (and other cell types), which is in line with the small elastic moduli of the F-actin network close to the leading edge found in measurements of the force motile cells are able to exert.

The actin cytoskeleton of chemotactic amoebae operates close to the onset of oscillations

NASA Astrophysics Data System (ADS)

Westendorf, Christian; Negrete, Jose, Jr.; Bae, Albert; Sandmann, Rabea; Bodenschatz, Eberhard; Beta, Carsten

2013-03-01

We report evidence that the actin machinery of chemotactic Dictyostelium cells operates close to an oscillatory instability. The averaged F-actin response of many cells to a short-time pulse of cAMP is reminiscent of a damped oscillation. At the single-cell level, however, the response dynamics ranged from short, strongly damped responses to slowly decaying, weakly damped oscillations. Furthermore, in a small subpopulation, we observed self-sustained oscillations in the cortical F-actin concentration. We systematically exposed a large number of cells to periodic pulse trains. The results indicate a resonance peak at periodic inputs of around 20 s. We propose a delayed feedback model that explains our experimental findings based on a time-delay in the actin regulatory network. To quantitatively test the model, we performed stimulation experiments with cells that express GFP-tagged fusion proteins of Coronin and Aip1. These served as markers of the F-actin disassembly process and thus allow us to estimate the delay time. Based on this independent estimate, our model predicts an intrinsic period of 20 s, which agrees with the resonance observed experimentally. Financial support by the Max-Planck Society and the DFG (SFB 937).

Microscale force response and morphology of tunable co-polymerized cytoskeleton networks

NASA Astrophysics Data System (ADS)

Ricketts, Shea; Yadav, Vikrant; Ross, Jennifer L.; Robertson-Anderson, Rae M.

The cytoskeleton is largely comprised of actin and microtubules that entangle and crosslink to form complex networks and structures, giving rise to nonlinear multifunctional mechanics in cells. The relative concentrations of semiflexible actin filaments and rigid microtubules tune cytoskeleton function, allowing cells to move and divide while maintaining rigidity and resilience. To elucidate this complex tunability, we create in vitro composites of co-polymerized actin and microtubules with actin:microtubule molar ratios of 0:1-1:0. We use optical tweezers and confocal microscopy to characterize the nonlinear microscale force response and morphology of the composites. We optically drag a microsphere 30 μm through varying actin-microtubule networks at 10 μm/s and 20 μm/s, and measure the force the networks exerts to resist the strain and the force relaxation following strain. We use dual-color confocal microscopy to image distinctly-labeled filaments in the networks, and characterize the integration of actin and microtubules, network connectivity, and filament rigidity. We find that increasing the fraction of microtubules in networks non-monotonically increases elasticity and stiffness, and hinders force relaxation by suppressing network mobility and fluctuations. NSF CAREER Award (DMR-1255446), Scialog Collaborative Innovation Award funded by Research Corporation for Scientific Advancement (Grant No. 24192).

ERK reinforces actin polymerization to power persistent edge protrusion during motility

PubMed Central

Mendoza, Michelle C.; Vilela, Marco; Juarez, Jesus E.; Blenis, John; Danuser, Gaudenz

2016-01-01

Cells move through perpetual protrusion and retraction cycles at the leading edge. These cycles are coordinated with substrate adhesion and retraction of the cell rear. Here, we tracked spatial and temporal fluctuations in the molecular activities of individual moving cells to elucidate how extracellular regulated kinase (ERK) signaling controlled the dynamics of protrusion and retraction cycles. ERK is activated by many cell-surface receptors and we found that ERK signaling specifically reinforced cellular protrusions so that they translated into rapid, sustained forward motion of the leading edge. Using quantitative fluorescent speckle microscopy (qFSM) and cross-correlation analysis, we showed that ERK controlled the rate and timing of actin polymerization by promoting the recruitment of the actin nucleator Arp2/3 to the leading edge. Arp2/3 activity generates branched actin networks that can produce pushing force. These findings support a model in which surges in ERK activity induced by extracellular cues enhance Arp2/3-mediated actin polymerization to generate protrusion power phases with enough force to counteract increasing membrane tension and to promote sustained motility. PMID:25990957

Characterization of biomechanical properties of cells through dielectrophoresis-based cell stretching and actin cytoskeleton modeling.

PubMed

Bai, Guohua; Li, Ying; Chu, Henry K; Wang, Kaiqun; Tan, Qiulin; Xiong, Jijun; Sun, Dong

2017-04-04

Cytoskeleton is a highly dynamic network that helps to maintain the rigidity of a cell, and the mechanical properties of a cell are closely related to many cellular functions. This paper presents a new method to probe and characterize cell mechanical properties through dielectrophoresis (DEP)-based cell stretching manipulation and actin cytoskeleton modeling. Leukemia NB4 cells were used as cell line, and changes in their biological properties were examined after chemotherapy treatment with doxorubicin (DOX). DEP-integrated microfluidic chip was utilized as a low-cost and efficient tool to study the deformability of cells. DEP forces used in cell stretching were first evaluated through computer simulation, and the results were compared with modeling equations and with the results of optical stretching (OT) experiments. Structural parameters were then extracted by fitting the experimental data into the actin cytoskeleton model, and the underlying mechanical properties of the cells were subsequently characterized. The DEP forces generated under different voltage inputs were calculated and the results from different approaches demonstrate good approximations to the force estimation. Both DEP and OT stretching experiments confirmed that DOX-treated NB4 cells were stiffer than the untreated cells. The structural parameters extracted from the model and the confocal images indicated significant change in actin network after DOX treatment. The proposed DEP method combined with actin cytoskeleton modeling is a simple engineering tool to characterize the mechanical properties of cells.

Cross-Linking Molecules Modify Composite Actin Networks Independently

NASA Astrophysics Data System (ADS)

Schmoller, K. M.; Lieleg, O.; Bausch, A. R.

2008-09-01

While cells make use of many actin binding proteins (ABPs) simultaneously to tailor the mechanical properties of the cytoskeleton, the detailed interplay of different ABPs is not understood. By a combination of macrorheological measurements and confocal microscopy, we show that the ABPs fascin and filamin modify the structural and viscoelastic properties of composite in vitro actin networks independently. The outnumbering ABP dictates the local network structure and therefore also dominates the macromechanical network response.

Apical constriction: themes and variations on a cellular mechanism driving morphogenesis

PubMed Central

Martin, Adam C.; Goldstein, Bob

2014-01-01

Apical constriction is a cell shape change that promotes tissue remodeling in a variety of homeostatic and developmental contexts, including gastrulation in many organisms and neural tube formation in vertebrates. In recent years, progress has been made towards understanding how the distinct cell biological processes that together drive apical constriction are coordinated. These processes include the contraction of actin-myosin networks, which generates force, and the attachment of actin networks to cell-cell junctions, which allows forces to be transmitted between cells. Different cell types regulate contractility and adhesion in unique ways, resulting in apical constriction with varying dynamics and subcellular organizations, as well as a variety of resulting tissue shape changes. Understanding both the common themes and the variations in apical constriction mechanisms promises to provide insight into the mechanics that underlie tissue morphogenesis. PMID:24803648

Actin-mediated bacterial propulsion: comet profile, velocity pulsations.

PubMed

Benza, V G

2008-05-23

The propulsion of bacteria under the action of an actin gel network is examined in terms of gel concentration dynamics. The model includes the elasticity of the network, the gel-bacterium interaction, the bulk and interface polymerization. A formula for the cruise velocity is obtained where the contributions to bacterial motility arising from elasticity and polymerization are made explicit. Higher velocities correspond to lower concentration peaks and longer tails, in agreement with experimental results. The condition for the onset of motion is explicitly given. The behavior of the system is explored by varying the growth rates and the gel elasticity. At steady state two regimes are found, respectively, of constant and pulsating velocity; in the latter case, the velocity undergoes sudden accelerations and subsequent recoveries. The transition to the pulsating regime is obtained by increasing the elastic response of the gel.

Cytoskeleton in motion: the dynamics of keratin intermediate filaments in epithelia.

PubMed

Windoffer, Reinhard; Beil, Michael; Magin, Thomas M; Leube, Rudolf E

2011-09-05

Epithelia are exposed to multiple forms of stress. Keratin intermediate filaments are abundant in epithelia and form cytoskeletal networks that contribute to cell type-specific functions, such as adhesion, migration, and metabolism. A perpetual keratin filament turnover cycle supports these functions. This multistep process keeps the cytoskeleton in motion, facilitating rapid and protein biosynthesis-independent network remodeling while maintaining an intact network. The current challenge is to unravel the molecular mechanisms underlying the regulation of the keratin cycle in relation to actin and microtubule networks and in the context of epithelial tissue function.

Cytoskeleton in motion: the dynamics of keratin intermediate filaments in epithelia

PubMed Central

Windoffer, Reinhard; Beil, Michael; Magin, Thomas M.

2011-01-01

Epithelia are exposed to multiple forms of stress. Keratin intermediate filaments are abundant in epithelia and form cytoskeletal networks that contribute to cell type–specific functions, such as adhesion, migration, and metabolism. A perpetual keratin filament turnover cycle supports these functions. This multistep process keeps the cytoskeleton in motion, facilitating rapid and protein biosynthesis–independent network remodeling while maintaining an intact network. The current challenge is to unravel the molecular mechanisms underlying the regulation of the keratin cycle in relation to actin and microtubule networks and in the context of epithelial tissue function. PMID:21893596

Cortical actin networks induce spatio-temporal confinement of phospholipids in the plasma membrane - a minimally invasive investigation by STED-FCS

NASA Astrophysics Data System (ADS)

Andrade, Débora M.; Clausen, Mathias P.; Keller, Jan; Mueller, Veronika; Wu, Congying; Bear, James E.; Hell, Stefan W.; Lagerholm, B. Christoffer; Eggeling, Christian

2015-06-01

Important discoveries in the last decades have changed our view of the plasma membrane organisation. Specifically, the cortical cytoskeleton has emerged as a key modulator of the lateral diffusion of membrane proteins. Cytoskeleton-dependent compartmentalised lipid diffusion has been proposed, but this concept remains controversial because this phenomenon has thus far only been observed with artefact-prone probes in combination with a single technique: single particle tracking. In this paper, we report the first direct observation of compartmentalised phospholipid diffusion in the plasma membrane of living cells using a minimally invasive, fluorescent dye labelled lipid analogue. These observations were made using optical STED nanoscopy in combination with fluorescence correlation spectroscopy (STED-FCS), a technique which allows the study of membrane dynamics on a sub-millisecond time-scale and with a spatial resolution of down to 40 nm. Specifically, we find that compartmentalised phospholipid diffusion depends on the cortical actin cytoskeleton, and that this constrained diffusion is directly dependent on the F-actin branching nucleator Arp2/3. These findings provide solid evidence that the Arp2/3-dependent cortical actin cytoskeleton plays a pivotal role in the dynamic organisation of the plasma membrane, potentially regulating fundamental cellular processes.

Cortical actin networks induce spatio-temporal confinement of phospholipids in the plasma membrane--a minimally invasive investigation by STED-FCS.

PubMed

Andrade, Débora M; Clausen, Mathias P; Keller, Jan; Mueller, Veronika; Wu, Congying; Bear, James E; Hell, Stefan W; Lagerholm, B Christoffer; Eggeling, Christian

2015-06-29

Important discoveries in the last decades have changed our view of the plasma membrane organisation. Specifically, the cortical cytoskeleton has emerged as a key modulator of the lateral diffusion of membrane proteins. Cytoskeleton-dependent compartmentalised lipid diffusion has been proposed, but this concept remains controversial because this phenomenon has thus far only been observed with artefact-prone probes in combination with a single technique: single particle tracking. In this paper, we report the first direct observation of compartmentalised phospholipid diffusion in the plasma membrane of living cells using a minimally invasive, fluorescent dye labelled lipid analogue. These observations were made using optical STED nanoscopy in combination with fluorescence correlation spectroscopy (STED-FCS), a technique which allows the study of membrane dynamics on a sub-millisecond time-scale and with a spatial resolution of down to 40 nm. Specifically, we find that compartmentalised phospholipid diffusion depends on the cortical actin cytoskeleton, and that this constrained diffusion is directly dependent on the F-actin branching nucleator Arp2/3. These findings provide solid evidence that the Arp2/3-dependent cortical actin cytoskeleton plays a pivotal role in the dynamic organisation of the plasma membrane, potentially regulating fundamental cellular processes.

Cortical actin networks induce spatio-temporal confinement of phospholipids in the plasma membrane – a minimally invasive investigation by STED-FCS

PubMed Central

Andrade, Débora M.; Clausen, Mathias P.; Keller, Jan; Mueller, Veronika; Wu, Congying; Bear, James E.; Hell, Stefan W.; Lagerholm, B. Christoffer; Eggeling, Christian

2015-01-01

Important discoveries in the last decades have changed our view of the plasma membrane organisation. Specifically, the cortical cytoskeleton has emerged as a key modulator of the lateral diffusion of membrane proteins. Cytoskeleton-dependent compartmentalised lipid diffusion has been proposed, but this concept remains controversial because this phenomenon has thus far only been observed with artefact-prone probes in combination with a single technique: single particle tracking. In this paper, we report the first direct observation of compartmentalised phospholipid diffusion in the plasma membrane of living cells using a minimally invasive, fluorescent dye labelled lipid analogue. These observations were made using optical STED nanoscopy in combination with fluorescence correlation spectroscopy (STED-FCS), a technique which allows the study of membrane dynamics on a sub-millisecond time-scale and with a spatial resolution of down to 40 nm. Specifically, we find that compartmentalised phospholipid diffusion depends on the cortical actin cytoskeleton, and that this constrained diffusion is directly dependent on the F-actin branching nucleator Arp2/3. These findings provide solid evidence that the Arp2/3-dependent cortical actin cytoskeleton plays a pivotal role in the dynamic organisation of the plasma membrane, potentially regulating fundamental cellular processes. PMID:26118385

Course 6: Physics of Composite Cell Membrane and Actin Based Cytoskeleton

NASA Astrophysics Data System (ADS)

Sackmann, E.; Bausch, A. R.; Vonna, L.

1 Architecture of composite cell membranes 1.1 The lipid/protein bilayer is a multicomponent smectic phase with mosaic like architecture 1.2 The spectrin/actin cytoskeleton as hyperelastic cell stabilizer 1.3 The actin cortex: Architecture and function 2 Physics of the actin based cytoskeleton 2.1 Actin is a living semiflexible polymer 2.2 Actin network as viscoelastic body 2.3 Correlation between macroscopic viscoelasticity and molecular 3 Heterogeneous actin gels in cells and biological function 3.1 Manipulation of actin gels 3.2 Control of organization and function of actin cortex by cell signalling 4 Micromechanics and microrheometry of cells 5 Activation of endothelial cells: On the possibility of formation of stress fibers as phase transition of actin-network triggered by cell signalling pathways 6 On cells as adaptive viscoplastic bodies 7 Controll of cellular protrusions controlled by actin/myosin cortex

The availability of filament ends modulates actin stochastic dynamics in live plant cells

PubMed Central

Li, Jiejie; Staiger, Benjamin H.; Henty-Ridilla, Jessica L.; Abu-Abied, Mohamad; Sadot, Einat; Blanchoin, Laurent; Staiger, Christopher J.

2014-01-01

A network of individual filaments that undergoes incessant remodeling through a process known as stochastic dynamics comprises the cortical actin cytoskeleton in plant epidermal cells. From images at high spatial and temporal resolution, it has been inferred that the regulation of filament barbed ends plays a central role in choreographing actin organization and turnover. How this occurs at a molecular level, whether different populations of ends exist in the array, and how individual filament behavior correlates with the overall architecture of the array are unknown. Here we develop an experimental system to modulate the levels of heterodimeric capping protein (CP) and examine the consequences for actin dynamics, architecture, and cell expansion. Significantly, we find that all phenotypes are the opposite for CP-overexpression (OX) cells compared with a previously characterized cp-knockdown line. Specifically, CP OX lines have fewer filament–filament annealing events, as well as reduced filament lengths and lifetimes. Further, cp-knockdown and OX lines demonstrate the existence of a subpopulation of filament ends sensitive to CP concentration. Finally, CP levels correlate with the biological process of axial cell expansion; for example, epidermal cells from hypocotyls with reduced CP are longer than wild-type cells, whereas CP OX lines have shorter cells. On the basis of these and other genetic studies in this model system, we hypothesize that filament length and lifetime positively correlate with the extent of axial cell expansion in dark-grown hypocotyls. PMID:24523291

Mechanical stress and network structure drive protein dynamics during cytokinesis.

PubMed

Srivastava, Vasudha; Robinson, Douglas N

2015-03-02

Cell-shape changes associated with processes like cytokinesis and motility proceed on several-second timescales but are derived from molecular events, including protein-protein interactions, filament assembly, and force generation by molecular motors, all of which occur much faster [1-4]. Therefore, defining the dynamics of such molecular machinery is critical for understanding cell-shape regulation. In addition to signaling pathways, mechanical stresses also direct cytoskeletal protein accumulation [5-7]. A myosin-II-based mechanosensory system controls cellular contractility and shape during cytokinesis and under applied stress [6, 8]. In Dictyostelium, this system tunes myosin II accumulation by feedback through the actin network, particularly through the crosslinker cortexillin I. Cortexillin-binding IQGAPs are major regulators of this system. Here, we defined the short timescale dynamics of key cytoskeletal proteins during cytokinesis and under mechanical stress, using fluorescence recovery after photobleaching and fluorescence correlation spectroscopy, to examine the dynamic interplay between these proteins. Equatorially enriched proteins including cortexillin I, IQGAP2, and myosin II recovered much more slowly than actin and polar crosslinkers. The mobility of equatorial proteins was greatly reduced at the furrow compared to the interphase cortex, suggesting their stabilization during cytokinesis. This mobility shift did not arise from a single biochemical event, but rather from a global inhibition of protein dynamics by mechanical-stress-associated changes in the cytoskeletal structure. Mechanical tuning of contractile protein dynamics provides robustness to the cytoskeletal framework responsible for regulating cell shape and contributes to cytokinesis fidelity. Copyright © 2015 Elsevier Ltd. All rights reserved.

ELMO recruits actin cross-linking family 7 (ACF7) at the cell membrane for microtubule capture and stabilization of cellular protrusions.

PubMed

Margaron, Yoran; Fradet, Nadine; Côté, Jean-François

2013-01-11

ELMO and DOCK180 proteins form an evolutionarily conserved module controlling Rac GTPase signaling during cell migration, phagocytosis, and myoblast fusion. Here, we identified the microtubule and actin-binding spectraplakin ACF7 as a novel ELMO-interacting partner. A C-terminal polyproline segment in ELMO and the last spectrin repeat of ACF7 mediate a direct interaction between these proteins. Co-expression of ELMO1 with ACF7 promoted the formation of long membrane protrusions during integrin-mediated cell spreading. Quantification of membrane dynamics established that coupling of ELMO and ACF7 increases the persistence of the protruding activity. Mechanistically, we uncovered a role for ELMO in the recruitment of ACF7 to the membrane to promote microtubule capture and stability. Functionally, these effects of ELMO and ACF7 on cytoskeletal dynamics required the Rac GEF DOCK180. In conclusion, our findings support a role for ELMO in protrusion stability by acting at the interface between the actin cytoskeleton and the microtubule network.

Diverse roles of actin in C. elegans early embryogenesis

PubMed Central

Velarde, Nathalie; Gunsalus, Kristin C; Piano, Fabio

2007-01-01

Background The actin cytoskeleton plays critical roles in early development in Caenorhabditis elegans. To further understand the complex roles of actin in early embryogenesis we use RNAi and in vivo imaging of filamentous actin (F-actin) dynamics. Results Using RNAi, we found processes that are differentially sensitive to levels of actin during early embryogenesis. Mild actin depletion shows defects in cortical ruffling, pseudocleavage, and establishment of polarity, while more severe depletion shows defects in polar body extrusion, cytokinesis, chromosome segregation, and eventually, egg production. These defects indicate that actin is required for proper oocyte development, fertilization, and a wide range of important events during early embryogenesis, including proper chromosome segregation. In vivo visualization of the cortical actin cytoskeleton shows dynamics that parallel but are distinct from the previously described myosin dynamics. Two distinct types of actin organization are observed at the cortex. During asymmetric polarization to the anterior, or the establishment phase (Phase I), actin forms a meshwork of microfilaments and focal accumulations throughout the cortex, while during the anterior maintenance phase (Phase II) it undergoes a morphological transition to asymmetrically localized puncta. The proper asymmetric redistribution is dependent on the PAR proteins, while both asymmetric redistribution and morphological transitions are dependent upon PFN-1 and NMY-2. Just before cytokinesis, actin disappears from most of the cortex and is only found around the presumptive cytokinetic furrow. Finally, we describe dynamic actin-enriched comets in the early embryo. Conclusion During early C. elegans embryogenesis actin plays more roles and its organization is more dynamic than previously described. Morphological transitions of F-actin, from meshwork to puncta, as well as asymmetric redistribution, are regulated by the PAR proteins. Results from this study indicate new insights into the cellular and developmental roles of the actin cytoskeleton. PMID:18157918

Interconnected network motifs control podocyte morphology and kidney function.

PubMed

Azeloglu, Evren U; Hardy, Simon V; Eungdamrong, Narat John; Chen, Yibang; Jayaraman, Gomathi; Chuang, Peter Y; Fang, Wei; Xiong, Huabao; Neves, Susana R; Jain, Mohit R; Li, Hong; Ma'ayan, Avi; Gordon, Ronald E; He, John Cijiang; Iyengar, Ravi

2014-02-04

Podocytes are kidney cells with specialized morphology that is required for glomerular filtration. Diseases, such as diabetes, or drug exposure that causes disruption of the podocyte foot process morphology results in kidney pathophysiology. Proteomic analysis of glomeruli isolated from rats with puromycin-induced kidney disease and control rats indicated that protein kinase A (PKA), which is activated by adenosine 3',5'-monophosphate (cAMP), is a key regulator of podocyte morphology and function. In podocytes, cAMP signaling activates cAMP response element-binding protein (CREB) to enhance expression of the gene encoding a differentiation marker, synaptopodin, a protein that associates with actin and promotes its bundling. We constructed and experimentally verified a β-adrenergic receptor-driven network with multiple feedback and feedforward motifs that controls CREB activity. To determine how the motifs interacted to regulate gene expression, we mapped multicompartment dynamical models, including information about protein subcellular localization, onto the network topology using Petri net formalisms. These computational analyses indicated that the juxtaposition of multiple feedback and feedforward motifs enabled the prolonged CREB activation necessary for synaptopodin expression and actin bundling. Drug-induced modulation of these motifs in diseased rats led to recovery of normal morphology and physiological function in vivo. Thus, analysis of regulatory motifs using network dynamics can provide insights into pathophysiology that enable predictions for drug intervention strategies to treat kidney disease.

Interconnected Network Motifs Control Podocyte Morphology and Kidney Function

PubMed Central

Azeloglu, Evren U.; Hardy, Simon V.; Eungdamrong, Narat John; Chen, Yibang; Jayaraman, Gomathi; Chuang, Peter Y.; Fang, Wei; Xiong, Huabao; Neves, Susana R.; Jain, Mohit R.; Li, Hong; Ma’ayan, Avi; Gordon, Ronald E.; He, John Cijiang; Iyengar, Ravi

2014-01-01

Podocytes are kidney cells with specialized morphology that is required for glomerular filtration. Diseases, such as diabetes, or drug exposure that causes disruption of the podocyte foot process morphology results in kidney pathophysiology. Proteomic analysis of glomeruli isolated from rats with puromycin-induced kidney disease and control rats indicated that protein kinase A (PKA), which is activated by adenosine 3′,5′-monophosphate (cAMP), is a key regulator of podocyte morphology and function. In podocytes, cAMP signaling activates cAMP response element–binding protein (CREB) to enhance expression of the gene encoding a differentiation marker, synaptopodin, a protein that associates with actin and promotes its bundling. We constructed and experimentally verified a β-adrenergic receptor–driven network with multiple feedback and feedforward motifs that controls CREB activity. To determine how the motifs interacted to regulate gene expression, we mapped multicompartment dynamical models, including information about protein subcellular localization, onto the network topology using Petri net formalisms. These computational analyses indicated that the juxtaposition of multiple feedback and feedforward motifs enabled the prolonged CREB activation necessary for synaptopodin expression and actin bundling. Drug-induced modulation of these motifs in diseased rats led to recovery of normal morphology and physiological function in vivo. Thus, analysis of regulatory motifs using network dynamics can provide insights into pathophysiology that enable predictions for drug intervention strategies to treat kidney disease. PMID:24497609

The atypical Rho GTPase RhoD is a regulator of actin cytoskeleton dynamics and directed cell migration

DOE Office of Scientific and Technical Information (OSTI.GOV)

Blom, Magdalena; Reis, Katarina; Heldin, Johan

RhoD belongs to the Rho GTPases, a protein family responsible for the regulation and organization of the actin cytoskeleton, and, consequently, many cellular processes like cell migration, cell division and vesicle trafficking. Here, we demonstrate that the actin cytoskeleton is dynamically regulated by increased or decreased protein levels of RhoD. Ectopic expression of RhoD has previously been shown to give an intertwined weave of actin filaments. We show that this RhoD-dependent effect is detected in several cell types and results in a less dynamic actin filament system. In contrast, RhoD depletion leads to increased actin filament-containing structures, such as corticalmore » actin, stress fibers and edge ruffles. Moreover, vital cellular functions such as cell migration and proliferation are defective when RhoD is silenced. Taken together, we present data suggesting that RhoD is an important component in the control of actin dynamics and directed cell migration. - Highlights: • Increased RhoD expression leads to loss of actin structures, e.g. stress fibers and gives rise to decreased actin dynamics. • RhoD knockdown induces various actin-containing structures such as edge ruffles, stress fibers and cortical actin, in a cell-type specific manner. • RhoD induces specific actin rearrangements depending on its subcellular localization. • RhoD knockdown has effects on cellular processes, such as directed cell migration and proliferation.« less

Biological role and structural mechanism of twinfilin–capping protein interaction

PubMed Central

Falck, Sandra; Paavilainen, Ville O; Wear, Martin A; Grossmann, J Günter; Cooper, John A; Lappalainen, Pekka

2004-01-01

Twinfilin and capping protein (CP) are highly conserved actin-binding proteins that regulate cytoskeletal dynamics in organisms from yeast to mammals. Twinfilin binds actin monomer, while CP binds the barbed end of the actin filament. Remarkably, twinfilin and CP also bind directly to each other, but the mechanism and role of this interaction in actin dynamics are not defined. Here, we found that the binding of twinfilin to CP does not affect the binding of either protein to actin. Furthermore, site-directed mutagenesis studies revealed that the CP-binding site resides in the conserved C-terminal tail region of twinfilin. The solution structure of the twinfilin–CP complex supports these conclusions. In vivo, twinfilin's binding to both CP and actin monomer was found to be necessary for twinfilin's role in actin assembly dynamics, based on genetic studies with mutants that have defined biochemical functions. Our results support a novel model for how sequential interactions between actin monomers, twinfilin, CP, and actin filaments promote cytoskeletal dynamics. PMID:15282541

Stochastic Severing of Actin Filaments by Actin Depolymerizing Factor/Cofilin Controls the Emergence of a Steady Dynamical Regime

PubMed Central

Roland, Jeremy; Berro, Julien; Michelot, Alphée; Blanchoin, Laurent; Martiel, Jean-Louis

2008-01-01

Actin dynamics (i.e., polymerization/depolymerization) powers a large number of cellular processes. However, a great deal remains to be learned to explain the rapid actin filament turnover observed in vivo. Here, we developed a minimal kinetic model that describes key details of actin filament dynamics in the presence of actin depolymerizing factor (ADF)/cofilin. We limited the molecular mechanism to 1), the spontaneous growth of filaments by polymerization of actin monomers, 2), the ageing of actin subunits in filaments, 3), the cooperative binding of ADF/cofilin to actin filament subunits, and 4), filament severing by ADF/cofilin. First, from numerical simulations and mathematical analysis, we found that the average filament length, 〈L〉, is controlled by the concentration of actin monomers (power law: 5/6) and ADF/cofilin (power law: −2/3). We also showed that the average subunit residence time inside the filament, 〈T〉, depends on the actin monomer (power law: −1/6) and ADF/cofilin (power law: −2/3) concentrations. In addition, filament length fluctuations are ∼20% of the average filament length. Moreover, ADF/cofilin fragmentation while modulating filament length keeps filaments in a high molar ratio of ATP- or ADP-Pi versus ADP-bound subunits. This latter property has a protective effect against a too high severing activity of ADF/cofilin. We propose that the activity of ADF/cofilin in vivo is under the control of an affinity gradient that builds up dynamically along growing actin filaments. Our analysis shows that ADF/cofilin regulation maintains actin filaments in a highly dynamical state compatible with the cytoskeleton dynamics observed in vivo. PMID:18065447

Spectraplakins: Master orchestrators of cytoskeletal dynamics

PubMed Central

Suozzi, Kathleen C.; Wu, Xiaoyang

2012-01-01

The dynamics of different cytoskeletal networks are coordinated to bring about many fundamental cellular processes, from neuronal pathfinding to cell division. Increasing evidence points to the importance of spectraplakins in integrating cytoskeletal networks. Spectraplakins are evolutionarily conserved giant cytoskeletal cross-linkers, which belong to the spectrin superfamily. Their genes consist of multiple promoters and many exons, yielding a vast array of differential splice forms with distinct functions. Spectraplakins are also unique in their ability to associate with all three elements of the cytoskeleton: F-actin, microtubules, and intermediate filaments. Recent studies have begun to unveil their role in a wide range of processes, from cell migration to tissue integrity. PMID:22584905

A functional interplay between the small GTPase Rab11a and mitochondria-shaping proteins regulates mitochondrial positioning and polarization of the actin cytoskeleton downstream of Src family kinases.

PubMed

Landry, Marie-Claude; Champagne, Claudia; Boulanger, Marie-Chloé; Jetté, Alexandra; Fuchs, Margit; Dziengelewski, Claire; Lavoie, Josée N

2014-01-24

It is believed that mitochondrial dynamics is coordinated with endosomal traffic rates during cytoskeletal remodeling, but the mechanisms involved are largely unknown. The adenovirus early region 4 ORF4 protein (E4orf4) subverts signaling by Src family kinases (SFK) to perturb cellular morphology, membrane traffic, and organellar dynamics and to trigger cell death. Using E4orf4 as a model, we uncovered a functional connection between mitochondria-shaping proteins and the small GTPase Rab11a, a key regulator of polarized transport via recycling endosomes. We found that E4orf4 induced dramatic changes in the morphology of mitochondria along with their mobilization at the vicinity of a polarized actin network typifying E4orf4 action, in a manner controlled by SFK and Rab11a. Mitochondrial remodeling was associated with increased proximity between Rab11a and mitochondrial membranes, changes in fusion-fission dynamics, and mitochondrial relocalization of the fission factor dynamin-related protein 1 (Drp1), which was regulated by the Rab11a effector protein FIP1/RCP. Knockdown of FIP1/RCP or inhibition of Drp1 markedly impaired mitochondrial remodeling and actin assembly, involving Rab11a-mediated mitochondrial dynamics in E4orf4-induced signaling. A similar mobilization of mitochondria near actin-rich structures was mediated by Rab11 and Drp1 in viral Src-transformed cells and contributed to the biogenesis of podosome rosettes. These findings suggest a role for Rab11a in the trafficking of Drp1 to mitochondria upon SFK activation and unravel a novel functional interplay between Rab11a and mitochondria during reshaping of the cell cytoskeleton, which would facilitate mitochondria redistribution near energy-requiring actin-rich structures.

Hypertrophic Stimulation Increases β-actin Dynamics in Adult Feline Cardiomyocytes

PubMed Central

Balasubramanian, Sundaravadivel; Mani, Santhosh K.; Kasiganesan, Harinath; Baicu, Catalin C.; Kuppuswamy, Dhandapani

2010-01-01

The myocardium responds to hemodynamic stress through cellular growth and organ hypertrophy. The impact of cytoskeletal elements on this process, however, is not fully understood. While α-actin in cardiomyocytes governs muscle contraction in combination with the myosin motor, the exact role of β-actin has not been established. We hypothesized that in adult cardiomyocytes, as in non-myocytes, β-actin can facilitate cytoskeletal rearrangement within cytoskeletal structures such as Z-discs. Using a feline right ventricular pressure overload (RVPO) model, we measured the level and distribution of β-actin in normal and pressure overloaded myocardium. Resulting data demonstrated enriched levels of β-actin and enhanced translocation to the Triton-insoluble cytoskeletal and membrane skeletal complexes. In addition, RVPO in vivo and in vitro hypertrophic stimulation with endothelin (ET) or insulin in isolated adult cardiomyocytes enhanced the content of polymerized fraction (F-actin) of β-actin. To determine the localization and dynamics of β-actin, we adenovirally expressed GFP-tagged β-actin in isolated adult cardiomyocytes. The ectopically expressed β-actin-GFP localized to the Z-discs, costameres, and cell termini. Fluorescence recovery after photobleaching (FRAP) measurements of β-actin dynamics revealed that β-actin at the Z-discs is constantly being exchanged with β-actin from cytoplasmic pools and that this exchange is faster upon hypertrophic stimulation with ET or insulin. In addition, in electrically stimulated isolated adult cardiomyocytes, while β-actin overexpression improved cardiomyocyte contractility, immunoneutralization of β-actin resulted in a reduced contractility suggesting that β-actin could be important for the contractile function of adult cardiomyocytes. These studies demonstrate the presence and dynamics of β-actin in the adult cardiomyocyte and reinforce its usefulness in measuring cardiac cytoskeletal rearrangement during hypertrophic stimulation. PMID:20635003

Hypertrophic stimulation increases beta-actin dynamics in adult feline cardiomyocytes.

PubMed

Balasubramanian, Sundaravadivel; Mani, Santhosh K; Kasiganesan, Harinath; Baicu, Catalin C; Kuppuswamy, Dhandapani

2010-07-12

The myocardium responds to hemodynamic stress through cellular growth and organ hypertrophy. The impact of cytoskeletal elements on this process, however, is not fully understood. While alpha-actin in cardiomyocytes governs muscle contraction in combination with the myosin motor, the exact role of beta-actin has not been established. We hypothesized that in adult cardiomyocytes, as in non-myocytes, beta-actin can facilitate cytoskeletal rearrangement within cytoskeletal structures such as Z-discs. Using a feline right ventricular pressure overload (RVPO) model, we measured the level and distribution of beta-actin in normal and pressure overloaded myocardium. Resulting data demonstrated enriched levels of beta-actin and enhanced translocation to the Triton-insoluble cytoskeletal and membrane skeletal complexes. In addition, RVPO in vivo and in vitro hypertrophic stimulation with endothelin (ET) or insulin in isolated adult cardiomyocytes enhanced the content of polymerized fraction (F-actin) of beta-actin. To determine the localization and dynamics of beta-actin, we adenovirally expressed GFP-tagged beta-actin in isolated adult cardiomyocytes. The ectopically expressed beta-actin-GFP localized to the Z-discs, costameres, and cell termini. Fluorescence recovery after photobleaching (FRAP) measurements of beta-actin dynamics revealed that beta-actin at the Z-discs is constantly being exchanged with beta-actin from cytoplasmic pools and that this exchange is faster upon hypertrophic stimulation with ET or insulin. In addition, in electrically stimulated isolated adult cardiomyocytes, while beta-actin overexpression improved cardiomyocyte contractility, immunoneutralization of beta-actin resulted in a reduced contractility suggesting that beta-actin could be important for the contractile function of adult cardiomyocytes. These studies demonstrate the presence and dynamics of beta-actin in the adult cardiomyocyte and reinforce its usefulness in measuring cardiac cytoskeletal rearrangement during hypertrophic stimulation.

The C-terminus SH3-binding domain of Kv1.3 is required for the actin-mediated immobilization of the channel via cortactin

PubMed Central

Hajdu, Peter; Martin, Geoffrey V.; Chimote, Ameet A.; Szilagyi, Orsolya; Takimoto, Koichi; Conforti, Laura

2015-01-01

Kv1.3 channels play a pivotal role in the activation and migration of T-lymphocytes. These functions are accompanied by the channels' polarization, which is essential for associated downstream events. However, the mechanisms that govern the membrane movement of Kv1.3 channels remain unclear. F-actin polymerization occurs concomitantly to channel polarization, implicating the actin cytoskeleton in this process. Here we show that cortactin, a factor initiating the actin network, controls the membrane mobilization of Kv1.3 channels. FRAP with EGFP-tagged Kv1.3 channels demonstrates that knocking down cortactin decreases the actin-based immobilization of the channels. Using various deletion and mutation constructs, we show that the SH3 motif of Kv1.3 mediates the channel immobilization. Proximity ligation assays indicate that deletion or mutation of the SH3 motif also disrupts interaction of the channel with cortactin. In T-lymphocytes, the interaction between HS1 (the cortactin homologue) and Kv1.3 occurs at the immune synapse and requires the channel's C-terminal domain. These results show that actin dynamics regulates the membrane motility of Kv1.3 channels. They also provide evidence that the SH3 motif of the channel and cortactin plays key roles in this process. PMID:25739456

Discovery of functional interactions among actin regulators by analysis of image fluctuations in an unperturbed motile cell system.

PubMed

Isogai, Tadamoto; Danuser, Gaudenz

2018-05-26

Cell migration is driven by propulsive forces derived from polymerizing actin that pushes and extends the plasma membrane. The underlying actin network is constantly undergoing adaptation to new mechano-chemical environments and intracellular conditions. As such, mechanisms that regulate actin dynamics inherently contain multiple feedback loops and redundant pathways. Given the highly adaptable nature of such a system, studies that use only perturbation experiments (e.g. knockdowns, overexpression, pharmacological activation/inhibition, etc.) are challenged by the nonlinearity and redundancy of the pathway. In these pathway configurations, perturbation experiments at best describe the function(s) of a molecular component in an adapting (e.g. acutely drug-treated) or fully adapted (e.g. permanent gene silenced) cell system, where the targeted component now resides in a non-native equilibrium. Here, we propose how quantitative live-cell imaging and analysis of constitutive fluctuations of molecular activities can overcome these limitations. We highlight emerging actin filament barbed-end biology as a prime example of a complex, nonlinear molecular process that requires a fluctuation analytic approach, especially in an unperturbed cellular system, to decipher functional interactions of barbed-end regulators, actin polymerization and membrane protrusion.This article is part of the theme issue 'Self-organization in cell biology'. © 2018 The Author(s).

Two-Phase Acto-Cytosolic Fluid Flow in a Moving Keratocyte: A 2D Continuum Model.

PubMed

Nikmaneshi, M R; Firoozabadi, B; Saidi, M S

2015-09-01

The F-actin network and cytosol in the lamellipodia of crawling cells flow in a centripetal pattern and spout-like form, respectively. We have numerically studied this two-phase flow in the realistic geometry of a moving keratocyte. Cytosol has been treated as a low viscosity Newtonian fluid flowing through the high viscosity porous medium of F-actin network. Other involved phenomena including myosin activity, adhesion friction, and interphase interaction are also discussed to provide an overall view of this problem. Adopting a two-phase coupled model by myosin concentration, we have found new accurate perspectives of acto-cytosolic flow and pressure fields, myosin distribution, as well as the distribution of effective forces across the lamellipodia of a keratocyte with stationary shape. The order of magnitude method is also used to determine the contribution of forces in the internal dynamics of lamellipodia.

Molecular Motor-Induced Instabilities and Cross Linkers Determine Biopolymer Organization

PubMed Central

Smith, D.; Ziebert, F.; Humphrey, D.; Duggan, C.; Steinbeck, M.; Zimmermann, W.; Käs, J.

2007-01-01

All eukaryotic cells rely on the active self-organization of protein filaments to form a responsive intracellular cytoskeleton. The necessity of motility and reaction to stimuli additionally requires pathways that quickly and reversibly change cytoskeletal organization. While thermally driven order-disorder transitions are, from the viewpoint of physics, the most obvious method for controlling states of organization, the timescales necessary for effective cellular dynamics would require temperatures exceeding the physiologically viable temperature range. We report a mechanism whereby the molecular motor myosin II can cause near-instantaneous order-disorder transitions in reconstituted cytoskeletal actin solutions. When motor-induced filament sliding diminishes, the actin network structure rapidly and reversibly self-organizes into various assemblies. Addition of stable cross linkers was found to alter the architectures of ordered assemblies. These isothermal transitions between dynamic disorder and self-assembled ordered states illustrate that the interplay between passive crosslinking and molecular motor activity plays a substantial role in dynamic cellular organization. PMID:17604319

Spatiotemporal dynamics of actin remodeling and endomembrane trafficking in alveolar epithelial type I cell wound healing

PubMed Central

Godin, Lindsay M.; Vergen, Jorge; Prakash, Y. S.; Pagano, Richard E.

2011-01-01

Alveolar epithelial type I cell (ATI) wounding is prevalent in ventilator-injured lungs and likely contributes to pathogenesis of “barotrauma” and “biotrauma.” In experimental models most wounded alveolar cells repair plasma membrane (PM) defects and survive insults. Considering the force balance between edge energy at the PM wound margins and adhesive interactions of the lipid bilayer with the underlying cytoskeleton (CSK), we tested the hypothesis that subcortical actin depolymerization is a key facilitator of PM repair. Using real-time fluorescence imaging of primary rat ATI transfected with a live cell actin-green fluorescent protein construct (Lifeact-GFP) and loaded with N-rhodamine phosphatidylethanolamine (PE), we examined the spatial and temporal coordination between cytoskeletal remodeling and PM repair following micropuncture. Membrane integrity was inferred from the fluorescence intensity profiles of the cytosolic label calcein AM. Wounding led to rapid depolymerization of the actin CSK near the wound site, concurrent with accumulation of endomembrane-derived N-rhodamine PE. Both responses were sustained until PM integrity was reestablished, which typically occurs between ∼10 and 40 s after micropuncture. Only thereafter did the actin CSK near the wound begin to repolymerize, while the rate of endomembrane lipid accumulation decreased. Between 60 and 90 s after successful PM repair, after translocation of the actin nucleation factor cortactin, a dense actin fiber network formed. In cells that did not survive micropuncture injury, actin remodeling did not occur. These novel results highlight the importance of actin remodeling in ATI cell repair and suggest molecular targets for modulating the repair process. PMID:21216977

Initiation of DNA replication requires actin dynamics and formin activity.

PubMed

Parisis, Nikolaos; Krasinska, Liliana; Harker, Bethany; Urbach, Serge; Rossignol, Michel; Camasses, Alain; Dewar, James; Morin, Nathalie; Fisher, Daniel

2017-11-02

Nuclear actin regulates transcriptional programmes in a manner dependent on its levels and polymerisation state. This dynamics is determined by the balance of nucleocytoplasmic shuttling, formin- and redox-dependent filament polymerisation. Here, using Xenopus egg extracts and human somatic cells, we show that actin dynamics and formins are essential for DNA replication. In proliferating cells, formin inhibition abolishes nuclear transport and initiation of DNA replication, as well as general transcription. In replicating nuclei from transcriptionally silent Xenopus egg extracts, we identified numerous actin regulators, and disruption of actin dynamics abrogates nuclear transport, preventing NLS (nuclear localisation signal)-cargo release from RanGTP-importin complexes. Nuclear formin activity is further required to promote loading of cyclin-dependent kinase (CDK) and proliferating cell nuclear antigen (PCNA) onto chromatin, as well as initiation and elongation of DNA replication. Therefore, actin dynamics and formins control DNA replication by multiple direct and indirect mechanisms. © 2017 The Authors.

Diffusion of GPI-anchored proteins is influenced by the activity of dynamic cortical actin

PubMed Central

Saha, Suvrajit; Lee, Il-Hyung; Polley, Anirban; Groves, Jay T.; Rao, Madan; Mayor, Satyajit

2015-01-01

Molecular diffusion at the surface of living cells is believed to be predominantly driven by thermal kicks. However, there is growing evidence that certain cell surface molecules are driven by the fluctuating dynamics of cortical cytoskeleton. Using fluorescence correlation spectroscopy, we measure the diffusion coefficient of a variety of cell surface molecules over a temperature range of 24–37°C. Exogenously incorporated fluorescent lipids with short acyl chains exhibit the expected increase of diffusion coefficient over this temperature range. In contrast, we find that GPI-anchored proteins exhibit temperature-independent diffusion over this range and revert to temperature-dependent diffusion on cell membrane blebs, in cells depleted of cholesterol, and upon acute perturbation of actin dynamics and myosin activity. A model transmembrane protein with a cytosolic actin-binding domain also exhibits the temperature-independent behavior, directly implicating the role of cortical actin. We show that diffusion of GPI-anchored proteins also becomes temperature dependent when the filamentous dynamic actin nucleator formin is inhibited. However, changes in cortical actin mesh size or perturbation of branched actin nucleator Arp2/3 do not affect this behavior. Thus cell surface diffusion of GPI-anchored proteins and transmembrane proteins that associate with actin is driven by active fluctuations of dynamic cortical actin filaments in addition to thermal fluctuations, consistent with expectations from an “active actin-membrane composite” cell surface. PMID:26378258

Live-cell imaging of G-actin dynamics using sequential FDAP

PubMed Central

Kiuchi, Tai; Nagai, Tomoaki; Ohashi, Kazumasa; Watanabe, Naoki; Mizuno, Kensaku

2011-01-01

Various microscopic techniques have been developed to understand the mechanisms that spatiotemporally control actin filament dynamics in live cells. Kinetic data on the processes of actin assembly and disassembly on F-actin have been accumulated. However, the kinetics of cytoplasmic G-actin, a key determinant for actin polymerization, has remained unclear because of a lack of appropriate methods to measure the G-actin concentration quantitatively. We have developed two new microscopic techniques based on the fluorescence decay after photoactivation (FDAP) time-lapse imaging of photoswitchable Dronpa-labeled actin. These techniques, sequential FDAP (s-FDAP) and multipoint FDAP, were used to measure the time-dependent changes in and spatial distribution of the G-actin concentration in live cells. Use of s-FDAP provided data on changes in the G-actin concentration with high temporal resolution; these data were useful for the model analysis of actin assembly processes in live cells. The s-FDAP analysis also provided evidence that the cytoplasmic G-actin concentration substantially decreases after cell stimulation and that the extent of stimulus-induced actin assembly and cell size extension are linearly correlated with the G-actin concentration before cell stimulation. The advantages of using s-FDAP and multipoint FDAP to measure spatiotemporal G-actin dynamics and the roles of G-actin concentration and ADF/cofilin in stimulus-induced actin assembly and lamellipodium extension in live cells are discussed. PMID:22754616

AtFH1 formin mutation affects actin filament and microtubule dynamics in Arabidopsis thaliana.

PubMed

Rosero, Amparo; Žársky, Viktor; Cvrčková, Fatima

2013-01-01

Plant cell growth and morphogenesis depend on remodelling of both actin and microtubule cytoskeletons. AtFH1 (At5g25500), the main housekeeping Arabidopsis formin, is targeted to membranes and known to nucleate and bundle actin. The effect of mutations in AtFH1 on root development and cytoskeletal dynamics was examined. Consistent with primarily actin-related formin function, fh1 mutants showed increased sensitivity to the actin polymerization inhibitor latrunculin B (LatB). LatB-treated mutants had thicker, shorter roots than wild-type plants. Reduced cell elongation and morphological abnormalities were observed in both trichoblasts and atrichoblasts. Fluorescently tagged cytoskeletal markers were used to follow cytoskeletal dynamics in wild-type and mutant plants using confocal microscopy and VAEM (variable-angle epifluorescence microscopy). Mutants exhibited more abundant but less dynamic F-actin bundles and more dynamic microtubules than wild-type seedlings. Treatment of wild-type seedlings with a formin inhibitor, SMIFH2, mimicked the root growth and cell expansion phenotypes and cytoskeletal structure alterations observed in fh1 mutants. The results suggest that besides direct effects on actin organization, the in vivo role of AtFH1 also includes modulation of microtubule dynamics, possibly mediated by actin-microtubule cross-talk.

Plant actin cytoskeleton re-modeling by plant parasitic nematodes.

PubMed

Engler, Janice de Almeida; Rodiuc, Natalia; Smertenko, Andrei; Abad, Pierre

2010-03-01

The cytoskeleton is an important component of the plant's defense mechanism against the attack of pathogenic organisms. Plants however, are defenseless against parasitic root-knot and cyst nematodes and respond to the invasion by the development of a special feeding site that supplies the parasite with nutrients required for the completion of its life cycle. Recent studies of nematode invasion under treatment with cytoskeletal drugs and in mutant plants where normal functions of the cytoskeleton have been affected, demonstrate the importance of the cytoskeleton in the establishment of a feeding site and successful nematode reproduction. It appears that in the case of microfilaments, nematodes hijack the intracellular machinery that regulates actin dynamics and modulate the organization and properties of the actin filament network. Intervening with this process reduces the nematode infection efficiency and inhibits its life cycle. This discovery uncovers a new pathway that can be exploited for the protection of plants against nematodes.

The Abl-related gene (Arg) requires its F-actin-microtubule cross-linking activity to regulate lamellipodial dynamics during fibroblast adhesion.

PubMed

Miller, Ann L; Wang, Yinxiang; Mooseker, Mark S; Koleske, Anthony J

2004-05-10

Microtubules (MTs) help establish and maintain cell polarity by promoting actin-dependent membrane protrusion at the leading edge of the cell, but the molecular mechanisms that mediate cross-talk between actin and MTs during this process are unclear. We demonstrate that the Abl-related gene (Arg) nonreceptor tyrosine kinase is required for dynamic lamellipodial protrusions after adhesion to fibronectin. arg-/- fibroblasts exhibit reduced lamellipodial dynamics as compared with wild-type fibroblasts, and this defect can be rescued by reexpression of an Arg-yellow fluorescent protein fusion. We show that Arg can bind MTs with high affinity and cross-link filamentous actin (F-actin) bundles and MTs in vitro. MTs concentrate and insert into Arg-induced F-actin-rich cell protrusions. Arg requires both its F-actin-binding domains and its MT-binding domain to rescue the defects in lamellipodial dynamics of arg-/- fibroblasts. These findings demonstrate that Arg can mediate physical contact between F-actin and MTs at the cell periphery and that this cross-linking activity is required for Arg to regulate lamellipodial dynamics in fibroblasts. Copyright the Rockefeller University Press

Changes in actin structural transitions associated with oxidative inhibition of muscle contraction.

PubMed

Prochniewicz, Ewa; Spakowicz, Daniel; Thomas, David D

2008-11-11

We have used transient phosphorescence anisotropy (TPA) to detect changes in actin structural dynamics associated with oxidative inhibition of muscle contraction. Contractility of skinned rabbit psoas muscle fibers was inhibited by treatment with 50 mM H 2O 2, which induced oxidative modifications in the myosin head and in actin, as previously reported. Using proteins purified from oxidized and unoxidized muscle, we used TPA to measure the effects of weakly (+ATP) and strongly (no ATP) bound myosin heads (S1) on the microsecond dynamics of actin labeled at Cys374 with erythrosine iodoacetamide. Oxidative modification of S1 had no effect on actin dynamics in the absence of ATP (strong binding complex), but restricted the dynamics in the presence of ATP (weakly bound complex). In contrast, oxidative modification of actin did not have a significant effect on the weak-to-strong transitions. Thus, we concluded that (1) the effects of oxidation on the dynamics of actin in the actomyosin complex are predominantly determined by oxidation-induced changes in S1, and (2) changes in weak-to-strong structural transitions in actin and myosin are coupled to each other and are associated with oxidative inhibition of muscle contractility.

Changes in actin structural transitions associated with oxidative inhibition of muscle contraction

PubMed Central

Prochniewicz, Ewa; Spakowicz, Daniel; Thomas, David D.

2011-01-01

We have used transient phosphorescence anisotropy (TPA) to detect changes in actin structural dynamics associated with oxidative inhibition of muscle contraction. Contractility of skinned rabbit psoas muscle fibers was inhibited by treatment with 50 mM H2O2, which induced oxidative modifications in the myosin head and in actin, as previously reported. Using proteins purified from oxidized and unoxidized muscle, we used TPA to measure the effects of weakly (+ATP) and strongly (no ATP) bound myosin heads (S1) on the microsecond dynamics of actin labeled at Cys374 with erythrosine iodoacetamide. Oxidative modification of S1 had no effect on actin dynamics in the absence of ATP (strong binding complex), but restricted the dynamics in the presence of ATP (weakly bound complex). In contrast, oxidative modification of actin did not have a significant effect on the weak-to-strong transitions. Thus, we concluded that (1) the effects of oxidation on the dynamics of actin in the actomyosin complex are predominantly determined by oxidation-induced changes in S1, and (2) changes in weak-to-strong structural transitions in actin and myosin are coupled to each other and are associated with oxidative inhibition of muscle contractility. PMID:18855423

Smooth muscle length adaptation and actin filament length: a network model of the cytoskeletal dysregulation.

PubMed

Silveira, Paulo S P; Fredberg, Jeffrey J

2005-10-01

Length adaptation of the airway smooth muscle cell is attributable to cytoskeletal remodeling. It has been proposed that dysregulated actin filaments may become longer in asthma, and that such elongation would prevent a parallel-to-series transition of contractile units, thus precluding the well-known beneficial effects of deep inspirations and tidal breathing. To test the potential effect that actin filament elongation could have in overall muscle mechanics, we present an extremely simple model. The cytoskeleton is represented as a 2-D network of links (contractile filaments) connecting nodes (adhesion plaques). Such a network evolves in discrete time steps by forming and dissolving links in a stochastic fashion. Links are formed by idealized contractile units whose properties are either those from normal or elongated actin filaments. Oscillations were then imposed on the network to evaluate both the effects of breathing and length adaptation. In response to length oscillation, a network with longer actin filaments showed smaller decreases of force, smaller increases in compliance, and higher shortening velocities. Taken together, these changes correspond to a network that is refractory to the effects of breathing and therefore approximates an asthmatic scenario. Thus, an extremely simple model seems to capture some relatively complex mechanics of airway smooth muscle, supporting the idea that dysregulation of actin filament length may contribute to excessive airway narrowing.

3D Actin Network Centerline Extraction with Multiple Active Contours

PubMed Central

Xu, Ting; Vavylonis, Dimitrios; Huang, Xiaolei

2013-01-01

Fluorescence microscopy is frequently used to study two and three dimensional network structures formed by cytoskeletal polymer fibers such as actin filaments and actin cables. While these cytoskeletal structures are often dilute enough to allow imaging of individual filaments or bundles of them, quantitative analysis of these images is challenging. To facilitate quantitative, reproducible and objective analysis of the image data, we propose a semi-automated method to extract actin networks and retrieve their topology in 3D. Our method uses multiple Stretching Open Active Contours (SOACs) that are automatically initialized at image intensity ridges and then evolve along the centerlines of filaments in the network. SOACs can merge, stop at junctions, and reconfigure with others to allow smooth crossing at junctions of filaments. The proposed approach is generally applicable to images of curvilinear networks with low SNR. We demonstrate its potential by extracting the centerlines of synthetic meshwork images, actin networks in 2D Total Internal Reflection Fluorescence Microscopy images, and 3D actin cable meshworks of live fission yeast cells imaged by spinning disk confocal microscopy. Quantitative evaluation of the method using synthetic images shows that for images with SNR above 5.0, the average vertex error measured by the distance between our result and ground truth is 1 voxel, and the average Hausdorff distance is below 10 voxels. PMID:24316442

Nonequilibrium dynamics of probe filaments in actin-myosin networks

NASA Astrophysics Data System (ADS)

Gladrow, J.; Broedersz, C. P.; Schmidt, C. F.

2017-08-01

Active dynamic processes of cells are largely driven by the cytoskeleton, a complex and adaptable semiflexible polymer network, motorized by mechanoenzymes. Small dimensions, confined geometries, and hierarchical structures make it challenging to probe dynamics and mechanical response of such networks. Embedded semiflexible probe polymers can serve as nonperturbing multiscale probes to detect force distributions in active polymer networks. We show here that motor-induced forces transmitted to the probe polymers are reflected in nonequilibrium bending dynamics, which we analyze in terms of spatial eigenmodes of an elastic beam under steady-state conditions. We demonstrate how these active forces induce correlations among the mode amplitudes, which furthermore break time-reversal symmetry. This leads to a breaking of detailed balance in this mode space. We derive analytical predictions for the magnitude of resulting probability currents in mode space in the white-noise limit of motor activity. We relate the structure of these currents to the spatial profile of motor-induced forces along the probe polymers and provide a general relation for observable currents on two-dimensional hyperplanes.

Actin filaments as tension sensors.

PubMed

Galkin, Vitold E; Orlova, Albina; Egelman, Edward H

2012-02-07

The field of mechanobiology has witnessed an explosive growth over the past several years as interest has greatly increased in understanding how mechanical forces are transduced by cells and how cells migrate, adhere and generate traction. Actin, a highly abundant and anomalously conserved protein, plays a large role in forming the dynamic cytoskeleton that is so essential for cell form, motility and mechanosensitivity. While the actin filament (F-actin) has been viewed as dynamic in terms of polymerization and depolymerization, new results suggest that F-actin itself may function as a highly dynamic tension sensor. This property may help explain the unusual conservation of actin's sequence, as well as shed further light on actin's essential role in structures from sarcomeres to stress fibers. Copyright © 2012 Elsevier Ltd. All rights reserved.

In Silico Reconstitution of Actin-Based Symmetry Breaking and Motility

PubMed Central

Dayel, Mark J.; Akin, Orkun; Landeryou, Mark; Risca, Viviana; Mogilner, Alex; Mullins, R. Dyche

2009-01-01

Eukaryotic cells assemble viscoelastic networks of crosslinked actin filaments to control their shape, mechanical properties, and motility. One important class of actin network is nucleated by the Arp2/3 complex and drives both membrane protrusion at the leading edge of motile cells and intracellular motility of pathogens such as Listeria monocytogenes. These networks can be reconstituted in vitro from purified components to drive the motility of spherical micron-sized beads. An Elastic Gel model has been successful in explaining how these networks break symmetry, but how they produce directed motile force has been less clear. We have combined numerical simulations with in vitro experiments to reconstitute the behavior of these motile actin networks in silico using an Accumulative Particle-Spring (APS) model that builds on the Elastic Gel model, and demonstrates simple intuitive mechanisms for both symmetry breaking and sustained motility. The APS model explains observed transitions between smooth and pulsatile motion as well as subtle variations in network architecture caused by differences in geometry and conditions. Our findings also explain sideways symmetry breaking and motility of elongated beads, and show that elastic recoil, though important for symmetry breaking and pulsatile motion, is not necessary for smooth directional motility. The APS model demonstrates how a small number of viscoelastic network parameters and construction rules suffice to recapture the complex behavior of motile actin networks. The fact that the model not only mirrors our in vitro observations, but also makes novel predictions that we confirm by experiment, suggests that the model captures much of the essence of actin-based motility in this system. PMID:19771152

Dissecting the contribution of actin and vimentin intermediate filaments to mechanical phenotype of suspended cells using high-throughput deformability measurements and computational modeling.

PubMed

Gladilin, Evgeny; Gonzalez, Paula; Eils, Roland

2014-08-22

Mechanical cell properties play an important role in many basic biological functions, including motility, adhesion, proliferation and differentiation. There is a growing body of evidence that the mechanical cell phenotype can be used for detection and, possibly, treatment of various diseases, including cancer. Understanding of pathological mechanisms requires investigation of the relationship between constitutive properties and major structural components of cells, i.e., the nucleus and cytoskeleton. While the contribution of actin und microtubules to cellular rheology has been extensively studied in the past, the role of intermediate filaments has been scarcely investigated up to now. Here, for the first time we compare the effects of drug-induced disruption of actin and vimentin intermediate filaments on mechanical properties of suspended NK cells using high-throughput deformability measurements and computational modeling. Although, molecular mechanisms of actin and vimentin disruption by the applied cytoskeletal drugs, Cytochalasin-D and Withaferin-A, are different, cell softening in both cases can be attributed to reduction of the effective density and stiffness of filament networks. Our experimental data suggest that actin and vimentin deficient cells exhibit, in average, 41% and 20% higher deformability in comparison to untreated control. 3D Finite Element simulation is performed to quantify the contribution of cortical actin and perinuclear vimentin to mechanical phenotype of the whole cell. Our simulation provides quantitative estimates for decreased filament stiffness in drug-treated cells and predicts more than two-fold increase of the strain magnitude in the perinuclear vimentin layer of actin deficient cells relatively to untreated control. Thus, the mechanical function of vimentin becomes particularly essential in motile and proliferating cells that have to dynamically remodel the cortical actin network. These insights add functional cues to frequently observed overexpression of vimentin in diverse types of cancer and underline the role of vimentin targeting drugs, such as Withaferin-A, as a potent cancerostatic supplement. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

NETWORKED 3B: a novel protein in the actin cytoskeleton-endoplasmic reticulum interaction.

PubMed

Wang, Pengwei; Hussey, Patrick J

2017-03-01

In plants movement of the endoplasmic reticulum (ER) is dependent on the actin cytoskeleton. However little is known about proteins that link the ER membrane and the actin cytoskeleton. Here we identified a novel protein, NETWORKED 3B (NET3B), which is associated with the ER and actin cytoskeleton in vivo. NET3B belongs to a superfamily of plant specific actin binding proteins, the NETWORKED family. NET3B associates with the actin cytoskeleton in vivo through an N-terminal NET actin binding (NAB) domain, which has been well-characterized in other members of the NET family. A three amino acid insertion, Val-Glu-Asp, in the NAB domain of NET3B appears to lower its ability to localize to the actin cytoskeleton compared with NET1A, the founding member of the NET family. The C-terminal domain of NET3B links the protein to the ER. Overexpression of NET3B enhanced the association between the ER and the actin cytoskeleton, and the extent of this association was dependent on the amount of NET3B available. Another effect of NET3B overexpression was a reduction in ER membrane diffusion. In conclusion, our results revealed that NET3B modulates ER and actin cytoskeleton interactions in higher plants. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Simultaneous quantification of actin monomer and filament dynamics with modeling-assisted analysis of photoactivation

PubMed Central

Kapustina, Maryna; Read, Tracy-Ann

2016-01-01

ABSTRACT Photoactivation allows one to pulse-label molecules and obtain quantitative data about their behavior. We have devised a new modeling-based analysis for photoactivatable actin experiments that simultaneously measures properties of monomeric and filamentous actin in a three-dimensional cellular environment. We use this method to determine differences in the dynamic behavior of β- and γ-actin isoforms, showing that both inhabit filaments that depolymerize at equal rates but that β-actin exists in a higher monomer-to-filament ratio. We also demonstrate that cofilin (cofilin 1) equally accelerates depolymerization of filaments made from both isoforms, but is only required to maintain the β-actin monomer pool. Finally, we used modeling-based analysis to assess actin dynamics in axon-like projections of differentiating neuroblastoma cells, showing that the actin monomer concentration is significantly depleted as the axon develops. Importantly, these results would not have been obtained using traditional half-time analysis. Given that parameters of the publicly available modeling platform can be adjusted to suit the experimental system of the user, this method can easily be used to quantify actin dynamics in many different cell types and subcellular compartments. PMID:27831495

Long-range self-organization of cytoskeletal myosin II filament stacks.

PubMed

Hu, Shiqiong; Dasbiswas, Kinjal; Guo, Zhenhuan; Tee, Yee-Han; Thiagarajan, Visalatchi; Hersen, Pascal; Chew, Teng-Leong; Safran, Samuel A; Zaidel-Bar, Ronen; Bershadsky, Alexander D

2017-02-01

Although myosin II filaments are known to exist in non-muscle cells, their dynamics and organization are incompletely understood. Here, we combined structured illumination microscopy with pharmacological and genetic perturbations, to study the process of actomyosin cytoskeleton self-organization into arcs and stress fibres. A striking feature of the myosin II filament organization was their 'registered' alignment into stacks, spanning up to several micrometres in the direction orthogonal to the parallel actin bundles. While turnover of individual myosin II filaments was fast (characteristic half-life time 60 s) and independent of actin filament turnover, the process of stack formation lasted a longer time (in the range of several minutes) and required myosin II contractility, as well as actin filament assembly/disassembly and crosslinking (dependent on formin Fmnl3, cofilin1 and α-actinin-4). Furthermore, myosin filament stack formation involved long-range movements of individual myosin filaments towards each other suggesting the existence of attractive forces between myosin II filaments. These forces, possibly transmitted via mechanical deformations of the intervening actin filament network, may in turn remodel the actomyosin cytoskeleton and drive its self-organization.

Expanded experimental parameter space of semiflexible polymer assemblies through programmable nanomaterials

NASA Astrophysics Data System (ADS)

Smith, David; Schuldt, Carsten; Lorenz, Jessica; Tschirner, Teresa; Moebius-Winkler, Maximilian; Kaes, Josef; Glaser, Martin; Haendler, Tina; Schnauss, Joerg

2015-03-01

Biologically evolved materials are often used as inspiration in the development of new materials as well as examinations into the underlying physical principles governing their behavior. For instance, the biopolymer constituents of the highly dynamic cellular cytoskeleton such as actin have inspired a deep understanding of soft polymer-based materials. However, the molecular toolbox provided by biological systems has been evolutionarily optimized to carry out the necessary functions of cells, and the inability modify basic properties such as biopolymer stiffness hinders a meticulous examination of parameter space. Using actin as inspiration, we circumvent these limitations using model systems assembled from programmable materials such as DNA. Nanorods with comparable, but controllable dimensions and mechanical properties as actin can be constructed from small sets of specially designed DNA strands. In entangled gels, these allow us to systematically determine the dependence of network mechanical properties on parameters such as persistence length and crosslink strength. At higher concentrations in the presence of local attractive forces, we see a transition to highly-ordered bundled and ``aster'' phases similar to those previously characterized in systems of actin or microtubules.

Membrane related dynamics and the formation of actin in cells growing on micro-topographies: a spatial computational model.

PubMed

Bittig, Arne T; Matschegewski, Claudia; Nebe, J Barbara; Stählke, Susanne; Uhrmacher, Adelinde M

2014-09-09

Intra-cellular processes of cells at the interface to an implant surface are influenced significantly by their extra-cellular surrounding. Specifically, when growing osteoblasts on titanium surfaces with regular micro-ranged geometry, filaments are shorter, less aligned and they concentrate at the top of the geometric structures. Changes to the cytoskeleton network, i. e., its localization, alignment, orientation, and lengths of the filaments, as well as the overall concentration and distribution of key-actors are induced. For example, integrin is distributed homogeneously, whereas integrin in activated state and vinculin, both components of focal adhesions, have been found clustered on the micro-ranged geometries. Also, the concentration of Rho, an intracellular signaling protein related to focal adhesion regulation, was significantly lower. To explore whether regulations associated with the focal adhesion complex can be responsible for the changed actin filament patterns, a spatial computational model has been developed using ML-Space, a rule-based model description language, and its associated Brownian-motion-based simulator. The focus has been on the deactivation of cofilin in the vicinity of the focal adhesion complex. The results underline the importance of sensing mechanisms to support a clustering of actin filament nucleations on the micro-ranged geometries, and of intracellular diffusion processes, which lead to spatially heterogeneous distributions of active (dephosphorylated) cofilin, which in turn influences the organization of the actin network. We find, for example, that the spatial heterogeneity of key molecular actors can explain the difference in filament lengths in cells on different micro-geometries partly, but to explain the full extent, further model assumptions need to be added and experimentally validated. In particular, our findings and hypothesis referring to the role, distribution, and amount of active cofilin have still to be verified in wet-lab experiments. Letting cells grow on surface structures is a possibility to shed new light on the intricate mechanisms that relate membrane and actin related dynamics in the cell. Our results demonstrate the need for declarative expressive spatial modeling approaches that allow probing different hypotheses, and the central role of the focal adhesion complex not only for nucleating actin filaments, but also for regulating possible severing agents locally.

Membrane related dynamics and the formation of actin in cells growing on micro-topographies: a spatial computational model

PubMed Central

2014-01-01

Background Intra-cellular processes of cells at the interface to an implant surface are influenced significantly by their extra-cellular surrounding. Specifically, when growing osteoblasts on titanium surfaces with regular micro-ranged geometry, filaments are shorter, less aligned and they concentrate at the top of the geometric structures. Changes to the cytoskeleton network, i. e., its localization, alignment, orientation, and lengths of the filaments, as well as the overall concentration and distribution of key-actors are induced. For example, integrin is distributed homogeneously, whereas integrin in activated state and vinculin, both components of focal adhesions, have been found clustered on the micro-ranged geometries. Also, the concentration of Rho, an intracellular signaling protein related to focal adhesion regulation, was significantly lower. Results To explore whether regulations associated with the focal adhesion complex can be responsible for the changed actin filament patterns, a spatial computational model has been developed using ML-Space, a rule-based model description language, and its associated Brownian-motion-based simulator. The focus has been on the deactivation of cofilin in the vicinity of the focal adhesion complex. The results underline the importance of sensing mechanisms to support a clustering of actin filament nucleations on the micro-ranged geometries, and of intracellular diffusion processes, which lead to spatially heterogeneous distributions of active (dephosphorylated) cofilin, which in turn influences the organization of the actin network. We find, for example, that the spatial heterogeneity of key molecular actors can explain the difference in filament lengths in cells on different micro-geometries partly, but to explain the full extent, further model assumptions need to be added and experimentally validated. In particular, our findings and hypothesis referring to the role, distribution, and amount of active cofilin have still to be verified in wet-lab experiments. Conclusion Letting cells grow on surface structures is a possibility to shed new light on the intricate mechanisms that relate membrane and actin related dynamics in the cell. Our results demonstrate the need for declarative expressive spatial modeling approaches that allow probing different hypotheses, and the central role of the focal adhesion complex not only for nucleating actin filaments, but also for regulating possible severing agents locally. PMID:25200251

Cofilin 1-Mediated Biphasic F-Actin Dynamics of Neuronal Cells Affect Herpes Simplex Virus 1 Infection and Replication

PubMed Central

Xiang, Yangfei; Zheng, Kai; Ju, Huaiqiang; Wang, Shaoxiang; Pei, Ying; Ding, Weichao; Chen, Zhenping; Wang, Qiaoli; Qiu, Xianxiu; Zhong, Meigong; Zeng, Fanli; Ren, Zhe; Qian, Chuiwen; Liu, Ge

2012-01-01

Herpes simplex virus 1 (HSV-1) invades the nervous system and causes pathological changes. In this study, we defined the remodeling of F-actin and its possible mechanisms during HSV-1 infection of neuronal cells. HSV-1 infection enhanced the formation of F-actin-based structures in the early stage of infection, which was followed by a continuous decrease in F-actin during the later stages of infection. The disruption of F-actin dynamics by chemical inhibitors significantly reduced the efficiency of viral infection and intracellular HSV-1 replication. The active form of the actin-depolymerizing factor cofilin 1 was found to increase at an early stage of infection and then to continuously decrease in a manner that corresponded to the remodeling pattern of F-actin, suggesting that cofilin 1 may be involved in the biphasic F-actin dynamics induced by HSV-1 infection. Knockdown of cofilin 1 impaired HSV-1-induced F-actin assembly during early infection and inhibited viral entry; however, overexpression of cofilin 1 did not affect F-actin assembly or viral entry during early infection but decreased intracellular viral reproduction efficiently. Our results, for the first time, demonstrated the biphasic F-actin dynamics in HSV-1 neuronal infection and confirmed the association of F-actin with the changes in the expression and activity of cofilin 1. These results may provide insight into the mechanism by which HSV-1 productively infects neuronal cells and causes pathogenesis. PMID:22623803

Quantitative Analysis of Filament Branch Orientation in Listeria Actin Comet Tails.

PubMed

Jasnin, Marion; Crevenna, Alvaro H

2016-02-23

Several bacterial and viral pathogens hijack the host actin cytoskeleton machinery to facilitate spread and infection. In particular, Listeria uses Arp2/3-mediated actin filament nucleation at the bacterial surface to generate a branched network that will help propel the bacteria. However, the mechanism of force generation remains elusive due to the lack of high-resolution three-dimensional structural data on the spatial organization of the actin mother and daughter (i.e., branch) filaments within this network. Here, we have explored the three-dimensional structure of Listeria actin tails in Xenopus laevis egg extracts using cryo-electron tomography. We found that the architecture of Listeria actin tails is shared between those formed in cells and in cell extracts. Both contained nanoscopic bundles along the plane of the substrate, where the bacterium lies, and upright filaments (also called Z filaments), both oriented tangentially to the bacterial cell wall. Here, we were able to identify actin filament intersections, which likely correspond to branches, within the tails. A quantitative analysis of putative Arp2/3-mediated branches in the actin network showed that mother filaments lie on the plane of the substrate, whereas daughter filaments have random deviations out of this plane. Moreover, the analysis revealed that branches are randomly oriented with respect to the bacterial surface. Therefore, the actin filament network does not push directly toward the surface but rather accumulates, building up stress around the Listeria surface. Our results favor a mechanism of force generation for Listeria movement where the stress is released into propulsive motion. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Actin filaments-A target for redox regulation.

PubMed

Wilson, Carlos; Terman, Jonathan R; González-Billault, Christian; Ahmed, Giasuddin

2016-10-01

Actin and its ability to polymerize into dynamic filaments is critical for the form and function of cells throughout the body. While multiple proteins have been characterized as affecting actin dynamics through noncovalent means, actin and its protein regulators are also susceptible to covalent modifications of their amino acid residues. In this regard, oxidation-reduction (Redox) intermediates have emerged as key modulators of the actin cytoskeleton with multiple different effects on cellular form and function. Here, we review work implicating Redox intermediates in post-translationally altering actin and discuss what is known regarding how these alterations affect the properties of actin. We also focus on two of the best characterized enzymatic sources of these Redox intermediates-the NADPH oxidase NOX and the flavoprotein monooxygenase MICAL-and detail how they have both been identified as altering actin, but share little similarity and employ different means to regulate actin dynamics. Finally, we discuss the role of these enzymes and redox signaling in regulating the actin cytoskeleton in vivo and highlight their importance for neuronal form and function in health and disease. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Actin filaments – a target for redox regulation

PubMed Central

Wilson, Carlos; Terman, Jonathan R.; González-Billault, Christian; Ahmed, Giasuddin

2016-01-01

Actin and its ability to polymerize into dynamic filaments is critical for the form and function of cells throughout the body. While multiple proteins have been characterized as affecting actin dynamics through non-covalent means, actin and its protein regulators are also susceptible to covalent modifications of their amino acid residues. In this regard, oxidation-reduction (Redox) intermediates have emerged as key modulators of the actin cytoskeleton with multiple different effects on cellular form and function. Here, we review work implicating Redox intermediates in post-translationally altering actin and discuss what is known regarding how these alterations affect the properties of actin. We also focus on two of the best characterized enzymatic sources of these Redox intermediates – the NADPH oxidase NOX and the flavoprotein monooxygenase MICAL – and detail how they have both been identified as altering actin, but share little similarity and employ different means to regulate actin dynamics. Finally, we discuss the role of these enzymes and redox signaling in regulating the actin cytoskeleton in vivo and highlight their importance for neuronal form and function in health and disease. PMID:27309342

Actin Polymerization is Stimulated by Actin Crosslinking Protein Palladin

PubMed Central

Gurung, Ritu; Yadav, Rahul; Brungardt, Joseph G.; Orlova, Albina; Egelman, Edward H.; Beck, Moriah R.

2016-01-01

The actin scaffold protein palladin regulates both normal cell migration and invasive cell motility, processes that require the coordinated regulation of actin dynamics. However, the potential effect of palladin on actin dynamics has remained elusive. Here we show that the actin binding immunoglobulin-like domain of palladin, which is directly responsible for both actin binding and bundling, also stimulates actin polymerization in vitro. Palladin eliminated the lag phase that is characteristic of the slow nucleation step of actin polymerization. Furthermore, palladin dramatically reduced depolymerization, slightly enhanced the elongation rate, and did not alter the critical concentration. Microscopy and in vitro crosslinking assays reveal differences in actin bundle architecture when palladin is incubated with actin before or after polymerization. These results suggest a model whereby palladin stimulates a polymerization-competent form of G-actin, akin to metal ions, either through charge neutralization or conformational changes. PMID:26607837

3D Filament Network Segmentation with Multiple Active Contours

NASA Astrophysics Data System (ADS)

Xu, Ting; Vavylonis, Dimitrios; Huang, Xiaolei

2014-03-01

Fluorescence microscopy is frequently used to study two and three dimensional network structures formed by cytoskeletal polymer fibers such as actin filaments and microtubules. While these cytoskeletal structures are often dilute enough to allow imaging of individual filaments or bundles of them, quantitative analysis of these images is challenging. To facilitate quantitative, reproducible and objective analysis of the image data, we developed a semi-automated method to extract actin networks and retrieve their topology in 3D. Our method uses multiple Stretching Open Active Contours (SOACs) that are automatically initialized at image intensity ridges and then evolve along the centerlines of filaments in the network. SOACs can merge, stop at junctions, and reconfigure with others to allow smooth crossing at junctions of filaments. The proposed approach is generally applicable to images of curvilinear networks with low SNR. We demonstrate its potential by extracting the centerlines of synthetic meshwork images, actin networks in 2D TIRF Microscopy images, and 3D actin cable meshworks of live fission yeast cells imaged by spinning disk confocal microscopy.

Internal dynamics of F-actin and myosin subfragment-1 studied by quasielastic neutron scattering

DOE Office of Scientific and Technical Information (OSTI.GOV)

Matsuo, Tatsuhito; Arata, Toshiaki; Oda, Toshiro

2015-04-10

Various biological functions related to cell motility are driven by the interaction between the partner proteins, actin and myosin. To obtain insights into how this interaction occurs, the internal dynamics of F-actin and myosin subfragment-1 (S1) were characterized by the quasielastic neutron scattering measurements on the solution samples of F-actin and S1. Contributions of the internal motions of the proteins to the scattering spectra were separated from those of the global macromolecular diffusion. Analysis of the spectra arising from the internal dynamics showed that the correlation times of the atomic motions were about two times shorter for F-actin than formore » S1, suggesting that F-actin fluctuates more rapidly than S1. It was also shown that the fraction of the immobile atoms is larger for S1 than for F-actin. These results suggest that F-actin actively facilitates the binding of myosin by utilizing the more frequent conformational fluctuations than those of S1. - Highlights: • We studied the internal dynamics of F-actin and myosin S1 by neutron scattering. • The correlation times of the atomic motions were smaller for F-actin than for S1. • The fraction of the immobile atoms was also smaller for F-actin than for S1. • Our results suggest that mobility of atoms in F-actin is higher than that in S1. • We propose that high flexibility of F-actin facilitates the binding of myosin.« less

Surface adsorption and hopping cause probe-size-dependent microrheology of actin networks

NASA Astrophysics Data System (ADS)

He, Jun; Tang, Jay X.

2011-04-01

A network of filaments formed primarily by the abundant cytoskeletal protein actin gives animal cells their shape and elasticity. The rheological properties of reconstituted actin networks have been studied by tracking micron-sized probe beads embedded within the networks. We investigate how microrheology depends on surface properties of probe particles by varying the stickiness of their surface. For this purpose, we chose carboxylate polystyrene (PS) beads, silica beads, bovine serum albumin (BSA) -coated PS beads, and polyethylene glycol (PEG) -grafted PS beads, which show descending stickiness to actin filaments, characterized by confocal imaging and microrheology. Probe size dependence of microrheology is observed for all four types of beads. For the slippery PEG beads, particle-tracking microrheology detects weaker networks using smaller beads, which tend to diffuse through the network by hopping from one confinement “cage” to another. This trend is reversed for the other three types of beads, for which microrheology measures stiffer networks for smaller beads due to physisorption of nearby filaments to the bead surface. We explain the probe size dependence with two simple models. We also evaluate depletion effect near nonadsorption bead surface using quantitative image analysis and discuss the possible impact of depletion on microrheology. Analysis of these effects is necessary in order to accurately define the actin network rheology both in vitro and in vivo.

Nonlinear photomechanics of nematic networks: upscaling microscopic behaviour to macroscopic deformation

NASA Astrophysics Data System (ADS)

Chung, Hayoung; Choi, Joonmyung; Yun, Jung-Hoon; Cho, Maenghyo

2016-02-01

A liquid crystal network whose chromophores are functionalized by photochromic dye exhibits light-induced mechanical behaviour. As a result, the micro-scaled thermotropic traits of the network and the macroscopic phase behaviour are both influenced as light alternates the shape of the dyes. In this paper, we present an analysis of this photomechanical behaviour based on the proposed multiscale framework, which incorporates the molecular details of microstate evolution into a continuum-based understanding. The effects of trans-to-cis photoisomerization driven by actinic light irradiation are first examined using molecular dynamics simulations, and are compared against the predictions of the classical dilution model; this reveals certain characteristics of mesogenic interaction upon isomerization, followed by changes in the polymeric structure. We then upscale the thermotropic phase-related information with the aid of a nonlinear finite element analysis; macroscopic deflection with respect to the wide ranges of temperature and actinic light intensity are thereby examined, which reveals that the classical model underestimates the true deformation. This work therefore provides measures for analysing photomechanics in general by bridging the gap between the micro- and macro-scales.

The Tyrosine Kinase Activity of c-Src Regulates Actin Dynamics and Organization of Podosomes in Osteoclasts

PubMed Central

Destaing, Olivier; Sanjay, Archana; Itzstein, Cecile; Horne, William C.; Toomre, Derek

2008-01-01

Podosomes are dynamic actin-rich structures composed of a dense F-actin core surrounded by a cloud of more diffuse F-actin. Src performs one or more unique functions in osteoclasts (OCLs), and podosome belts and bone resorption are impaired in the absence of Src. Using Src−/− OCLs, we investigated the specific functions of Src in the organization and dynamics of podosomes. We found that podosome number and the podosome-associated actin cloud were decreased in Src−/− OCLs. Videomicroscopy and fluorescence recovery after photobleaching analysis revealed that the life span of Src−/− podosomes was increased fourfold and that the rate of actin flux in the core was decreased by 40%. Thus, Src regulates the formation, structure, life span, and rate of actin polymerization in podosomes and in the actin cloud. Rescue of Src−/− OCLs with Src mutants showed that both the kinase activity and either the SH2 or the SH3 binding domain are required for Src to restore normal podosome organization and dynamics. Moreover, inhibition of Src family kinase activities in Src−/− OCLs by Src inhibitors or by expressing dominant-negative SrcK295M induced the formation of abnormal podosomes. Thus, Src is an essential regulator of podosome structure, dynamics and organization. PMID:17978100

Myosin II Motor Activity in the Lateral Amygdala Is Required for Fear Memory Consolidation

ERIC Educational Resources Information Center

Gavin, Cristin F.; Rubio, Maria D.; Young, Erica; Miller, Courtney; Rumbaugh, Gavin

2012-01-01

Learning induces dynamic changes to the actin cytoskeleton that are required to support memory formation. However, the molecular mechanisms that mediate filamentous actin (F-actin) dynamics during learning and memory are poorly understood. Myosin II motors are highly expressed in actin-rich growth structures including dendritic spines, and we have…

Mutations in actin used for structural studies partially disrupt β-thymosin/WH2 domains interaction.

PubMed

Deville, Célia; Girard-Blanc, Christine; Assrir, Nadine; Nhiri, Naïma; Jacquet, Eric; Bontems, François; Renault, Louis; Petres, Stéphane; van Heijenoort, Carine

2016-10-01

Understanding the structural basis of actin cytoskeleton remodeling requires stabilization of actin monomers, oligomers, and filaments in complex with partner proteins, using various biochemical strategies. Here, we report a dramatic destabilization of the dynamic interaction with a model β-thymosin/WH2 domain induced by mutations in actin. This result underlines that mutant actins should be used with prudence to characterize interactions with intrinsically disordered partners as destabilization of dynamic interactions, although identifiable by NMR, may be invisible to other structural techniques. It also highlights how both β-thymosin/WH2 domains and actin tune local structure and dynamics in regulatory processes involving intrinsically disordered domains. © 2016 Federation of European Biochemical Societies.

How actin crosslinking and bundling proteins cooperate to generate an enhanced cell mechanical response

NASA Technical Reports Server (NTRS)

Tseng, Yiider; Kole, Thomas P.; Lee, Jerry S H.; Fedorov, Elena; Almo, Steven C.; Schafer, Benjamin W.; Wirtz, Denis

2005-01-01

Actin-crosslinking proteins organize actin filaments into dynamic and complex subcellular scaffolds that orchestrate important mechanical functions, including cell motility and adhesion. Recent mutation studies have shown that individual crosslinking proteins often play seemingly non-essential roles, leading to the hypothesis that they have considerable redundancy in function. We report live-cell, in vitro, and theoretical studies testing the mechanical role of the two ubiquitous actin-crosslinking proteins, alpha-actinin and fascin, which co-localize to stress fibers and the basis of filopodia. Using live-cell particle tracking microrheology, we show that the addition of alpha-actinin and fascin elicits a cell mechanical response that is significantly greater than that originated by alpha-actinin or fascin alone. These live-cell measurements are supported by quantitative rheological measurements with reconstituted actin filament networks containing pure proteins that show that alpha-actinin and fascin can work in concert to generate enhanced cell stiffness. Computational simulations using finite element modeling qualitatively reproduce and explain the functional synergy of alpha-actinin and fascin. These findings highlight the cooperative activity of fascin and alpha-actinin and provide a strong rationale that an evolutionary advantage might be conferred by the cooperative action of multiple actin-crosslinking proteins with overlapping but non-identical biochemical properties. Thus the combination of structural proteins with similar function can provide the cell with unique properties that are required for biologically optimal responses.

An Actin Depolymerizing Factor from the Halophyte Smooth Cordgrass, Spartina alterniflora (SaADF2) is Superior to its Rice homolog (OsADF2) in Conferring Drought and Salt Tolerance when Constitutively Overexpressed in Rice.

PubMed

Sengupta, Sonali; Mangu, Venkata; Sanchez, Luis; Bedre, Renesh; Joshi, Rohit; Rajasekaran, Kanniah; Baisakh, Niranjan

2018-05-31

Actin depolymerizing factors (ADFs) maintain the cellular actin network dynamics by regulating severing and disassembly of actin filaments in response to environmental cues. An ADF isolated from a monocot halophyte, Spartina alterniflora (SaADF2) imparted significantly higher level of drought and salinity tolerance when expressed in rice than its rice homologue OsADF2. SaADF2 differs from OsADF2 by a few amino acid residues, including a substitution in the regulatory phosphorylation site serine-6, which accounted for its weak interaction with OsCDPK6 (calcium dependent protein kinase), thus resulting in an increased efficacy of SaADF2 and enhanced cellular actin dynamics. SaADF2 overexpression preserved the actin filament organization better in rice protoplasts under desiccation stress. The predicted tertiary structure of SaADF2 showed a longer F-loop than OsADF2 that could have contributed to higher actin-binding affinity and rapid F-actin depolymerization in vitro by SaADF2. Rice transgenics constitutively overexpressing SaADF2 (SaADF2-OE) showed better growth, relative water content, and photosynthetic and agronomic yield under drought conditions than wild-type (WT) and OsADF2 overexpressers (OsADF2-OE). SaADF2-OE preserved intact grana structure after prolonged drought stress, whereas WT and OsADF2-OE presented highly damaged and disorganized grana stacking. The possible role of ADF2 in transactivation was hypothesized from the comparative transcriptome analyses, which showed significant differential expression of stress-related genes including interacting partners of ADF2 in overexpressers. Identification of a complex, differential interactome decorating or regulating stress-modulated cytoskeleton driven by ADF isoforms will lead us to key pathways that could be potential target for genome engineering to improve abiotic stress tolerance in agricultural crops. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

AtFH1 formin mutation affects actin filament and microtubule dynamics in Arabidopsis thaliana

PubMed Central

Cvrčková, Fatima

2013-01-01

Plant cell growth and morphogenesis depend on remodelling of both actin and microtubule cytoskeletons. AtFH1 (At5g25500), the main housekeeping Arabidopsis formin, is targeted to membranes and known to nucleate and bundle actin. The effect of mutations in AtFH1 on root development and cytoskeletal dynamics was examined. Consistent with primarily actin-related formin function, fh1 mutants showed increased sensitivity to the actin polymerization inhibitor latrunculin B (LatB). LatB-treated mutants had thicker, shorter roots than wild-type plants. Reduced cell elongation and morphological abnormalities were observed in both trichoblasts and atrichoblasts. Fluorescently tagged cytoskeletal markers were used to follow cytoskeletal dynamics in wild-type and mutant plants using confocal microscopy and VAEM (variable-angle epifluorescence microscopy). Mutants exhibited more abundant but less dynamic F-actin bundles and more dynamic microtubules than wild-type seedlings. Treatment of wild-type seedlings with a formin inhibitor, SMIFH2, mimicked the root growth and cell expansion phenotypes and cytoskeletal structure alterations observed in fh1 mutants. The results suggest that besides direct effects on actin organization, the in vivo role of AtFH1 also includes modulation of microtubule dynamics, possibly mediated by actin–microtubule cross-talk. PMID:23202131

Mobilization of HIV Spread by Diaphanous 2 Dependent Filopodia in Infected Dendritic Cells

PubMed Central

Aggarwal, Anupriya; Iemma, Tina L.; Shih, Ivy; Newsome, Timothy P.; McAllery, Samantha; Cunningham, Anthony L.; Turville, Stuart G.

2012-01-01

Paramount to the success of persistent viral infection is the ability of viruses to navigate hostile environments en route to future targets. In response to such obstacles, many viruses have developed the ability of establishing actin rich-membrane bridges to aid in future infections. Herein through dynamic imaging of HIV infected dendritic cells, we have observed how viral high-jacking of the actin/membrane network facilitates one of the most efficient forms of HIV spread. Within infected DC, viral egress is coupled to viral filopodia formation, with more than 90% of filopodia bearing immature HIV on their tips at extensions of 10 to 20 µm. Live imaging showed HIV filopodia routinely pivoting at their base, and projecting HIV virions at µm.sec−1 along repetitive arc trajectories. HIV filopodial dynamics lead to up to 800 DC to CD4 T cell contacts per hour, with selection of T cells culminating in multiple filopodia tethering and converging to envelope the CD4 T-cell membrane with budding HIV particles. Long viral filopodial formation was dependent on the formin diaphanous 2 (Diaph2), and not a dominant Arp2/3 filopodial pathway often associated with pathogenic actin polymerization. Manipulation of HIV Nef reduced HIV transfer 25-fold by reducing viral filopodia frequency, supporting the potency of DC HIV transfer was dependent on viral filopodia abundance. Thus our observations show HIV corrupts DC to CD4 T cell interactions by physically embedding at the leading edge contacts of long DC filopodial networks. PMID:22685410

The pros and cons of common actin labeling tools for visualizing actin dynamics during Drosophila oogenesis

PubMed Central

Spracklen, Andrew J.; Fagan, Tiffany N.; Lovander, Kaylee E.; Tootle, Tina L.

2015-01-01

Dynamic remodeling of the actin cytoskeleton is required for both development and tissue homeostasis. While fixed image analysis has provided significant insight into such events, a complete understanding of cytoskeletal dynamics requires live imaging. Numerous tools for the live imaging of actin have been generated by fusing the actin-binding domain from an actin-interacting protein to a fluorescent protein. Here we comparatively assess the utility of three such tools – Utrophin, Lifeact, and F-tractin – for characterizing the actin remodeling events occurring within the germline-derived nurse cells during Drosophila mid-oogenesis or follicle development. Specifically, we used the UAS/GAL4 system to express these tools at different levels and in different cells, and analyzed these tools for effects on fertility, alterations in the actin cytoskeleton, and ability to label filamentous actin (F-actin) structures by both fixed and live imaging. While both Utrophin and Lifeact robustly label F-actin structures within the Drosophila germline, when strongly expressed they cause sterility and severe actin defects including cortical actin breakdown resulting in multi-nucleate nurse cells, early F-actin filament and aggregate formation during stage 9 (S9), and disorganized parallel actin filament bundles during stage 10B (S10B). However, by using a weaker germline GAL4 driver in combination with a higher temperature, Utrophin can label F-actin with minimal defects. Additionally, strong Utrophin expression within the germline causes F-actin formation in the nurse cell nuclei and germinal vesicle during mid-oogenesis. Similarly, Lifeact expression results in nuclear F-actin only within the germinal vesicle. F-tractin expresses at a lower level than the other two labeling tools, but labels cytoplasmic F-actin structures well without causing sterility or striking actin defects. Together these studies reveal how critical it is to evaluate the utility of each actin labeling tool within the tissue and cell type of interest in order to identify the tool that represents the best compromise between acceptable labeling and minimal disruption of the phenomenon being observed. In this case, we find that F-tractin, and perhaps Utrophin, when Utrophin expression levels are optimized to label efficiently without causing actin defects, can be used to study F-actin dynamics within the Drosophila nurse cells. PMID:24995797

Surface-induced polymerization of actin.

PubMed Central

Renault, A; Lenne, P F; Zakri, C; Aradian, A; Vénien-Bryan, C; Amblard, F

1999-01-01

Living cells contain a very large amount of membrane surface area, which potentially influences the direction, the kinetics, and the localization of biochemical reactions. This paper quantitatively evaluates the possibility that a lipid monolayer can adsorb actin from a nonpolymerizing solution, induce its polymerization, and form a 2D network of individual actin filaments, in conditions that forbid bulk polymerization. G- and F-actin solutions were studied beneath saturated Langmuir monolayers containing phosphatidylcholine (PC, neutral) and stearylamine (SA, a positively charged surfactant) at PC:SA = 3:1 molar ratio. Ellipsometry, tensiometry, shear elastic measurements, electron microscopy, and dark-field light microscopy were used to characterize the adsorption kinetics and the interfacial polymerization of actin. In all cases studied, actin follows a monoexponential reaction-limited adsorption with similar time constants (approximately 10(3) s). At a longer time scale the shear elasticity of the monomeric actin adsorbate increases only in the presence of lipids, to a 2D shear elastic modulus of mu approximately 30 mN/m, indicating the formation of a structure coupled to the monolayer. Electron microscopy shows the formation of a 2D network of actin filaments at the PC:SA surface, and several arguments strongly suggest that this network is indeed causing the observed elasticity. Adsorption of F-actin to PC:SA leads more quickly to a slightly more rigid interface with a modulus of mu approximately 50 mN/m. PMID:10049338

Coactosin accelerates cell dynamism by promoting actin polymerization.

PubMed

Hou, Xubin; Katahira, Tatsuya; Ohashi, Kazumasa; Mizuno, Kensaku; Sugiyama, Sayaka; Nakamura, Harukazu

2013-07-01

During development, cells dynamically move or extend their processes, which are achieved by actin dynamics. In the present study, we paid attention to Coactosin, an actin binding protein, and studied its role in actin dynamics. Coactosin was associated with actin and Capping protein in neural crest cells and N1E-115 neuroblastoma cells. Accumulation of Coactosin to cellular processes and its association with actin filaments prompted us to reveal the effect of Coactosin on cell migration. Coactosin overexpression induced cellular processes in cultured neural crest cells. In contrast, knock-down of Coactosin resulted in disruption of actin polymerization and of neural crest cell migration. Importantly, Coactosin was recruited to lamellipodia and filopodia in response to Rac signaling, and mutated Coactosin that cannot bind to F-actin did not react to Rac signaling, nor support neural crest cell migration. It was also shown that deprivation of Rac signaling from neural crest cells by dominant negative Rac1 (DN-Rac1) interfered with neural crest cell migration, and that co-transfection of DN-Rac1 and Coactosin restored neural crest cell migration. From these results we have concluded that Coactosin functions downstream of Rac signaling and that it is involved in neurite extension and neural crest cell migration by actively participating in actin polymerization. Copyright © 2013 Elsevier Inc. All rights reserved.

[Cytoskeletal actin and its associated proteins. Some examples in Protista].

PubMed

Guillén, N; Carlier, M F; Brugerolle, G; Tardieux, I; Ausseil, J

1998-06-01

Many processes, cell motility being an example, require cells to remodel the actin cytoskeleton in response to both intracellular and extracellular signals. Reorganization of the actin cytoskeleton involves the rapid disassembly and reassembly of actin filaments, a phenomenon regulated by the action of particular actin-binding proteins. In recent years, an interest in studying actin regulation in unicellular organisms has arisen. Parasitic protozoan are among these organisms and studies of the cytoskeleton functions of these protozoan are relevant related to either cell biology or pathogenicity. To discuss recent data in this field, a symposium concerning "Actin and actin-binding proteins in protists" was held on May 8-11 in Paris, France, during the XXXV meeting of the French Society of Protistology. As a brief summary of the symposium we report here findings concerning the in vitro actin dynamic assembly, as well as the characterization of several actin-binding proteins from the parasitic protozoan Entamoeba histolytica, Trichomonas vaginalis and Plasmodium knowlesi. In addition, localization of actin in non-pathogen protists such as Prorocentrum micans and Crypthecodinium cohnii is also presented. The data show that some actin-binding proteins facilitate organization of filaments into higher order structures as pseudopods, while others have regulatory functions, indicating very particular roles for actin-binding proteins. One of the proteins discussed during the symposium, the actin depolymerizing factor ADF, was shown to enhance the treadmilling rate of actin filaments. In vitro, ADF binds to the ADP-bound forms of G-actin and F-actin, thereby participating in and changing the rate of actin assembly. Biochemical approaches allowed the identification of a protein complex formed by HSP/C70-cap32-34 which might also be involved in depolymerization of F-actin in P. knowlesi. Molecular and cellular approaches were used to identify proteins such as ABP-120 and myosin IB at the leading edge of E. histolytica. ABP-120 organizes F-actin in a network and myosin IB participates in the pseudopod formation. Similar approaches using T. vaginalis resulted in the discovery of an actin-binding protein that participate in the F-actin reorganization during adhesion of parasites to target cells. This protein is homologous to alpha-actinin from other eukaryotic cells. Finally, by using cell biology approaches, F-actin was observed in the cytoplasm as well as in the nucleus of Dinoflagellates. The recent developments in the molecular genetics of protozoa will provide new insights to understand the roles of actin-binding proteins during cytoskeleton activities.

Dynamic actin filaments control the mechanical behavior of the human red blood cell membrane

PubMed Central

Gokhin, David S.; Nowak, Roberta B.; Khoory, Joseph A.; de la Piedra, Alfonso; Ghiran, Ionita C.; Fowler, Velia M.

2015-01-01

Short, uniform-length actin filaments function as structural nodes in the spectrin-actin membrane skeleton to optimize the biomechanical properties of red blood cells (RBCs). Despite the widespread assumption that RBC actin filaments are not dynamic (i.e., do not exchange subunits with G-actin in the cytosol), this assumption has never been rigorously tested. Here we show that a subpopulation of human RBC actin filaments is indeed dynamic, based on rhodamine-actin incorporation into filaments in resealed ghosts and fluorescence recovery after photobleaching (FRAP) analysis of actin filament mobility in intact RBCs (∼25–30% of total filaments). Cytochalasin-D inhibition of barbed-end exchange reduces rhodamine-actin incorporation and partially attenuates FRAP recovery, indicating functional interaction between actin subunit turnover at the single-filament level and mobility at the membrane-skeleton level. Moreover, perturbation of RBC actin filament assembly/disassembly with latrunculin-A or jasplakinolide induces an approximately twofold increase or ∼60% decrease, respectively, in soluble actin, resulting in altered membrane deformability, as determined by alterations in RBC transit time in a microfluidic channel assay, as well as by abnormalities in spontaneous membrane oscillations (flickering). These experiments identify a heretofore-unrecognized but functionally important subpopulation of RBC actin filaments, whose properties and architecture directly control the biomechanical properties of the RBC membrane. PMID:25717184

Regulation of the actin cytoskeleton-plasma membrane interplay by phosphoinositides.

PubMed

Saarikangas, Juha; Zhao, Hongxia; Lappalainen, Pekka

2010-01-01

The plasma membrane and the underlying cortical actin cytoskeleton undergo continuous dynamic interplay that is responsible for many essential aspects of cell physiology. Polymerization of actin filaments against cellular membranes provides the force for a number of cellular processes such as migration, morphogenesis, and endocytosis. Plasma membrane phosphoinositides (especially phosphatidylinositol bis- and trisphosphates) play a central role in regulating the organization and dynamics of the actin cytoskeleton by acting as platforms for protein recruitment, by triggering signaling cascades, and by directly regulating the activities of actin-binding proteins. Furthermore, a number of actin-associated proteins, such as BAR domain proteins, are capable of directly deforming phosphoinositide-rich membranes to induce plasma membrane protrusions or invaginations. Recent studies have also provided evidence that the actin cytoskeleton-plasma membrane interactions are misregulated in a number of pathological conditions such as cancer and during pathogen invasion. Here, we summarize the wealth of knowledge on how the cortical actin cytoskeleton is regulated by phosphoinositides during various cell biological processes. We also discuss the mechanisms by which interplay between actin dynamics and certain membrane deforming proteins regulate the morphology of the plasma membrane.

Organization of actin in the leading edge of cultured cells: influence of osmium tetroxide and dehydration on the ultrastructure of actin meshworks

PubMed Central

1981-01-01

The ordered structure of the leading edge (lamellipodium) of cultured fibroblasts is readily revealed in cells extracted briefly in Triton X- 100-glutaraldehyde mixtures, fixed further in glutaraldehyde, and then negatively stained for electron microscopy. By this procedure, the leading edge regions show a highly organised, three-dimensional network of actin filaments together with variable numbers of radiating actin filament bundles or microspikes. The use of Phalloidin after glutaraldehyde fixation resulted in a marginal improvement in filament order. Processing of the cytoskeletons though the additional steps generally employed for conventional electron microscopy resulted in a marked deterioration or complete disruption of the order of the actin filament networks. In contrast, the actin filaments of the stress fiber bundles were essentially unaffected. Thus, postfixation in osmium tetroxide (1% for 7 min at room temperature) transformed the networks to a reticulum of kinked fibers, resembling those produced by the exposure of muscle F-actin to OsO4 in vitro (P. Maupin-Szamier and T. D. Pollard. 1978. J. Cell Biol. 77:837--852). While limited exposure to OsO4 (0.2+ for 20 min at 0 degrees C) obviated this destruction, dehydration in acetone or ethanol, with or without post-osmication, caused a further and unavoidable disordering and aggregation of the meshwork filaments. The meshwork regions of the leading edge then showed a striking resemblance to the networks hitherto described in critical point-dried preparations of cultured cells. I conclude that much of the "microtrabecular lattice" described by Wolosewick and Porter (1979. J. Cell Biol. 82:114--139) in the latter preparations constitutes actin meshworks and actin filament arrays, with their associated components, that have been distorted and aggregated by the preparative procedures employed. PMID:6799521

Actin filaments regulate the adhesion between the plasma membrane and the cell wall of tobacco guard cells.

PubMed

Yu, Qin; Ren, Jing-Jing; Kong, Lan-Jing; Wang, Xiu-Ling

2018-01-01

During the opening and closing of stomata, guard cells undergo rapid and reversible changes in their volume and shape, which affects the adhesion of the plasma membrane (PM) to the cell wall (CW). The dynamics of actin filaments in guard cells are involved in stomatal movement by regulating structural changes and intracellular signaling. However, it is unclear whether actin dynamics regulate the adhesion of the PM to the CW. In this study, we investigated the relationship between actin dynamics and PM-CW adhesion by the hyperosmotic-induced plasmolysis of tobacco guard cells. We found that actin filaments in guard cells were depolymerized during mannitol-induced plasmolysis. The inhibition of actin dynamics by treatment with latrunculin B or jasplakinolide and the disruption of the adhesion between the PM and the CW by treatment with RGDS peptide (Arg-Gly-Asp-Ser) enhanced guard cell plasmolysis. However, treatment with latrunculin B alleviated the RGDS peptide-induced plasmolysis and endocytosis. Our results reveal that the actin depolymerization is involved in the regulation of the PW-CW adhesion during hyperosmotic-induced plasmolysis in tobacco guard cells.

Impact of branching on the elasticity of actin networks

PubMed Central

Pujol, Thomas; du Roure, Olivia; Fermigier, Marc; Heuvingh, Julien

2012-01-01

Actin filaments play a fundamental role in cell mechanics: assembled into networks by a large number of partners, they ensure cell integrity, deformability, and migration. Here we focus on the mechanics of the dense branched network found at the leading edge of a crawling cell. We develop a new technique based on the dipolar attraction between magnetic colloids to measure mechanical properties of branched actin gels assembled around the colloids. This technique allows us to probe a large number of gels and, through the study of different networks, to access fundamental relationships between their microscopic structure and their mechanical properties. We show that the architecture does regulate the elasticity of the network: increasing both capping and branching concentrations strongly stiffens the networks. These effects occur at protein concentrations that can be regulated by the cell. In addition, the dependence of the elastic modulus on the filaments’ flexibility and on increasing internal stress has been studied. Our overall results point toward an elastic regime dominated by enthalpic rather than entropic deformations. This result strongly differs from the elasticity of diluted cross-linked actin networks and can be explained by the dense dendritic structure of lamellipodium-like networks. PMID:22689953

A new link between the retrograde actin flow and focal adhesions.

PubMed

Yamashiro, Sawako; Watanabe, Naoki

2014-11-01

The retrograde actin flow, continuous centripetal movement of the cell peripheral actin networks, is widely observed in adherent cells. The retrograde flow is believed to facilitate cell migration when linked to cell adhesion molecules. In this review, we summarize our current knowledge regarding the functional relationship between the retrograde actin flow and focal adhesions (FAs). We also introduce our recent study in which single-molecule speckle (SiMS) microscopy dissected the complex interactions between FAs and the local actin flow. FAs do not simply impede the actin flow, but actively attract and remodel the local actin network. Our findings provide a new insight into the mechanisms for protrusion and traction force generation at the cell leading edge. Furthermore, we discuss possible roles of the actin flow-FA interaction based on the accumulated knowledge and our SiMS study. © The Authors 2014. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Regulation of the Pollen-Specific Actin-Depolymerizing Factor LlADF1

PubMed Central

Allwood, Ellen G.; Anthony, Richard G.; Smertenko, Andrei P.; Reichelt, Stefanie; Drobak, Bjorn K.; Doonan, John H.; Weeds, Alan G.; Hussey, Patrick J.

2002-01-01

Pollen tube growth is dependent on a dynamic actin cytoskeleton, suggesting that actin-regulating proteins are involved. We have examined the regulation of the lily pollen-specific actin-depolymerizing factor (ADF) LlADF1. Its actin binding and depolymerizing activity is pH sensitive, inhibited by certain phosphoinositides, but not controlled by phosphorylation. Compared with its F-actin binding properties, its low activity in depolymerization assays has been used to explain why pollen ADF decorates F-actin in pollen grains. This low activity is incompatible with a role in increasing actin dynamics necessary to promote pollen tube growth. We have identified a plant homolog of actin-interacting protein, AIP1, which enhances the depolymerization of F-actin in the presence of LlADF1 by ∼60%. Both pollen ADF and pollen AIP1 bind F-actin in pollen grains but are mainly cytoplasmic in pollen tubes. Our results suggest that together these proteins remodel actin filaments as pollen grains enter and exit dormancy. PMID:12417710

Actin cable distribution and dynamics arising from cross-linking, motor pulling, and filament turnover

PubMed Central

Tang, Haosu; Laporte, Damien; Vavylonis, Dimitrios

2014-01-01

The growth of fission yeast relies on the polymerization of actin filaments nucleated by formin For3p, which localizes at tip cortical sites. These actin filaments bundle to form actin cables that span the cell and guide the movement of vesicles toward the cell tips. A big challenge is to develop a quantitative understanding of these cellular actin structures. We used computer simulations to study the spatial and dynamical properties of actin cables. We simulated individual actin filaments as semiflexible polymers in three dimensions composed of beads connected with springs. Polymerization out of For3p cortical sites, bundling by cross-linkers, pulling by type V myosin, and severing by cofilin are simulated as growth, cross-linking, pulling, and turnover of the semiflexible polymers. With the foregoing mechanisms, the model generates actin cable structures and dynamics similar to those observed in live-cell experiments. Our simulations reproduce the particular actin cable structures in myoVΔ cells and predict the effect of increased myosin V pulling. Increasing cross-linking parameters generates thicker actin cables. It also leads to antiparallel and parallel phases with straight or curved cables, consistent with observations of cells overexpressing α-actinin. Finally, the model predicts that clustering of formins at cell tips promotes actin cable formation. PMID:25103242

Myosin IIA interacts with the spectrin-actin membrane skeleton to control red blood cell membrane curvature and deformability.

PubMed

Smith, Alyson S; Nowak, Roberta B; Zhou, Sitong; Giannetto, Michael; Gokhin, David S; Papoin, Julien; Ghiran, Ionita C; Blanc, Lionel; Wan, Jiandi; Fowler, Velia M

2018-05-08

The biconcave disk shape and deformability of mammalian RBCs rely on the membrane skeleton, a viscoelastic network of short, membrane-associated actin filaments (F-actin) cross-linked by long, flexible spectrin tetramers. Nonmuscle myosin II (NMII) motors exert force on diverse F-actin networks to control cell shapes, but a function for NMII contractility in the 2D spectrin-F-actin network of RBCs has not been tested. Here, we show that RBCs contain membrane skeleton-associated NMIIA puncta, identified as bipolar filaments by superresolution fluorescence microscopy. MgATP disrupts NMIIA association with the membrane skeleton, consistent with NMIIA motor domains binding to membrane skeleton F-actin and contributing to membrane mechanical properties. In addition, the phosphorylation of the RBC NMIIA heavy and light chains in vivo indicates active regulation of NMIIA motor activity and filament assembly, while reduced heavy chain phosphorylation of membrane skeleton-associated NMIIA indicates assembly of stable filaments at the membrane. Treatment of RBCs with blebbistatin, an inhibitor of NMII motor activity, decreases the number of NMIIA filaments associated with the membrane and enhances local, nanoscale membrane oscillations, suggesting decreased membrane tension. Blebbistatin-treated RBCs also exhibit elongated shapes, loss of membrane curvature, and enhanced deformability, indicating a role for NMIIA contractility in promoting membrane stiffness and maintaining RBC biconcave disk cell shape. As structures similar to the RBC membrane skeleton exist in many metazoan cell types, these data demonstrate a general function for NMII in controlling specialized membrane morphology and mechanical properties through contractile interactions with short F-actin in spectrin-F-actin networks.

Mechanisms of the cytopathic action of actin-ADP-ribosylating toxins.

PubMed

Aktories, K; Wegner, A

1992-10-01

Clostridium botulinum C2 toxin, Clostridium perfringens iota toxin, and Clostridium spiroforme toxin ADP-ribosylate actin monomers. Toxin-induced ADP-ribosylation disturbs the cellular equilibrium between monomeric and polymeric actin and traps monomeric actin in its unpolymerized form, thereby depolymerizing actin filaments and destroying the microfilament network. Furthermore, the toxins ADP-ribosylate gelsolin actin complexes. These modifications may contribute to the cytopathic action of the toxins.

Actin Hydrophobic Loop (262-274) and Filament Nucleation and Elongation

PubMed Central

Shvetsov, Alexander; Galkin, Vitold E.; Orlova, Albina; Phillips, Martin; Bergeron, Sarah E.; Rubenstein, Peter A.; Egelman, Edward H.; Reisler, Emil

2014-01-01

Summary The importance of actin hydrophobic loop 262-274 dynamics to actin polymerization and filament stability has been shown recently using a yeast actin mutant, L180C/L269C/C374A, in which the hydrophobic loop could be locked in a “parked” conformation by a disulfide bond between C180 and C269. Such a cross-linked G-actin does not form filaments, suggesting nucleation and/or elongation inhibition. To determine the role of loop dynamics in filament nucleation and/or elongation, we studied the polymerization of the cross-linked actin in the presence of cofilin - to assist with actin nucleation - and with phalloidin, to stabilize the elongating filament segments. We demonstrate here that together, but not alone, phalloidin and cofilin co-rescue the polymerization of cross-linked actin. The polymerization was also rescued by filament seeds added together with phalloidin but not with cofilin. Thus, loop immobilization via cross-linking inhibits both filament nucleation and elongation. Nevertheless, the conformational changes needed to catalyze ATP hydrolysis by actin occur in the cross-linked actin. When actin filaments are fully decorated by cofilin the helical twist of F-actin changes by ~ 5° per subunit. Electron microscopic analysis of filaments rescued by cofilin and phalloidin revealed a dense contact between opposite strands in F-actin, and a change of twist by ~ 1° per subunit, indicating either partial or disordered attachment of cofilin to F-actin and/or a competition between cofilin and phalloidin to alter F-actin symmetry. Our findings show an importance of the hydrophobic loop conformational dynamics to both actin nucleation and elongation and reveal that the inhibition of these two steps in the cross-linked actin can be relieved by appropriate factors. PMID:18037437

Organization and dynamics of the actin cytoskeleton during dendritic spine morphological remodeling.

PubMed

Chazeau, Anaël; Giannone, Grégory

2016-08-01

In the central nervous system, most excitatory post-synapses are small subcellular structures called dendritic spines. Their structure and morphological remodeling are tightly coupled to changes in synaptic transmission. The F-actin cytoskeleton is the main driving force of dendritic spine remodeling and sustains synaptic plasticity. It is therefore essential to understand how changes in synaptic transmission can regulate the organization and dynamics of actin binding proteins (ABPs). In this review, we will provide a detailed description of the organization and dynamics of F-actin and ABPs in dendritic spines and will discuss the current models explaining how the actin cytoskeleton sustains both structural and functional synaptic plasticity.

Tropomyosin inhibits ADF/cofilin-dependent actin filament dynamics.

PubMed

Ono, Shoichiro; Ono, Kanako

2002-03-18

Tropomyosin binds to actin filaments and is implicated in stabilization of actin cytoskeleton. We examined biochemical and cell biological properties of Caenorhabditis elegans tropomyosin (CeTM) and obtained evidence that CeTM is antagonistic to ADF/cofilin-dependent actin filament dynamics. We purified CeTM, actin, and UNC-60B (a muscle-specific ADF/cofilin isoform), all of which are derived from C. elegans, and showed that CeTM and UNC-60B bound to F-actin in a mutually exclusive manner. CeTM inhibited UNC-60B-induced actin depolymerization and enhancement of actin polymerization. Within isolated native thin filaments, actin and CeTM were detected as major components, whereas UNC-60B was present at a trace amount. Purified UNC-60B was unable to interact with the native thin filaments unless CeTM and other associated proteins were removed by high-salt extraction. Purified CeTM was sufficient to restore the resistance of the salt-extracted filaments from UNC-60B. In muscle cells, CeTM and UNC-60B were localized in different patterns. Suppression of CeTM by RNA interference resulted in disorganized actin filaments and paralyzed worms in wild-type background. However, in an ADF/cofilin mutant background, suppression of CeTM did not worsen actin organization and worm motility. These results suggest that tropomyosin is a physiological inhibitor of ADF/cofilin-dependent actin dynamics.

Diffusion of GPI-anchored proteins is influenced by the activity of dynamic cortical actin.

PubMed

Saha, Suvrajit; Lee, Il-Hyung; Polley, Anirban; Groves, Jay T; Rao, Madan; Mayor, Satyajit

2015-11-05

Molecular diffusion at the surface of living cells is believed to be predominantly driven by thermal kicks. However, there is growing evidence that certain cell surface molecules are driven by the fluctuating dynamics of cortical cytoskeleton. Using fluorescence correlation spectroscopy, we measure the diffusion coefficient of a variety of cell surface molecules over a temperature range of 24-37 °C. Exogenously incorporated fluorescent lipids with short acyl chains exhibit the expected increase of diffusion coefficient over this temperature range. In contrast, we find that GPI-anchored proteins exhibit temperature-independent diffusion over this range and revert to temperature-dependent diffusion on cell membrane blebs, in cells depleted of cholesterol, and upon acute perturbation of actin dynamics and myosin activity. A model transmembrane protein with a cytosolic actin-binding domain also exhibits the temperature-independent behavior, directly implicating the role of cortical actin. We show that diffusion of GPI-anchored proteins also becomes temperature dependent when the filamentous dynamic actin nucleator formin is inhibited. However, changes in cortical actin mesh size or perturbation of branched actin nucleator Arp2/3 do not affect this behavior. Thus cell surface diffusion of GPI-anchored proteins and transmembrane proteins that associate with actin is driven by active fluctuations of dynamic cortical actin filaments in addition to thermal fluctuations, consistent with expectations from an "active actin-membrane composite" cell surface. © 2015 Saha et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Profilin-Dependent Nucleation and Assembly of Actin Filaments Controls Cell Elongation in Arabidopsis1[OPEN

PubMed Central

Cao, Lingyan; Blanchoin, Laurent; Staiger, Christopher J.

2016-01-01

Actin filaments in plant cells are incredibly dynamic; they undergo incessant remodeling and assembly or disassembly within seconds. These dynamic events are choreographed by a plethora of actin-binding proteins, but the exact mechanisms are poorly understood. Here, we dissect the contribution of Arabidopsis (Arabidopsis thaliana) PROFILIN1 (PRF1), a conserved actin monomer-binding protein, to actin organization and single filament dynamics during axial cell expansion of living epidermal cells. We found that reduced PRF1 levels enhanced cell and organ growth. Surprisingly, we observed that the overall frequency of nucleation events in prf1 mutants was dramatically decreased and that a subpopulation of actin filaments that assemble at high rates was reduced. To test whether profilin cooperates with plant formin proteins to execute actin nucleation and rapid filament elongation in cells, we used a pharmacological approach. Here, we used Small Molecule Inhibitor of Formin FH2 (SMIFH2), after validating its mode of action on a plant formin in vitro, and observed a reduced nucleation frequency of actin filaments in live cells. Treatment of wild-type epidermal cells with SMIFH2 mimicked the phenotype of prf1 mutants, and the nucleation frequency in prf1-2 mutant was completely insensitive to these treatments. Our data provide compelling evidence that PRF1 coordinates the stochastic dynamic properties of actin filaments by modulating formin-mediated actin nucleation and assembly during plant cell expansion. PMID:26574597

Side-binding proteins modulate actin filament dynamics

PubMed Central

Crevenna, Alvaro H; Arciniega, Marcelino; Dupont, Aurélie; Mizuno, Naoko; Kowalska, Kaja; Lange, Oliver F; Wedlich-Söldner, Roland; Lamb, Don C

2015-01-01

Actin filament dynamics govern many key physiological processes from cell motility to tissue morphogenesis. A central feature of actin dynamics is the capacity of filaments to polymerize and depolymerize at their ends in response to cellular conditions. It is currently thought that filament kinetics can be described by a single rate constant for each end. In this study, using direct visualization of single actin filament elongation, we show that actin polymerization kinetics at both filament ends are strongly influenced by the binding of proteins to the lateral filament surface. We also show that the pointed-end has a non-elongating state that dominates the observed filament kinetic asymmetry. Estimates of flexibility as well as effects on fragmentation and growth suggest that the observed kinetic diversity arises from structural alteration. Tuning elongation kinetics by exploiting the malleability of the filament structure may be a ubiquitous mechanism to generate a rich variety of cellular actin dynamics. DOI: http://dx.doi.org/10.7554/eLife.04599.001 PMID:25706231

ACF7: an essential integrator of microtubule dynamics.

PubMed

Kodama, Atsuko; Karakesisoglou, Iakowos; Wong, Ellen; Vaezi, Alec; Fuchs, Elaine

2003-10-31

ACF7 is a member of the spectraplakin family of cytoskeletal crosslinking proteins possessing actin and microtubule binding domains. Here, we show that ACF7 is an essential integrator of MT-actin dynamics. In endodermal cells, ACF7 binds along microtubules but concentrates at their distal ends and at cell borders when polarized. In ACF7's absence, microtubules still bind EB1 and CLIP170, but they no longer grow along polarized actin bundles, nor do they pause and tether to actin-rich cortical sites. The consequences are less stable, long microtubules with skewed cytoplasmic trajectories and altered dynamic instability. In response to wounding, ACF7 null cultures activate polarizing signals, but fail to maintain them and coordinate migration. Rescue of these defects requires ACF7's actin and microtubule binding domains. Thus, spectraplakins are important for controlling microtubule dynamics and reinforcing links between microtubules and polarized F-actin, so that cellular polarization and coordinated cell movements can be sustained.

Actin is an essential component of plant gravitropic signaling pathways

NASA Astrophysics Data System (ADS)

Braun, Markus; Hauslage, Jens; Limbach, Christoph

2003-08-01

A role of the actin cytoskeleton in the different phases of gravitropism in higher plant organs seems obvious, but experimental evidence is still inconclusive and contradictory. In gravitropically tip-growing rhizoids and protonemata, however, it is well documented that actin is an essential component of the tip-growth machinery and is involved either in the cellular mechanisms that lead to gravity sensing and in the processes of the graviresponses that result in the reorientation of the growth direction. All these processes depend on a complexly organized and highly dynamic organization of actin filaments whose diverse functions are coordinated by numerous associated proteins. Actin filaments and myosins mediate the transport of secretory vehicles to the growing tip and precisely control the delivery of cell wall material. In addition, both cell types use a very efficient actomyosin-based system to control and correct the position of their statoliths and to direct sedimenting statoliths to confined graviperception sites at the plasma membrane. The studies presented in this paper provide evidence for the essential role of actin in plant gravity sensing and the gravitropic responses. A unique actin-organizing center exists in the tip of characean rhizoids and protonemata which is associated with and dynamically regulated by a specific set of actin-dynamizing proteins. It is concluded that this highly dynamic apical actin array is an essential prerequisite for gravity sensing and gravity-oriented tip growth.

Computational Study of the Binding Mechanism of Actin-Depolymerizing Factor 1 with Actin in Arabidopsis thaliana.

PubMed

Du, Juan; Wang, Xue; Dong, Chun-Hai; Yang, Jian Ming; Yao, Xiao Jun

2016-01-01

Actin is a highly conserved protein. It plays important roles in cellular function and exists either in the monomeric (G-actin) or polymeric form (F-actin). Members of the actin-depolymerizing factor (ADF)/cofilin protein family bind to both G-actin and F-actin and play vital roles in actin dynamics by manipulating the rates of filament polymerization and depolymerization. It has been reported that the S6D and R98A/K100A mutants of actin-depolymerizing factor 1 (ADF1) in Arabidopsis thaliana decreased the binding affinity of ADF for the actin monomer. To investigate the binding mechanism and dynamic behavior of the ADF1-actin complex, we constructed a homology model of the AtADF1-actin complex based on the crystal structure of AtADF1 and the twinfilin C-terminal ADF-H domain in a complex with a mouse actin monomer. The model was then refined for subsequent molecular dynamics simulations. Increased binding energy of the mutated system was observed using the Molecular Mechanics Generalized Born Surface Area and Poisson-Boltzmann Surface Area (MM-GB/PBSA) methods. To determine the residues that make decisive contributions to the ADF1 actin-binding affinity, per-residue decomposition and computational alanine scanning analyses were performed, which provided more detailed information on the binding mechanism. Root-mean-square fluctuation and principal component analyses confirmed that the S6D and R98A/K100A mutants induced an increased conformational flexibility. The comprehensive molecular insight gained from this study is of great importance for understanding the binding mechanism of ADF1 and G-actin.

Computational Study of the Binding Mechanism of Actin-Depolymerizing Factor 1 with Actin in Arabidopsis thaliana

PubMed Central

Wang, Xue; Dong, Chun-Hai; Yang, Jian Ming; Yao, Xiao Jun

2016-01-01

Actin is a highly conserved protein. It plays important roles in cellular function and exists either in the monomeric (G-actin) or polymeric form (F-actin). Members of the actin-depolymerizing factor (ADF)/cofilin protein family bind to both G-actin and F-actin and play vital roles in actin dynamics by manipulating the rates of filament polymerization and depolymerization. It has been reported that the S6D and R98A/K100A mutants of actin-depolymerizing factor 1 (ADF1) in Arabidopsis thaliana decreased the binding affinity of ADF for the actin monomer. To investigate the binding mechanism and dynamic behavior of the ADF1–actin complex, we constructed a homology model of the AtADF1–actin complex based on the crystal structure of AtADF1 and the twinfilin C-terminal ADF-H domain in a complex with a mouse actin monomer. The model was then refined for subsequent molecular dynamics simulations. Increased binding energy of the mutated system was observed using the Molecular Mechanics Generalized Born Surface Area and Poisson–Boltzmann Surface Area (MM-GB/PBSA) methods. To determine the residues that make decisive contributions to the ADF1 actin-binding affinity, per-residue decomposition and computational alanine scanning analyses were performed, which provided more detailed information on the binding mechanism. Root-mean-square fluctuation and principal component analyses confirmed that the S6D and R98A/K100A mutants induced an increased conformational flexibility. The comprehensive molecular insight gained from this study is of great importance for understanding the binding mechanism of ADF1 and G-actin. PMID:27414648

Critical forces for actin filament buckling and force transmission influence transport in actomyosin networks

NASA Astrophysics Data System (ADS)

Stam, Samantha; Gardel, Margaret

Viscoelastic networks of biopolymers coordinate the motion of intracellular objects during transport. These networks have nonlinear mechanical properties due to events such as filament buckling or breaking of cross-links. The influence of such nonlinear properties on the time and length scales of transport is not understood. Here, we use in vitro networks of actin and the motor protein myosin II to clarify how intracellular forces regulate active diffusion. We observe two transitions in the mean-squared displacement of cross-linked actin with increasing motor concentration. The first is a sharp transition from initially subdiffusive to diffusive-like motion that requires filament buckling but does not cause net contraction of the network. Further increase of the motor density produces a second transition to network rupture and ballistic actin transport. This corresponds with an increase in the correlation of motion and thus may be caused when forces propagate far enough for global motion. We conclude that filament buckling and overall network contraction require different amounts of force and produce distinct transport properties. These nonlinear transitions may act as mechanical switches that can be turned on to produce observed motion within cells.

Force Exertion and Transmission in Cross-Linked Actin Networks

NASA Astrophysics Data System (ADS)

Stam, Samantha

Cells are responsive to external cues in their environment telling them to proliferate or migrate within their surrounding tissue. Sensing of cues that are mechanical in nature, such stiffness of a tissue or forces transmitted from other cells, is believed to involve the cytoskeleton of a cell. The cytoskeleton is a complex network of proteins consisting of polymers that provide structural support, motor proteins that remodel these structures, and many others. We do not yet have a complete understanding of how cytoskeletal components respond to either internal or external mechanical force and stiffness. Such an understanding should involve mechanisms by which constituent molecules, such as motor proteins, are responsive to mechanics. Additionally, physical models of how forces are transmitted through biopolymer networks are necessary. My research has focused on networks formed by the cytoskeletal filament actin and the molecular motor protein myosin II. Actin filaments form networks and bundles that form a structural framework of the cell, and myosin II slides actin filaments. In this thesis, we show that stiffness of an elastic load that opposes myosin-generated actin sliding has a very sharp effect on the myosin force output in simulations. Secondly, we show that the stiffness and connectivity of cytoskeletal filaments regulates the contractility and anisotropy of network deformations that transmit force on material length scales. Together, these results have implications for predicting and interpreting the deformations and forces in biopolymeric active materials.

Changes in actin dynamics are involved in salicylic acid signaling pathway.

PubMed

Matoušková, Jindřiška; Janda, Martin; Fišer, Radovan; Sašek, Vladimír; Kocourková, Daniela; Burketová, Lenka; Dušková, Jiřina; Martinec, Jan; Valentová, Olga

2014-06-01

Changes in actin cytoskeleton dynamics are one of the crucial players in many physiological as well as non-physiological processes in plant cells. Positioning of actin filament arrays is necessary for successful establishment of primary lines of defense toward pathogen attack, depolymerization leads very often to the enhanced susceptibility to the invading pathogen. On the other hand it was also shown that the disruption of actin cytoskeleton leads to the induction of defense response leading to the expression of PATHOGENESIS RELATED proteins (PR). In this study we show that pharmacological actin depolymerization leads to the specific induction of genes in salicylic acid pathway but not that involved in jasmonic acid signaling. Life imaging of leafs of Arabidopsis thaliana with GFP-tagged fimbrin (GFP-fABD2) treated with 1 mM salicylic acid revealed rapid disruption of actin filaments resembling the pattern viewed after treatment with 200 nM latrunculin B. The effect of salicylic acid on actin filament fragmentation was prevented by exogenous addition of phosphatidic acid, which binds to the capping protein and thus promotes actin polymerization. The quantitative evaluation of actin filament dynamics is also presented. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

A novel role for WAVE1 in controlling actin network growth rate and architecture

PubMed Central

Sweeney, Meredith O.; Collins, Agnieszka; Padrick, Shae B.; Goode, Bruce L.

2015-01-01

Branched actin filament networks in cells are assembled through the combined activities of Arp2/3 complex and different WASP/WAVE proteins. Here we used TIRF and electron microscopy to directly compare for the first time the assembly kinetics and architectures of actin filament networks produced by Arp2/3 complex and dimerized VCA regions of WAVE1, WAVE2, or N-WASP. WAVE1 produced strikingly different networks from WAVE2 or N-WASP, which comprised unexpectedly short filaments. Further analysis showed that the WAVE1-specific activity stemmed from an inhibitory effect on filament elongation both in the presence and absence of Arp2/3 complex, which was observed even at low stoichiometries of WAVE1 to actin monomers, precluding an effect from monomer sequestration. Using a series of VCA chimeras, we mapped the elongation inhibitory effects of WAVE1 to its WH2 (“V”) domain. Further, mutating a single conserved lysine residue potently disrupted WAVE1's inhibitory effects. Taken together, our results show that WAVE1 has unique activities independent of Arp2/3 complex that can govern both the growth rates and architectures of actin filament networks. Such activities may underlie previously observed differences between the cellular functions of WAVE1 and WAVE2. PMID:25473116

The Cdc42 guanine nucleotide exchange factor FGD6 coordinates cell polarity and endosomal membrane recycling in osteoclasts.

PubMed

Steenblock, Charlotte; Heckel, Tobias; Czupalla, Cornelia; Espírito Santo, Ana Isabel; Niehage, Christian; Sztacho, Martin; Hoflack, Bernard

2014-06-27

The initial step of bone digestion is the adhesion of osteoclasts onto bone surfaces and the assembly of podosomal belts that segregate the bone-facing ruffled membrane from other membrane domains. During bone digestion, membrane components of the ruffled border also need to be recycled after macropinocytosis of digested bone materials. How osteoclast polarity and membrane recycling are coordinated remains unknown. Here, we show that the Cdc42-guanine nucleotide exchange factor FGD6 coordinates these events through its Src-dependent interaction with different actin-based protein networks. At the plasma membrane, FGD6 couples cell adhesion and actin dynamics by regulating podosome formation through the assembly of complexes comprising the Cdc42-interactor IQGAP1, the Rho GTPase-activating protein ARHGAP10, and the integrin interactors Talin-1/2 or Filamin A. On endosomes and transcytotic vesicles, FGD6 regulates retromer-dependent membrane recycling through its interaction with the actin nucleation-promoting factor WASH. These results provide a mechanism by which a single Cdc42-exchange factor controlling different actin-based processes coordinates cell adhesion, cell polarity, and membrane recycling during bone degradation. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Dynamic maintenance of asymmetric meiotic spindle position through Arp2/3 complex-driven cytoplasmic streaming in mouse oocytes

PubMed Central

Yi, Kexi; Unruh, Jay R.; Deng, Manqi; Slaughter, Brian D.; Rubinstein, Boris; Li, Rong

2012-01-01

Mature mammalian oocytes are poised for the completion of second polar body extrusion upon fertilization by positioning the metaphase spindle in close proximity to an actomyosin-rich cortical cap. Loss of this spindle position asymmetry is often associated with poor oocyte quality and infertility 1–3. Here, we report a novel role for the Arp2/3 actin nucleation complex in the maintenance of asymmetric spindle position in mature mouse oocytes. The Arp2/3 complex localizes to the cortical cap in a Ran GTPase-dependent manner and accounts for the nucleation of the majority of actin filaments in both the cortical cap and a cytoplasmic actin network. Inhibition of Arp2/3 complex activity or localization leads to rapid dissociation of the spindle from the cortex. High resolution live imaging and spatiotemporal image correlation spectroscopy (STICS) analysis reveal that in normal oocytes actin filaments flow continuously away from the Arp2/3-rich cortex, generating a cytoplamic streaming that results in a net pushing force on the spindle toward the actomyosin cap. Arp2/3 inhibition not only diminishes this actin flow and cytoplamic streaming but also enables a reverse streaming driven by myosin-II-based cortical contraction, leading to spindle movement away from the cortex. We conclude that the Arp2/3 complex maintains asymmetric meiotic spindle position by generating an actin polymerization-driven cytoplamic streaming and by suppressing a counteracting force from myosin-II-based contractility. PMID:21874009

Dynamic contact guidance of migrating cells

NASA Astrophysics Data System (ADS)

Losert, Wolfgang; Sun, Xiaoyu; Guven, Can; Driscoll, Meghan; Fourkas, John

2014-03-01

We investigate the effects of nanotopographical surfaces on the cell migration and cell shape dynamics of the amoeba Dictyostelium discoideum. Amoeboid motion exhibits significant contact guidance along surfaces with nanoscale ridges or grooves. We show quantitatively that nanoridges spaced 1.5 μm apart exhibit the greatest contact guidance efficiency. Using principal component analysis, we characterize the dynamics of the cell shape modulated by the coupling between the cell membrane and ridges. We show that motion parallel to the ridges is enhanced, while the turning, at the largest spatial scales, is suppressed. Since protrusion dynamics are principally governed by actin dynamics, we imaged the actin polymerization of cells on ridges. We found that actin polymerization occurs preferentially along nanoridges in a ``monorail'' like fashion. The ridges then provide us with a tool to study actin dynamics in an effectively reduced dimensional system.

Actin, microtubules, and vimentin intermediate filaments cooperate for elongation of invadopodia

PubMed Central

Goldman, Robert D.; Louvard, Daniel

2010-01-01

Invasive cancer cells are believed to breach the basement membrane (BM) using specialized protrusions called invadopodia. We found that the crossing of a native BM is a three-stage process: invadopodia indeed form and perforate the BM, elongate into mature invadopodia, and then guide the cell toward the stromal compartment. We studied the remodeling of cytoskeleton networks during invadopodia formation and elongation using ultrastructural analysis, spatial distribution of molecular markers, and RNA interference silencing of protein expression. We show that formation of invadopodia requires only the actin cytoskeleton and filopodia- and lamellipodia-associated proteins. In contrast, elongation of invadopodia is mostly dependent on filopodial actin machinery. Moreover, intact microtubules and vimentin intermediate filament networks are required for further growth. We propose that invadopodia form by assembly of dendritic/diagonal and bundled actin networks and then mature by elongation of actin bundles, followed by the entry of microtubules and vimentin filaments. These findings provide a link between the epithelial to mesenchymal transition and BM transmigration. PMID:20421424

Broken Detailed Balance of Filament Dynamics in Active Networks

NASA Astrophysics Data System (ADS)

Gladrow, J.; Fakhri, N.; MacKintosh, F. C.; Schmidt, C. F.; Broedersz, C. P.

2016-06-01

Myosin motor proteins drive vigorous steady-state fluctuations in the actin cytoskeleton of cells. Endogenous embedded semiflexible filaments such as microtubules, or added filaments such as single-walled carbon nanotubes are used as novel tools to noninvasively track equilibrium and nonequilibrium fluctuations in such biopolymer networks. Here, we analytically calculate shape fluctuations of semiflexible probe filaments in a viscoelastic environment, driven out of equilibrium by motor activity. Transverse bending fluctuations of the probe filaments can be decomposed into dynamic normal modes. We find that these modes no longer evolve independently under nonequilibrium driving. This effective mode coupling results in nonzero circulatory currents in a conformational phase space, reflecting a violation of detailed balance. We present predictions for the characteristic frequencies associated with these currents and investigate how the temporal signatures of motor activity determine mode correlations, which we find to be consistent with recent experiments on microtubules embedded in cytoskeletal networks.

Stochastic Simulation of Actin Dynamics Reveals the Role of Annealing and Fragmentation

PubMed Central

Fass, Joseph; Pak, Chi; Bamburg, James; Mogilner, Alex

2008-01-01

Recent observations of F-actin dynamics call for theoretical models to interpret and understand the quantitative data. A number of existing models rely on simplifications and do not take into account F-actin fragmentation and annealing. We use Gillespie’s algorithm for stochastic simulations of the F-actin dynamics including fragmentation and annealing. The simulations vividly illustrate that fragmentation and annealing have little influence on the shape of the polymerization curve and on nucleotide profiles within filaments but drastically affect the F-actin length distribution, making it exponential. We find that recent surprising measurements of high length diffusivity at the critical concentration cannot be explained by fragmentation and annealing events unless both fragmentation rates and frequency of undetected fragmentation and annealing events are greater than previously thought. The simulations compare well with experimentally measured actin polymerization data and lend additional support to a number of existing theoretical models. PMID:18279896

Differential effects of LifeAct-GFP and actin-GFP on cell mechanics assessed using micropipette aspiration.

PubMed

Sliogeryte, Kristina; Thorpe, Stephen D; Wang, Zhao; Thompson, Clare L; Gavara, Nuria; Knight, Martin M

2016-01-25

The actin cytoskeleton forms a dynamic structure involved in many fundamental cellular processes including the control of cell morphology, migration and biomechanics. Recently LifeAct-GFP (green fluorescent protein) has been proposed for visualising actin structure and dynamics in live cells as an alternative to actin-GFP which has been shown to affect cell mechanics. Here we compare the two approaches in terms of their effect on cellular mechanical behaviour. Human mesenchymal stem cells (hMSCs) were analysed using micropipette aspiration and the effective cellular equilibrium and instantaneous moduli calculated using the standard linear solid model. We show that LifeAct-GFP provides clearer visualisation of F-actin organisation and dynamics. Furthermore, LifeAct-GFP does not alter effective cellular mechanical properties whereas actin-GFP expression causes an increase in the cell modulus. Interestingly, LifeAct-GFP expression did produce a small (~10%) increase in the percentage of cells exhibiting aspiration-induced membrane bleb formation, whilst actin-GFP expression reduced blebbing. Further studies examined the influence of LifeAct-GFP in other cell types, namely chondrogenically differentiated hMSCs and murine chondrocytes. LifeAct-GFP also had no effect on the moduli of these non-blebbing cells for which mechanical properties are largely dependent on the actin cortex. In conclusion we show that LifeAct-GFP enables clearer visualisation of actin organisation and dynamics without disruption of the biomechanical properties of either the whole cell or the actin cortex. Thus the study provides new evidence supporting the use of LifeAct-GFP rather than actin-GFP for live cell microscopy and the study of cellular mechanobiology. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

System-wide organization of actin cytoskeleton determines organelle transport in hypocotyl plant cells

PubMed Central

Nowak, Jacqueline; Ivakov, Alexander; Somssich, Marc; Persson, Staffan; Nikoloski, Zoran

2017-01-01

The actin cytoskeleton is an essential intracellular filamentous structure that underpins cellular transport and cytoplasmic streaming in plant cells. However, the system-level properties of actin-based cellular trafficking remain tenuous, largely due to the inability to quantify key features of the actin cytoskeleton. Here, we developed an automated image-based, network-driven framework to accurately segment and quantify actin cytoskeletal structures and Golgi transport. We show that the actin cytoskeleton in both growing and elongated hypocotyl cells has structural properties facilitating efficient transport. Our findings suggest that the erratic movement of Golgi is a stable cellular phenomenon that might optimize distribution efficiency of cell material. Moreover, we demonstrate that Golgi transport in hypocotyl cells can be accurately predicted from the actin network topology alone. Thus, our framework provides quantitative evidence for system-wide coordination of cellular transport in plant cells and can be readily applied to investigate cytoskeletal organization and transport in other organisms. PMID:28655850

Spontaneous symmetry breaking for geometrical trajectories of actin-based motility in three dimensions

NASA Astrophysics Data System (ADS)

Wen, Fu-Lai; Leung, Kwan-tai; Chen, Hsuan-Yi

2016-07-01

Actin-based motility is important for many cellular processes. In this article we extend our previous studies of an actin-propelled circular disk in two dimensions to an actin-propelled spherical bead in three dimensions. We find that for an achiral load the couplings between the motion of the load and the actin network induce a series of bifurcations, starting with a transition from rest to moving state, followed by a transition from straight to planar curves, and finally a further transition from motion in a plane to one with torsion. To address the intriguing, experimentally observed chiral motility of the bacterium Listeria monocytogenes, we also study the motility of a spherical load with a built-in chirality. For such a chiral load, stable circular trajectories are no longer found in numerical simulations. Instead, helical trajectories with handedness that depends on the chirality of the load are found. Our results reveal the relation between the symmetry of actin network and the trajectories of actin-propelled loads.

The activities of the C-terminal regions of the formin protein disheveled-associated activator of morphogenesis (DAAM) in actin dynamics.

PubMed

Vig, Andrea Teréz; Földi, István; Szikora, Szilárd; Migh, Ede; Gombos, Rita; Tóth, Mónika Ágnes; Huber, Tamás; Pintér, Réka; Talián, Gábor Csaba; Mihály, József; Bugyi, Beáta

2017-08-18

Disheveled-associated activator of morphogenesis (DAAM) is a diaphanous-related formin protein essential for the regulation of actin cytoskeleton dynamics in diverse biological processes. The conserved formin homology 1 and 2 (FH1-FH2) domains of DAAM catalyze actin nucleation and processively mediate filament elongation. These activities are indirectly regulated by the N- and C-terminal regions flanking the FH1-FH2 domains. Recently, the C-terminal diaphanous-autoregulatory domain (DAD) and the C terminus (CT) of formins have also been shown to regulate actin assembly by directly interacting with actin. Here, to better understand the biological activities of DAAM, we studied the role of DAD-CT regions of Drosophila DAAM in its interaction with actin with in vitro biochemical and in vivo genetic approaches. We found that the DAD-CT region binds actin in vitro and that its main actin-binding element is the CT region, which does not influence actin dynamics on its own. However, we also found that it can tune the nucleating activity and the filament end-interaction properties of DAAM in an FH2 domain-dependent manner. We also demonstrate that DAD-CT makes the FH2 domain more efficient in antagonizing with capping protein. Consistently, in vivo data suggested that the CT region contributes to DAAM-mediated filopodia formation and dynamics in primary neurons. In conclusion, our results demonstrate that the CT region of DAAM plays an important role in actin assembly regulation in a biological context. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Co-transcriptional nuclear actin dynamics

PubMed Central

Percipalle, Piergiorgio

2013-01-01

Actin is a key player for nuclear structure and function regulating both chromosome organization and gene activity. In the cell nucleus actin interacts with many different proteins. Among these proteins several studies have identified classical nuclear factors involved in chromatin structure and function, transcription and RNA processing as well as proteins that are normally involved in controlling the actin cytoskeleton. These discoveries have raised the possibility that nuclear actin performs its multi task activities through tight interactions with different sets of proteins. This high degree of promiscuity in the spectrum of protein-to-protein interactions correlates well with the conformational plasticity of actin and the ability to undergo regulated changes in its polymerization states. Several of the factors involved in controlling head-to-tail actin polymerization have been shown to be in the nucleus where they seem to regulate gene activity. By focusing on the multiple tasks performed by actin and actin-binding proteins, possible models of how actin dynamics controls the different phases of the RNA polymerase II transcription cycle are being identified. PMID:23138849

Mechanically tunable actin networks using programmable DNA based cross-linkers

NASA Astrophysics Data System (ADS)

Schnauss, Joerg; Lorenz, Jessica; Schuldt, Carsten; Kaes, Josef; Smith, David

Cells employ multiple cross-linkers with very different properties. Studies of the entire phase space, however, were infeasible since they were restricted to naturally occurring cross-linkers. These components cannot be controllably varied and differ in many parameters. We resolve this limitation by forming artificial actin cross-linkers, which can be controllably varied. The basic building block is DNA enabling a well-defined length variation. DNA can be attached to actin binding peptides with known binding affinities. We used bulk rheology to investigate mechanical properties of these networks. We were able to reproduce mechanical features of actin networks cross-linked by fascin by using a short version of our artificial complex with a high binding affinity. Additionally, we were able to resemble findings for the cross-linker alpha-actinin by employing a long cross-linker with a low binding affinity. Between these natural limits we investigated three different cross-linker lengths each with two different binding affinities. With these controlled variations we are able to precisely screen the phase space of cross-linked actin networks by changing only one specific parameter and not the entire set of properties as in the case of naturally occurring cross-linking complexes.

Actin growth profile in clathrin-mediated endocytosis

NASA Astrophysics Data System (ADS)

Tweten, D. J.; Bayly, P. V.; Carlsson, A. E.

2017-05-01

Clathrin-mediated endocytosis in yeast is driven by a protein patch containing close to 100 different types of proteins. Among the proteins are 5000 -10 000 copies of polymerized actin, and successful endocytosis requires growth of the actin network. Since it is not known exactly how actin network growth drives endocytosis, we calculate the spatial distribution of actin growth required to generate the force that drives the process. First, we establish the force distribution that must be supplied by actin growth, by combining membrane-bending profiles obtained via electron microscopy with established theories of membrane mechanics. Next, we determine the profile of actin growth, using a continuum mechanics approach and an iterative procedure starting with an actin growth profile obtained from a linear analysis. The profile has fairly constant growth outside a central hole of radius 45-50 nm, but very little growth in this hole. This growth profile can reproduce the required forces if the actin shear modulus exceeds 80 kPa, and the growing filaments can exert very large polymerization forces. The growth profile prediction could be tested via electron-microscopy or super-resolution experiments in which the turgor pressure is suddenly turned off.

Computational spatiotemporal analysis identifies WAVE2 and Cofilin as joint regulators of costimulation-mediated T cell actin dynamics

PubMed Central

Roybal, Kole T.; Buck, Taráz E.; Ruan, Xiongtao; Cho, Baek Hwan; Clark, Danielle J.; Ambler, Rachel; Tunbridge, Helen M.; Zhang, Jianwei; Verkade, Paul; Wülfing, Christoph; Murphy, Robert F.

2016-01-01

Fluorescence microscopy is one of the most important tools in cell biology research and it provides spatial and temporal information to investigate regulatory systems inside cells. This technique can generate data in the form of signal intensities at thousands of positions resolved inside individual live cells; however, given extensive cell-to-cell variation, methods do not currently exist to assemble these data into three- or four-dimensional maps of protein concentration that can be compared across different cells and conditions. Here, we have developed one such method and applied it to investigate actin dynamics in T cell activation. Antigen recognition in T cells by the T cell receptor (TCR) is amplified by engagement of the costimulatory receptor CD28 and we have determined how CD28 modulates actin dynamics. We imaged actin and eight core actin regulators under conditions where CD28 in the context of a strong TCR signal was engaged or blocked to yield over a thousand movies. Our computational analysis identified diminished recruitment of the activator of actin nucleation WAVE2 and the actin severing protein cofilin to F-actin as the dominant difference upon costimulation blockade. Reconstitution of WAVE2 and cofilin activity restored the defect in actin signaling dynamics upon costimulation blockade. Thus we have developed and validated an approach to quantify protein distributions in time and space for analysis of complex regulatory systems. PMID:27095595

A theoretical and computational framework for mechanics of the cortex

NASA Astrophysics Data System (ADS)

Torres-SáNchez, Alejandro; Arroyo, Marino

The cell cortex is a thin network of actin filaments lying beneath the cell surface of animal cells. Myosin motors exert contractile forces in this network leading to active stresses, which play a key role in processes such as cytokinesis or cell migration. Thus, understanding the mechanics of the cortex is fundamental to understand the mechanics of animal cells. Due to the dynamic remodeling of the actin network, the cortex behaves as a viscoelastic fluid. Furthermore, due to the difference between its thickness (tens of nanometers) and its dimensions (tens of microns), the cortex can be regarded a surface. Thus, we can model the cortex as a viscoelastic fluid, confined to a surface, that generates active stresses. Interestingly, geometric confinement results in the coupling between shape generation and material flows. In this work we present a theoretical framework to model the mechanics of the cortex that couples elasticity, hydrodynamics and force generation. We complement our theoretical description with a computational setting to simulate the resulting non-linear equations. We use this methodology to understand different processes such as asymmetric cell division or experimental probing of the rheology of the cortex We acknowledge the support of the Europen Research Council through Grant ERC CoG-681434.

Bacterial subversion of host actin dynamics at the plasma membrane.

PubMed

Carabeo, Rey

2011-10-01

Invasion of non-phagocytic cells by a number of bacterial pathogens involves the subversion of the actin cytoskeletal remodelling machinery to produce actin-rich cell surface projections designed to engulf the bacteria. The signalling that occurs to induce these actin-rich structures has considerable overlap among a diverse group of bacteria. The molecular organization within these structures act in concert to internalize the invading pathogen. This dynamic process could be subdivided into three acts - actin recruitment, engulfment, and finally, actin disassembly/internalization. This review will present the current state of knowledge of the molecular processes involved in each stage of bacterial invasion, and provide a perspective that highlights the temporal and spatial control of actin remodelling that occurs during bacterial invasion. © 2011 Blackwell Publishing Ltd.

Liquid behavior of cross-linked actin bundles.

PubMed

Weirich, Kimberly L; Banerjee, Shiladitya; Dasbiswas, Kinjal; Witten, Thomas A; Vaikuntanathan, Suriyanarayanan; Gardel, Margaret L

2017-02-28

The actin cytoskeleton is a critical regulator of cytoplasmic architecture and mechanics, essential in a myriad of physiological processes. Here we demonstrate a liquid phase of actin filaments in the presence of the physiological cross-linker, filamin. Filamin condenses short actin filaments into spindle-shaped droplets, or tactoids, with shape dynamics consistent with a continuum model of anisotropic liquids. We find that cross-linker density controls the droplet shape and deformation timescales, consistent with a variable interfacial tension and viscosity. Near the liquid-solid transition, cross-linked actin bundles show behaviors reminiscent of fluid threads, including capillary instabilities and contraction. These data reveal a liquid droplet phase of actin, demixed from the surrounding solution and dominated by interfacial tension. These results suggest a mechanism to control organization, morphology, and dynamics of the actin cytoskeleton.

A Missense Mutation in the Capza3 Gene and Disruption of F-actin Organization in Spermatids of repro32 Infertile Male Mice

PubMed Central

Geyer, Christopher B.; Inselman, Amy L.; Sunman, Jeffrey A.; Bornstein, Sheila; Handel, Mary Ann; Eddy, Edward M.

2009-01-01

Males homozygous for the repro32 ENU-induced mutation produced by the Reproductive Genomics program at The Jackson Laboratory are infertile, have low epididymal sperm concentrations, and produce sperm with abnormally shaped heads and poor motility. The purpose of the present study was to identify the mutated gene in repro32 mice and to define the structural and functional changes causing infertility and the aberrant sperm phenotype. In repro32/repro32 mice, we discovered a failure to shed excess cytoplasm and disorganization of the middle piece of the flagellum at spermiation, resulting in the outer dense fibers being wrapped around the sperm head within a bag of cytoplasm. Using a candidate-gene approach, a mutation was identified in the spermatid-specific “capping protein (actin filament) muscle Z-line, alpha 3” gene (Capza3). CAPZA3 protein localization was altered in spermatids concurrent with altered localization of a unique CAPZB variant isoform and disruption of the filamentous actin (F-actin) network. These observations strongly suggest the missense mutation in Capza3 is responsible for the mutant phenotype of repro32/repro32 sperm and regulation of F-actin dynamics by a spermatogenic cell-specific CAPZ heterodimer is essential for removal of the cytoplasm and maintenance of midpiece integrity during spermiation in the mouse. PMID:19341723

Traveling waves in actin dynamics and cell motility

PubMed Central

Allard, Jun; Mogilner, Alex

2012-01-01

Much of current understanding of cell motility arose from studying steady treadmilling of actin arrays. Recently, there have been a growing number of observations of a more complex, non-steady, actin behavior, including self-organized waves. It is becoming clear that these waves result from activation and inhibition feedbacks in actin dynamics acting on different scales, but the exact molecular nature of these feedbacks and respective roles of biomechanics and biochemistry are still unclear. Here, we review recent advances achieved in experimental and theoretical studies of actin waves and discuss mechanisms and physiological significance of wavy protrusions. PMID:22985541

Live-cell imaging of actin dynamics reveals mechanisms of stereocilia length regulation in the inner ear

PubMed Central

Drummond, Meghan C.; Barzik, Melanie; Bird, Jonathan E.; Zhang, Duan-Sun; Lechene, Claude P.; Corey, David P.; Cunningham, Lisa L.; Friedman, Thomas B.

2015-01-01

The maintenance of sensory hair cell stereocilia is critical for lifelong hearing; however, mechanisms of structural homeostasis remain poorly understood. Conflicting models propose that stereocilia F-actin cores are either continually renewed every 24–48 h via a treadmill or are stable, exceptionally long-lived structures. Here to distinguish between these models, we perform an unbiased survey of stereocilia actin dynamics in more than 500 utricle hair cells. Live-imaging EGFP-β-actin or dendra2-β-actin reveal stable F-actin cores with turnover and elongation restricted to stereocilia tips. Fixed-cell microscopy of wild-type and mutant β-actin demonstrates that incorporation of actin monomers into filaments is required for localization to stereocilia tips. Multi-isotope imaging mass spectrometry and live imaging of single differentiating hair cells capture stereociliogenesis and explain uniform incorporation of 15N-labelled protein and EGFP-β-actin into nascent stereocilia. Collectively, our analyses support a model in which stereocilia actin cores are stable structures that incorporate new F-actin only at the distal tips. PMID:25898120

The unusual dynamics of parasite actin result from isodesmic polymerization

PubMed Central

Skillman, Kristen M.; Ma, Christopher I.; Fremont, Daved H.; Diraviyam, Karthikeyan; Cooper, John A.; Sept, David; Sibley, L. David

2013-01-01

Previous reports have indicated that parasite actins are short and inherently unstable, despite being required for motility. Here, we re-examine the polymerization properties of actin in Toxoplasma gondii (TgACTI), unexpectedly finding that it exhibits isodesmic polymerization in contrast to the conventional nucleation-elongation process of all previously studied actins from both eukaryotes and bacteria. TgACTI polymerization kinetics lacks both a lag phase and critical concentration, normally characteristic of actins. Unique among actins, the kinetics of assembly can be fit with a single set of rate constants for all subunit interactions, without need for separate nucleation and elongation rates. This isodesmic model accurately predicts the assembly, disassembly, and the size distribution of TgACTI filaments in vitro, providing a mechanistic explanation for actin dynamics in vivo. Our findings expand the repertoire of mechanisms by which actin polymerization is governed and offer clues about the evolution of self-assembling, stabilized protein polymers. PMID:23921463

TWISTED DWARF1 Mediates the Action of Auxin Transport Inhibitors on Actin Cytoskeleton Dynamics

PubMed Central

Bailly, Aurelien; Zwiewka, Marta; Sovero, Valpuri; Ge, Pei; Aryal, Bibek; Hao, Pengchao; Linnert, Miriam; Burgardt, Noelia Inés; Lücke, Christian; Weiwad, Matthias; Michel, Max; Weiergräber, Oliver H.; Pollmann, Stephan; Azzarello, Elisa; Fukao, Yoichiro; Hoffmann, Céline; Wedlich-Söldner, Roland

2016-01-01

Plant growth and architecture is regulated by the polar distribution of the hormone auxin. Polarity and flexibility of this process is provided by constant cycling of auxin transporter vesicles along actin filaments, coordinated by a positive auxin-actin feedback loop. Both polar auxin transport and vesicle cycling are inhibited by synthetic auxin transport inhibitors, such as 1-N-naphthylphthalamic acid (NPA), counteracting the effect of auxin; however, underlying targets and mechanisms are unclear. Using NMR, we map the NPA binding surface on the Arabidopsis thaliana ABCB chaperone TWISTED DWARF1 (TWD1). We identify ACTIN7 as a relevant, although likely indirect, TWD1 interactor, and show TWD1-dependent regulation of actin filament organization and dynamics and that TWD1 is required for NPA-mediated actin cytoskeleton remodeling. The TWD1-ACTIN7 axis controls plasma membrane presence of efflux transporters, and as a consequence act7 and twd1 share developmental and physiological phenotypes indicative of defects in auxin transport. These can be phenocopied by NPA treatment or by chemical actin (de)stabilization. We provide evidence that TWD1 determines downstream locations of auxin efflux transporters by adjusting actin filament debundling and dynamizing processes and mediating NPA action on the latter. This function appears to be evolutionary conserved since TWD1 expression in budding yeast alters actin polarization and cell polarity and provides NPA sensitivity. PMID:27053424

The Dynamic Actin Cytoskeleton in Smooth Muscle.

PubMed

Tang, Dale D

2018-01-01

Smooth muscle contraction requires both myosin activation and actin cytoskeletal remodeling. Actin cytoskeletal reorganization facilitates smooth muscle contraction by promoting force transmission between the contractile unit and the extracellular matrix (ECM), and by enhancing intercellular mechanical transduction. Myosin may be viewed to serve as an "engine" for smooth muscle contraction whereas the actin cytoskeleton may function as a "transmission system" in smooth muscle. The actin cytoskeleton in smooth muscle also undergoes restructuring upon activation with growth factors or the ECM, which controls smooth muscle cell proliferation and migration. Abnormal smooth muscle contraction, cell proliferation, and motility contribute to the development of vascular and pulmonary diseases. A number of actin-regulatory proteins including protein kinases have been discovered to orchestrate actin dynamics in smooth muscle. In particular, Abelson tyrosine kinase (c-Abl) is an important molecule that controls actin dynamics, contraction, growth, and motility in smooth muscle. Moreover, c-Abl coordinates the regulation of blood pressure and contributes to the pathogenesis of airway hyperresponsiveness and vascular/airway remodeling in vivo. Thus, c-Abl may be a novel pharmacological target for the development of new therapy to treat smooth muscle diseases such as hypertension and asthma. © 2018 Elsevier Inc. All rights reserved.

A hemidesmosomal protein regulates actin dynamics and traction forces in motile keratinocytes

PubMed Central

Hiroyasu, Sho; Colburn, Zachary T.; Jones, Jonathan C. R.

2016-01-01

During wound healing of the skin, keratinocytes disassemble hemidesmosomes and reorganize their actin cytoskeletons in order to exert traction forces on and move directionally over the dermis. Nonetheless, the transmembrane hemidesmosome component collagen XVII (ColXVII) is found in actin-rich lamella, situated behind the lamellipodium. A set of actin bundles, along which ColXVII colocalizes with actinin4, is present at each lamella. Knockdown of either ColXVII or actinin4 not only inhibits directed migration of keratinocytes but also relieves constraints on actin bundle retrograde movement at the site of lamella, such that actin bundle movement is enhanced more than 5-fold. Moreover, whereas control keratinocytes move in a stepwise fashion over a substrate by generating alternating traction forces, of up to 1.4 kPa, at each flank of the lamellipodium, ColXVII knockdown keratinocytes fail to do so. In summary, our data indicate that ColXVII-actinin4 complexes at the lamella of a moving keratinocyte regulate actin dynamics, thereby determining the direction of cell movement.—Hiroyasu, S., Colburn, Z. T., Jones, J. C. R. A hemidesmosomal protein regulates actin dynamics and traction forces in motile keratinocytes. PMID:26936359

Myosin isoform determines the conformational dynamics and cooperativity of actin filaments in the strongly bound actomyosin complex

PubMed Central

Prochniewicz, Ewa; Chin, Harvey F.; Henn, Arnon; Hannemann, Diane E.; Olivares, Adrian O.; Thomas, David D.; De La Cruz, Enrique M.

2010-01-01

SUMMARY We have used transient phosphorescence anisotropy (TPA) to detect the microsecond rotational dynamics of erythrosin iodoacetamide (ErIA)-labeled actin strongly bound to single-headed fragments of muscle myosin (muscle S1) and non-muscle myosin V (MV). The conformational dynamics of actin filaments in solution are markedly influenced by the isoform of bound myosin. Both myosins increase the final anisotropy of actin at sub-stoichiometric binding densities, indicating long-range, non-nearest neighbor cooperative restriction of filament rotational dynamics amplitude, but the cooperative unit is larger with MV than muscle S1. Both myosin isoforms also cooperatively affect the actin filament rotational correlation time, but with opposite effects; muscle S1 decreases rates of intrafilament torsional motion, while binding of MV increases the rates of motion. The cooperative effects on the rates of intrafilament motions correlate with the kinetics of myosin binding to actin filaments such that MV binds more rapidly, and muscle myosin more slowly, to partially decorated filaments than to bare filaments. The two isoforms also differ in their effects on the phosphorescence lifetime of the actin-bound ErIA; while muscle S1 increases the lifetime, suggesting decreased aqueous exposure of the probe, MV does not induce a significant change. We conclude that the dynamics and structure of actin in the strongly bound actomyosin complex is determined by the isoform of the bound myosin, in a manner likely to accommodate the diverse functional roles of actomyosin in muscle and non-muscle cells. PMID:19962990

Stochastic Ratcheting on a Funneled Energy Landscape Is Necessary for Highly Efficient Contractility of Actomyosin Force Dipoles

NASA Astrophysics Data System (ADS)

Komianos, James E.; Papoian, Garegin A.

2018-04-01

Current understanding of how contractility emerges in disordered actomyosin networks of nonmuscle cells is still largely based on the intuition derived from earlier works on muscle contractility. In addition, in disordered networks, passive cross-linkers have been hypothesized to percolate force chains in the network, hence, establishing large-scale connectivity between local contractile clusters. This view, however, largely overlooks the free energy of cross-linker binding at the microscale, which, even in the absence of active fluctuations, provides a thermodynamic drive towards highly overlapping filamentous states. In this work, we use stochastic simulations and mean-field theory to shed light on the dynamics of a single actomyosin force dipole—a pair of antiparallel actin filaments interacting with active myosin II motors and passive cross-linkers. We first show that while passive cross-linking without motor activity can produce significant contraction between a pair of actin filaments, driven by thermodynamic favorability of cross-linker binding, a sharp onset of kinetic arrest exists at large cross-link binding energies, greatly diminishing the effectiveness of this contractility mechanism. Then, when considering an active force dipole containing nonmuscle myosin II, we find that cross-linkers can also serve as a structural ratchet when the motor dissociates stochastically from the actin filaments, resulting in significant force amplification when both molecules are present. Our results provide predictions of how actomyosin force dipoles behave at the molecular level with respect to filament boundary conditions, passive cross-linking, and motor activity, which can explicitly be tested using an optical trapping experiment.

Thymosin-beta(4) changes the conformation and dynamics of actin monomers.

PubMed Central

De La Cruz, E M; Ostap, E M; Brundage, R A; Reddy, K S; Sweeney, H L; Safer, D

2000-01-01

Thymosin-beta(4) (Tbeta(4)) binds actin monomers stoichiometrically and maintains the bulk of the actin monomer pool in metazoan cells. Tbeta(4) binding quenches the fluorescence of N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (AEDANS) conjugated to Cys(374) of actin monomers. The K(d) of the actin-Tbeta(4) complex depends on the cation and nucleotide bound to actin but is not affected by the AEDANS probe. The different stabilities are determined primarily by the rates of dissociation. At 25 degrees C, the free energy of Tbeta(4) binding MgATP-actin is primarily enthalpic in origin but entropic for CaATP-actin. Binding is coupled to the dissociation of bound water molecules, which is greater for CaATP-actin than MgATP-actin monomers. Proteolysis of MgATP-actin, but not CaATP-actin, at Gly(46) on subdomain 2 is >12 times faster when Tbeta(4) is bound. The C terminus of Tbeta(4) contacts actin near this cleavage site, at His(40). By tritium exchange, Tbeta(4) slows the exchange rate of approximately eight rapidly exchanging amide protons on actin. We conclude that Tbeta(4) changes the conformation and structural dynamics ("breathing") of actin monomers. The conformational change may reflect the unique ability of Tbeta(4) to sequester actin monomers and inhibit nucleotide exchange. PMID:10777749

Palmitoylation of LIM Kinase-1 ensures spine-specific actin polymerization and morphological plasticity

PubMed Central

George, Joju; Soares, Cary; Montersino, Audrey; Beique, Jean-Claude; Thomas, Gareth M

2015-01-01

Precise regulation of the dendritic spine actin cytoskeleton is critical for neurodevelopment and neuronal plasticity, but how neurons spatially control actin dynamics is not well defined. Here, we identify direct palmitoylation of the actin regulator LIM kinase-1 (LIMK1) as a novel mechanism to control spine-specific actin dynamics. A conserved palmitoyl-motif is necessary and sufficient to target LIMK1 to spines and to anchor LIMK1 in spines. ShRNA knockdown/rescue experiments reveal that LIMK1 palmitoylation is essential for normal spine actin polymerization, for spine-specific structural plasticity and for long-term spine stability. Palmitoylation is critical for LIMK1 function because this modification not only controls LIMK1 targeting, but is also essential for LIMK1 activation by its membrane-localized upstream activator PAK. These novel roles for palmitoylation in the spatial control of actin dynamics and kinase signaling provide new insights into structural plasticity mechanisms and strengthen links between dendritic spine impairments and neuropathological conditions. DOI: http://dx.doi.org/10.7554/eLife.06327.001 PMID:25884247

A WAVE2–Arp2/3 actin nucleator apparatus supports junctional tension at the epithelial zonula adherens

PubMed Central

Verma, Suzie; Han, Siew Ping; Michael, Magdalene; Gomez, Guillermo A.; Yang, Zhe; Teasdale, Rohan D.; Ratheesh, Aparna; Kovacs, Eva M.; Ali, Radiya G.; Yap, Alpha S.

2012-01-01

The epithelial zonula adherens (ZA) is a specialized adhesive junction where actin dynamics and myosin-driven contractility coincide. The junctional cytoskeleton is enriched in myosin II, which generates contractile force to support junctional tension. It is also enriched in dynamic actin filaments, which are replenished by ongoing actin assembly. In this study we sought to pursue the relationship between actin assembly and junctional contractility. We demonstrate that WAVE2–Arp2/3 is a major nucleator of actin assembly at the ZA and likely acts in response to junctional Rac signaling. Furthermore, WAVE2–Arp2/3 is necessary for junctional integrity and contractile tension at the ZA. Maneuvers that disrupt the function of either WAVE2 or Arp2/3 reduced junctional tension and compromised the ability of cells to buffer side-to-side forces acting on the ZA. WAVE2–Arp2/3 disruption depleted junctions of both myosin IIA and IIB, suggesting that dynamic actin assembly may support junctional tension by facilitating the local recruitment of myosin. PMID:23051739

A WAVE2-Arp2/3 actin nucleator apparatus supports junctional tension at the epithelial zonula adherens.

PubMed

Verma, Suzie; Han, Siew Ping; Michael, Magdalene; Gomez, Guillermo A; Yang, Zhe; Teasdale, Rohan D; Ratheesh, Aparna; Kovacs, Eva M; Ali, Radiya G; Yap, Alpha S

2012-12-01

The epithelial zonula adherens (ZA) is a specialized adhesive junction where actin dynamics and myosin-driven contractility coincide. The junctional cytoskeleton is enriched in myosin II, which generates contractile force to support junctional tension. It is also enriched in dynamic actin filaments, which are replenished by ongoing actin assembly. In this study we sought to pursue the relationship between actin assembly and junctional contractility. We demonstrate that WAVE2-Arp2/3 is a major nucleator of actin assembly at the ZA and likely acts in response to junctional Rac signaling. Furthermore, WAVE2-Arp2/3 is necessary for junctional integrity and contractile tension at the ZA. Maneuvers that disrupt the function of either WAVE2 or Arp2/3 reduced junctional tension and compromised the ability of cells to buffer side-to-side forces acting on the ZA. WAVE2-Arp2/3 disruption depleted junctions of both myosin IIA and IIB, suggesting that dynamic actin assembly may support junctional tension by facilitating the local recruitment of myosin.

Growing Actin Networks Form Lamellipodium and Lamellum by Self-Assembly

PubMed Central

Huber, Florian; Käs, Josef; Stuhrmann, Björn

2008-01-01

Many different cell types are able to migrate by formation of a thin actin-based cytoskeletal extension. Recently, it became evident that this extension consists of two distinct substructures, designated lamellipodium and lamellum, which differ significantly in their kinetic and kinematic properties as well as their biochemical composition. We developed a stochastic two-dimensional computer simulation that includes chemical reaction kinetics, G-actin diffusion, and filament transport to investigate the formation of growing actin networks in migrating cells. Model parameters were chosen based on experimental data or theoretical considerations. In this work, we demonstrate the system's ability to form two distinct networks by self-organization. We found a characteristic transition in mean filament length as well as a distinct maximum in depolymerization flux, both within the first 1–2 μm. The separation into two distinct substructures was found to be extremely robust with respect to initial conditions and variation of model parameters. We quantitatively investigated the complex interplay between ADF/cofilin and tropomyosin and propose a plausible mechanism that leads to spatial separation of, respectively, ADF/cofilin- or tropomyosin-dominated compartments. Tropomyosin was found to play an important role in stabilizing the lamellar actin network. Furthermore, the influence of filament severing and annealing on the network properties is explored, and simulation data are compared to existing experimental data. PMID:18708450

A cardiomyocyte-specific Wdr1 knockout demonstrates essential functional roles for actin disassembly during myocardial growth and maintenance in mice.

PubMed

Yuan, Baiyin; Wan, Ping; Chu, Dandan; Nie, Junwei; Cao, Yunshan; Luo, Wen; Lu, Shuangshuang; Chen, Jiong; Yang, Zhongzhou

2014-07-01

Actin dynamics are critical for muscle development and function, and mutations leading to deregulation of actin dynamics cause various forms of heritable muscle diseases. AIP1 is a major cofactor of the actin depolymerizing factor/cofilin in eukaryotes, promoting actin depolymerizing factor/cofilin-mediated actin disassembly. Its function in vertebrate muscle has been unknown. To investigate functional roles of AIP1 in myocardium, we generated conditional knockout (cKO) mice with cardiomyocyte-specific deletion of Wdr1, the mammalian homolog of yeast AIP1. Wdr1 cKO mice began to die at postnatal day 13 (P13), and none survived past P24. At P12, cKO mice exhibited cardiac hypertrophy and impaired contraction of the left ventricle. Electrocardiography revealed reduced heart rate, abnormal P wave, and abnormal T wave at P10 and prolonged QT interval at P12. Actin filament (F-actin) accumulations began at P10 and became prominent at P12 in the myocardium of cKO mice. Within regions of F-actin accumulation in myofibrils, the sarcomeric components α-actinin and tropomodulin-1 exhibited disrupted patterns, indicating that F-actin accumulations caused by Wdr1 deletion result in disruption of sarcomeric structure. Ectopic cofilin colocalized with F-actin aggregates. In adult mice, Wdr1 deletion resulted in similar but much milder phenotypes of heart hypertrophy, F-actin accumulations within myofibrils, and lethality. Taken together, these results demonstrate that AIP1-regulated actin dynamics play essential roles in heart function in mice. Copyright © 2014 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Geometrical Determinants of Neuronal Actin Waves.

PubMed

Tomba, Caterina; Braïni, Céline; Bugnicourt, Ghislain; Cohen, Floriane; Friedrich, Benjamin M; Gov, Nir S; Villard, Catherine

2017-01-01

Hippocampal neurons produce in their early stages of growth propagative, actin-rich dynamical structures called actin waves. The directional motion of actin waves from the soma to the tip of neuronal extensions has been associated with net forward growth, and ultimately with the specification of neurites into axon and dendrites. Here, geometrical cues are used to control actin wave dynamics by constraining neurons on adhesive stripes of various widths. A key observable, the average time between the production of consecutive actin waves, or mean inter-wave interval (IWI), was identified. It scales with the neurite width, and more precisely with the width of the proximal segment close to the soma. In addition, the IWI is independent of the total number of neurites. These two results suggest a mechanistic model of actin wave production, by which the material conveyed by actin waves is assembled in the soma until it reaches the threshold leading to the initiation and propagation of a new actin wave. Based on these observations, we formulate a predictive theoretical description of actin wave-driven neuronal growth and polarization, which consistently accounts for different sets of experiments.

Geometrical Determinants of Neuronal Actin Waves

PubMed Central

Tomba, Caterina; Braïni, Céline; Bugnicourt, Ghislain; Cohen, Floriane; Friedrich, Benjamin M.; Gov, Nir S.; Villard, Catherine

2017-01-01

Hippocampal neurons produce in their early stages of growth propagative, actin-rich dynamical structures called actin waves. The directional motion of actin waves from the soma to the tip of neuronal extensions has been associated with net forward growth, and ultimately with the specification of neurites into axon and dendrites. Here, geometrical cues are used to control actin wave dynamics by constraining neurons on adhesive stripes of various widths. A key observable, the average time between the production of consecutive actin waves, or mean inter-wave interval (IWI), was identified. It scales with the neurite width, and more precisely with the width of the proximal segment close to the soma. In addition, the IWI is independent of the total number of neurites. These two results suggest a mechanistic model of actin wave production, by which the material conveyed by actin waves is assembled in the soma until it reaches the threshold leading to the initiation and propagation of a new actin wave. Based on these observations, we formulate a predictive theoretical description of actin wave-driven neuronal growth and polarization, which consistently accounts for different sets of experiments. PMID:28424590

Coupled biopolymer networks

NASA Astrophysics Data System (ADS)

Schwarz, J. M.; Zhang, Tao

2015-03-01

The actin cytoskeleton provides the cell with structural integrity and allows it to change shape to crawl along a surface, for example. The actin cytoskeleton can be modeled as a semiflexible biopolymer network that modifies its morphology in response to both external and internal stimuli. Just inside the inner nuclear membrane of a cell exists a network of filamentous lamin that presumably protects the heart of the cell nucleus--the DNA. Lamins are intermediate filaments that can also be modeled as semiflexible biopolymers. It turns out that the actin cytoskeletal biopolymer network and the lamin biopolymer network are coupled via a sequence of proteins that bridge the outer and inner nuclear membranes. We, therefore, probe the consequences of such a coupling via numerical simulations to understand the resulting deformations in the lamin network in response to perturbations in the cytoskeletal network. Such study could have implications for mechanical mechanisms of the regulation of transcription, since DNA--yet another semiflexible polymer--contains lamin-binding domains, and, thus, widen the field of epigenetics.

Mechanisms of leading edge protrusion in interstitial migration

PubMed Central

Wilson, Kerry; Lewalle, Alexandre; Fritzsche, Marco; Thorogate, Richard; Duke, Tom; Charras, Guillaume

2013-01-01

While the molecular and biophysical mechanisms underlying cell protrusion on two-dimensional substrates are well understood, our knowledge of the actin structures driving protrusion in three-dimensional environments is poor, despite relevance to inflammation, development and cancer. Here we report that, during chemotactic migration through microchannels with 5 μm × 5 μm cross-sections, HL60 neutrophil-like cells assemble an actin-rich slab filling the whole channel cross-section at their front. This leading edge comprises two distinct F-actin networks: an adherent network that polymerizes perpendicular to cell-wall interfaces and a ‘free’ network that grows from the free membrane at the cell front. Each network is polymerized by a distinct nucleator and, due to their geometrical arrangement, the networks interact mechanically. On the basis of our experimental data, we propose that, during interstitial migration, medial growth of the adherent network compresses the free network preventing its retrograde movement and enabling new polymerization to be converted into forward protrusion. PMID:24305616

State transitions of actin cortices in vitro and in vivo

NASA Astrophysics Data System (ADS)

Tan, Tzer Han; Keren, Kinneret; Mackintosh, Fred; Schmidt, Christoph; Fakhri, Nikta

Most animal cells are enveloped by a thin layer of actin cortex which governs the cell mechanics. A functional cortex must be rigid to provide mechanical support while being flexible to allow for rapid restructuring events such as cell division. To satisfy these requirements, the actin cortex is highly dynamic with fast actin turnover and myosin-driven contractility. The regulatory mechanism responsible for the transition between a mechanically stable state and a restructuring state is not well understood. Here, we develop a technique to map the dynamics of reconstituted actin cortices in emulsion droplets using IR fluorescent single-walled carbon nanotubes (SWNTs). By increasing crosslinker concentration, we find that a homogeneous cortex transitions to an intermediate state with broken rotational symmetry and a globally contractile state which further breaks translational symmetry. We apply this new dynamic mapping technique to cortices of live starfish oocytes in various developmental stages. To identify the regulatory mechanism for steady state transitions, we subject the oocytes to actin and myosin disrupting drugs.

Water molecules in the nucleotide binding cleft of actin: effects on subunit conformation and implications for ATP hydrolysis.

PubMed

Saunders, Marissa G; Voth, Gregory A

2011-10-14

In the monomeric actin crystal structure, the positions of a highly organized network of waters are clearly visible within the active site. However, the recently proposed models of filamentous actin (F-actin) did not extend to including these waters. Since the water network is important for ATP hydrolysis, information about water position is critical to understanding the increased rate of catalysis upon filament formation. Here, we show that waters in the active site are essential for intersubdomain rotational flexibility and that they organize the active-site structure. Including the crystal structure waters during simulation setup allows us to observe distinct changes in the active-site structure upon the flattening of the actin subunit, as proposed in the Oda model for F-actin. We identify changes in both protein position and water position relative to the phosphate tail that suggest a mechanism for accelerating the rate of nucleotide hydrolysis in F-actin by stabilizing charge on the β-phosphate and by facilitating deprotonation of catalytic water. Copyright © 2011 Elsevier Ltd. All rights reserved.

Identification of cation-binding sites on actin that drive polymerization and modulate bending stiffness

PubMed Central

Kang, Hyeran; Bradley, Michael J.; McCullough, Brannon R.; Pierre, Anaëlle; Grintsevich, Elena E.; Reisler, Emil; De La Cruz, Enrique M.

2012-01-01

The assembly of actin monomers into filaments and networks plays vital roles throughout eukaryotic biology, including intracellular transport, cell motility, cell division, determining cellular shape, and providing cells with mechanical strength. The regulation of actin assembly and modulation of filament mechanical properties are critical for proper actin function. It is well established that physiological salt concentrations promote actin assembly and alter the overall bending mechanics of assembled filaments and networks. However, the molecular origins of these salt-dependent effects, particularly if they involve nonspecific ionic strength effects or specific ion-binding interactions, are unknown. Here, we demonstrate that specific cation binding at two discrete sites situated between adjacent subunits along the long-pitch helix drive actin polymerization and determine the filament bending rigidity. We classify the two sites as “polymerization” and “stiffness” sites based on the effects that mutations at the sites have on salt-dependent filament assembly and bending mechanics, respectively. These results establish the existence and location of the cation-binding sites that confer salt dependence to the assembly and mechanics of actin filaments. PMID:23027950

Disruption of the actin cytoskeleton results in the promotion of gravitropism in inflorescence stems and hypocotyls of Arabidopsis

NASA Technical Reports Server (NTRS)

Yamamoto, Kazuyoshi; Kiss, John Z.

2002-01-01

The actin cytoskeleton is hypothesized to play a major role in gravity perception and transduction mechanisms in roots of plants. To determine whether actin microfilaments (MFs) are involved in these processes in stem-like organs, we studied gravitropism in Arabidopsis inflorescence stems and hypocotyls. Localization studies using Alexa Fluor-phalloidin in conjugation with confocal microscopy demonstrated a longitudinally and transversely oriented actin MF network in endodermal cells of stems and hypocotyls. Latrunculin B (Lat-B) treatment of hypocotyls caused depolymerization of actin MFs in endodermal cells and a significant reduction of hypocotyl growth rates. Actin MFs in Lat-B-treated inflorescence stems also were disrupted, but growth rates were not affected. Despite disruption of the actin cytoskeleton in these two organs, Lat-B-treated stems and hypocotyls exhibited a promotion of gravitropic curvature in response to reorientation. In contrast, Lat-B reduced gravitropic curvature in roots but also reduced the growth rate. Thus, in contrast to prevailing hypotheses, our results suggest that actin MFs are not a necessary component of gravitropism in inflorescence stems and hypocotyls. Furthermore, this is the first study to demonstrate a prominent actin MF network in endodermal cells in the putative gravity-perceiving cells in stems.

Disruption of the Actin Cytoskeleton Results in the Promotion of Gravitropism in Inflorescence Stems and Hypocotyls of Arabidopsis1

PubMed Central

Yamamoto, Kazuyoshi; Kiss, John Z.

2002-01-01

The actin cytoskeleton is hypothesized to play a major role in gravity perception and transduction mechanisms in roots of plants. To determine whether actin microfilaments (MFs) are involved in these processes in stem-like organs, we studied gravitropism in Arabidopsis inflorescence stems and hypocotyls. Localization studies using Alexa Fluor-phalloidin in conjugation with confocal microscopy demonstrated a longitudinally and transversely oriented actin MF network in endodermal cells of stems and hypocotyls. Latrunculin B (Lat-B) treatment of hypocotyls caused depolymerization of actin MFs in endodermal cells and a significant reduction of hypocotyl growth rates. Actin MFs in Lat-B-treated inflorescence stems also were disrupted, but growth rates were not affected. Despite disruption of the actin cytoskeleton in these two organs, Lat-B-treated stems and hypocotyls exhibited a promotion of gravitropic curvature in response to reorientation. In contrast, Lat-B reduced gravitropic curvature in roots but also reduced the growth rate. Thus, in contrast to prevailing hypotheses, our results suggest that actin MFs are not a necessary component of gravitropism in inflorescence stems and hypocotyls. Furthermore, this is the first study to demonstrate a prominent actin MF network in endodermal cells in the putative gravity-perceiving cells in stems. PMID:11842170

Scale dependence of the mechanics of active gels with increasing motor concentration.

PubMed

Sonn-Segev, Adar; Bernheim-Groswasser, Anne; Roichman, Yael

2017-10-18

Actin is a protein that plays an essential role in maintaining the mechanical integrity of cells. In response to strong external stresses, it can assemble into large bundles, but it grows into a fine branched network to induce cell motion. In some cases, the self-organization of actin fibers and networks involves the action of bipolar filaments of the molecular motor myosin. Such self-organization processes mediated by large myosin bipolar filaments have been studied extensively in vitro. Here we create active gels, composed of single actin filaments and small myosin bipolar filaments. The active steady state in these gels persists long enough to enable the characterization of their mechanical properties using one and two point microrheology. We study the effect of myosin concentration on the mechanical properties of this model system for active matter, for two different motor assembly sizes. In contrast to previous studies of networks with large motor assemblies, we find that the fluctuations of tracer particles embedded in the network decrease in amplitude as motor concentration increases. Nonetheless, we show that myosin motors stiffen the actin networks, in accordance with bulk rheology measurements of networks containing larger motor assemblies. This implies that such stiffening is of universal nature and may be relevant to a wider range of cytoskeleton-based structures.

Dynamics in steady state in vitro acto-myosin networks

NASA Astrophysics Data System (ADS)

Sonn-Segev, Adar; Bernheim-Groswasser, Anne; Roichman, Yael

2017-04-01

It is well known that many biochemical processes in the cell such as gene regulation, growth signals and activation of ion channels, rely on mechanical stimuli. However, the mechanism by which mechanical signals propagate through cells is not as well understood. In this review we focus on stress propagation in a minimal model for cell elasticity, actomyosin networks, which are comprised of a sub-family of cytoskeleton proteins. After giving an overview of th actomyosin network components, structure and evolution we review stress propagation in these materials as measured through the correlated motion of tracer beads. We also discuss the possibility to extract structural features of these networks from the same experiments. We show that stress transmission through these networks has two pathways, a quickly dissipative one through the bulk, and a long ranged weakly dissipative one through the pre-stressed actin network.

Cofilin-2 controls actin filament length in muscle sarcomeres

PubMed Central

Kremneva, Elena; Makkonen, Maarit H.; Skwarek-Maruszewska, Aneta; Gateva, Gergana; Michelot, Alphee; Dominguez, Roberto; Lappalainen, Pekka

2014-01-01

SUMMARY ADF/cofilins drive cytoskeletal dynamics by promoting the disassembly of ‘aged’ ADP-actin filaments. Mammals express several ADF/cofilin isoforms, but their specific biochemical activities and cellular functions have not been studied in detail. Here we demonstrate that the muscle-specific isoform cofilin-2 promotes actin filament disassembly in sarcomeres to control the precise length of thin filaments in the contractile apparatus. In contrast to other isoforms, cofilin-2 efficiently binds and disassembles both ADP- and ATP/ADP-Pi-actin filaments. We mapped surface-exposed cofilin-2-specific residues required for ATP-actin binding and propose that these residues function as an ‘actin nucleotide-state sensor’ among ADF/cofilins. The results suggest that cofilin-2 evolved specific biochemical and cellular properties allowing it to control actin dynamics in sarcomeres, where filament pointed ends may contain a mixture of ADP- and ATP/ADP-Pi-actin subunits. Our findings also offer a rationale for why cofilin-2 mutations in humans lead to myopathies. PMID:25373779

Divergent regulation of the sarcomere and the cytoskeleton.

PubMed

Schevzov, Galina; Fath, Thomas; Vrhovski, Bernadette; Vlahovich, Nicole; Rajan, Sudarsan; Hook, Jeff; Joya, Josephine E; Lemckert, Frances; Puttur, Franz; Lin, Jim J-C; Hardeman, Edna C; Wieczorek, David F; O'Neill, Geraldine M; Gunning, Peter W

2008-01-04

The existence of a feedback mechanism regulating the precise amounts of muscle structural proteins, such as actin and the actin-associated protein tropomyosin (Tm), in the sarcomeres of striated muscles is well established. However, the regulation of nonmuscle or cytoskeletal actin and Tms in nonmuscle cell structures has not been elucidated. Unlike the thin filaments of striated muscles, the actin cytoskeleton in nonmuscle cells is intrinsically dynamic. Given the differing requirements for the structural integrity of the actin thin filaments of the sarcomere compared with the requirement for dynamicity of the actin cytoskeleton in nonmuscle cells, we postulated that different regulatory mechanisms govern the expression of sarcomeric versus cytoskeletal Tms, as key regulators of the properties of the actin cytoskeleton. Comprehensive analyses of tissues from transgenic and knock-out mouse lines that overexpress the cytoskeletal Tms, Tm3 and Tm5NM1, and a comparison with sarcomeric Tms provide evidence for this. Moreover, we show that overexpression of a cytoskeletal Tm drives the amount of filamentous actin.

The interplay between neuronal activity and actin dynamics mimic the setting of an LTD synaptic tag

PubMed Central

Szabó, Eszter C.; Manguinhas, Rita; Fonseca, Rosalina

2016-01-01

Persistent forms of plasticity, such as long-term depression (LTD), are dependent on the interplay between activity-dependent synaptic tags and the capture of plasticity-related proteins. We propose that the synaptic tag represents a structural alteration that turns synapses permissive to change. We found that modulation of actin dynamics has different roles in the induction and maintenance of LTD. Inhibition of either actin depolymerisation or polymerization blocks LTD induction whereas only the inhibition of actin depolymerisation blocks LTD maintenance. Interestingly, we found that actin depolymerisation and CaMKII activation are involved in LTD synaptic-tagging and capture. Moreover, inhibition of actin polymerisation mimics the setting of a synaptic tag, in an activity-dependent manner, allowing the expression of LTD in non-stimulated synapses. Suspending synaptic activation also restricts the time window of synaptic capture, which can be restored by inhibiting actin polymerization. Our results support our hypothesis that modulation of the actin cytoskeleton provides an input-specific signal for synaptic protein capture. PMID:27650071

Palytoxins and cytoskeleton: An overview.

PubMed

Louzao, M Carmen; Ares, Isabel R; Cagide, Eva; Espiña, Begoña; Vilariño, Natalia; Alfonso, Amparo; Vieytes, Mercedes R; Botana, Luis M

2011-03-01

Cytoskeleton is a dynamic structure essential for a wide variety of normal cellular processes, including the maintenance of cell shape and morphology, volume regulation, membrane dynamics and signal transduction. Cytoskeleton is organized into microtubules, actin meshwork and intermediate filaments. Actin has been identified as a major target for destruction during apoptosis and is also important under pathological conditions such as cancers. Several natural compounds actively modulate actin organization by specific signaling cascades being useful tools to study cytoskeleton dynamics. Palytoxin is a large bioactive compound, first isolated from zoanthids, with a complex structure and different analogs such as ostreocin-D or ovatoxin-a. This toxin has been identified as a potent tumor promoter and cytotoxic molecule, which leads to actin filament distortion and triggers cell death or apoptosis. In this review we report the findings on the involvement of palytoxin and analogues modulating the actin cytoskeleton within different cellular models. Copyright © 2010 Elsevier Ltd. All rights reserved.

SGR9, a RING type E3 ligase, modulates amyloplast dynamics important for gravity sensing.

NASA Astrophysics Data System (ADS)

Morita, Miyo T.; Nakamura, Moritaka; Tasaka, Masao

Gravitropism is triggered when the directional change of gravity is sensed in the specific cells, called statocytes. In higher plants, statocytes contain sinking heavier amyloplasts which are particular plastids accumulating starch granules. The displacement of amyloplasts within the statocytes is thought to be the initial event of gravity perception. We have demonstrated that endodermal cells are most likely to be the statocytes in Arabidop-sis shoots. Live cell imaging of the endodermal cell of stem has shown that most amyloplasts are sediment to the direction of gravity but they are not static. Several amyloplasts move dynamically in an actin filament (F-actin) dependent manner. In the presence of actin poly-merization inhibitor, all amyloplasts become static and sediment to the direction of gravity. In addition, stems treated with the inhibitor can exhibit gravitropism. These results suggest that F-actin-dependent dynamic movement of amyloplasts is not essential for gravity sensing. sgr (shoot gravitropism) 9 mutant exhibits greatly reduced shoot gravitropism. In endodermal cells of sgr9, dynamic amyloplast movement was predominantly observed and amyloplasts did not sediment to the direction of gravity. Interestingly, inhibition of actin polymerization re-stored both gravitropism and amyloplast sedimentation in sgr9. The SGR9 encodes a novel RING finger protein, which is localized to amyloplasts in endodermal cells. SGR9 showed ubiq-uitin E3 ligase activity in vitro. Together with live cell imaging of amyloplasts and F-actin, our data suggest that SGR9 modulate interaction between amyloplasts and F-actin on amylo-plasts. SGR9 positively act on amyloplasts sedimentation, probably by releasing amyloplasts from F-actin. SGR9 that is localized to amyloplast, possibly degrades unknown substrates by its E3 ligase activity, and this might promote release of amyloplasts from F-actin.

Exploring the human mesenchymal stem cell tubule communication network through electron microscopy.

PubMed

Valente, Sabrina; Rossi, Roberta; Resta, Leonardo; Pasquinelli, Gianandrea

2015-04-01

Cells use several mechanisms to transfer information to other cells. In this study, we describe micro/nanotubular connections and exosome-like tubule fragments in multipotent mesenchymal stem cells (MSCs) from human arteries. Scanning and transmission electron microscopy allowed characterization of sinusoidal microtubular projections (700 nm average size, 200 µm average length, with bulging mitochondria and actin microfilaments); short, uniform, variously shaped nanotubular projections (100 nm, bidirectional communication); and tubule fragments (50 nm). This is the first study demonstrating that MSCs from human arteries constitutively interact through an articulate and dynamic tubule network allowing long-range cell to cell communication.

Actin Filaments and Myosin I Alpha Cooperate with Microtubules for the Movement of LysosomesV⃞

PubMed Central

Cordonnier, Marie-Neige; Dauzonne, Daniel; Louvard, Daniel; Coudrier, Evelyne

2001-01-01

An earlier report suggested that actin and myosin I alpha (MMIα), a myosin associated with endosomes and lysosomes, were involved in the delivery of internalized molecules to lysosomes. To determine whether actin and MMIα were involved in the movement of lysosomes, we analyzed by time-lapse video microscopy the dynamic of lysosomes in living mouse hepatoma cells (BWTG3 cells), producing green fluorescent protein actin or a nonfunctional domain of MMIα. In GFP-actin cells, lysosomes displayed a combination of rapid long-range directional movements dependent on microtubules, short random movements, and pauses, sometimes on actin filaments. We showed that the inhibition of the dynamics of actin filaments by cytochalasin D increased pauses of lysosomes on actin structures, while depolymerization of actin filaments using latrunculin A increased the mobility of lysosomes but impaired the directionality of their long-range movements. The production of a nonfunctional domain of MMIα impaired the intracellular distribution of lysosomes and the directionality of their long-range movements. Altogether, our observations indicate for the first time that both actin filaments and MMIα contribute to the movement of lysosomes in cooperation with microtubules and their associated molecular motors. PMID:11739797

3D Model of Cytokinetic Contractile Ring Assembly: Node-Mediated and Backup Pathways

NASA Astrophysics Data System (ADS)

Bidone, Tamara; Vavylonis, Dimitrios

Cytokinetic ring assembly in model organism fission yeast is a dynamic process, involving condensation of a network of actin filaments and myosin motors bound to the cell membrane through cortical nodes. A 3D computational model of ring assembly illustrates how the combined activities of myosin motors, filament crosslinkers and actin turnover lead to robust ring formation [Bidone et al. Biophys. J, 2014]. We modeled the importance of the physical properties of node movement along the cell membrane and of myosin recruitment to nodes. Experiments by D. Zhang (Temasek Life Sciences) show that tethering of the cortical endoplasmic reticulum (ER) to the plasma membrane modulates the speed of node condensation and the degree of node clumping. We captured the trend observed in these experiments by changes in the node drag coefficient and initial node distribution in simulations PM. The model predicted that reducing crosslinking activities in ER tethering mutants with faster node speed enhances actomyosin clumping. We developed a model of how tilted and/or misplaced rings assemble in cells that lack the node structural component anillin-like Mid1 and thus fail to recruit myosin II to nodes independently of actin. If actin-dependent binding of diffusive myosin to the cortex is incorporated into the model, it generates progressively elongating cortical actomyosin strands with fluctuating actin bundles at the tails. These stands often close into a ring, similar to observations by the group of J.Q. Wu (The Ohio State University). NIH R01GM098430.

F5-peptide induces aspermatogenesis by disrupting organization of actin- and microtubule-based cytoskeletons in the testis

PubMed Central

Gao, Ying; Mruk, Dolores D.; Lui, Wing-yee; Lee, Will M.; Cheng, C. Yan

2016-01-01

During the release of sperm at spermiation, a biologically active F5-peptide, which can disrupt the Sertoli cell tight junction (TJ) permeability barrier, is produced at the site of the degenerating apical ES (ectoplasmic specialization). This peptide coordinates the events of spermiation and blood-testis barrier (BTB) remodeling at stage VIII of the epithelial cycle, creating a local apical ES-BTB axis to coordinate cellular events across the epithelium. The mechanism(s) by which F5-peptide perturbs BTB restructuring, and its involvement in apical ES dynamics remain unknown. F5-peptide, besides perturbing BTB integrity, was shown to induce germ cell release from the epithelium following its efficient in vivo overexpression in the testis. Overexpression of F5-peptide caused disorganization of actin- and microtubule (MT)-based cytoskeletons, mediated by altering the spatiotemporal expression of actin binding/regulatory proteins in the seminiferous epithelium. F5-peptide perturbed the ability of actin microfilaments and/or MTs from converting between their bundled and unbundled/defragmented configuration, thereby perturbing adhesion between spermatids and Sertoli cells. Since apical ES and basal ES/BTB are interconnected through the underlying cytoskeletal networks, this thus provides an efficient and novel mechanism to coordinate different cellular events across the epithelium during spermatogenesis through changes in the organization of actin microfilaments and MTs. These findings also illustrate the potential of F5-peptide being a male contraceptive peptide for men. PMID:27611949

Deciphering Interplay between Salmonella Invasion Effectors

PubMed Central

Koronakis, Vassilis

2008-01-01

Bacterial pathogens have evolved a specialized type III secretion system (T3SS) to translocate virulence effector proteins directly into eukaryotic target cells. Salmonellae deploy effectors that trigger localized actin reorganization to force their own entry into non-phagocytic host cells. Six effectors (SipC, SipA, SopE/2, SopB, SptP) can individually manipulate actin dynamics at the plasma membrane, which acts as a ‘signaling hub’ during Salmonella invasion. The extent of crosstalk between these spatially coincident effectors remains unknown. Here we describe trans and cis binary entry effector interplay (BENEFIT) screens that systematically examine functional associations between effectors following their delivery into the host cell. The results reveal extensive ordered synergistic and antagonistic relationships and their relative potency, and illuminate an unexpectedly sophisticated signaling network evolved through longstanding pathogen–host interaction. PMID:18389058

Cell Protrusion and Retraction Driven by Fluctuations in Actin Polymerization: A Two-Dimensional Model

PubMed Central

Ryan, Gillian L.; Holz, Danielle; Yamashiro, Sawako; Taniguchi, Daisuke; Watanabe, Naoki; Vavylonis, Dimitrios

2017-01-01

Animal cells that spread onto a surface often rely on actin-rich lamellipodial extensions to execute protrusion. Many cell types recently adhered on a two-dimensional substrate exhibit protrusion and retraction of their lamellipodia, even though the cell is not translating. Traveling waves of protrusion have also been observed, similar to those observed in crawling cells. These regular patterns of protrusion and retraction allow quantitative analysis for comparison to mathematical models. The periodic fluctuations in leading edge position of XTC cells have been linked to excitable actin dynamics using a one-dimensional model of actin dynamics, as a function of arc-length along the cell. In this work we extend this earlier model of actin dynamics into two dimensions (along the arc-length and radial directions of the cell) and include a model membrane that protrudes and retracts in response to the changing number of free barbed ends of actin filaments near the membrane. We show that if the polymerization rate at the barbed ends changes in response to changes in their local concentration at the leading edge and/or the opposing force from the cell membrane, the model can reproduce the patterns of membrane protrusion and retraction seen in experiment. We investigate both Brownian ratchet and switch-like force-velocity relationships between the membrane load forces and actin polymerization rate. The switch-like polymerization dynamics recover the observed patterns of protrusion and retraction as well as the fluctuations in F-actin concentration profiles. The model generates predictions for the behavior of cells after local membrane tension perturbations. PMID:28752950

Dynamics of the actin cytoskeleton mediates receptor cross talk: An emerging concept in tuning receptor signaling

PubMed Central

Mattila, Pieta K.; Batista, Facundo D.

2016-01-01

Recent evidence implicates the actin cytoskeleton in the control of receptor signaling. This may be of particular importance in the context of immune receptors, such as the B cell receptor, where dysregulated signaling can result in autoimmunity and malignancy. Here, we discuss the role of the actin cytoskeleton in controlling receptor compartmentalization, dynamics, and clustering as a means to regulate receptor signaling through controlling the interactions with protein partners. We propose that the actin cytoskeleton is a point of integration for receptor cross talk through modulation of protein dynamics and clustering. We discuss the implication of this cross talk via the cytoskeleton for both ligand-induced and low-level constitutive (tonic) signaling necessary for immune cell survival. PMID:26833785

Arp2/3 Complex from Acanthamoeba Binds Profilin and Cross-links Actin Filaments

PubMed Central

Mullins, R. Dyche; Kelleher, Joseph F.; Xu, James; Pollard, Thomas D.

1998-01-01

The Arp2/3 complex was first purified from Acanthamoeba castellanii by profilin affinity chromatography. The mechanism of interaction with profilin was unknown but was hypothesized to be mediated by either Arp2 or Arp3. Here we show that the Arp2 subunit of the complex can be chemically cross-linked to the actin-binding site of profilin. By analytical ultracentrifugation, rhodamine-labeled profilin binds Arp2/3 complex with a Kd of 7 μM, an affinity intermediate between the low affinity of profilin for barbed ends of actin filaments and its high affinity for actin monomers. These data suggest the barbed end of Arp2 is exposed, but Arp2 and Arp3 are not packed together in the complex exactly like two actin monomers in a filament. Arp2/3 complex also cross-links actin filaments into small bundles and isotropic networks, which are mechanically stiffer than solutions of actin filaments alone. Arp2/3 complex is concentrated at the leading edge of motile Acanthamoeba, and its localization is distinct from that of α-actinin, another filament cross-linking protein. Based on localization and actin filament nucleation and cross-linking activities, we propose a role for Arp2/3 in determining the structure of the actin filament network at the leading edge of motile cells. PMID:9529382

Actin Turnover-Mediated Gravity Response in Maize Root Apices

PubMed Central

Mancuso, Stefano; Barlow, Peter W; Volkmann, Dieter

2006-01-01

The dynamic actin cytoskeleton has been proposed to be linked to gravity sensing in plants but the mechanistic understanding of these processes remains unknown. We have performed detailed pharmacological analyses of the role of the dynamic actin cytoskeleton in gravibending of maize (Zea mays) root apices. Depolymerization of actin filaments with two drugs having different mode of their actions, cytochalasin D and latrunculin B, stimulated root gravibending. By contrast, drug-induced stimulation of actin polymerization and inhibition of actin turnover, using two different agents phalloidin and jasplakinolide, compromised the root gravibending. Importantly, all these actin drugs inhibited root growth to similar extents suggesting that high actin turnover is essential for the gravity-related growth responses rather than for the general growth process. Both latrunculin B and cytochalasin D treatments inhibited root growth but restored gravibending of the decapped root apices, indicating that there is a strong potential for effective actin-mediated gravity sensing outside the cap. This elusive gravity sensing outside the root cap is dependent not only on the high rate of actin turnover but also on weakening of myosin activities, as general inhibition of myosin ATPases induced stimulation of gravibending of the decapped root apices. Collectively, these data provide evidence for the actin turnover-mediated gravity sensing outside the root cap. PMID:19521476

Alteration of the Cortical Actin Cytoskeleton Deregulates Ca2+ Signaling, Monospermic Fertilization, and Sperm Entry

PubMed Central

Puppo, A.; Chun, Jong T.; Gragnaniello, Giovanni; Garante, Ezio; Santella, Luigia

2008-01-01

Background When preparing for fertilization, oocytes undergo meiotic maturation during which structural changes occur in the endoplasmic reticulum (ER) that lead to a more efficient calcium response. During meiotic maturation and subsequent fertilization, the actin cytoskeleton also undergoes dramatic restructuring. We have recently observed that rearrangements of the actin cytoskeleton induced by actin-depolymerizing agents, or by actin-binding proteins, strongly modulate intracellular calcium (Ca2+) signals during the maturation process. However, the significance of the dynamic changes in F-actin within the fertilized egg has been largely unclear. Methodology/Principal Findings We have measured changes in intracellular Ca2+ signals and F-actin structures during fertilization. We also report the unexpected observation that the conventional antagonist of the InsP3 receptor, heparin, hyperpolymerizes the cortical actin cytoskeleton in postmeiotic eggs. Using heparin and other pharmacological agents that either hypo- or hyperpolymerize the cortical actin, we demonstrate that nearly all aspects of the fertilization process are profoundly affected by the dynamic restructuring of the egg cortical actin cytoskeleton. Conclusions/Significance Our findings identify important roles for subplasmalemmal actin fibers in the process of sperm-egg interaction and in the subsequent events related to fertilization: the generation of Ca2+ signals, sperm penetration, cortical granule exocytosis, and the block to polyspermy. PMID:18974786

Azadirachtin(A) distinctively modulates subdomain 2 of actin - novel mechanism to induce depolymerization revealed by molecular dynamics study.

PubMed

Pravin Kumar, R; Roopa, L; Sudheer Mohammed, M M; Kulkarni, Naveen

2016-12-01

Azadirachtin(A) (AZA), a potential insecticide from neem, binds to actin and induces depolymerization in Drosophila. AZA binds to the pocket same as that of Latrunculin A (LAT), but LAT inhibits actin polymerization by stiffening the actin structure and affects the ADP-ATP exchange. The mechanism by which AZA induces actin depolymerization is not clearly understood. Therefore, different computational experiments were conducted to delineate the precise mechanism of AZA-induced actin depolymerization. Molecular dynamics studies showed that AZA strongly interacted with subdomain 2 and destabilized the interactions between subdomain 2 of one actin and subdomains 1 and 4 of the adjacent actin, causing the separation of actin subunits. The separation was observed between subdomain 3 of subunit n and subdomain 4 of subunit n + 2. However, the specific triggering point for the separation of the subunits was the destabilization of direct interactions between subdomain 2 of subunit n (Arg39, Val45, Gly46 and Arg62) and subdomain 4 of subunit n + 2 (Asp286, Ile287, Asp288, Ile289, Asp244 and Lys291). These results reveal a unique mechanism of an actin filament modulator that induces depolymerization. This mechanism of AZA can be used to design similar molecules against mammalian actins for cancer therapy.

Microtubule-Actin Cross-Linking Factor 1: Domains, Interaction Partners, and Tissue-Specific Functions.

PubMed

Goryunov, Dmitry; Liem, Ronald K H

2016-01-01

The cytoskeleton of most eukaryotic cells is composed of three principal filamentous components: actin filaments, microtubules (MTs), and intermediate filaments. It is a highly dynamic system that plays crucial roles in a wide range of cellular processes, including migration, adhesion, cytokinesis, morphogenesis, intracellular traffic and signaling, and structural flexibility. Among the large number of cytoskeleton-associated proteins characterized to date, microtubule-actin cross-linking factor 1 (MACF1) is arguably the most versatile integrator and modulator of cytoskeleton-related processes. MACF1 belongs to the plakin family of proteins, and within it, to the spectraplakin subfamily. These proteins are characterized by the ability to bridge MT and actin cytoskeletal networks in a dynamic fashion, which underlies their involvement in the regulation of cell migration, axonal extension, and vesicular traffic. Studying MACF1 functions has provided insights not only into the regulation of the cytoskeleton but also into molecular mechanisms of both normal cellular physiology and cellular pathology. Multiple MACF1 isoforms exist, composed of a large variety of alternatively spliced domains. Each of these domains mediates a specific set of interactions and functions. These functions are manifested in tissue and cell-specific phenotypes observed in conditional MACF1 knockout mice. The conditional models described to date reveal critical roles of MACF1 in mammalian skin, nervous system, heart muscle, and intestinal epithelia. Complete elimination of MACF1 is early embryonic lethal, indicating an essential role for MACF1 in early development. Further studies of MACF1 domains and their interactions will likely reveal multiple new roles of this protein in various tissues. © 2016 Elsevier Inc. All rights reserved.

F-Actin Dynamics in Neurospora crassa ▿ †

PubMed Central

Berepiki, Adokiye; Lichius, Alexander; Shoji, Jun-Ya; Tilsner, Jens; Read, Nick D.

2010-01-01

This study demonstrates the utility of Lifeact for the investigation of actin dynamics in Neurospora crassa and also represents the first report of simultaneous live-cell imaging of the actin and microtubule cytoskeletons in filamentous fungi. Lifeact is a 17-amino-acid peptide derived from the nonessential Saccharomyces cerevisiae actin-binding protein Abp140p. Fused to green fluorescent protein (GFP) or red fluorescent protein (TagRFP), Lifeact allowed live-cell imaging of actin patches, cables, and rings in N. crassa without interfering with cellular functions. Actin cables and patches localized to sites of active growth during the establishment and maintenance of cell polarity in germ tubes and conidial anastomosis tubes (CATs). Recurrent phases of formation and retrograde movement of complex arrays of actin cables were observed at growing tips of germ tubes and CATs. Two populations of actin patches exhibiting slow and fast movement were distinguished, and rapid (1.2 μm/s) saltatory transport of patches along cables was observed. Actin cables accumulated and subsequently condensed into actin rings associated with septum formation. F-actin organization was markedly different in the tip regions of mature hyphae and in germ tubes. Only mature hyphae displayed a subapical collar of actin patches and a concentration of F-actin within the core of the Spitzenkörper. Coexpression of Lifeact-TagRFP and β-tubulin–GFP revealed distinct but interrelated localization patterns of F-actin and microtubules during the initiation and maintenance of tip growth. PMID:20139238

Leading-process actomyosin coordinates organelle positioning and adhesion receptor dynamics in radially migrating cerebellar granule neurons

DOE PAGES

Trivedi, Niraj; Ramahi, Joseph S.; Karakaya, Mahmut; ...

2014-12-02

During brain development, neurons migrate from germinal zones to their final positions to assemble neural circuits. A unique saltatory cadence involving cyclical organelle movement (e.g., centrosome motility) and leading-process actomyosin enrichment prior to nucleokinesis organizes neuronal migration. While functional evidence suggests that leading-process actomyosin is essential for centrosome motility, the role of the actin-enriched leading process in globally organizing organelle transport or traction forces remains unexplored. Our results show that myosin ii motors and F-actin dynamics are required for Golgi apparatus positioning before nucleokinesis in cerebellar granule neurons (CGNs) migrating along glial fibers. Moreover, we show that primary cilia aremore » motile organelles, localized to the leading-process F-actin-rich domain and immobilized by pharmacological inhibition of myosin ii and F-actin dynamics. Finally, leading process adhesion dynamics are dependent on myosin ii and F-actin. In conclusion, we propose that actomyosin coordinates the overall polarity of migrating CGNs by controlling asymmetric organelle positioning and cell-cell contacts as these cells move along their glial guides.« less

Leading-process actomyosin coordinates organelle positioning and adhesion receptor dynamics in radially migrating cerebellar granule neurons

DOE Office of Scientific and Technical Information (OSTI.GOV)

Trivedi, Niraj; Ramahi, Joseph S.; Karakaya, Mahmut

During brain development, neurons migrate from germinal zones to their final positions to assemble neural circuits. A unique saltatory cadence involving cyclical organelle movement (e.g., centrosome motility) and leading-process actomyosin enrichment prior to nucleokinesis organizes neuronal migration. While functional evidence suggests that leading-process actomyosin is essential for centrosome motility, the role of the actin-enriched leading process in globally organizing organelle transport or traction forces remains unexplored. Our results show that myosin ii motors and F-actin dynamics are required for Golgi apparatus positioning before nucleokinesis in cerebellar granule neurons (CGNs) migrating along glial fibers. Moreover, we show that primary cilia aremore » motile organelles, localized to the leading-process F-actin-rich domain and immobilized by pharmacological inhibition of myosin ii and F-actin dynamics. Finally, leading process adhesion dynamics are dependent on myosin ii and F-actin. In conclusion, we propose that actomyosin coordinates the overall polarity of migrating CGNs by controlling asymmetric organelle positioning and cell-cell contacts as these cells move along their glial guides.« less

TIPsy tour guides: how microtubule plus-end tracking proteins (+TIPs) facilitate axon guidance

PubMed Central

Bearce, Elizabeth A.; Erdogan, Burcu; Lowery, Laura Anne

2015-01-01

The growth cone is a dynamic cytoskeletal vehicle, which drives the end of a developing axon. It serves to interpret and navigate through the complex landscape and guidance cues of the early nervous system. The growth cone’s distinctive cytoskeletal organization offers a fascinating platform to study how extracellular cues can be translated into mechanical outgrowth and turning behaviors. While many studies of cell motility highlight the importance of actin networks in signaling, adhesion, and propulsion, both seminal and emerging works in the field have highlighted a unique and necessary role for microtubules (MTs) in growth cone navigation. Here, we focus on the role of singular pioneer MTs, which extend into the growth cone periphery and are regulated by a diverse family of microtubule plus-end tracking proteins (+TIPs). These +TIPs accumulate at the dynamic ends of MTs, where they are well-positioned to encounter and respond to key signaling events downstream of guidance receptors, catalyzing immediate changes in microtubule stability and actin cross-talk, that facilitate both axonal outgrowth and turning events. PMID:26175669

A Prestressed Cable Network Model of the Adherent Cell Cytoskeleton

PubMed Central

Coughlin, Mark F.; Stamenović, Dimitrije

2003-01-01

A prestressed cable network is used to model the deformability of the adherent cell actin cytoskeleton. The overall and microstructural model geometries and cable mechanical properties were assigned values based on observations from living cells and mechanical measurements on isolated actin filaments, respectively. The models were deformed to mimic cell poking (CP), magnetic twisting cytometry (MTC) and magnetic bead microrheometry (MBM) measurements on living adherent cells. The models qualitatively and quantitatively captured the fibroblast cell response to the deformation imposed by CP while exhibiting only some qualitative features of the cell response to MTC and MBM. The model for CP revealed that the tensed peripheral actin filaments provide the key resistance to indentation. The actin filament tension that provides mechanical integrity to the network was estimated at ∼158 pN, and the nonlinear mechanical response during CP originates from filament kinematics. The MTC and MBM simulations revealed that the model is incomplete, however, these simulations show cable tension as a key determinant of the model response. PMID:12547813

A prestressed cable network model of the adherent cell cytoskeleton.

PubMed

Coughlin, Mark F; Stamenović, Dimitrije

2003-02-01

A prestressed cable network is used to model the deformability of the adherent cell actin cytoskeleton. The overall and microstructural model geometries and cable mechanical properties were assigned values based on observations from living cells and mechanical measurements on isolated actin filaments, respectively. The models were deformed to mimic cell poking (CP), magnetic twisting cytometry (MTC) and magnetic bead microrheometry (MBM) measurements on living adherent cells. The models qualitatively and quantitatively captured the fibroblast cell response to the deformation imposed by CP while exhibiting only some qualitative features of the cell response to MTC and MBM. The model for CP revealed that the tensed peripheral actin filaments provide the key resistance to indentation. The actin filament tension that provides mechanical integrity to the network was estimated at approximately 158 pN, and the nonlinear mechanical response during CP originates from filament kinematics. The MTC and MBM simulations revealed that the model is incomplete, however, these simulations show cable tension as a key determinant of the model response.

Deconvolution of subcellular protrusion heterogeneity and the underlying actin regulator dynamics from live cell imaging.

PubMed

Wang, Chuangqi; Choi, Hee June; Kim, Sung-Jin; Desai, Aesha; Lee, Namgyu; Kim, Dohoon; Bae, Yongho; Lee, Kwonmoo

2018-04-27

Cell protrusion is morphodynamically heterogeneous at the subcellular level. However, the mechanism of cell protrusion has been understood based on the ensemble average of actin regulator dynamics. Here, we establish a computational framework called HACKS (deconvolution of heterogeneous activity in coordination of cytoskeleton at the subcellular level) to deconvolve the subcellular heterogeneity of lamellipodial protrusion from live cell imaging. HACKS identifies distinct subcellular protrusion phenotypes based on machine-learning algorithms and reveals their underlying actin regulator dynamics at the leading edge. Using our method, we discover "accelerating protrusion", which is driven by the temporally ordered coordination of Arp2/3 and VASP activities. We validate our finding by pharmacological perturbations and further identify the fine regulation of Arp2/3 and VASP recruitment associated with accelerating protrusion. Our study suggests HACKS can identify specific subcellular protrusion phenotypes susceptible to pharmacological perturbation and reveal how actin regulator dynamics are changed by the perturbation.

Cross-Linker Unbinding and Self-Similarity in Bundled Cytoskeletal Networks

NASA Astrophysics Data System (ADS)

Lieleg, O.; Bausch, A. R.

2007-10-01

The macromechanical properties of purely bundled in vitro actin networks are not only determined by the micromechanical properties of individual bundles but also by molecular unbinding events of the actin-binding protein (ABP) fascin. Under high mechanical load the network elasticity depends on the forced unbinding of individual ABPs in a rate dependent manner. Cross-linker unbinding in combination with the structural self-similarity of the network enables the introduction of a concentration-time superposition principle—broadening the mechanically accessible frequency range over 8 orders of magnitude.

A Second Las17 Monomeric Actin-Binding Motif Functions in Arp2/3-Dependent Actin Polymerization During Endocytosis

PubMed Central

Feliciano, Daniel; Tolsma, Thomas O.; Farrell, Kristen B.; Aradi, Al; Di Pietro, Santiago M.

2018-01-01

During clathrin-mediated endocytosis (CME), actin assembly provides force to drive vesicle internalization. Members of the Wiskott–Aldrich syndrome protein (WASP) family play a fundamental role stimulating actin assembly. WASP family proteins contain a WH2 motif that binds globular actin (G-actin) and a central-acidic motif that binds the Arp2/3 complex, thus promoting the formation of branched actin filaments. Yeast WASP (Las17) is the strongest of five factors promoting Arp2/3-dependent actin polymerization during CME. It was suggested that this strong activity may be caused by a putative second G-actin-binding motif in Las17. Here, we describe the in vitro and in vivo characterization of such Las17 G-actin-binding motif (LGM) and its dependence on a group of conserved arginine residues. Using the yeast two-hybrid system, GST-pulldown, fluorescence polarization and pyrene-actin polymerization assays, we show that LGM binds G-actin and is necessary for normal Arp2/3-mediated actin polymerization in vitro. Live-cell fluorescence microscopy experiments demonstrate that LGM is required for normal dynamics of actin polymerization during CME. Further, LGM is necessary for normal dynamics of endocytic machinery components that are recruited at early, intermediate and late stages of endocytosis, as well as for optimal endocytosis of native CME cargo. Both in vitro and in vivo experiments show that LGM has relatively lower potency compared to the previously known Las17 G-actin-binding motif, WH2. These results establish a second G-actin-binding motif in Las17 and advance our knowledge on the mechanism of actin assembly during CME. PMID:25615019

The Actin Depolymerizing Factor (ADF)/Cofilin Signaling Pathway and DNA Damage Responses in Cancer

PubMed Central

Chang, Chun-Yuan; Leu, Jyh-Der; Lee, Yi-Jang

2015-01-01

The actin depolymerizing factor (ADF)/cofilin protein family is essential for actin dynamics, cell division, chemotaxis and tumor metastasis. Cofilin-1 (CFL-1) is a primary non-muscle isoform of the ADF/cofilin protein family accelerating the actin filamental turnover in vitro and in vivo. In response to environmental stimulation, CFL-1 enters the nucleus to regulate the actin dynamics. Although the purpose of this cytoplasm-nucleus transition remains unclear, it is speculated that the interaction between CFL-1 and DNA may influence various biological responses, including DNA damage repair. In this review, we will discuss the possible involvement of CFL-1 in DNA damage responses (DDR) induced by ionizing radiation (IR), and the implications for cancer radiotherapy. PMID:25689427

Actin dynamics regulate immediate PAR-2-dependent responses to acute epidermal permeability barrier abrogation.

PubMed

Roelandt, Truus; Heughebaert, Carol; Verween, Gunther; Giddelo, Christina; Verbeken, Gilbert; Pirnay, Jean-Paul; Devos, Daniel; Crumrine, Debra; Roseeuw, Diane; Elias, Peter M; Hachem, Jean-Pierre

2011-02-01

Lamellar body (LB) secretion and terminal differentiation of stratum granulosum (SG) cells are signaled by both protease activated receptor-2 (PAR-2) and caveolin-1 (cav-1). To address the early dynamics of LB secretion, we examined cytoskeletal remodeling of keratinocytes in 3 mouse models following acute barrier abrogation: hairless mice, PAR-2 knockout (-/-) and cav-1 -/-. Under basal conditions, globular (G)-actin accumulates in SG cells cytosol, while filamentous (F)-actin is restricted to peri-membrane domains. Barrier abrogation induces the apical movement of F-actin and the retreat of the SG-G-actin front, paralleled by upstream cytoskeletal kinases activation. This phenomenon was both enhanced by PAR-2 agonist, and inhibited by cytochalasin-D and in PAR-2 knockout mice. We found that plasma membrane conformational changes causing LB secretion are controlled by PAR-2-dependent cytoskeletal rearrangements. We next addressed the interaction dynamics between cytoskeleton and plasma membrane following PAR-2-induced actin stress fiber formation in both cav-1 -/- and wildtype cells. Actin stress fiber formation is increased in cav-1 -/- cells prior to and following PAR-2 agonist peptide-treatment, while absence of cav-1 inhibits E-cadherin-mediated cell-to-cell adhesion. PAR-2 drives cytoskeletal/plasma membrane dynamics that regulate early LB secretion following barrier abrogation, stress fiber formation and keratinocyte adhesion. Copyright © 2010 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

Releasing the brakes while hanging on: Cortactin effects on actin-driven motility.

PubMed

Gov, Nir S; Bernheim-Groswasser, Anne

2012-01-01

Actin polymerization plays a major role in many cellular processes, including cell motility, vesicle trafficking, and pathogen propulsion. The transformation of the (protrusive) polymerization forces into directed motion requires that the growing filaments are positioned next to the surface. This is achieved by localization of surface actin nucleators (WASP), which then activate Arp2/3 complex to form new actin branches. Yet, the same surface-bound WASP molecule which initiates the nucleation of new actin branches, also inherently prevents the translation of the polymerization forces into motion, essentially because the WASP molecule has to be in contact with the network during the formation of the new branch. In our recent paper we show that cortactin relaxes this internal inhibition by enhancing the release of WASP-VCA molecule from the new branching site after nucleation is initiated. We show that this enhanced release has two major effects; it increases the turnover rate of branching per WASP molecule, and it decreases the friction-like force caused by the binding of the moving surface with respect to the growing actin network.

Region-Specific Involvement of Actin Rearrangement-Related Synaptic Structure Alterations in Conditioned Taste Aversion Memory

ERIC Educational Resources Information Center

Bi, Ai-Ling; Wang, Yue; Li, Bo-Qin; Wang, Qian-Qian; Ma, Ling; Yu, Hui; Zhao, Ling; Chen, Zhe-Yu

2010-01-01

Actin rearrangement plays an essential role in learning and memory; however, the spatial and temporal regulation of actin dynamics in different phases of associative memory has not been fully understood. Here, using the conditioned taste aversion (CTA) paradigm, we investigated the region-specific involvement of actin rearrangement-related…

Allosteric Coupling of CARMIL and V-1 Binding to Capping Protein Revealed by Hydrogen-Deuterium Exchange.

PubMed

Johnson, Britney; McConnell, Patrick; Kozlov, Alex G; Mekel, Marlene; Lohman, Timothy M; Gross, Michael L; Amarasinghe, Gaya K; Cooper, John A

2018-05-29

Actin assembly is important for cell motility. The ability of actin subunits to join or leave filaments via the barbed end is critical to actin dynamics. Capping protein (CP) binds to barbed ends to prevent subunit gain and loss and is regulated by proteins that include V-1 and CARMIL. V-1 inhibits CP by sterically blocking one binding site for actin. CARMILs bind at a distal site and decrease the affinity of CP for actin, suggested to be caused by conformational changes. We used hydrogen-deuterium exchange with mass spectrometry (HDX-MS) to probe changes in structural dynamics induced by V-1 and CARMIL binding to CP. V-1 and CARMIL induce changes in both proteins' binding sites on the surface of CP, along with a set of internal residues. Both also affect the conformation of CP's ββ subunit "tentacle," a second distal actin-binding site. Concerted regulation of actin assembly by CP occurs through allosteric couplings between CP modulator and actin binding sites. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Direct Microtubule-Binding by Myosin-10 Orients Centrosomes toward Retraction Fibers and Subcortical Actin Clouds.

PubMed

Kwon, Mijung; Bagonis, Maria; Danuser, Gaudenz; Pellman, David

2015-08-10

Positioning of centrosomes is vital for cell division and development. In metazoan cells, spindle positioning is controlled by a dynamic pool of subcortical actin that organizes in response to the position of retraction fibers. These actin "clouds" are proposed to generate pulling forces on centrosomes and mediate spindle orientation. However, the motors that pull astral microtubules toward these actin structures are not known. Here, we report that the unconventional myosin, Myo10, couples actin-dependent forces from retraction fibers and subcortical actin clouds to centrosomes. Myo10-mediated centrosome positioning requires its direct microtubule binding. Computational image analysis of large microtubule populations reveals a direct effect of Myo10 on microtubule dynamics and microtubule-cortex interactions. Myo10's role in centrosome positioning is distinct from, but overlaps with, that of dynein. Thus, Myo10 plays a key role in integrating the actin and microtubule cytoskeletons to position centrosomes and mitotic spindles. Copyright © 2015 Elsevier Inc. All rights reserved.

Direct Microtubule-Binding by Myosin-10 Orients Centrosomes toward Retraction Fibers and Subcortical Actin Clouds

PubMed Central

Kwon, Mijung; Bagonis, Maria; Danuser, Gaudenz; Pellman, David

2015-01-01

SUMMARY Positioning of centrosomes is vital for cell division and development. In metazoan cells, spindle positioning is controlled by a dynamic pool of subcortical actin that organizes in response to the position of retraction fibers. These actin “clouds” are proposed to generate pulling forces on centrosomes and mediate spindle orientation. However, the motors that pull astral microtubules toward these actin structures are not known. Here, we report that the unconventional myosin, Myo10, couples actin-dependent forces from retraction fibers and subcortical actin clouds to centrosomes. Myo10-mediated centrosome positioning requires its direct microtubule binding. Computational image analysis of large microtubule populations reveals a direct effect of Myo10 on microtubule dynamics and microtubule-cortex interactions. Myo10’s role in centrosome positioning is distinct from, but overlaps with, that of dynein. Thus, Myo10 plays a key role in integrating the actin and microtubule cytoskeletons to position centrosomes and mitotic spindles. PMID:26235048

Molecular organization of cytokinesis nodes and contractile rings by super-resolution fluorescence microscopy of live fission yeast

PubMed Central

Laplante, Caroline; Huang, Fang; Tebbs, Irene R.; Bewersdorf, Joerg; Pollard, Thomas D.

2016-01-01

Cytokinesis in animals, fungi, and amoebas depends on the constriction of a contractile ring built from a common set of conserved proteins. Many fundamental questions remain about how these proteins organize to generate the necessary tension for cytokinesis. Using quantitative high-speed fluorescence photoactivation localization microscopy (FPALM), we probed this question in live fission yeast cells at unprecedented resolution. We show that nodes, protein assembly precursors to the contractile ring, are discrete structural units with stoichiometric ratios and distinct distributions of constituent proteins. Anillin Mid1p, Fes/CIP4 homology-Bin/amphiphysin/Rvs (F-BAR) Cdc15p, IQ motif containing GTPase-activating protein (IQGAP) Rng2p, and formin Cdc12p form the base of the node that anchors the ends of myosin II tails to the plasma membrane, with myosin II heads extending into the cytoplasm. This general node organization persists in the contractile ring where nodes move bidirectionally during constriction. We observed the dynamics of the actin network during cytokinesis, starting with the extension of short actin strands from nodes, which sometimes connected neighboring nodes. Later in cytokinesis, a broad network of thick bundles coalesced into a tight ring around the equator of the cell. The actin ring was ∼125 nm wide and ∼125 nm thick. These observations establish the organization of the proteins in the functional units of a cytokinetic contractile ring. PMID:27647921

Oligomerization of coronin: Implication on actin filament length in Leishmania.

PubMed

Srivastava, Rashmi; Prasadareddy Kajuluri, Lova; Pathak, Neelam; Gupta, Chhitar M; Sahasrabuddhe, Amogh A

2015-12-01

Coronin proteins bind with actin filaments and participate in regulation of actin-dependent processes. These proteins contain a coiled-coil domain at their C-terminus, which is responsible for their dimeric or trimeric forms. However, the functional significance of these oligomeric configurations in organizing the actin cytoskeleton is obscure. Here, we report that the Leishmania coronin exists in a higher oligomeric form through its coiled-coil domain, the truncation of which ablates the ability of Leishmania coronin to assist actin-filament formation. F-actin co-sedimentation assay using purified proteins shows that the coiled-coil domain does not interact with actin-filaments and its absence does not abrogate actin-coronin interaction. Furthermore, it was shown that unlike other coronins, Leishmania coronin interacts with actin-filaments through its unique region. These results provided important insights into the role of coronin oligomerization in modulating actin-network. © 2015 Wiley Periodicals, Inc.

A novel role for WAVE1 in controlling actin network growth rate and architecture.

PubMed

Sweeney, Meredith O; Collins, Agnieszka; Padrick, Shae B; Goode, Bruce L

2015-02-01

Branched actin filament networks in cells are assembled through the combined activities of Arp2/3 complex and different WASP/WAVE proteins. Here we used TIRF and electron microscopy to directly compare for the first time the assembly kinetics and architectures of actin filament networks produced by Arp2/3 complex and dimerized VCA regions of WAVE1, WAVE2, or N-WASP. WAVE1 produced strikingly different networks from WAVE2 or N-WASP, which comprised unexpectedly short filaments. Further analysis showed that the WAVE1-specific activity stemmed from an inhibitory effect on filament elongation both in the presence and absence of Arp2/3 complex, which was observed even at low stoichiometries of WAVE1 to actin monomers, precluding an effect from monomer sequestration. Using a series of VCA chimeras, we mapped the elongation inhibitory effects of WAVE1 to its WH2 ("V") domain. Further, mutating a single conserved lysine residue potently disrupted WAVE1's inhibitory effects. Taken together, our results show that WAVE1 has unique activities independent of Arp2/3 complex that can govern both the growth rates and architectures of actin filament networks. Such activities may underlie previously observed differences between the cellular functions of WAVE1 and WAVE2. © 2015 Sweeney et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Motion in partially and fully cross-linked F-actin networks

NASA Astrophysics Data System (ADS)

Morris, Eliza; Ehrlicher, Allen; Weitz, David

2012-02-01

Single molecule experiments have measured stall forces and procession rates of molecular motors on isolated cytoskeletal fibers in Newtonian fluids. But in the cell, these motors are transporting cargo through a highly complex cytoskeletal network. To compare these single molecule results to the forces exerted by motors within the cell, an evaluation of the response of the cytoskeletal network is needed. Using magnetic tweezers and fluorescence confocal microscopy we observe and quantify the relationship between bead motion and filament response in F-actin networks both partially and fully cross-linked with filamin We find that when the transition from full to partial cross-linking is brought about by a decrease in cross-linker concentration there is a simultaneous decline in the elasticity of the network, but the response of the bead remains qualitatively similar. However, when the cross-linking is reduced through a shortening of the F-actin filaments the bead response is completely altered. The characteristics of the altered bead response will be discussed here.

Actin Depolymerizing Factor (ADF/Cofilin) Enhances the Rate of Filament Turnover: Implication in Actin-based Motility

PubMed Central

Carlier, Marie-France; Laurent, Valérie; Santolini, Jérôme; Melki, Ronald; Didry, Dominique; Xia, Gui-Xian; Hong, Yan; Chua, Nam-Hai; Pantaloni, Dominique

1997-01-01

Actin-binding proteins of the actin depolymerizing factor (ADF)/cofilin family are thought to control actin-based motile processes. ADF1 from Arabidopsis thaliana appears to be a good model that is functionally similar to other members of the family. The function of ADF in actin dynamics has been examined using a combination of physical–chemical methods and actin-based motility assays, under physiological ionic conditions and at pH 7.8. ADF binds the ADPbound forms of G- or F-actin with an affinity two orders of magnitude higher than the ATP- or ADP-Pi– bound forms. A major property of ADF is its ability to enhance the in vitro turnover rate (treadmilling) of actin filaments to a value comparable to that observed in vivo in motile lamellipodia. ADF increases the rate of propulsion of Listeria monocytogenes in highly diluted, ADF-limited platelet extracts and shortens the actin tails. These effects are mediated by the participation of ADF in actin filament assembly, which results in a change in the kinetic parameters at the two ends of the actin filament. The kinetic effects of ADF are end specific and cannot be accounted for by filament severing. The main functionally relevant effect is a 25-fold increase in the rate of actin dissociation from the pointed ends, while the rate of dissociation from the barbed ends is unchanged. This large increase in the rate-limiting step of the monomer-polymer cycle at steady state is responsible for the increase in the rate of actin-based motile processes. In conclusion, the function of ADF is not to sequester G-actin. ADF uses ATP hydrolysis in actin assembly to enhance filament dynamics. PMID:9087445

Single-point ACT2 gene mutation in the Arabidopsis root hair mutant der1-3 affects overall actin organization, root growth and plant development.

PubMed

Vaškebová, L; Šamaj, J; Ovecka, M

2017-12-27

The actin cytoskeleton forms a dynamic network in plant cells. A single-point mutation in the DER1 (deformed root hairs1) locus located in the sequence of ACTIN2, a gene for major actin in vegetative tissues of Arabidopsis thaliana, leads to impaired root hair development (Ringli C, Baumberger N, Diet A, Frey B, Keller B. 2002. ACTIN2 is essential for bulge site selection and tip growth during root hair development of Arabidopsis. Plant Physiology129: 1464-1472). Only root hair phenotypes have been described so far in der1 mutants, but here we demonstrate obvious aberrations in the organization of the actin cytoskeleton and overall plant development. Organization of the actin cytoskeleton in epidermal cells of cotyledons, hypocotyls and roots was studied qualitatively and quantitatively by live-cell imaging of transgenic lines carrying the GFP-FABD2 fusion protein and in fixed cells after phalloidin labelling. Patterns of root growth were characterized by FM4-64 vital staining, light-sheet microscopy imaging and microtubule immunolabelling. Plant phenotyping included analyses of germination, root growth and plant biomass. Speed of germination, plant fresh weight and total leaf area were significantly reduced in the der1-3 mutant in comparison with the C24 wild-type. Actin filaments in root, hypocotyl and cotyledon epidermal cells of the der1-3 mutant were shorter, thinner and arranged in more random orientations, while actin bundles were shorter and had altered orientations. The wavy pattern of root growth in der1-3 mutant was connected with higher frequencies of shifted cell division planes (CDPs) in root cells, which was consistent with the shifted positioning of microtubule-based preprophase bands and phragmoplasts. The organization of cortical microtubules in the root cells of the der1-3 mutant, however, was not altered. Root growth rate of the der1-3 mutant is not reduced, but changes in the actin cytoskeleton organization can induce a wavy root growth pattern through deregulation of CDP orientation. The results suggest that the der1-3 mutation in the ACT2 gene does not influence solely root hair formation process, but also has more general effects on the actin cytoskeleton, plant growth and development. © The Author(s) 2017. Published by Oxford University Press on behalf of the Annals of Botany Company.

2,4-Dichlorophenoxyacetic acid promotes S-nitrosylation and oxidation of actin affecting cytoskeleton and peroxisomal dynamics.

PubMed

Rodríguez-Serrano, M; Pazmiño, D M; Sparkes, I; Rochetti, A; Hawes, C; Romero-Puertas, M C; Sandalio, L M

2014-09-01

2,4-Dichlorophenoxyacetic acid (2,4-D) is a synthetic auxin used as a herbicide to control weeds in agriculture. A high concentration of 2,4-D promotes leaf epinasty and cell death. In this work, the molecular mechanisms involved in the toxicity of this herbicide are studied by analysing in Arabidopsis plants the accumulation of reactive oxygen species (ROS) and nitric oxide (NO), and their effect on cytoskeleton structure and peroxisome dynamics. 2,4-D (23 mM) promotes leaf epinasty, whereas this process was prevented by EDTA, which can reduce ·OH accumulation. The analysis of ROS accumulation by confocal microscopy showed a 2,4-D-dependent increase in both H2O2 and O2·(-), whereas total NO was not affected by the treatment. The herbicide promotes disturbances on the actin cytoskeleton structure as a result of post-translational modification of actin by oxidation and S-nitrosylation, which could disturb actin polymerization, as suggested by the reduction of the F-actin/G-actin ratio. These effects were reduced by EDTA, and the reduction of ROS production in Arabidopsis mutants deficient in xanthine dehydrogenase (Atxdh) gave rise to a reduction in actin oxidation. Also, 2,4-D alters the dynamics of the peroxisome, slowing the speed and shortening the distances by which these organelles are displaced. It is concluded that 2,4-D promotes oxidative and nitrosative stress, causing disturbances in the actin cytoskeleton, thereby affecting the dynamics of peroxisomes and some other organelles such as the mitochondria, with xanthine dehydrogenase being involved in ROS production under these conditions. These structural changes in turn appear to be responsible for the leaf epinasty. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.

2,4-Dichlorophenoxyacetic acid promotes S-nitrosylation and oxidation of actin affecting cytoskeleton and peroxisomal dynamics

PubMed Central

Rodríguez-Serrano, M.; Pazmiño, D. M.; Sparkes, I.; Rochetti, A.; Hawes, C.; Romero-Puertas, M. C.; Sandalio, L. M.

2014-01-01

2,4-Dichlorophenoxyacetic acid (2,4-D) is a synthetic auxin used as a herbicide to control weeds in agriculture. A high concentration of 2,4-D promotes leaf epinasty and cell death. In this work, the molecular mechanisms involved in the toxicity of this herbicide are studied by analysing in Arabidopsis plants the accumulation of reactive oxygen species (ROS) and nitric oxide (NO), and their effect on cytoskeleton structure and peroxisome dynamics. 2,4-D (23mM) promotes leaf epinasty, whereas this process was prevented by EDTA, which can reduce ·OH accumulation. The analysis of ROS accumulation by confocal microscopy showed a 2,4-D-dependent increase in both H2O2 and O2·–, whereas total NO was not affected by the treatment. The herbicide promotes disturbances on the actin cytoskeleton structure as a result of post-translational modification of actin by oxidation and S-nitrosylation, which could disturb actin polymerization, as suggested by the reduction of the F-actin/G-actin ratio. These effects were reduced by EDTA, and the reduction of ROS production in Arabidopsis mutants deficient in xanthine dehydrogenase (Atxdh) gave rise to a reduction in actin oxidation. Also, 2,4-D alters the dynamics of the peroxisome, slowing the speed and shortening the distances by which these organelles are displaced. It is concluded that 2,4-D promotes oxidative and nitrosative stress, causing disturbances in the actin cytoskeleton, thereby affecting the dynamics of peroxisomes and some other organelles such as the mitochondria, with xanthine dehydrogenase being involved in ROS production under these conditions. These structural changes in turn appear to be responsible for the leaf epinasty. PMID:24913628

Mechanism of deep-sea fish α-actin pressure tolerance investigated by molecular dynamics simulations.

PubMed

Wakai, Nobuhiko; Takemura, Kazuhiro; Morita, Takami; Kitao, Akio

2014-01-01

The pressure tolerance of monomeric α-actin proteins from the deep-sea fish Coryphaenoides armatus and C. yaquinae was compared to that of non-deep-sea fish C. acrolepis, carp, and rabbit/human/chicken actins using molecular dynamics simulations at 0.1 and 60 MPa. The amino acid sequences of actins are highly conserved across a variety of species. The actins from C. armatus and C. yaquinae have the specific substitutions Q137K/V54A and Q137K/L67P, respectively, relative to C. acrolepis, and are pressure tolerant to depths of at least 6000 m. At high pressure, we observed significant changes in the salt bridge patterns in deep-sea fish actins, and these changes are expected to stabilize ATP binding and subdomain arrangement. Salt bridges between ATP and K137, formed in deep-sea fish actins, are expected to stabilize ATP binding even at high pressure. At high pressure, deep-sea fish actins also formed a greater total number of salt bridges than non-deep-sea fish actins owing to the formation of inter-helix/strand and inter-subdomain salt bridges. Free energy analysis suggests that deep-sea fish actins are stabilized to a greater degree by the conformational energy decrease associated with pressure effect.

Mechanism of Deep-Sea Fish α-Actin Pressure Tolerance Investigated by Molecular Dynamics Simulations

PubMed Central

Wakai, Nobuhiko; Takemura, Kazuhiro; Morita, Takami; Kitao, Akio

2014-01-01

The pressure tolerance of monomeric α-actin proteins from the deep-sea fish Coryphaenoides armatus and C. yaquinae was compared to that of non-deep-sea fish C. acrolepis, carp, and rabbit/human/chicken actins using molecular dynamics simulations at 0.1 and 60 MPa. The amino acid sequences of actins are highly conserved across a variety of species. The actins from C. armatus and C. yaquinae have the specific substitutions Q137K/V54A and Q137K/L67P, respectively, relative to C. acrolepis, and are pressure tolerant to depths of at least 6000 m. At high pressure, we observed significant changes in the salt bridge patterns in deep-sea fish actins, and these changes are expected to stabilize ATP binding and subdomain arrangement. Salt bridges between ATP and K137, formed in deep-sea fish actins, are expected to stabilize ATP binding even at high pressure. At high pressure, deep-sea fish actins also formed a greater total number of salt bridges than non-deep-sea fish actins owing to the formation of inter-helix/strand and inter-subdomain salt bridges. Free energy analysis suggests that deep-sea fish actins are stabilized to a greater degree by the conformational energy decrease associated with pressure effect. PMID:24465747

Electrostatics Control Actin Filament Nucleation and Elongation Kinetics*

PubMed Central

Crevenna, Alvaro H.; Naredi-Rainer, Nikolaus; Schönichen, André; Dzubiella, Joachim; Barber, Diane L.; Lamb, Don C.; Wedlich-Söldner, Roland

2013-01-01

The actin cytoskeleton is a central mediator of cellular morphogenesis, and rapid actin reorganization drives essential processes such as cell migration and cell division. Whereas several actin-binding proteins are known to be regulated by changes in intracellular pH, detailed information regarding the effect of pH on the actin dynamics itself is still lacking. Here, we combine bulk assays, total internal reflection fluorescence microscopy, fluorescence fluctuation spectroscopy techniques, and theory to comprehensively characterize the effect of pH on actin polymerization. We show that both nucleation and elongation are strongly enhanced at acidic pH, with a maximum close to the pI of actin. Monomer association rates are similarly affected by pH at both ends, although dissociation rates are differentially affected. This indicates that electrostatics control the diffusional encounter but not the dissociation rate, which is critical for the establishment of actin filament asymmetry. A generic model of protein-protein interaction, including electrostatics, explains the observed pH sensitivity as a consequence of charge repulsion. The observed pH effect on actin in vitro agrees with measurements of Listeria propulsion in pH-controlled cells. pH regulation should therefore be considered as a modulator of actin dynamics in a cellular environment. PMID:23486468

Dynamic F-actin movement is essential for fertilization in Arabidopsis thaliana

PubMed Central

Kawashima, Tomokazu; Maruyama, Daisuke; Shagirov, Murat; Li, Jing; Hamamura, Yuki; Yelagandula, Ramesh; Toyama, Yusuke; Berger, Frédéric

2014-01-01

In animals, microtubules and centrosomes direct the migration of gamete pronuclei for fertilization. By contrast, flowering plants have lost essential components of the centrosome, raising the question of how flowering plants control gamete nuclei migration during fertilization. Here, we use Arabidopsis thaliana to document a novel mechanism that regulates F-actin dynamics in the female gametes and is essential for fertilization. Live imaging shows that F-actin structures assist the male nucleus during its migration towards the female nucleus. We identify a female gamete-specific Rho-GTPase that regulates F-actin dynamics and further show that actin–myosin interactions are also involved in male gamete nucleus migration. Genetic analyses and imaging indicate that microtubules are dispensable for migration and fusion of male and female gamete nuclei. The innovation of a novel actin-based mechanism of fertilization during plant evolution might account for the complete loss of the centrosome in flowering plants. DOI: http://dx.doi.org/10.7554/eLife.04501.001 PMID:25303363

Vacuolar and cytoskeletal dynamics during elicitor-induced programmed cell death in tobacco BY-2 cells.

PubMed

Higaki, Takumi; Kadota, Yasuhiro; Goh, Tatsuaki; Hayashi, Teruyuki; Kutsuna, Natsumaro; Sano, Toshio; Hasezawa, Seiichiro; Kuchitsu, Kazuyuki

2008-09-01

Responses of plant cells to environmental stresses often involve morphological changes, differentiation and redistribution of various organelles and cytoskeletal network. Tobacco BY-2 cells provide excellent model system for in vivo imaging of these intracellular events. Treatment of the cell cycle-synchronized BY-2 cells with a proteinaceous oomycete elicitor, cryptogein, induces highly synchronous programmed cell death (PCD) and provide a model system to characterize vacuolar and cytoskeletal dynamics during the PCD. Sequential observation revealed dynamic reorganization of the vacuole and actin microfilaments during the execution of the PCD. We further characterized the effects cryptogein on mitotic microtubule organization in cell cycle-synchronized cells. Cryptogein treatment at S phase inhibited formation of the preprophase band, a cortical microtubule band that predicts the cell division site. Cortical microtubules kept their random orientation till their disruption that gradually occurred during the execution of the PCD twelve hours after the cryptogein treatment. Possible molecular mechanisms and physiological roles of the dynamic behavior of the organelles and cytoskeletal network in the pathogenic signal-induced PCD are discussed.

Actin-membrane interactions mediated by NETWORKED2 in Arabidopsis pollen tubes through associations with Pollen Receptor-Like Kinase 4 and 5.

PubMed

Duckney, Patrick; Deeks, Michael J; Dixon, Martin R; Kroon, Johan; Hawkins, Timothy J; Hussey, Patrick J

2017-12-01

During fertilization, Pollen Receptor-Like Kinases (PRKs) control pollen tube growth through the pistil in response to extracellular signals, and regulate the actin cytoskeleton at the tube apex to drive tip growth. We investigated a novel link between membrane-integral PRKs and the actin cytoskeleton, mediated through interactions between PRKs and NET2A; a pollen-specific member of the NETWORKED superfamily of actin-binding proteins. We characterize NET2A as a novel actin-associated protein that localizes to punctae at the plasma membrane of the pollen tube shank, which are stably associated with cortical longitudinal actin cables. NET2A was demonstrated to interact specifically with PRK4 and PRK5 in Nicotiana benthamiana transient expression assays, and associated at discreet foci at the shank membrane of Arabidopsis pollen tubes. Our data indicate that NET2A is recruited to the plasma membrane by PRK4 and PRK5, and that PRK kinase activity is important in facilitating its interaction with NET2A. We conclude that NET2A-PRK interactions mediate discreet sites of stable interactions between the cortical longitudinal actin cables and plasma membrane in the shank region of growing pollen tubes, which we have termed Actin-Membrane Contact Sites (AMCSs). Interactions between PRKs and NET2A implicate a role for NET2A in signal transduction to the actin cytoskeleton during fertilization. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

Imbalance in mitochondrial dynamics and apoptosis in pregnancies among HIV-infected women on HAART with obstetric complications.

PubMed

Guitart-Mampel, Mariona; Hernandez, A Sandra; Moren, Constanza; Catalan-Garcia, Marc; Tobias, Ester; Gonzalez-Casacuberta, Ingrid; Juarez-Flores, Diana L; Gatell, Josep M; Cardellach, Francesc; Milisenda, Jose C; Grau, Josep M; Gratacos, Eduard; Figueras, Francesc; Garrabou, Gloria

2017-09-01

HIV infection and HAART trigger genetic and functional mitochondrial alterations leading to cell death and adverse clinical manifestations. Mitochondrial dynamics enable mitochondrial turnover and degradation of damaged mitochondria, which may lead to apoptosis. To evaluate markers of mitochondrial dynamics and apoptosis in pregnancies among HIV-infected women on HAART and determine their potential association with obstetric complications. This controlled, single-site, observational study without intervention included 26 HIV-infected pregnant women on HAART and 18 control pregnancies and their newborns. Maternal PBMCs and neonatal cord blood mononuclear cells (CBMCs) were isolated at the first trimester of gestation and at delivery. The placenta was homogenized at 5% w/v. Mitochondrial dynamics, fusion events [mitofusin 2 (Mfn2)/β-actin] and fission events [dynamin-related protein 1 (Drp1/β-actin)] and apoptosis (caspase 3/β-actin) were assessed by western blot analysis. Obstetric complications were significantly more frequent in pregnancies among HIV-infected women [OR 5.00 (95% CI 1.21-20.70)]. Mfn2/β-actin levels in PBMCs from controls significantly decreased during pregnancy (202.13 ± 57.45%), whereas cases maintained reduced levels from the first trimester of pregnancy and no differences were observed in CBMCs. Mfn2/β-actin and Drp1/β-actin contents significantly decreased in the placenta of cases. Caspase 3/β-actin levels significantly increased during pregnancy in PBMCs of cases (50.00 ± 7.89%), remaining significantly higher than in controls. No significant differences in caspase 3/β-actin content of neonatal CBMCs were observed, but there was a slight increased trend in placenta from cases. HIV- and HAART-mediated mitochondrial damage may be enhanced by decreased mitochondrial dynamics and increased apoptosis in maternal and placental compartments but not in the uninfected fetus. However, direct effects on mitochondrial dynamics and implication of apoptosis were not demonstrated in adverse obstetric outcomes. © The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Tropomodulin 1 Regulation of Actin Is Required for the Formation of Large Paddle Protrusions Between Mature Lens Fiber Cells.

PubMed

Cheng, Catherine; Nowak, Roberta B; Biswas, Sondip K; Lo, Woo-Kuen; FitzGerald, Paul G; Fowler, Velia M

2016-08-01

To elucidate the proteins required for specialized small interlocking protrusions and large paddle domains at lens fiber cell tricellular junctions (vertices), we developed a novel method to immunostain single lens fibers and studied changes in cell morphology due to loss of tropomodulin 1 (Tmod1), an F-actin pointed end-capping protein. We investigated F-actin and F-actin-binding protein localization in interdigitations of Tmod1+/+ and Tmod1-/- single mature lens fibers. F-actin-rich small protrusions and large paddles were present along cell vertices of Tmod1+/+ mature fibers. In contrast, Tmod1-/- mature fiber cells lack normal paddle domains, while small protrusions were unaffected. In Tmod1+/+ mature fibers, Tmod1, β2-spectrin, and α-actinin are localized in large puncta in valleys between paddles; but in Tmod1-/- mature fibers, β2-spectrin was dispersed while α-actinin was redistributed at the base of small protrusions and rudimentary paddles. Fimbrin and Arp3 (actin-related protein 3) were located in puncta at the base of small protrusions, while N-cadherin and ezrin outlined the cell membrane in both Tmod1+/+ and Tmod1-/- mature fibers. These results suggest that distinct F-actin organizations are present in small protrusions versus large paddles. Formation and/or maintenance of large paddle domains depends on a β2-spectrin-actin network stabilized by Tmod1. α-Actinin-crosslinked F-actin bundles are enhanced in absence of Tmod1, indicating altered cytoskeleton organization. Formation of small protrusions is likely facilitated by Arp3-branched and fimbrin-bundled F-actin networks, which do not depend on Tmod1. This is the first work to reveal the F-actin-associated proteins required for the formation of paddles between lens fibers.

Soft viscoelastic properties of nuclear actin age oocytes due to gravitational creep

PubMed Central

Feric, Marina; Broedersz, Chase P.; Brangwynne, Clifford P.

2015-01-01

The actin cytoskeleton helps maintain structural organization within living cells. In large X. laevis oocytes, gravity becomes a dominant force and is countered by a nuclear actin network that prevents liquid-like nuclear bodies from immediate sedimentation and coalescence. However, nuclear actin’s mechanical properties, and how they facilitate the stabilization of nuclear bodies, remain unknown. Using active microrheology, we find that nuclear actin forms a weak viscoelastic network, with a modulus of roughly 0.1 Pa. Embedded probe particles subjected to a constant force exhibit continuous displacement, due to viscoelastic creep. Gravitational forces also cause creep displacement of nuclear bodies, resulting in their asymmetric nuclear distribution. Thus, nuclear actin does not indefinitely support the emulsion of nuclear bodies, but only kinetically stabilizes them by slowing down gravitational creep to ~2 months. This is similar to the viability time of large oocytes, suggesting gravitational creep ages oocytes, with fatal consequences on long timescales. PMID:26577186

Force transmission in migrating cells

PubMed Central

Sauser, Roger; Ambrosi, Davide; Meister, Jean-Jacques; Verkhovsky, Alexander B.

2010-01-01

During cell migration, forces generated by the actin cytoskeleton are transmitted through adhesion complexes to the substrate. To investigate the mechanism of force generation and transmission, we analyzed the relationship between actin network velocity and traction forces at the substrate in a model system of persistently migrating fish epidermal keratocytes. Front and lateral sides of the cell exhibited much stronger coupling between actin motion and traction forces than the trailing cell body. Further analysis of the traction–velocity relationship suggested that the force transmission mechanisms were different in different cell regions: at the front, traction was generated by a gripping of the actin network to the substrate, whereas at the sides and back, it was produced by the network’s slipping over the substrate. Treatment with inhibitors of the actin–myosin system demonstrated that the cell body translocation could be powered by either of the two different processes, actomyosin contraction or actin assembly, with the former associated with significantly larger traction forces than the latter. PMID:20100912

Self-assembly of actin monomers into long filaments: Brownian dynamics simulations

NASA Astrophysics Data System (ADS)

Guo, Kunkun; Shillcock, Julian; Lipowsky, Reinhard

2009-07-01

Brownian dynamics simulations are used to study the dynamical process of self-assembly of actin monomers into long filaments containing up to 1000 actin protomers. In order to overcome the large separation of time scales between the diffusive motion of the free monomers and the relatively slow attachment and detachment processes at the two ends of the filaments, we introduce a novel rescaling procedure by which we speed all dynamical processes related to actin polymerization and depolymerization up by the same factor. In general, the actin protomers within a filament can attain three different states corresponding to a bound adenosine triphosphate (ATP), adenosine diphosphate with inorganic phosphate (ADP/P), and ADP molecule. The simplest situation that has been studied experimentally is provided by the polymerization of ADP-actin, for which all protomers are identical. This case is used to unravel certain relations between the filament's physical properties and the model parameters such as the attachment rate constant and the size of the capture zone, the detachment rate and the probability of the detached event, as well as the growth rate and waiting times between two successive attachment/detachment events. When a single filament is allowed to grow in a bath of constant concentration of free ADP-actin monomers, its growth rate increases linearly with the free monomer concentration in quantitative agreement with in vitro experiments. The results also show that the waiting time is governed by exponential distributions and that the two ends of a filament undergo biased random walks. The filament length fluctuations are described by a length diffusion constant that is found to attain a constant value at low ADP-actin concentration and to increase linearly with this concentration. It is straightforward to apply our simulation code to more complex processes such as polymerization of ATP-actin coupled to ATP hydrolysis, force generation by filaments, formation of filament bundles, and filament-membrane interactions.

Actin-interacting Protein 1 Promotes Disassembly of Actin-depolymerizing Factor/Cofilin-bound Actin Filaments in a pH-dependent Manner.

PubMed

Nomura, Kazumi; Hayakawa, Kimihide; Tatsumi, Hitoshi; Ono, Shoichiro

2016-03-04

Actin-interacting protein 1 (AIP1) is a conserved WD repeat protein that promotes disassembly of actin filaments when actin-depolymerizing factor (ADF)/cofilin is present. Although AIP1 is known to be essential for a number of cellular events involving dynamic rearrangement of the actin cytoskeleton, the regulatory mechanism of the function of AIP1 is unknown. In this study, we report that two AIP1 isoforms from the nematode Caenorhabditis elegans, known as UNC-78 and AIPL-1, are pH-sensitive in enhancement of actin filament disassembly. Both AIP1 isoforms only weakly enhance disassembly of ADF/cofilin-bound actin filaments at an acidic pH but show stronger disassembly activity at neutral and basic pH values. However, a severing-defective mutant of UNC-78 shows pH-insensitive binding to ADF/cofilin-decorated actin filaments, suggesting that the process of filament severing or disassembly, but not filament binding, is pH-dependent. His-60 of AIP1 is located near the predicted binding surface for the ADF/cofilin-actin complex, and an H60K mutation of AIP1 partially impairs its pH sensitivity, suggesting that His-60 is involved in the pH sensor for AIP1. These biochemical results suggest that pH-dependent changes in AIP1 activity might be a novel regulatory mechanism of actin filament dynamics. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Highly Dynamic Host Actin Reorganization around Developing Plasmodium Inside Hepatocytes

PubMed Central

Gomes-Santos, Carina S. S.; Itoe, Maurice A.; Afonso, Cristina; Henriques, Ricardo; Gardner, Rui; Sepúlveda, Nuno; Simões, Pedro D.; Raquel, Helena; Almeida, António Paulo; Moita, Luis F.; Frischknecht, Friedrich; Mota, Maria M.

2012-01-01

Plasmodium sporozoites are transmitted by Anopheles mosquitoes and infect hepatocytes, where a single sporozoite replicates into thousands of merozoites inside a parasitophorous vacuole. The nature of the Plasmodium-host cell interface, as well as the interactions occurring between these two organisms, remains largely unknown. Here we show that highly dynamic hepatocyte actin reorganization events occur around developing Plasmodium berghei parasites inside human hepatoma cells. Actin reorganization is most prominent between 10 to 16 hours post infection and depends on the actin severing and capping protein, gelsolin. Live cell imaging studies also suggest that the hepatocyte cytoskeleton may contribute to parasite elimination during Plasmodium development in the liver. PMID:22238609

Hippocampal Dendritic Spines Are Segregated Depending on Their Actin Polymerization

PubMed Central

Domínguez-Iturza, Nuria; Calvo, María; Benoist, Marion; Esteban, José Antonio; Morales, Miguel

2016-01-01

Dendritic spines are mushroom-shaped protrusions of the postsynaptic membrane. Spines receive the majority of glutamatergic synaptic inputs. Their morphology, dynamics, and density have been related to synaptic plasticity and learning. The main determinant of spine shape is filamentous actin. Using FRAP, we have reexamined the actin dynamics of individual spines from pyramidal hippocampal neurons, both in cultures and in hippocampal organotypic slices. Our results indicate that, in cultures, the actin mobile fraction is independently regulated at the individual spine level, and mobile fraction values do not correlate with either age or distance from the soma. The most significant factor regulating actin mobile fraction was the presence of astrocytes in the culture substrate. Spines from neurons growing in the virtual absence of astrocytes have a more stable actin cytoskeleton, while spines from neurons growing in close contact with astrocytes show a more dynamic cytoskeleton. According to their recovery time, spines were distributed into two populations with slower and faster recovery times, while spines from slice cultures were grouped into one population. Finally, employing fast lineal acquisition protocols, we confirmed the existence of loci with high polymerization rates within the spine. PMID:26881098

Spire and Formin 2 synergize and antagonize in regulating actin assembly in meiosis by a ping-pong mechanism.

PubMed

Montaville, Pierre; Jégou, Antoine; Pernier, Julien; Compper, Christel; Guichard, Bérengère; Mogessie, Binyam; Schuh, Melina; Romet-Lemonne, Guillaume; Carlier, Marie-France

2014-02-01

In mammalian oocytes, three actin binding proteins, Formin 2 (Fmn2), Spire, and profilin, synergistically organize a dynamic cytoplasmic actin meshwork that mediates translocation of the spindle toward the cortex and is required for successful fertilization. Here we characterize Fmn2 and elucidate the molecular mechanism for this synergy, using bulk solution and individual filament kinetic measurements of actin assembly dynamics. We show that by capping filament barbed ends, Spire recruits Fmn2 and facilitates its association with barbed ends, followed by rapid processive assembly and release of Spire. In the presence of actin, profilin, Spire, and Fmn2, filaments display alternating phases of rapid processive assembly and arrested growth, driven by a "ping-pong" mechanism, in which Spire and Fmn2 alternately kick off each other from the barbed ends. The results are validated by the effects of injection of Spire, Fmn2, and their interacting moieties in mouse oocytes. This original mechanism of regulation of a Rho-GTPase-independent formin, recruited by Spire at Rab11a-positive vesicles, supports a model for modulation of a dynamic actin-vesicle meshwork in the oocyte at the origin of asymmetric positioning of the meiotic spindle.

Lis1 controls dynamics of neuronal filopodia and spines to impact synaptogenesis and social behaviour

PubMed Central

Sudarov, Anamaria; Gooden, Frank; Tseng, Debbie; Gan, Wen-Biao; Ross, Margaret Elizabeth

2013-01-01

LIS1 (PAFAH1B1) mutation can impair neuronal migration, causing lissencephaly in humans. LIS1 loss is associated with dynein protein motor dysfunction, and disrupts the actin cytoskeleton through disregulated RhoGTPases. Recently, LIS1 was implicated as an important protein-network interaction node with high-risk autism spectrum disorder genes expressed in the synapse. How LIS1 might participate in this disorder has not been investigated. We examined the role of LIS1 in synaptogenesis of post-migrational neurons and social behaviour in mice. Two-photon imaging of actin-rich dendritic filopodia and spines in vivo showed significant reductions in elimination and turnover rates of dendritic protrusions of layer V pyramidal neurons in adolescent Lis1+/− mice. Lis1+/− filopodia on immature hippocampal neurons in vitro exhibited reduced density, length and RhoA dependent impaired dynamics compared to Lis1+/+. Moreover, Lis1+/− adolescent mice exhibited deficits in social interaction. Lis1 inactivation restricted to the postnatal hippocampus resulted in similar deficits in dendritic protrusion density and social interactions. Thus, LIS1 plays prominently in dendritic filopodia dynamics and spine turnover implicating reduced dendritic spine plasticity as contributing to developmental autistic-like behaviour. PMID:23483716

Mutations in KPTN Cause Macrocephaly, Neurodevelopmental Delay, and Seizures

PubMed Central

Baple, Emma L.; Maroofian, Reza; Chioza, Barry A.; Izadi, Maryam; Cross, Harold E.; Al-Turki, Saeed; Barwick, Katy; Skrzypiec, Anna; Pawlak, Robert; Wagner, Karin; Coblentz, Roselyn; Zainy, Tala; Patton, Michael A.; Mansour, Sahar; Rich, Phillip; Qualmann, Britta; Hurles, Matt E.; Kessels, Michael M.; Crosby, Andrew H.

2014-01-01

The proper development of neuronal circuits during neuromorphogenesis and neuronal-network formation is critically dependent on a coordinated and intricate series of molecular and cellular cues and responses. Although the cortical actin cytoskeleton is known to play a key role in neuromorphogenesis, relatively little is known about the specific molecules important for this process. Using linkage analysis and whole-exome sequencing on samples from families from the Amish community of Ohio, we have demonstrated that mutations in KPTN, encoding kaptin, cause a syndrome typified by macrocephaly, neurodevelopmental delay, and seizures. Our immunofluorescence analyses in primary neuronal cell cultures showed that endogenous and GFP-tagged kaptin associates with dynamic actin cytoskeletal structures and that this association is lost upon introduction of the identified mutations. Taken together, our studies have identified kaptin alterations responsible for macrocephaly and neurodevelopmental delay and define kaptin as a molecule crucial for normal human neuromorphogenesis. PMID:24239382

The Sla1 adaptor-clathrin interaction regulates coat formation and progression of endocytosis.

PubMed

Tolsma, Thomas O; Cuevas, Lena M; Di Pietro, Santiago M

2018-06-01

Clathrin-mediated endocytosis is a fundamental transport pathway that depends on numerous protein-protein interactions. Testing the importance of the adaptor protein-clathrin interaction for coat formation and progression of endocytosis in vivo has been difficult due to experimental constrains. Here, we addressed this question using the yeast clathrin adaptor Sla1, which is unique in showing a cargo endocytosis defect upon substitution of 3 amino acids in its clathrin-binding motif (sla1 AAA ) that disrupt clathrin binding. Live-cell imaging showed an impaired Sla1-clathrin interaction causes reduced clathrin levels but increased Sla1 levels at endocytic sites. Moreover, the rate of Sla1 recruitment was reduced indicating proper dynamics of both clathrin and Sla1 depend on their interaction. sla1 AAA cells showed a delay in progression through the various stages of endocytosis. The Arp2/3-dependent actin polymerization machinery was present for significantly longer time before actin polymerization ensued, revealing a link between coat formation and activation of actin polymerization. Ultimately, in sla1 AAA cells a larger than normal actin network was formed, dramatically higher levels of various machinery proteins other than clathrin were recruited, and the membrane profile of endocytic invaginations was longer. Thus, the Sla1-clathrin interaction is important for coat formation, regulation of endocytic progression and membrane bending. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Membrane tension and cytoskeleton organization in cell motility.

PubMed

Sens, Pierre; Plastino, Julie

2015-07-15

Cell membrane shape changes are important for many aspects of normal biological function, such as tissue development, wound healing and cell division and motility. Various disease states are associated with deregulation of how cells move and change shape, including notably tumor initiation and cancer cell metastasis. Cell motility is powered, in large part, by the controlled assembly and disassembly of the actin cytoskeleton. Much of this dynamic happens in close proximity to the plasma membrane due to the fact that actin assembly factors are membrane-bound, and thus actin filaments are generally oriented such that their growth occurs against or near the membrane. For a long time, the membrane was viewed as a relatively passive scaffold for signaling. However, results from the last five years show that this is not the whole picture, and that the dynamics of the actin cytoskeleton are intimately linked to the mechanics of the cell membrane. In this review, we summarize recent findings concerning the role of plasma membrane mechanics in cell cytoskeleton dynamics and architecture, showing that the cell membrane is not just an envelope or a barrier for actin assembly, but is a master regulator controlling cytoskeleton dynamics and cell polarity.

Active microrheology of entangled biopolymer composites link polymer flexibility and length to molecular force response

NASA Astrophysics Data System (ADS)

Fitzpatrick, Robert; Hauer, Cole; Kyrillos, Carl; McGorty, Ryan; Robertson-Anderson, Rae

Entangled polymers have complex viscoelastic properties that are tuned by polymer lengths and flexibilities. Entangled composites of distinct polymers offer added versatility and display nonlinear mechanics, serving as a platform for multifunctional materials. To determine the role of flexibility and length in polymer composites we use optical tweezers and confocal microscopy to measure mechanical and structural properties of co-entangled actin and DNA. Actin filaments have lengths of 5-20 μm, comparable to their persistence length, while DNA of similar lengths have hundreds of persistence lengths per chain. To characterize the nonlinear mechanics of actin-DNA composites, we optically drive a microsphere through the composite and measure the induced force during and following strain. We characterize viscoelasticity and relaxation timescales; and determine the dependence of these quantities on the actin:DNA ratio (0:1-1:0) and DNA length (4-100 μm). We use confocal microscopy to image distinctly labeled co-entangled actin and DNA and characterize network homogeneity and fluctuations. Initial results show actin and DNA are well-integrated and form structurally homogenous networks that exhibit stiffness and relaxation times that increase nonlinearly with increased actin. NSF Career Award (DMR-1254340), AFOSR Young Investigator Program Award (FA95550-12-1-0315), Scialog Collaborative Innovation Award funed by Research Corp. for Scientific Advancement (24192).

Green fluorescent protein fusions to Arabidopsis fimbrin 1 for spatio-temporal imaging of F-actin dynamics in roots.

PubMed

Wang, Yuh-Shuh; Motes, Christy M; Mohamalawari, Deepti R; Blancaflor, Elison B

2004-10-01

The visualization of green fluorescent protein (GFP) fusions with microtubule or actin filament (F-actin) binding proteins has provided new insights into the function of the cytoskeleton during plant development. For studies on actin, GFP fusions to talin have been the most generally used reporters. Although GFP-Talin has allowed in vivo F-actin imaging in a variety of plant cells, its utility in monitoring F-actin in stably transformed plants is limited particularly in developing roots where interesting actin dependent cell processes are occurring. In this study, we created a variety of GFP fusions to Arabidopsis Fimbrin 1 (AtFim1) to explore their utility for in vivo F-actin imaging in root cells and to better understand the actin binding properties of AtFim1 in living plant cells. Translational fusions of GFP to full-length AtFim1 or to some truncated variants of AtFim1 showed filamentous labeling in transient expression assays. One truncated fimbrin-GFP fusion was capable of labeling distinct filaments in stably transformed Arabidopsis roots. The filaments decorated by this construct were highly dynamic in growing root hairs and elongating root cells and were sensitive to actin disrupting drugs. Therefore, the fimbrin-GFP reporters we describe in this study provide additional tools for studying the actin cytoskeleton during root cell development. Moreover, the localization of AtFim1-GFP offers insights into the regulation of actin organization in developing roots by this class of actin cross-linking proteins. Copyright 2004 Wiley-Liss, Inc.

A Ca2+-dependent remodelled actin network directs vesicle trafficking to build wall ingrowth papillae in transfer cells.

PubMed

Zhang, Hui-Ming; Colyvas, Kim; Patrick, John W; Offler, Christina E

2017-10-13

The transport function of transfer cells is conferred by an enlarged plasma membrane area, enriched in nutrient transporters, that is supported on a scaffold of wall ingrowth (WI) papillae. Polarized plumes of elevated cytosolic Ca2+ define loci at which WI papillae form in developing adaxial epidermal transfer cells of Vicia faba cotyledons that are induced to trans-differentiate when the cotyledons are placed on culture medium. We evaluated the hypothesis that vesicle trafficking along a Ca2+-regulated remodelled actin network is the mechanism that underpins this outcome. Polarized to the outer periclinal cytoplasm, a Ca2+-dependent remodelling of long actin bundles into short, thin bundles was found to be essential for assembling WI papillae but not the underlying uniform wall layer. The remodelled actin network directed polarized vesicle trafficking to sites of WI papillae construction, and a pharmacological study indicated that both exo- and endocytosis contributed to assembly of the papillae. Potential candidates responsible for the Ca2+-dependent actin remodelling, along with those underpinning polarized exo- and endocyotosis, were identified in a transcriptome RNAseq database generated from the trans-differentiating epidermal cells. Of most significance, endocytosis was controlled by up-regulated expression of a dynamin-like isoform. How a cycle of localized exo- and endocytosis, regulated by Ca2+-dependent actin remodelling, assembles WI papillae is discussed. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Cysteine shotgun–mass spectrometry (CS-MS) reveals dynamic sequence of protein structure changes within mutant and stressed cells

PubMed Central

Krieger, Christine C.; An, Xiuli; Tang, Hsin-Yao; Mohandas, Narla; Speicher, David W.; Discher, Dennis E.

2011-01-01

Questions of if and when protein structures change within cells pervade biology and include questions of how the cytoskeleton sustains stresses on cells—particularly in mutant versus normal cells. Cysteine shotgun labeling with fluorophores is analyzed here with mass spectrometry of the spectrin–actin membrane skeleton in sheared red blood cell ghosts from normal and diseased mice. Sheared samples are compared to static samples at 37 °C in terms of cell membrane intensity in fluorescence microscopy, separated protein fluorescence, and tryptic peptide modification in liquid chromatography–tandem mass spectrometry (LC-MS/MS). Spectrin labeling proves to be the most sensitive to shear, whereas binding partners ankyrin and actin exhibit shear thresholds in labeling and both the ankyrin-binding membrane protein band 3 and the spectrin–actin stabilizer 4.1R show minimal differential labeling. Cells from 4.1R-null mice differ significantly from normal in the shear-dependent labeling of spectrin, ankyrin, and band 3: Decreased labeling of spectrin reveals less stress on the mutant network as spectrin dissociates from actin. Mapping the stress-dependent labeling kinetics of α- and β-spectrin by LC-MS/MS identifies Cys in these antiparallel chains that are either force-enhanced or force-independent in labeling, with structural analyses indicating the force-enhanced sites are sequestered either in spectrin’s triple-helical domains or in interactions with actin or ankyrin. Shear-sensitive sites identified comprehensively here in both spectrin and ankyrin appear consistent with stress relief through forced unfolding followed by cytoskeletal disruption. PMID:21527722

Mechanism of Actin-Based Motility

NASA Astrophysics Data System (ADS)

Pantaloni, Dominique; Le Clainche, Christophe; Carlier, Marie-France

2001-05-01

Spatially controlled polymerization of actin is at the origin of cell motility and is responsible for the formation of cellular protrusions like lamellipodia. The pathogens Listeria monocytogenes and Shigella flexneri, which undergo actin-based propulsion, are acknowledged models of the leading edge of lamellipodia. Actin-based motility of the bacteria or of functionalized microspheres can be reconstituted in vitro from only five pure proteins. Movement results from the regulated site-directed treadmilling of actin filaments, consistent with observations of actin dynamics in living motile cells and with the biochemical properties of the components of the synthetic motility medium.

Fragility of foot process morphology in kidney podocytes arises from chaotic spatial propagation of cytoskeletal instability

PubMed Central

Deerinck, Thomas J.; Chen, Yibang; He, John C.; Ellisman, Mark H.; Iyengar, Ravi

2017-01-01

Kidney podocytes’ function depends on fingerlike projections (foot processes) that interdigitate with those from neighboring cells to form the glomerular filtration barrier. The integrity of the barrier depends on spatial control of dynamics of actin cytoskeleton in the foot processes. We determined how imbalances in regulation of actin cytoskeletal dynamics could result in pathological morphology. We obtained 3-D electron microscopy images of podocytes and used quantitative features to build dynamical models to investigate how regulation of actin dynamics within foot processes controls local morphology. We find that imbalances in regulation of actin bundling lead to chaotic spatial patterns that could impair the foot process morphology. Simulation results are consistent with experimental observations for cytoskeletal reconfiguration through dysregulated RhoA or Rac1, and they predict compensatory mechanisms for biochemical stability. We conclude that podocyte morphology, optimized for filtration, is intrinsically fragile, whereby local transient biochemical imbalances may lead to permanent morphological changes associated with pathophysiology. PMID:28301477

The Formin Diaphanous Regulates Myoblast Fusion through Actin Polymerization and Arp2/3 Regulation

PubMed Central

Deng, Su; Bothe, Ingo; Baylies, Mary K.

2015-01-01

The formation of multinucleated muscle cells through cell-cell fusion is a conserved process from fruit flies to humans. Numerous studies have shown the importance of Arp2/3, its regulators, and branched actin for the formation of an actin structure, the F-actin focus, at the fusion site. This F-actin focus forms the core of an invasive podosome-like structure that is required for myoblast fusion. In this study, we find that the formin Diaphanous (Dia), which nucleates and facilitates the elongation of actin filaments, is essential for Drosophila myoblast fusion. Following cell recognition and adhesion, Dia is enriched at the myoblast fusion site, concomitant with, and having the same dynamics as, the F-actin focus. Through analysis of Dia loss-of-function conditions using mutant alleles but particularly a dominant negative Dia transgene, we demonstrate that reduction in Dia activity in myoblasts leads to a fusion block. Significantly, no actin focus is detected, and neither branched actin regulators, SCAR or WASp, accumulate at the fusion site when Dia levels are reduced. Expression of constitutively active Dia also causes a fusion block that is associated with an increase in highly dynamic filopodia, altered actin turnover rates and F-actin distribution, and mislocalization of SCAR and WASp at the fusion site. Together our data indicate that Dia plays two roles during invasive podosome formation at the fusion site: it dictates the level of linear F-actin polymerization, and it is required for appropriate branched actin polymerization via localization of SCAR and WASp. These studies provide new insight to the mechanisms of cell-cell fusion, the relationship between different regulators of actin polymerization, and invasive podosome formation that occurs in normal development and in disease. PMID:26295716

Mechanical output of myosin II motors is regulated by myosin filament size and actin network mechanics

NASA Astrophysics Data System (ADS)

Stam, Samantha; Alberts, Jonathan; Gardel, Margaret; Munro, Edwin

2013-03-01

The interactions of bipolar myosin II filaments with actin arrays are a predominate means of generating forces in numerous physiological processes including muscle contraction and cell migration. However, how the spatiotemporal regulation of these forces depends on motor mechanochemistry, bipolar filament size, and local actin mechanics is unknown. Here, we simulate myosin II motors with an agent-based model in which the motors have been benchmarked against experimental measurements. Force generation occurs in two distinct regimes characterized either by stable tension maintenance or by stochastic buildup and release; transitions between these regimes occur by changes to duty ratio and myosin filament size. The time required for building force to stall scales inversely with the stiffness of a network and the actin gliding speed of a motor. Finally, myosin motors are predicted to contract a network toward stiffer regions, which is consistent with experimental observations. Our representation of myosin motors can be used to understand how their mechanical and biochemical properties influence their observed behavior in a variety of in vitro and in vivo contexts.

Desmoglein 3 acting as an upstream regulator of Rho GTPases, Rac-1/Cdc42 in the regulation of actin organisation and dynamics

PubMed Central

Man Tsang, Siu; Brown, Louise; Gadmor, Hanan; Gammon, Luke; Fortune, Farida; Wheeler, Ann; Wan, Hong

2012-01-01

Desmoglein 3 (Dsg3), a member of the desmoglein sub-family, serves as an adhesion molecule in desmosomes. Our previous study showed that overexpression of human Dsg3 in several epithelial lines induces formation of membrane protrusions, a phenotype suggestive of Rho GTPase activation. Here we examined the interaction between Dsg3 and actin in detail and showed that endogenous Dsg3 colocalises and interacts with actin, particularly the junctional actin in a Rac1-dependent manner. Ablation of Rac1 activity by dominant negative Rac1 mutant (N17Rac1) or the Rac1 specific inhibitor (NSC23766) directly disrupts the interaction between Dsg3 and actin. Assembly of the junctional actin at the cell borders is accompanied with enhanced levels of Dsg3, while inhibition of Dsg3 by RNAi results in profound changes in the organisation of actin cytoskeleton. In accordance, overexpression of Dsg3 results in a remarkable increase of Rac1 and Cdc42 activities and to a lesser extent, RhoA. The enhancements in Rho GTPases are accompanied by the pronounced actin-based membrane structures such as lamellipodia and filopodia, enhanced rate of actin turnover and cell polarisation. Together, our results reveal an important novel function for Dsg3 in promoting actin dynamics through regulating Rac1 and Cdc42 activation in epithelial cells. PMID:22796473

Myocardin-Related Transcription Factor A Activation by Competition with WH2 Domain Proteins for Actin Binding.

PubMed

Weissbach, Julia; Schikora, Franziska; Weber, Anja; Kessels, Michael; Posern, Guido

2016-05-15

The myocardin-related transcription factors (MRTFs) are coactivators of serum response factor (SRF)-mediated gene expression. Activation of MRTF-A occurs in response to alterations in actin dynamics and critically requires the dissociation of repressive G-actin-MRTF-A complexes. However, the mechanism leading to the release of MRTF-A remains unclear. Here we show that WH2 domains compete directly with MRTF-A for actin binding. Actin nucleation-promoting factors, such as N-WASP and WAVE2, as well as isolated WH2 domains, including those of Spire2 and Cobl, activate MRTF-A independently of changes in actin dynamics. Simultaneous inhibition of Arp2-Arp3 or mutation of the CA region only partially reduces MRTF-A activation by N-WASP and WAVE2. Recombinant WH2 domains and the RPEL domain of MRTF-A bind mutually exclusively to cellular and purified G-actin in vitro The competition by different WH2 domains correlates with MRTF-SRF activation. Following serum stimulation, nonpolymerizable actin dissociates from MRTF-A, and de novo formation of the G-actin-RPEL complex is impaired by a transferable factor. Our work demonstrates that WH2 domains activate MRTF-A and contribute to target gene regulation by a competitive mechanism, independently of their role in actin filament formation. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

A network of conserved formins, regulated by the guanine exchange factor EXC-5 and the GTPase CDC-42, modulates tubulogenesis in vivo.

PubMed

Shaye, Daniel D; Greenwald, Iva

2016-11-15

The C. elegans excretory cell (EC) is a powerful model for tubulogenesis, a conserved process that requires precise cytoskeletal regulation. EXC-6, an ortholog of the disease-associated formin INF2, coordinates cell outgrowth and lumen formation during EC tubulogenesis by regulating F-actin at the tip of the growing canal and the dynamics of basolateral microtubules. EXC-6 functions in parallel with EXC-5/FGD, a predicted activator of the Rho GTPase Cdc42. Here, we identify the parallel pathway: EXC-5 functions through CDC-42 to regulate two other formins: INFT-2, another INF2 ortholog, and CYK-1, the sole ortholog of the mammalian diaphanous (mDia) family of formins. We show that INFT-2 promotes F-actin accumulation in the EC, and that CYK-1 inhibits INFT-2 to regulate F-actin levels and EXC-6-promoted outgrowth. As INF2 and mDia physically interact and cross-regulate in cultured cells, our work indicates that a conserved EXC-5-CDC-42 pathway modulates this regulatory interaction and that it is functionally important in vivo during tubulogenesis. © 2016. Published by The Company of Biologists Ltd.

Correlative STED and Atomic Force Microscopy on Live Astrocytes Reveals Plasticity of Cytoskeletal Structure and Membrane Physical Properties during Polarized Migration

PubMed Central

Curry, Nathan; Ghézali, Grégory; Kaminski Schierle, Gabriele S.; Rouach, Nathalie; Kaminski, Clemens F.

2017-01-01

The plasticity of the cytoskeleton architecture and membrane properties is important for the establishment of cell polarity, adhesion and migration. Here, we present a method which combines stimulated emission depletion (STED) super-resolution imaging and atomic force microscopy (AFM) to correlate cytoskeletal structural information with membrane physical properties in live astrocytes. Using STED compatible dyes for live cell imaging of the cytoskeleton, and simultaneously mapping the cell surface topology with AFM, we obtain unprecedented detail of highly organized networks of actin and microtubules in astrocytes. Combining mechanical data from AFM with optical imaging of actin and tubulin further reveals links between cytoskeleton organization and membrane properties. Using this methodology we illustrate that scratch-induced migration induces cytoskeleton remodeling. The latter is caused by a polarization of actin and microtubule elements within astroglial cell processes, which correlates strongly with changes in cell stiffness. The method opens new avenues for the dynamic probing of the membrane structural and functional plasticity of living brain cells. It is a powerful tool for providing new insights into mechanisms of cell structural remodeling during physiological or pathological processes, such as brain development or tumorigenesis. PMID:28469559

The F-BAR protein Hof1 tunes formin activity to sculpt actin cables during polarized growth

PubMed Central

Graziano, Brian R.; Yu, Hoi-Ying E.; Alioto, Salvatore L.; Eskin, Julian A.; Ydenberg, Casey A.; Waterman, David P.; Garabedian, Mikael; Goode, Bruce L.

2014-01-01

Asymmetric cell growth and division rely on polarized actin cytoskeleton remodeling events, the regulation of which is poorly understood. In budding yeast, formins stimulate the assembly of an organized network of actin cables that direct polarized secretion. Here we show that the Fer/Cip4 homology–Bin amphiphysin Rvs protein Hof1, which has known roles in cytokinesis, also functions during polarized growth by directly controlling the activities of the formin Bnr1. A mutant lacking the C-terminal half of Hof1 displays misoriented and architecturally altered cables, along with impaired secretory vesicle traffic. In vitro, Hof1 inhibits the actin nucleation and elongation activities of Bnr1 without displacing the formin from filament ends. These effects depend on the Src homology 3 domain of Hof1, the formin homology 1 (FH1) domain of Bnr1, and Hof1 dimerization, suggesting a mechanism by which Hof1 “restrains” the otherwise flexible FH1-FH2 apparatus. In vivo, loss of inhibition does not alter actin levels in cables but, instead, cable shape and functionality. Thus Hof1 tunes formins to sculpt the actin cable network. PMID:24719456

Direct interaction of microtubule- and actin-based transport motors

NASA Technical Reports Server (NTRS)

Huang, J. D.; Brady, S. T.; Richards, B. W.; Stenolen, D.; Resau, J. H.; Copeland, N. G.; Jenkins, N. A.

1999-01-01

The microtubule network is thought to be used for long-range transport of cellular components in animal cells whereas the actin network is proposed to be used for short-range transport, although the mechanism(s) by which this transport is coordinated is poorly understood. For example, in sea urchins long-range Ca2+-regulated transport of exocytotic vesicles requires a microtubule-based motor, whereas an actin-based motor is used for short-range transport. In neurons, microtubule-based kinesin motor proteins are used for long-range vesicular transport but microtubules do not extend into the neuronal termini, where actin filaments form the cytoskeletal framework, and kinesins are rapidly degraded upon their arrival in neuronal termini, indicating that vesicles may have to be transferred from microtubules to actin tracks to reach their final destination. Here we show that an actin-based vesicle-transport motor, MyoVA, can interact directly with a microtubule-based transport motor, KhcU. As would be expected if these complexes were functional, they also contain kinesin light chains and the localization of MyoVA and KhcU overlaps in the cell. These results indicate that cellular transport is, in part, coordinated through the direct interaction of different motor molecules.

Short-term withdrawal from developmental exposure to cocaine activates the glucocorticoid receptor and alters spine dynamics.

PubMed

Caffino, Lucia; Giannotti, Giuseppe; Malpighi, Chiara; Racagni, Giorgio; Fumagalli, Fabio

2015-10-01

Although glucocorticoid receptors (GRs) contribute to the action of cocaine, their role following developmental exposure to the psychostimulant is still unknown. To address this issue, we exposed adolescent male rats to cocaine (20mg/kg/day) from post-natal day (PND) 28 to PND 42 and sacrificed them at PND 45 or 90. We studied the medial prefrontal cortex (mPFC), a brain region that is still developing during adolescence. In PND 45 rats we found enhanced GR transcription and translation as well as increased trafficking toward the nucleus of the receptor, with no alteration in plasma corticosterone levels. We also showed reduced expression of the GR co-chaperone FKBP51, that normally keeps the receptor in the cytoplasm, and increased expression of Src1, which cooperates in the activation of GR transcriptional activity, revealing that short withdrawal alters the finely tuned mechanisms regulating GR action. Since activation of GRs regulate dendritic spine morphology, we next investigated spine dynamics in cocaine-withdrawn rats. We found that PSD95, cofilin and F-actin, molecules regulating spine actin network, are reduced in the mPFC of PND 45 rats suggesting reduced spine density, confirmed by confocal imaging. Further, formation of filopodia, i.e. the inactive spines, is enhanced suggesting the formation of non-functional spines. Of note, no changes were found in molecules related to GR machinery or spine dynamics following long-term abstinence, i.e. in adult rats (PND 90). These findings demonstrate that short withdrawal promotes plastic changes in the developing brain via the dysregulation of the GR system and alterations in the spine network. Copyright © 2015 Elsevier B.V. and ECNP. All rights reserved.

The transcriptional repressor Sum1p counteracts Sir2p in regulation of the actin cytoskeleton, mitochondrial quality control and replicative lifespan in Saccharomyces cerevisiae.

PubMed

Higuchi-Sanabria, Ryo; Vevea, Jason D; Charalel, Joseph K; Sapar, Maria L; Pon, Liza A

2016-01-18

Increasing the stability or dynamics of the actin cytoskeleton can extend lifespan in C. elegans and S. cerevisiae . Actin cables of budding yeast, bundles of actin filaments that mediate cargo transport, affect lifespan control through effects on mitochondrial quality control. Sir2p, the founding member of the Sirtuin family of lifespan regulators, also affects actin cable dynamics, assembly, and function in mitochondrial quality control. Here, we obtained evidence for novel interactions between Sir2p and Sum1p, a transcriptional repressor that was originally identified through mutations that genetically suppress sir2 ∆ phenotypes unrelated to lifespan. We find that deletion of SUM1 in wild-type cells results in increased mitochondrial function and actin cable abundance. Furthermore, deletion of SUM1 suppresses defects in actin cables and mitochondria of sir2 ∆ yeast, and extends the replicative lifespan and cellular health span of sir2 ∆ cells. Thus, Sum1p suppresses Sir2p function in control of specific aging determinants and lifespan in budding yeast.

Geometrical and Mechanical Properties Control Actin Filament Organization

PubMed Central

Ennomani, Hajer; Théry, Manuel; Nedelec, Francois; Blanchoin, Laurent

2015-01-01

The different actin structures governing eukaryotic cell shape and movement are not only determined by the properties of the actin filaments and associated proteins, but also by geometrical constraints. We recently demonstrated that limiting nucleation to specific regions was sufficient to obtain actin networks with different organization. To further investigate how spatially constrained actin nucleation determines the emergent actin organization, we performed detailed simulations of the actin filament system using Cytosim. We first calibrated the steric interaction between filaments, by matching, in simulations and experiments, the bundled actin organization observed with a rectangular bar of nucleating factor. We then studied the overall organization of actin filaments generated by more complex pattern geometries used experimentally. We found that the fraction of parallel versus antiparallel bundles is determined by the mechanical properties of actin filament or bundles and the efficiency of nucleation. Thus nucleation geometry, actin filaments local interactions, bundle rigidity, and nucleation efficiency are the key parameters controlling the emergent actin architecture. We finally simulated more complex nucleation patterns and performed the corresponding experiments to confirm the predictive capabilities of the model. PMID:26016478

Letrozole regulates actin cytoskeleton polymerization dynamics in a SRC-1 dependent manner in the hippocampus of mice.

PubMed

Zhao, Yangang; Yu, Yanlan; Zhang, Yuanyuan; He, Li; Qiu, Linli; Zhao, Jikai; Liu, Mengying; Zhang, Jiqiang

2017-03-01

In the hippocampus, local estrogens (E 2 ) derived from testosterone that is catalyzed by aromatase play important roles in the regulation of hippocampal neural plasticity, but the underlying mechanisms remain unclear. The actin cytoskeleton contributes greatly to hippocampal synaptic plasticity; however, whether it is regulated by local E 2 and the related mechanisms remain to be elucidated. In this study, we first examined the postnatal developmental profiles of hippocampal aromatase and specific proteins responsible for actin cytoskeleton dynamics. Then we used aromatase inhibitor letrozole (LET) to block local E 2 synthesis and examined the changes of these proteins and steroid receptor coactivator-1 (SRC-1), the predominant coactivator for steroid nuclear receptors. Finally, SRC-1 specific RNA interference was used to examine the effects of SRC-1 on the expression of these actin remodeling proteins. The results showed a V-type profile for aromatase and increased profiles for actin cytoskeleton proteins in both male and female hippocampus without obvious sex differences. LET treatment dramatically decreased the F-actin/G-actin ratio, the expression of Rictor, phospho-AKT (ser473), Profilin-1, phospho-Cofilin (Ser3), and SRC-1 in a dose-dependent manner. In vitro studies demonstrated that LET induced downregulation of these proteins could be reversed by E 2 , and E 2 induced increase of these proteins were significantly suppressed by SRC-1 shRNA interference. These results for the first time clearly demonstrated that local E 2 inhibition could induce aberrant actin polymerization; they also showed an important role of SRC-1 in the mediation of local E 2 action on hippocampal synaptic plasticity by regulation of actin cytoskeleton dynamics. Copyright © 2016 Elsevier Ltd. All rights reserved.

Identifying the dynamics of actin and tubulin polymerization in iPSCs and in iPSC-derived neurons

PubMed Central

Magliocca, Valentina; Petrini, Stefania; Franchin, Tiziana; Borghi, Rossella; Niceforo, Alessia; Abbaszadeh, Zeinab; Bertini, Enrico; Compagnucci, Claudia

2017-01-01

The development of the nervous system requires cytoskeleton-mediated processes coordinating self-renewal, migration, and differentiation of neurons. It is not surprising that many neurodevelopmental problems and neurodegenerative disorders are caused by deficiencies in cytoskeleton-related genes. For this reason, we focus on the cytoskeletal dynamics in proliferating iPSCs and in iPSC-derived neurons to better characterize the underpinnings of cytoskeletal organization looking at actin and tubulin repolymerization studies using the cell permeable probes SiR-Actin and SiR-Tubulin. During neurogenesis, each neuron extends an axon in a complex and changing environment to reach its final target. The dynamic behavior of the growth cone and its capacity to respond to multiple spatial information allows it to find its correct target. We decided to characterize various parameters of the actin filaments and microtubules. Our results suggest that a rapid re-organization of the cytoskeleton occurs 45 minutes after treatments with de-polymerizing agents in iPSCs and 60 minutes in iPSC-derived neurons in both actin filaments and microtubules. The quantitative data confirm that the actin filaments have a primary role in the re-organization of the cytoskeleton soon after de-polymerization, while microtubules have a major function following cytoskeletal stabilization. In conclusion, we investigate the possibility that de-polymerization of the actin filaments may have an impact on microtubules organization and that de-polymerization of the microtubules may affect the stability of the actin filaments. Our results suggest that a reciprocal influence of the actin filaments occurs over the microtubules and vice versa in both in iPSCs and iPSC-derived neurons. PMID:29340040

HopW1 from Pseudomonas syringae disrupts the actin cytoskeleton to promote virulence in Arabidopsis.

PubMed

Kang, Yongsung; Jelenska, Joanna; Cecchini, Nicolas M; Li, Yujie; Lee, Min Woo; Kovar, David R; Greenberg, Jean T

2014-06-01

A central mechanism of virulence of extracellular bacterial pathogens is the injection into host cells of effector proteins that modify host cellular functions. HopW1 is an effector injected by the type III secretion system that increases the growth of the plant pathogen Pseudomonas syringae on the Columbia accession of Arabidopsis. When delivered by P. syringae into plant cells, HopW1 causes a reduction in the filamentous actin (F-actin) network and the inhibition of endocytosis, a known actin-dependent process. When directly produced in plants, HopW1 forms complexes with actin, disrupts the actin cytoskeleton and inhibits endocytosis as well as the trafficking of certain proteins to vacuoles. The C-terminal region of HopW1 can reduce the length of actin filaments and therefore solubilize F-actin in vitro. Thus, HopW1 acts by disrupting the actin cytoskeleton and the cell biological processes that depend on actin, which in turn are needed for restricting P. syringae growth in Arabidopsis.

Brownian dynamics simulations of interactions between aldolase and G- or F-actin.

PubMed Central

Ouporov, I V; Knull, H R; Thomasson, K A

1999-01-01

Compartmentation of proteins in cells is important to proper cell function. Interactions of F-actin and glycolytic enzymes is one mechanism by which glycolytic enzymes can compartment. Brownian dynamics (BD) simulations of the binding of the muscle form of the glycolytic enzyme fructose-1,6-bisphosphate aldolase (aldolase) to F- or G-actin provide first-encounter snapshots of these interactions. Using x-ray structures of aldolase, G-actin, and three-dimensional models of F-actin, the electrostatic potential about each protein was predicted by solving the linearized Poisson-Boltzmann equation for use in BD simulations. The BD simulations provided solution complexes of aldolase with F- or G-actin. All complexes demonstrate the close contacts between oppositely charged regions of the protein surfaces. Positively charged surface regions of aldolase (residues Lys 13, 27, 288, 293, and 341 and Arg 257) are attracted to the negatively charged amino terminus (Asp 1 and Glu 2 and 4) and other patches (Asp 24, 25, and 363 and Glu 361, 364, 99, and 100) of actin subunits. According to BD results, the most important factor for aldolase binding to actin is the quaternary structure of aldolase and actin. Two pairs of adjacent aldolase subunits greatly add to the positive electrostatic potential of each other creating a region of attraction for the negatively charged subdomain 1 of the actin subunit that is exposed to solvent in the quaternary F-actin structure. PMID:9876119

Cortical actin nanodynamics determines nitric oxide release in vascular endothelium.

PubMed

Fels, Johannes; Jeggle, Pia; Kusche-Vihrog, Kristina; Oberleithner, Hans

2012-01-01

The release of the main vasodilator nitric oxide (NO) by the endothelial NO synthase (eNOS) is a hallmark of endothelial function. We aim at elucidating the underlying mechanism how eNOS activity depends on cortical stiffness (К(cortex)) of living endothelial cells. It is hypothesized that cortical actin dynamics determines К(cortex) and directly influences eNOS activity. By combined atomic force microscopy and fluorescence imaging we generated mechanical and optical sections of single living cells. This approach allows the discrimination between К(cortex) and bulk cell stiffness (К(bulk)) and, additionally, the simultaneous analysis of submembranous actin web dynamics. We show that К(cortex) softens when cortical F-actin depolymerizes and that this shift from a gel-like stiff cortex to a soft G-actin rich layer, triggers the stiffness-sensitive eNOS activity. The results implicate that stiffness changes in the ∼100 nm phase of the submembranous actin web, without affecting К(bulk), regulate NO release and thus determines endothelial function.

Xenopus egg cytoplasm with intact actin.

PubMed

Field, Christine M; Nguyen, Phuong A; Ishihara, Keisuke; Groen, Aaron C; Mitchison, Timothy J

2014-01-01

We report optimized methods for preparing Xenopus egg extracts without cytochalasin D, that we term "actin-intact egg extract." These are undiluted egg cytoplasm that contains abundant organelles, and glycogen which supplies energy, and represents the least perturbed cell-free cytoplasm preparation we know of. We used this system to probe cell cycle regulation of actin and myosin-II dynamics (Field et al., 2011), and to reconstitute the large, interphase asters that organize early Xenopus embryos (Mitchison et al., 2012; Wühr, Tan, Parker, Detrich, & Mitchison, 2010). Actin-intact Xenopus egg extracts are useful for analysis of actin dynamics, and interaction of actin with other cytoplasmic systems, in a cell-free system that closely mimics egg physiology, and more generally for probing the biochemistry and biophysics of the egg, zygote, and early embryo. Detailed protocols are provided along with assays used to check cell cycle state and tips for handling and storing undiluted egg extracts. © 2014 Elsevier Inc. All rights reserved.

The Transcriptomic Signature of RacA Activation and Inactivation Provides New Insights into the Morphogenetic Network of Aspergillus niger

PubMed Central

Kwon, Min Jin; Nitsche, Benjamin M.; Arentshorst, Mark; Jørgensen, Thomas R.; Ram, Arthur F. J.; Meyer, Vera

2013-01-01

RacA is the main Rho GTPase in Aspergillus niger regulating polarity maintenance via controlling actin dynamics. Both deletion and dominant activation of RacA (RacG18V) provoke an actin localization defect and thereby loss of polarized tip extension, resulting in frequent dichotomous branching in the ΔracA strain and an apolar growing phenotype for RacG18V. In the current study the transcriptomics and physiological consequences of these morphological changes were investigated and compared with the data of the morphogenetic network model for the dichotomous branching mutant ramosa-1. This integrated approach revealed that polar tip growth is most likely orchestrated by the concerted activities of phospholipid signaling, sphingolipid signaling, TORC2 signaling, calcium signaling and CWI signaling pathways. The transcriptomic signatures and the reconstructed network model for all three morphology mutants (ΔracA, RacG18V, ramosa-1) imply that these pathways become integrated to bring about different physiological adaptations including changes in sterol, zinc and amino acid metabolism and changes in ion transport and protein trafficking. Finally, the fate of exocytotic (SncA) and endocytotic (AbpA, SlaB) markers in the dichotomous branching mutant ΔracA was followed, demonstrating that hyperbranching does not per se result in increased protein secretion. PMID:23894378

Actin Engine in Immunological Synapse

PubMed Central

Piragyte, Indre

2012-01-01

T cell activation and function require physical contact with antigen presenting cells at a specialized junctional structure known as the immunological synapse. Once formed, the immunological synapse leads to sustained T cell receptor-mediated signalling and stabilized adhesion. High resolution microscopy indeed had a great impact in understanding the function and dynamic structure of immunological synapse. Trends of recent research are now moving towards understanding the mechanical part of immune system, expanding our knowledge in mechanosensitivity, force generation, and biophysics of cell-cell interaction. Actin cytoskeleton plays inevitable role in adaptive immune system, allowing it to bear dynamic and precise characteristics at the same time. The regulation of mechanical engine seems very complicated and overlapping, but it enables cells to be very sensitive to external signals such as surface rigidity. In this review, we focus on actin regulators and how immune cells regulate dynamic actin rearrangement process to drive the formation of immunological synapse. PMID:22916042

Rotational dynamics of spin-labeled F-actin during activation of myosin S1 ATPase using caged ATP.

PubMed Central

Ostap, E. M.; Thomas, D. D.

1991-01-01

The most probable source of force generation in muscle fibers in the rotation of the myosin head when bound to actin. This laboratory has demonstrated that ATP induces microsecond rotational motions of spin-labeled myosin heads bound to actin (Berger, C. L. E. C. Svensson, and D. D. Thomas. 1989. Proc. Natl. Acad. Sci. USA. 86:8753-8757). Our goal is to determine whether the observed ATP-induced rotational motions of actin-bound heads are accompanied by changes in actin rotational motions. We have used saturation transfer electron paramagnetic resonance (ST-EPR) and laser-induced photolysis of caged ATP to monitor changes in the microsecond rotational dynamics of spin-labeled F-actin in the presence of myosin subfragment-1 (S1). A maleimide spin label was attached selectively to cys-374 on actin. In the absence of ATP (with or without caged ATP), the ST-EPR spectrum (corresponding to an effective rotational time of approximately 150 microseconds) was essentially the same as observed for the same spin label bound to cys-707 (SH1) on S1, indicating that S1 is rigidly bound to actin in rigor. At normal ionic strength (micro = 186 mM), a decrease in ST-EPR intensity (increase in microsecond F-actin mobility) was clearly indicated upon photolysis of 1 mM caged ATP with a 50-ms, 351-nm laser pulse. This increase in mobility is due to the complete dissociation of Si from the actin filament. At low ionic strength (micro, = 36 mM), when about half the Si heads remain bound during ATP hydrolysis, no change in the actin mobility was detected, despite much faster motions of labeled S1 bound to actin. Therefore, we conclude that the active interaction of Si, actin,and ATP induces rotation of myosin heads relative to actin, but does not affect the microsecond rotational motion of actin itself, as detected at cys-374 of actin. PMID:1651780

Computational spatiotemporal analysis identifies WAVE2 and cofilin as joint regulators of costimulation-mediated T cell actin dynamics.

PubMed

Roybal, Kole T; Buck, Taráz E; Ruan, Xiongtao; Cho, Baek Hwan; Clark, Danielle J; Ambler, Rachel; Tunbridge, Helen M; Zhang, Jianwei; Verkade, Paul; Wülfing, Christoph; Murphy, Robert F

2016-04-19

Fluorescence microscopy is one of the most important tools in cell biology research because it provides spatial and temporal information to investigate regulatory systems inside cells. This technique can generate data in the form of signal intensities at thousands of positions resolved inside individual live cells. However, given extensive cell-to-cell variation, these data cannot be readily assembled into three- or four-dimensional maps of protein concentration that can be compared across different cells and conditions. We have developed a method to enable comparison of imaging data from many cells and applied it to investigate actin dynamics in T cell activation. Antigen recognition in T cells by the T cell receptor (TCR) is amplified by engagement of the costimulatory receptor CD28. We imaged actin and eight core actin regulators to generate over a thousand movies of T cells under conditions in which CD28 was either engaged or blocked in the context of a strong TCR signal. Our computational analysis showed that the primary effect of costimulation blockade was to decrease recruitment of the activator of actin nucleation WAVE2 (Wiskott-Aldrich syndrome protein family verprolin-homologous protein 2) and the actin-severing protein cofilin to F-actin. Reconstitution of WAVE2 and cofilin activity restored the defect in actin signaling dynamics caused by costimulation blockade. Thus, we have developed and validated an approach to quantify protein distributions in time and space for the analysis of complex regulatory systems. Copyright © 2016, American Association for the Advancement of Science.

Dynamics of Mechanical Signal Transmission through Prestressed Stress Fibers

PubMed Central

Hwang, Yongyun; Barakat, Abdul I.

2012-01-01

Transmission of mechanical stimuli through the actin cytoskeleton has been proposed as a mechanism for rapid long-distance mechanotransduction in cells; however, a quantitative understanding of the dynamics of this transmission and the physical factors governing it remains lacking. Two key features of the actin cytoskeleton are its viscoelastic nature and the presence of prestress due to actomyosin motor activity. We develop a model of mechanical signal transmission through prestressed viscoelastic actin stress fibers that directly connect the cell surface to the nucleus. The analysis considers both temporally stationary and oscillatory mechanical signals and accounts for cytosolic drag on the stress fibers. To elucidate the physical parameters that govern mechanical signal transmission, we initially focus on the highly simplified case of a single stress fiber. The results demonstrate that the dynamics of mechanical signal transmission depend on whether the applied force leads to transverse or axial motion of the stress fiber. For transverse motion, mechanical signal transmission is dominated by prestress while fiber elasticity has a negligible effect. Conversely, signal transmission for axial motion is mediated uniquely by elasticity due to the absence of a prestress restoring force. Mechanical signal transmission is significantly delayed by stress fiber material viscosity, while cytosolic damping becomes important only for longer stress fibers. Only transverse motion yields the rapid and long-distance mechanical signal transmission dynamics observed experimentally. For simple networks of stress fibers, mechanical signals are transmitted rapidly to the nucleus when the fibers are oriented largely orthogonal to the applied force, whereas the presence of fibers parallel to the applied force slows down mechanical signal transmission significantly. The present results suggest that cytoskeletal prestress mediates rapid mechanical signal transmission and allows temporally oscillatory signals in the physiological frequency range to travel a long distance without significant decay due to material viscosity and/or cytosolic drag. PMID:22514731

Neuronal profilins in health and disease: Relevance for spine plasticity and Fragile X syndrome

PubMed Central

Michaelsen-Preusse, Kristin; Zessin, Sabine; Grigoryan, Gayane; Scharkowski, Franziska; Feuge, Jonas; Remus, Anita; Korte, Martin

2016-01-01

Learning and memory, to a large extent, depend on functional changes at synapses. Actin dynamics orchestrate the formation of synapses, as well as their stabilization, and the ability to undergo plastic changes. Hence, profilins are of key interest as they bind to G-actin and enhance actin polymerization. However, profilins also compete with actin nucleators, thereby restricting filament formation. Here, we provide evidence that the two brain isoforms, profilin1 (PFN1) and PFN2a, regulate spine actin dynamics in an opposing fashion, and that whereas both profilins are needed during synaptogenesis, only PFN2a is crucial for adult spine plasticity. This finding suggests that PFN1 is the juvenile isoform important during development, whereas PFN2a is mandatory for spine stability and plasticity in mature neurons. In line with this finding, only PFN1 levels are altered in the mouse model of the developmental neurological disorder Fragile X syndrome. This finding is of high relevance because Fragile X syndrome is the most common monogenetic cause for autism spectrum disorder. Indeed, the expression of recombinant profilins rescued the impairment in spinogenesis, a hallmark in Fragile X syndrome, thereby linking the regulation of actin dynamics to synapse development and possible dysfunction. PMID:26951674

Spire and Formin 2 Synergize and Antagonize in Regulating Actin Assembly in Meiosis by a Ping-Pong Mechanism

PubMed Central

Montaville, Pierre; Jégou, Antoine; Pernier, Julien; Compper, Christel; Guichard, Bérengère; Mogessie, Binyam; Schuh, Melina; Romet-Lemonne, Guillaume; Carlier, Marie-France

2014-01-01

In mammalian oocytes, three actin binding proteins, Formin 2 (Fmn2), Spire, and profilin, synergistically organize a dynamic cytoplasmic actin meshwork that mediates translocation of the spindle toward the cortex and is required for successful fertilization. Here we characterize Fmn2 and elucidate the molecular mechanism for this synergy, using bulk solution and individual filament kinetic measurements of actin assembly dynamics. We show that by capping filament barbed ends, Spire recruits Fmn2 and facilitates its association with barbed ends, followed by rapid processive assembly and release of Spire. In the presence of actin, profilin, Spire, and Fmn2, filaments display alternating phases of rapid processive assembly and arrested growth, driven by a “ping-pong” mechanism, in which Spire and Fmn2 alternately kick off each other from the barbed ends. The results are validated by the effects of injection of Spire, Fmn2, and their interacting moieties in mouse oocytes. This original mechanism of regulation of a Rho-GTPase–independent formin, recruited by Spire at Rab11a-positive vesicles, supports a model for modulation of a dynamic actin-vesicle meshwork in the oocyte at the origin of asymmetric positioning of the meiotic spindle. PMID:24586110

Novel actin crosslinker superfamily member identified by a two step degenerate PCR procedure.

PubMed

Byers, T J; Beggs, A H; McNally, E M; Kunkel, L M

1995-07-24

Actin-crosslinking proteins link F-actin into the bundles and networks that constitute the cytoskeleton. Dystrophin, beta-spectrin, alpha-actinin, ABP-120, ABP-280, and fimbrin share homologous actin-binding domains and comprise an actin crosslinker superfamily. We have identified a novel member of this superfamily (ACF7) using a degenerate primer-mediated PCR strategy that was optimized to resolve less-abundant superfamily sequences. The ACF7 gene is on human chromosome 1 and hybridizes to high molecular weight bands on northern blots. Sequence comparisons argue that ACF7 does not fit into one of the existing families, but represents a new class within the superfamily.

The nature of the globular- to fibrous-actin transition.

PubMed

Oda, Toshiro; Iwasa, Mitsusada; Aihara, Tomoki; Maéda, Yuichiro; Narita, Akihiro

2009-01-22

Actin plays crucial parts in cell motility through a dynamic process driven by polymerization and depolymerization, that is, the globular (G) to fibrous (F) actin transition. Although our knowledge about the actin-based cellular functions and the molecules that regulate the G- to F-actin transition is growing, the structural aspects of the transition remain enigmatic. We created a model of F-actin using X-ray fibre diffraction intensities obtained from well oriented sols of rabbit skeletal muscle F-actin to 3.3 A in the radial direction and 5.6 A along the equator. Here we show that the G- to F-actin conformational transition is a simple relative rotation of the two major domains by about 20 degrees. As a result of the domain rotation, the actin molecule in the filament is flat. The flat form is essential for the formation of stable, helical F-actin. Our F-actin structure model provides the basis for understanding actin polymerization as well as its molecular interactions with actin-binding proteins.

Vascular Endothelial Growth Factor (VEGF) Promotes Assembly of the p130Cas Interactome to Drive Endothelial Chemotactic Signaling and Angiogenesis.

PubMed

Evans, Ian M; Kennedy, Susan A; Paliashvili, Ketevan; Santra, Tapesh; Yamaji, Maiko; Lovering, Ruth C; Britton, Gary; Frankel, Paul; Kolch, Walter; Zachary, Ian C

2017-02-01

p130Cas is a polyvalent adapter protein essential for cardiovascular development, and with a key role in cell movement. In order to identify the pathways by which p130Cas exerts its biological functions in endothelial cells we mapped the p130Cas interactome and its dynamic changes in response to VEGF using high-resolution mass spectrometry and reconstruction of protein interaction (PPI) networks with the aid of multiple PPI databases. VEGF enriched the p130Cas interactome in proteins involved in actin cytoskeletal dynamics and cell movement, including actin-binding proteins, small GTPases and regulators or binders of GTPases. Detailed studies showed that p130Cas association of the GTPase-binding scaffold protein, IQGAP1, plays a key role in VEGF chemotactic signaling, endothelial polarization, VEGF-induced cell migration, and endothelial tube formation. These findings indicate a cardinal role for assembly of the p130Cas interactome in mediating the cell migratory response to VEGF in angiogenesis, and provide a basis for further studies of p130Cas in cell movement. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Enlazin, a Natural Fusion of Two Classes of Canonical Cytoskeletal Proteins, Contributes to Cytokinesis Dynamics

PubMed Central

Octtaviani, Edelyn; Effler, Janet C.

2006-01-01

Cytokinesis requires a complex network of equatorial and global proteins to regulate cell shape changes. Here, using interaction genetics, we report the first characterization of a novel protein, enlazin. Enlazin is a natural fusion of two canonical classes of actin-associated proteins, the ezrin-radixin-moesin family and fimbrin, and it is localized to actin-rich structures. A fragment of enlazin, enl-tr, was isolated as a genetic suppressor of the cytokinesis defect of cortexillin-I mutants. Expression of enl-tr disrupts expression of endogenous enlazin, indicating that enl-tr functions as a dominant-negative lesion. Enlazin is distributed globally during cytokinesis and is required for cortical tension and cell adhesion. Consistent with a role in cell mechanics, inhibition of enlazin in a cortexillin-I background restores cytokinesis furrowing dynamics and suppresses the growth-in-suspension defect. However, as expected for a role in cell adhesion, inhibiting enlazin in a myosin-II background induces a synthetic cytokinesis phenotype, frequently arresting furrow ingression at the dumbbell shape and/or causing recession of the furrow. Thus, enlazin has roles in cell mechanics and adhesion, and these roles seem to be differentially significant for cytokinesis, depending on the genetic background. PMID:17050732

Actin nucleator Spire 1 is a regulator of ectoplasmic specialization in the testis.

PubMed

Wen, Qing; Li, Nan; Xiao, Xiang; Lui, Wing-Yee; Chu, Darren S; Wong, Chris K C; Lian, Qingquan; Ge, Renshan; Lee, Will M; Silvestrini, Bruno; Cheng, C Yan

2018-02-12

Germ cell differentiation during the epithelial cycle of spermatogenesis is accompanied by extensive remodeling at the Sertoli cell-cell and Sertoli cell-spermatid interface to accommodate the transport of preleptotene spermatocytes and developing spermatids across the blood-testis barrier (BTB) and the adluminal compartment of the seminiferous epithelium, respectively. The unique cell junction in the testis is the actin-rich ectoplasmic specialization (ES) designated basal ES at the Sertoli cell-cell interface, and the apical ES at the Sertoli-spermatid interface. Since ES dynamics (i.e., disassembly, reassembly and stabilization) are supported by actin microfilaments, which rapidly converts between their bundled and unbundled/branched configuration to confer plasticity to the ES, it is logical to speculate that actin nucleation proteins play a crucial role to ES dynamics. Herein, we reported findings that Spire 1, an actin nucleator known to polymerize actins into long stretches of linear microfilaments in cells, is an important regulator of ES dynamics. Its knockdown by RNAi in Sertoli cells cultured in vitro was found to impede the Sertoli cell tight junction (TJ)-permeability barrier through changes in the organization of F-actin across Sertoli cell cytosol. Unexpectedly, Spire 1 knockdown also perturbed microtubule (MT) organization in Sertoli cells cultured in vitro. Biochemical studies using cultured Sertoli cells and specific F-actin vs. MT polymerization assays supported the notion that a transient loss of Spire 1 by RNAi disrupted Sertoli cell actin and MT polymerization and bundling activities. These findings in vitro were reproduced in studies in vivo by RNAi using Spire 1-specific siRNA duplexes to transfect testes with Polyplus in vivo-jetPEI as a transfection medium with high transfection efficiency. Spire 1 knockdown in the testis led to gross disruption of F-actin and MT organization across the seminiferous epithelium, thereby impeding the transport of spermatids and phagosomes across the epithelium and perturbing spermatogenesis. In summary, Spire 1 is an ES regulator to support germ cell development during spermatogenesis.

Sharing is Caring: The Role of Actin/Myosin-V in Synaptic Vesicle Transport between Synapses in vivo

NASA Astrophysics Data System (ADS)

Gramlich, Michael

Inter-synaptic vesicle sharing is an important but not well understood process of pre-synaptic function. Further, the molecular mechanisms that underlie this inter-synaptic exchange are not well known, and whether this inter-synaptic vesicle sharing is regulated by neural activity remains largely unexplored. I address these questions by studying CA1/CA3 Hippocampal neurons at the single synaptic vesicle level. Using high-resolution tracking of individual vesicles that have recently undergone endocytosis, I observe long-distance axonal transport of synaptic vesicles is partly mediated by the actin network. Further, the actin-dependent transport is predominantly carried out by Myosin-V. I develop a correlated-motion analysis to characterize the mechanics of how actin and Myosin-V affect vesicle transport. Lastly, I also observe that vesicle exit rates from the synapse to the axon and long-distance vesicle transport are both regulated by activity, but Myosin-V does not appear to mediate the activity dependence. These observations highlight the roles of the axonal actin network, and Myosin-V in particular, in regulating inter-synaptic vesicle exchange.

Zinc and Copper Effects on Stability of Tubulin and Actin Networks in Dendrites and Spines of Hippocampal Neurons.

PubMed

Perrin, Laura; Roudeau, Stéphane; Carmona, Asuncion; Domart, Florelle; Petersen, Jennifer D; Bohic, Sylvain; Yang, Yang; Cloetens, Peter; Ortega, Richard

2017-07-19

Zinc and copper ions can modulate the activity of glutamate receptors. However, labile zinc and copper ions likely represent only the tip of the iceberg and other neuronal functions are suspected for these metals in their bound state. We performed synchrotron X-ray fluorescence imaging with 30 nm resolution to image total biometals in dendrites and spines from hippocampal neurons. We found that zinc is distributed all along the dendrites while copper is mainly pinpointed within the spines. In spines, zinc content is higher within the spine head while copper is higher within the spine neck. Such specific distributions suggested metal interactions with cytoskeleton proteins. Zinc supplementation induced the increase of β-tubulin content in dendrites. Copper supplementation impaired the β-tubulin and F-actin networks. Copper chelation resulted in the decrease of F-actin content in dendrites, drastically reducing the number of F-actin protrusions. These results indicate that zinc is involved in microtubule stability whereas copper is essential for actin-dependent stability of dendritic spines, although copper excess can impair the dendritic cytoskeleton.

Regulation of Mitochondrial Structure and Dynamics by the Cytoskeleton and Mechanical Factors.

PubMed

Bartolák-Suki, Erzsébet; Imsirovic, Jasmin; Nishibori, Yuichiro; Krishnan, Ramaswamy; Suki, Béla

2017-08-21

Mitochondria supply cells with energy in the form of ATP, guide apoptosis, and contribute to calcium buffering and reactive oxygen species production. To support these diverse functions, mitochondria form an extensive network with smaller clusters that are able to move along microtubules aided by motor proteins. Mitochondria are also associated with the actin network, which is involved in cellular responses to various mechanical factors. In this review, we discuss mitochondrial structure and function in relation to the cytoskeleton and various mechanical factors influencing cell functions. We first summarize the morphological features of mitochondria with an emphasis on fission and fusion as well as how network properties govern function. We then review the relationship between the mitochondria and the cytoskeletal structures, including mechanical interactions. We also discuss how stretch and its dynamic pattern affect mitochondrial structure and function. Finally, we present preliminary data on how extracellular matrix stiffness influences mitochondrial morphology and ATP generation. We conclude by discussing the more general role that mitochondria may play in mechanobiology and how the mechanosensitivity of mitochondria may contribute to the development of several diseases and aging.

Regulation of Mitochondrial Structure and Dynamics by the Cytoskeleton and Mechanical Factors

PubMed Central

Bartolák-Suki, Erzsébet; Imsirovic, Jasmin; Nishibori, Yuichiro; Krishnan, Ramaswamy; Suki, Béla

2017-01-01

Mitochondria supply cells with energy in the form of ATP, guide apoptosis, and contribute to calcium buffering and reactive oxygen species production. To support these diverse functions, mitochondria form an extensive network with smaller clusters that are able to move along microtubules aided by motor proteins. Mitochondria are also associated with the actin network, which is involved in cellular responses to various mechanical factors. In this review, we discuss mitochondrial structure and function in relation to the cytoskeleton and various mechanical factors influencing cell functions. We first summarize the morphological features of mitochondria with an emphasis on fission and fusion as well as how network properties govern function. We then review the relationship between the mitochondria and the cytoskeletal structures, including mechanical interactions. We also discuss how stretch and its dynamic pattern affect mitochondrial structure and function. Finally, we present preliminary data on how extracellular matrix stiffness influences mitochondrial morphology and ATP generation. We conclude by discussing the more general role that mitochondria may play in mechanobiology and how the mechanosensitivity of mitochondria may contribute to the development of several diseases and aging. PMID:28825689

Temperature-induced sol-gel transition and microgel formation in α-actinin cross-linked actin networks: A rheological study

NASA Astrophysics Data System (ADS)

Tempel, M.; Isenberg, G.; Sackmann, E.

1996-08-01

We have studied the sol-gel transition, the viscoelastic and the structural properties of networks constituted of semiflexible actin filaments cross-linked by α-actinin. Cross-linking was regulated in a reversible way by varying the temperature through the association-dissociation equilibrium of the actin-α-actinin system. Viscoelastic parameters [shear storage modulus G'(ω), phase shift tan(Φ)(ω), creep compliance J(t)] were measured as a function of temperature and actin-to-cross-linker ratio by a magnetically driven rotating disc rheometer. G'(ω) and tan(Φ)(ω) were studied at a frequency ω corresponding to the elastic plateau regime of the G'(ω) versus ω spectrum of the purely entangled solution. The microstructure of the networks was viewed by negative staining electron microscopy (EM). The phase shift tan(Φ) (or equivalently the viscosity η) diverges and reaches a maximum when approaching the apparent gel point from lower and higher temperatures, and the maximum defines the gel point (temperature Tg). The elastic plateau modulus G'N diverges at temperatures beyond this gel point TTg. The cross-linking transition (corresponding to a sol-gel transition at zero frequency) is interpreted in terms of a percolation model and the divergence of G'N at TTg), (2) that microscopic segregation takes place at T<=Tg leading to local formation of clusters (a state termed microgel), and (3) that at low actin-α-actinin ratios (rAα<=10) and low temperatures (T<=10 °C) macroscopic segregation into bundles of cross-linked actin filaments and a diluted solution of actin filaments is observed. The three regimes of network structure are represented by an equivalent phase diagram.

Rapid Glucose Depletion Immobilizes Active Myosin-V on Stabilized Actin Cables

PubMed Central

Xu, Li; Bretscher, Anthony

2014-01-01

Summary Polarization of eukaryotic cells requires organelles and protein complexes to be transported to their proper destinations along the cytoskeleton [1]. When nutrients are abundant, budding yeast grows rapidly transporting secretory vesicles for localized growth and actively segregating organelles [2, 3]. This is mediated by myosin-Vs transporting cargos along F-actin bundles known as actin cables [4]. Actin cables are dynamic structures regulated by assembly, stabilization and disassembly [5]. Polarized growth and actin filament dynamics consume energy. For most organisms, glucose is the preferred energy source and generally represses alternative carbon source usage [6]. Thus upon abrupt glucose depletion, yeast shuts down pathways consuming large amounts of energy, including the vacuolar-ATPase [7, 8], translation [9] and phosphoinositide metabolism [10]. Here we show that glucose withdrawal rapidly (<1 min) depletes ATP levels and the yeast myosin V, Myo2, responds by relocalizing to actin cables, making it the fastest response documented. Myo2 immobilized on cables releases its secretory cargo, defining a new rigor-like state of a myosin-V in vivo. Only actively transporting Myo2 can be converted to the rigor-like state. Glucose depletion has differential effects on the actin cytoskeleton resulting in disassembly of actin patches with concomitant inhibition of endocytosis, and strong stabilization of actin cables, thereby revealing a selective and previously unappreciated ATP requirement for actin cable disassembly. A similar response is seen in HeLa cells to ATP depletion. These findings reveal a new fast-acting energy conservation strategy halting growth by immobilizing myosin-V in a newly described state on selectively stabilized actin cables. PMID:25308080

Aldolase sequesters WASP and affects WASP/Arp2/3-stimulated actin dynamics.

PubMed

Ritterson Lew, Carolyn; Tolan, Dean R

2013-08-01

In addition to its roles in sugar metabolism, fructose-1,6-bisphosphate aldolase (aldolase) has been implicated in cellular functions independent from these roles, termed "moonlighting functions." These moonlighting functions likely involve the known aldolase-actin interaction, as many proteins with which aldolase interacts are involved in actin-dependent processes. Specifically, aldolase interacts both in vitro and in cells with Wiskott-Aldrich Syndrome Protein (WASP), a protein involved in controlling actin dynamics, yet the function of this interaction remains unknown. Here, the effect of aldolase on WASP-dependent processes in vitro and in cells is investigated. Aldolase inhibits WASP/Arp2/3-dependent actin polymerization in vitro. In cells, knockdown of aldolase results in a decreased rate of cell motility and cell spreading, two WASP-dependent processes. Expression of exogenous aldolase rescues these defects. Whether these effects of aldolase on WASP-dependent processes were due to aldolase catalysis or moonlighting functions is tested using aldolase variants defective in either catalytic or actin-binding activity. While the actin-binding deficient aldolase variant is unable to inhibit actin polymerization in vitro and is unable to rescue cell motility defects in cells, the catalytically inactive aldolase is able to perform these functions, providing evidence that aldolase moonlighting plays a role in WASP-mediated processes. Copyright © 2013 Wiley Periodicals, Inc.

Nanoscale segregation of actin nucleation and elongation factors determines dendritic spine protrusion

PubMed Central

Chazeau, Anaël; Mehidi, Amine; Nair, Deepak; Gautier, Jérémie J; Leduc, Cécile; Chamma, Ingrid; Kage, Frieda; Kechkar, Adel; Thoumine, Olivier; Rottner, Klemens; Choquet, Daniel; Gautreau, Alexis; Sibarita, Jean-Baptiste; Giannone, Grégory

2014-01-01

Actin dynamics drive morphological remodeling of neuronal dendritic spines and changes in synaptic transmission. Yet, the spatiotemporal coordination of actin regulators in spines is unknown. Using single protein tracking and super-resolution imaging, we revealed the nanoscale organization and dynamics of branched F-actin regulators in spines. Branched F-actin nucleation occurs at the PSD vicinity, while elongation occurs at the tip of finger-like protrusions. This spatial segregation differs from lamellipodia where both branched F-actin nucleation and elongation occur at protrusion tips. The PSD is a persistent confinement zone for IRSp53 and the WAVE complex, an activator of the Arp2/3 complex. In contrast, filament elongators like VASP and formin-like protein-2 move outwards from the PSD with protrusion tips. Accordingly, Arp2/3 complexes associated with F-actin are immobile and surround the PSD. Arp2/3 and Rac1 GTPase converge to the PSD, respectively, by cytosolic and free-diffusion on the membrane. Enhanced Rac1 activation and Shank3 over-expression, both associated with spine enlargement, induce delocalization of the WAVE complex from the PSD. Thus, the specific localization of branched F-actin regulators in spines might be reorganized during spine morphological remodeling often associated with synaptic plasticity. PMID:25293574

Myocardin-Related Transcription Factor A Activation by Competition with WH2 Domain Proteins for Actin Binding

PubMed Central

Weissbach, Julia; Schikora, Franziska; Weber, Anja; Kessels, Michael

2016-01-01

The myocardin-related transcription factors (MRTFs) are coactivators of serum response factor (SRF)-mediated gene expression. Activation of MRTF-A occurs in response to alterations in actin dynamics and critically requires the dissociation of repressive G-actin–MRTF-A complexes. However, the mechanism leading to the release of MRTF-A remains unclear. Here we show that WH2 domains compete directly with MRTF-A for actin binding. Actin nucleation-promoting factors, such as N-WASP and WAVE2, as well as isolated WH2 domains, including those of Spire2 and Cobl, activate MRTF-A independently of changes in actin dynamics. Simultaneous inhibition of Arp2-Arp3 or mutation of the CA region only partially reduces MRTF-A activation by N-WASP and WAVE2. Recombinant WH2 domains and the RPEL domain of MRTF-A bind mutually exclusively to cellular and purified G-actin in vitro. The competition by different WH2 domains correlates with MRTF-SRF activation. Following serum stimulation, nonpolymerizable actin dissociates from MRTF-A, and de novo formation of the G-actin–RPEL complex is impaired by a transferable factor. Our work demonstrates that WH2 domains activate MRTF-A and contribute to target gene regulation by a competitive mechanism, independently of their role in actin filament formation. PMID:26976641

Roles of type II myosin and a tropomyosin isoform in retrograde actin flow in budding yeast

PubMed Central

Huckaba, Thomas M.; Lipkin, Thomas; Pon, Liza A.

2006-01-01

Retrograde flow of cortical actin networks and bundles is essential for cell motility and retrograde intracellular movement, and for the formation and maintenance of microvilli, stereocilia, and filopodia. Actin cables, which are F-actin bundles that serve as tracks for anterograde and retrograde cargo movement in budding yeast, undergo retrograde flow that is driven, in part, by actin polymerization and assembly. We find that the actin cable retrograde flow rate is reduced by deletion or delocalization of the type II myosin Myo1p, and by deletion or conditional mutation of the Myo1p motor domain. Deletion of the tropomyosin isoform Tpm2p, but not the Tpm1p isoform, increases the rate of actin cable retrograde flow. Pretreatment of F-actin with Tpm2p, but not Tpm1p, inhibits Myo1p binding to F-actin and Myo1p-dependent F-actin gliding. These data support novel, opposing roles of Myo1p and Tpm2 in regulating retrograde actin flow in budding yeast and an isoform-specific function of Tpm1p in promoting actin cable function in myosin-driven anterograde cargo transport. PMID:17178912

Adhesive F-actin Waves: A Novel Integrin-Mediated Adhesion Complex Coupled to Ventral Actin Polymerization

PubMed Central

Case, Lindsay B.; Waterman, Clare M.

2011-01-01

At the leading lamellipodium of migrating cells, protrusion of an Arp2/3-nucleated actin network is coupled to formation of integrin-based adhesions, suggesting that Arp2/3-mediated actin polymerization and integrin-dependent adhesion may be mechanistically linked. Arp2/3 also mediates actin polymerization in structures distinct from the lamellipodium, in “ventral F-actin waves” that propagate as spots and wavefronts along the ventral plasma membrane. Here we show that integrins engage the extracellular matrix downstream of ventral F-actin waves in several mammalian cell lines as well as in primary mouse embryonic fibroblasts. These “adhesive F-actin waves” require a cycle of integrin engagement and disengagement to the extracellular matrix for their formation and propagation, and exhibit morphometry and a hierarchical assembly and disassembly mechanism distinct from other integrin-containing structures. After Arp2/3-mediated actin polymerization, zyxin and VASP are co-recruited to adhesive F-actin waves, followed by paxillin and vinculin, and finally talin and integrin. Adhesive F-actin waves thus represent a previously uncharacterized integrin-based adhesion complex associated with Arp2/3-mediated actin polymerization. PMID:22069459

Structural basis of thymosin-β4/profilin exchange leading to actin filament polymerization

PubMed Central

Xue, Bo; Leyrat, Cedric; Grimes, Jonathan M.; Robinson, Robert C.

2014-01-01

Thymosin-β4 (Tβ4) and profilin are the two major sequestering proteins that maintain the pool of monomeric actin (G-actin) within cells of higher eukaryotes. Tβ4 prevents G-actin from joining a filament, whereas profilin:actin only supports barbed-end elongation. Here, we report two Tβ4:actin structures. The first structure shows that Tβ4 has two helices that bind at the barbed and pointed faces of G-actin, preventing the incorporation of the bound G-actin into a filament. The second structure displays a more open nucleotide binding cleft on G-actin, which is typical of profilin:actin structures, with a concomitant disruption of the Tβ4 C-terminal helix interaction. These structures, combined with biochemical assays and molecular dynamics simulations, show that the exchange of bound actin between Tβ4 and profilin involves both steric and allosteric components. The sensitivity of profilin to the conformational state of actin indicates a similar allosteric mechanism for the dissociation of profilin during filament elongation. PMID:25313062

Cofilin Changes the Twist of F-Actin: Implications for Actin Filament Dynamics and Cellular Function

PubMed Central

McGough, Amy; Pope, Brian; Chiu, Wah; Weeds, Alan

1997-01-01

Cofilin is an actin depolymerizing protein found widely distributed in animals and plants. We have used electron cryomicroscopy and helical reconstruction to identify its binding site on actin filaments. Cofilin binds filamentous (F)-actin cooperatively by bridging two longitudinally associated actin subunits. The binding site is centered axially at subdomain 2 of the lower actin subunit and radially at the cleft between subdomains 1 and 3 of the upper actin subunit. Our work has revealed a totally unexpected (and unique) property of cofilin, namely, its ability to change filament twist. As a consequence of this change in twist, filaments decorated with cofilin have much shorter ‘actin crossovers' (∼75% of those normally observed in F-actin structures). Although their binding sites are distinct, cofilin and phalloidin do not bind simultaneously to F-actin. This is the first demonstration of a protein that excludes another actin-binding molecule by changing filament twist. Alteration of F-actin structure by cofilin/ADF appears to be a novel mechanism through which the actin cytoskeleton may be regulated or remodeled. PMID:9265645

siRNA Screen Identifies Trafficking Host Factors that Modulate Alphavirus Infection

PubMed Central

Radoshitzky, Sheli R.; Pegoraro, Gianluca; Chī, Xiǎolì; Dǒng, Lián; Chiang, Chih-Yuan; Jozwick, Lucas; Clester, Jeremiah C.; Cooper, Christopher L.; Courier, Duane; Langan, David P.; Underwood, Knashka; Kuehl, Kathleen A.; Sun, Mei G.; Caì, Yíngyún; Yú, Shuǐqìng; Burk, Robin; Zamani, Rouzbeh; Kota, Krishna; Kuhn, Jens H.; Bavari, Sina

2016-01-01

Little is known about the repertoire of cellular factors involved in the replication of pathogenic alphaviruses. To uncover molecular regulators of alphavirus infection, and to identify candidate drug targets, we performed a high-content imaging-based siRNA screen. We revealed an actin-remodeling pathway involving Rac1, PIP5K1- α, and Arp3, as essential for infection by pathogenic alphaviruses. Infection causes cellular actin rearrangements into large bundles of actin filaments termed actin foci. Actin foci are generated late in infection concomitantly with alphavirus envelope (E2) expression and are dependent on the activities of Rac1 and Arp3. E2 associates with actin in alphavirus-infected cells and co-localizes with Rac1–PIP5K1-α along actin filaments in the context of actin foci. Finally, Rac1, Arp3, and actin polymerization inhibitors interfere with E2 trafficking from the trans-Golgi network to the cell surface, suggesting a plausible model in which transport of E2 to the cell surface is mediated via Rac1- and Arp3-dependent actin remodeling. PMID:27031835

RNase L Interacts with Filamin A To Regulate Actin Dynamics and Barrier Function for Viral Entry

PubMed Central

Siddiqui, Mohammad Adnan; Dayal, Shubham; Naji, Merna; Ezelle, Heather J.; Zeng, Chun; Zhou, Aimin; Hassel, Bret A.

2014-01-01

ABSTRACT The actin cytoskeleton and its network of associated proteins constitute a physical barrier that viruses must circumvent to gain entry into cells for productive infection. The mechanisms by which the physical signals of infection are sensed by the host to activate an innate immune response are not well understood. The antiviral endoribonuclease RNase L is ubiquitously expressed in a latent form and activated upon binding 2-5A, a unique oligoadenylate produced during viral infections. We provide evidence that RNase L in its inactive form interacts with the actin-binding protein Filamin A to modulate the actin cytoskeleton and inhibit virus entry. Cells lacking either RNase L or Filamin A displayed increased virus entry which was exacerbated in cells lacking both proteins. RNase L deletion mutants that reduced Filamin A interaction displayed a compromised ability to restrict virus entry, supporting the idea of an important role for the RNase L-Filamin A complex in barrier function. Remarkably, both the wild type and a catalytically inactive RNase L mutant were competent to reduce virus entry when transfected into RNase L-deficient cells, indicating that this novel function of RNase L is independent of its enzymatic activity. Virus infection and RNase L activation disrupt its association with Filamin A and release RNase L to mediate its canonical nuclease-dependent antiviral activities. The dual functions of RNase L as a constitutive component of the actin cytoskeleton and as an induced mediator of antiviral signaling and effector functions provide insights into its mechanisms of antiviral activity and opportunities for the development of novel antiviral agents. PMID:25352621

AlphaII-spectrin interacts with Tes and EVL, two actin-binding proteins located at cell contacts.

PubMed

Rotter, Björn; Bournier, Odile; Nicolas, Gael; Dhermy, Didier; Lecomte, Marie-Christine

2005-06-01

The spectrin-based membrane skeleton, a multi-protein scaffold attached to diverse cellular membranes, is presumed to be involved in the stabilization of membranes, the establishment of membrane domains as well as in vesicle trafficking and nuclear functions. Spectrin tetramers made of alpha- and beta-subunits are linked to actin microfilaments, forming a network that binds a multitude of proteins. The most prevalent alpha-spectrin subunit in non-erythroid cells, alphaII-spectrin, contains two particular spectrin repeats in its central region, alpha9 and alpha10, which host an Src homology 3 domain, a tissue-specific spliced sequence of 20 residues, a calmodulin-binding site and major cleavage sites for caspases and calpains. Using yeast two-hybrid screening of kidney libraries, we identified two partners of the alpha9-alpha10 repeats: the potential tumour suppressor Tes, an actin-binding protein mainly located at focal adhesions; and EVL (Ena/vasodilator-stimulated phosphoprotein-like protein), another actin-binding protein, equally recruited at focal adhesions. Interactions between spectrin and overexpressed Tes and EVL were confirmed by co-immunoprecipitation. In vitro studies showed that the interaction between Tes and spectrin is mediated by a LIM (Lin-11, Isl-1 and Mec3) domain of Tes and by the alpha10 repeat of alphaII-spectrin whereas EVL interacts with the Src homology 3 domain located within the alpha9 repeat. Moreover, we describe an in vitro interaction between Tes and EVL, and a co-localization of these two proteins at focal adhesions. These interactions between alphaII-spectrin, Tes and EVL indicate new functions for spectrin in actin dynamics and focal adhesions.

Mapping the Dynamics of Shear Stress—Induced Structural Changes in Endothelial Cells

PubMed Central

Mott, Rosalind E.; Helmke, Brian P.

2009-01-01

Hemodynamic shear stress regulates endothelial cell biochemical processes that govern cytoskeletal contractility, focal adhesion dynamics, and extracellular matrix assembly. Since shear stress causes rapid strain focusing at discrete locations in the cytoskeleton, we hypothesized that shear stress coordinately alters structural dynamics in the cytoskeleton, focal adhesion sites, and extracellular matrix on a time scale of minutes. Using multi-wavelength 4-D fluorescence microscopy, we measured the displacement of rhodamine-fibronectin and of GFP-labeled actin, vimentin, paxillin, and/or vinculin in aortic endothelial cells before and after onset of steady unidirectional shear stress. In the cytoskeleton, the onset of shear stress increased actin polymerization into lamellipodia, altered the angle of lateral displacement of actin stress fibers and vimentin filaments, and decreased centripetal remodeling of actin stress fibers in both subconfluent and confluent cell layers. Shear stress induced the formation of new focal complexes and reduced the centripetal remodeling of focal adhesions in regions of new actin polymerization. The structural dynamics of focal adhesions and the fibronectin matrix varied with cell density. In subconfluent cell layers, shear stress onset decreased the displacement of focal adhesions and fibronectin fibrils. In confluent monolayers, the direction of fibronectin and focal adhesion displacement shifted significantly towards the downstream direction within one minute after onset of shear stress. These spatially coordinated rapid changes in the structural dynamics of cytoskeleton, focal adhesions, and extracellular matrix are consistent with focusing of mechanical stress and/or strain near major sites of shear stress-mediated mechanotransduction. PMID:17855768

Fine-tuning of actin dynamics by the HSPB8-BAG3 chaperone complex facilitates cytokinesis and contributes to its impact on cell division.

PubMed

Varlet, Alice Anaïs; Fuchs, Margit; Luthold, Carole; Lambert, Herman; Landry, Jacques; Lavoie, Josée N

2017-07-01

The small heat shock protein HSPB8 and its co-chaperone BAG3 are proposed to regulate cytoskeletal proteostasis in response to mechanical signaling in muscle cells. Here, we show that in dividing cells, the HSPB8-BAG3 complex is instrumental to the accurate disassembly of the actin-based contractile ring during cytokinesis, a process required to allow abscission of daughter cells. Silencing of HSPB8 markedly decreased the mitotic levels of BAG3 in HeLa cells, supporting its crucial role in BAG3 mitotic functions. Cells depleted of HSPB8 were delayed in cytokinesis, remained connected via a disorganized intercellular bridge, and exhibited increased incidence of nuclear abnormalities that result from failed cytokinesis (i.e., bi- and multi-nucleation). Such phenotypes were associated with abnormal accumulation of F-actin at the intercellular bridge of daughter cells at telophase. Remarkably, the actin sequestering drug latrunculin A, like the inhibitor of branched actin polymerization CK666, normalized F-actin during cytokinesis and restored proper cell division in HSPB8-depleted cells, implicating deregulated actin dynamics as a cause of abscission failure. Moreover, this HSPB8-dependent phenotype could be corrected by rapamycin, an autophagy-promoting drug, whereas it was mimicked by drugs impairing lysosomal function. Together, the results further support a role for the HSPB8-BAG3 chaperone complex in quality control of actin-based structure dynamics that are put under high tension, notably during cell cytokinesis. They expand a so-far under-appreciated connection between selective autophagy and cellular morphodynamics that guide cell division.

A Robust Actin Filaments Image Analysis Framework

PubMed Central

Alioscha-Perez, Mitchel; Benadiba, Carine; Goossens, Katty; Kasas, Sandor; Dietler, Giovanni; Willaert, Ronnie; Sahli, Hichem

2016-01-01

The cytoskeleton is a highly dynamical protein network that plays a central role in numerous cellular physiological processes, and is traditionally divided into three components according to its chemical composition, i.e. actin, tubulin and intermediate filament cytoskeletons. Understanding the cytoskeleton dynamics is of prime importance to unveil mechanisms involved in cell adaptation to any stress type. Fluorescence imaging of cytoskeleton structures allows analyzing the impact of mechanical stimulation in the cytoskeleton, but it also imposes additional challenges in the image processing stage, such as the presence of imaging-related artifacts and heavy blurring introduced by (high-throughput) automated scans. However, although there exists a considerable number of image-based analytical tools to address the image processing and analysis, most of them are unfit to cope with the aforementioned challenges. Filamentous structures in images can be considered as a piecewise composition of quasi-straight segments (at least in some finer or coarser scale). Based on this observation, we propose a three-steps actin filaments extraction methodology: (i) first the input image is decomposed into a ‘cartoon’ part corresponding to the filament structures in the image, and a noise/texture part, (ii) on the ‘cartoon’ image, we apply a multi-scale line detector coupled with a (iii) quasi-straight filaments merging algorithm for fiber extraction. The proposed robust actin filaments image analysis framework allows extracting individual filaments in the presence of noise, artifacts and heavy blurring. Moreover, it provides numerous parameters such as filaments orientation, position and length, useful for further analysis. Cell image decomposition is relatively under-exploited in biological images processing, and our study shows the benefits it provides when addressing such tasks. Experimental validation was conducted using publicly available datasets, and in osteoblasts grown in two different conditions: static (control) and fluid shear stress. The proposed methodology exhibited higher sensitivity values and similar accuracy compared to state-of-the-art methods. PMID:27551746

Dynamic localization and interaction with other Bacillus subtilis actin-like proteins are important for the function of MreB.

PubMed

Defeu Soufo, Hervé Joël; Graumann, Peter L

2006-12-01

Bacterial actin-like proteins play a key role in cell morphology and in chromosome segregation. Many bacteria, like Bacillus subtilis, contain three genes encoding actin-like proteins, called mreB, mbl and mreBH in B. subtilis. We show that MreB and Mbl colocalize extensively within live cells, and that all three B. subtilis actin paralogues interact with each other underneath the cell membrane. A mutation in the phosphate 2 motif of MreB had a dominant negative effect on cell morphology and on chromosome segregation. Expression of this mutant allele of MreB interfered with the dynamic localization of Mbl. These experiments show that the interaction between MreB and Mbl has physiological significance. An mreB deletion strain can grow under special media conditions, however, depletion of Mbl in this mutant background abolished growth, indicating that actin paralogues can partially complement each other. The membrane protein MreC was found to interact with Mbl, but not with MreB, revealing a clear distinction between the function of the two paralogues. The phosphate 2 mutant MreB protein allowed for filament formation of mutant or wild-type MreB, but abolished the dynamic reorganization of the filaments. The latter mutation led to a strong reduction, but not complete loss, of function of MreB, both in terms of chromosome segregation and of cell morphology. Our work shows that that the dynamic localization of MreB is essential for the proper activity of the actin-like protein and that the interactions between MreB paralogues have important physiological significance.

Capu and Spire assemble a cytoplasmic actin mesh that maintains microtubule organization in the Drosophila oocyte.

PubMed

Dahlgaard, Katja; Raposo, Alexandre A S F; Niccoli, Teresa; St Johnston, Daniel

2007-10-01

Mutants in the actin nucleators Cappuccino and Spire disrupt the polarized microtubule network in the Drosophila oocyte that defines the anterior-posterior axis, suggesting that microtubule organization depends on actin. Here, we show that Cappuccino and Spire organize an isotropic mesh of actin filaments in the oocyte cytoplasm. capu and spire mutants lack this mesh, whereas overexpressed truncated Cappuccino stabilizes the mesh in the presence of Latrunculin A and partially rescues spire mutants. Spire overexpression cannot rescue capu mutants, but prevents actin mesh disassembly at stage 10B and blocks late cytoplasmic streaming. We also show that the actin mesh regulates microtubules indirectly, by inhibiting kinesin-dependent cytoplasmic flows. Thus, the Capu pathway controls alternative states of the oocyte cytoplasm: when active, it assembles an actin mesh that suppresses kinesin motility to maintain a polarized microtubule cytoskeleton. When inactive, unrestrained kinesin movement generates flows that wash microtubules to the cortex.

Requirement of Nck adaptors for actin dynamics and cell migration stimulated by platelet-derived growth factor B.

PubMed

Rivera, G M; Antoku, S; Gelkop, S; Shin, N Y; Hanks, S K; Pawson, T; Mayer, B J

2006-06-20

The Nck family of Src homology (SH) 2/SH3 domain adaptors functions to link tyrosine phosphorylation induced by extracellular signals with downstream regulators of actin dynamics. We investigated the role of mammalian Nck adaptors in signaling from the activated platelet-derived growth factor (PDGF) receptor (PDGFbetaR) to the actin cytoskeleton. We report here that Nck adaptors are required for cytoskeletal reorganization and chemotaxis stimulated by PDGF-B. Analysis of tyrosine-phosphorylated proteins demonstrated that Crk-associated substrate (p130(Cas)), not the activated PDGFbetaR itself, is the major Nck SH2 domain-binding protein in PDGF-B-stimulated cells. Both Nck- and p130(Cas)-deficient cells fail to display cytoskeletal rearrangements, including the formation of membrane ruffles and the disassembly of actin bundles, typically shown by their WT counterparts in response to PDGF-B. Furthermore, Nck and p130(Cas) colocalize in phosphotyrosine-enriched membrane ruffles induced by PDGF-B in NIH 3T3 cells. These results suggest that Nck adaptors play an essential role in linking the activated PDGFbetaR with actin dynamics through a pathway that involves p130(Cas).

Cell-cell and cell-extracellular matrix adhesions cooperate to organize actomyosin networks and maintain force transmission during dorsal closure.

PubMed

Goodwin, Katharine; Lostchuck, Emily E; Cramb, Kaitlyn M L; Zulueta-Coarasa, Teresa; Fernandez-Gonzalez, Rodrigo; Tanentzapf, Guy

2017-05-15

Tissue morphogenesis relies on the coordinated action of actin networks, cell-cell adhesions, and cell-extracellular matrix (ECM) adhesions. Such coordination can be achieved through cross-talk between cell-cell and cell-ECM adhesions. Drosophila dorsal closure (DC), a morphogenetic process in which an extraembryonic tissue called the amnioserosa contracts and ingresses to close a discontinuity in the dorsal epidermis of the embryo, requires both cell-cell and cell-ECM adhesions. However, whether the functions of these two types of adhesions are coordinated during DC is not known. Here we analyzed possible interdependence between cell-cell and cell-ECM adhesions during DC and its effect on the actomyosin network. We find that loss of cell-ECM adhesion results in aberrant distributions of cadherin-mediated adhesions and actin networks in the amnioserosa and subsequent disruption of myosin recruitment and dynamics. Moreover, loss of cell-cell adhesion caused up-regulation of cell-ECM adhesion, leading to reduced cell deformation and force transmission across amnioserosa cells. Our results show how interdependence between cell-cell and cell-ECM adhesions is important in regulating cell behaviors, force generation, and force transmission critical for tissue morphogenesis. © 2017 Goodwin, Lostchuck, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Expanding Actin Rings Zipper the Mouse Embryo for Blastocyst Formation.

PubMed

Zenker, Jennifer; White, Melanie D; Gasnier, Maxime; Alvarez, Yanina D; Lim, Hui Yi Grace; Bissiere, Stephanie; Biro, Maté; Plachta, Nicolas

2018-04-19

Transformation from morula to blastocyst is a defining event of preimplantation embryo development. During this transition, the embryo must establish a paracellular permeability barrier to enable expansion of the blastocyst cavity. Here, using live imaging of mouse embryos, we reveal an actin-zippering mechanism driving this embryo sealing. Preceding blastocyst stage, a cortical F-actin ring assembles at the apical pole of the embryo's outer cells. The ring structure forms when cortical actin flows encounter a network of polar microtubules that exclude F-actin. Unlike stereotypical actin rings, the actin rings of the mouse embryo are not contractile, but instead, they expand to the cell-cell junctions. Here, they couple to the junctions by recruiting and stabilizing adherens and tight junction components. Coupling of the actin rings triggers localized myosin II accumulation, and it initiates a tension-dependent zippering mechanism along the junctions that is required to seal the embryo for blastocyst formation. Copyright © 2018 Elsevier Inc. All rights reserved.

F-actin cross-linking enhances the stability of force generation in disordered actomyosin networks

NASA Astrophysics Data System (ADS)

Jung, Wonyeong; Murrell, Michael P.; Kim, Taeyoon

2015-12-01

Myosin molecular motors and actin cross-linking proteins (ACPs) are known to mediate the generation and transmission of mechanical forces within the cortical F-actin cytoskeleton that drive major cellular processes such as cell division and migration. However, how motors and ACPs interact collectively over diverse timescales to modulate the time-dependent mechanical properties of the cytoskeleton remains unclear. In this study, we present a three-dimensional agent-based computational model of the cortical actomyosin network to quantitatively determine the effects of motor activity and the density and kinetics of ACPs on the accumulation and maintenance of mechanical tension within a disordered actomyosin network. We found that motors accumulate large stress quickly by behaving as temporary cross-linkers although this stress is relaxed over time unless there are sufficient passive ACPs to stabilize the network. Stabilization by ACPs helps motors to generate forces up to their maximum potential, leading to significant enhancement of the efficiency and stability of stress generation. Thus, we demonstrated that the force-dependent kinetics of ACP dissociation plays a critical role for the accumulation and sustainment of stress and the structural remodeling of networks.

Encoding mechano-memories in filamentous-actin networks

NASA Astrophysics Data System (ADS)

Majumdar, Sayantan; Foucard, Louis; Levine, Alex; Gardel, Margaret L.

History-dependent adaptation is a central feature of learning and memory. Incorporating such features into `adaptable materials' that can modify their mechanical properties in response to external cues, remains an outstanding challenge in materials science. Here, we study a novel mechanism of mechano-memory in cross-linked F-actin networks, the essential determinants of the mechanical behavior of eukaryotic cells. We find that the non-linear mechanical response of such networks can be reversibly programmed through induction of mechano-memories. In particular, the direction, magnitude, and duration of previously applied shear stresses can be encoded into the network architecture. The `memory' of the forcing history is long-lived, but it can be erased by force applied in the opposite direction. These results demonstrate that F-actin networks can encode analog read-write mechano-memories which can be used for adaptation to mechanical stimuli. We further show that the mechano-memory arises from changes in the nematic order of the constituent filaments. Our results suggest a new mechanism of mechanical sensing in eukaryotic cells and provide a strategy for designing a novel class of materials. S.M. acknowledges U. Chicago MRSEC for support through a Kadanoff-Rice fellowship.

Gβ Regulates Coupling between Actin Oscillators for Cell Polarity and Directional Migration

PubMed Central

Cai, Huaqing; Sun, Yaohui; Huang, Chuan-Hsiang; Freyre, Mariel; Zhao, Min; Devreotes, Peter N.; Weiner, Orion D.

2016-01-01

For directional movement, eukaryotic cells depend on the proper organization of their actin cytoskeleton. This engine of motility is made up of highly dynamic nonequilibrium actin structures such as flashes, oscillations, and traveling waves. In Dictyostelium, oscillatory actin foci interact with signals such as Ras and phosphatidylinositol 3,4,5-trisphosphate (PIP3) to form protrusions. However, how signaling cues tame actin dynamics to produce a pseudopod and guide cellular motility is a critical open question in eukaryotic chemotaxis. Here, we demonstrate that the strength of coupling between individual actin oscillators controls cell polarization and directional movement. We implement an inducible sequestration system to inactivate the heterotrimeric G protein subunit Gβ and find that this acute perturbation triggers persistent, high-amplitude cortical oscillations of F-actin. Actin oscillators that are normally weakly coupled to one another in wild-type cells become strongly synchronized following acute inactivation of Gβ. This global coupling impairs sensing of internal cues during spontaneous polarization and sensing of external cues during directional motility. A simple mathematical model of coupled actin oscillators reveals the importance of appropriate coupling strength for chemotaxis: moderate coupling can increase sensitivity to noisy inputs. Taken together, our data suggest that Gβ regulates the strength of coupling between actin oscillators for efficient polarity and directional migration. As these observations are only possible following acute inhibition of Gβ and are masked by slow compensation in genetic knockouts, our work also shows that acute loss-of-function approaches can complement and extend the reach of classical genetics in Dictyostelium and likely other systems as well. PMID:26890004

Vasodilator-stimulated Phosphoprotein (VASP) Regulates Actin Polymerization and Contraction in Airway Smooth Muscle by a Vinculin-dependent Mechanism*

PubMed Central

Wu, Yidi; Gunst, Susan J.

2015-01-01

Vasodilator-stimulated phosphoprotein (VASP) can catalyze actin polymerization by elongating actin filaments. The elongation mechanism involves VASP oligomerization and its binding to profilin, a G-actin chaperone. Actin polymerization is required for tension generation during the contraction of airway smooth muscle (ASM); however, the role of VASP in regulating actin dynamics in ASM is not known. We stimulated ASM cells and tissues with the contractile agonist acetylcholine (ACh) or the adenylyl cyclase activator, forskolin (FSK), a dilatory agent. ACh and FSK stimulated VASP Ser157 phosphorylation by different kinases. Inhibition of VASP Ser157 phosphorylation by expression of the mutant VASP S157A in ASM tissues suppressed VASP phosphorylation and membrane localization in response to ACh, and also inhibited contraction and actin polymerization. ACh but not FSK triggered the formation of VASP-VASP complexes as well as VASP-vinculin and VASP-profilin complexes at membrane sites. VASP-VASP complex formation and the interaction of VASP with vinculin and profilin were inhibited by expression of the inactive vinculin mutant, vinculin Y1065F, but VASP phosphorylation and membrane localization were unaffected. We conclude that VASP phosphorylation at Ser157 mediates its localization at the membrane, but that VASP Ser157 phosphorylation and membrane localization are not sufficient to activate its actin catalytic activity. The interaction of VASP with activated vinculin at membrane adhesion sites is a necessary prerequisite for VASP-mediated molecular processes necessary for actin polymerization. Our results show that VASP is a critical regulator of actin dynamics and tension generation during the contractile activation of ASM. PMID:25759389

A Feedback Loop between Dynamin and Actin Recruitment during Clathrin-Mediated Endocytosis

PubMed Central

Taylor, Marcus J.; Lampe, Marko; Merrifield, Christien J.

2012-01-01

Clathrin-mediated endocytosis proceeds by a sequential series of reactions catalyzed by discrete sets of protein machinery. The final reaction in clathrin-mediated endocytosis is membrane scission, which is mediated by the large guanosine triophosphate hydrolase (GTPase) dynamin and which may involve the actin-dependent recruitment of N-terminal containing BIN/Amphiphysin/RVS domain containing (N-BAR) proteins. Optical microscopy has revealed a detailed picture of when and where particular protein types are recruited in the ∼20–30 s preceding scission. Nevertheless, the regulatory mechanisms and functions that underpin protein recruitment are not well understood. Here we used an optical assay to investigate the coordination and interdependencies between the recruitment of dynamin, the actin cytoskeleton, and N-BAR proteins to individual clathrin-mediated endocytic scission events. These measurements revealed that a feedback loop exists between dynamin and actin at sites of membrane scission. The kinetics of dynamin, actin, and N-BAR protein recruitment were modulated by dynamin GTPase activity. Conversely, acute ablation of actin dynamics using latrunculin-B led to a ∼50% decrease in the incidence of scission, an ∼50% decrease in the amplitude of dynamin recruitment, and abolished actin and N-BAR recruitment to scission events. Collectively these data suggest that dynamin, actin, and N-BAR proteins work cooperatively to efficiently catalyze membrane scission. Dynamin controls its own recruitment to scission events by modulating the kinetics of actin and N-BAR recruitment to sites of scission. Conversely actin serves as a dynamic scaffold that concentrates dynamin and N-BAR proteins at sites of scission. PMID:22505844

Defective Gpsm2/Gαi3 signalling disrupts stereocilia development and growth cone actin dynamics in Chudley-McCullough syndrome

NASA Astrophysics Data System (ADS)

Mauriac, Stephanie A.; Hien, Yeri E.; Bird, Jonathan E.; Carvalho, Steve Dos-Santos; Peyroutou, Ronan; Lee, Sze Chim; Moreau, Maite M.; Blanc, Jean-Michel; Geyser, Aysegul; Medina, Chantal; Thoumine, Olivier; Beer-Hammer, Sandra; Friedman, Thomas B.; Rüttiger, Lukas; Forge, Andrew; Nürnberg, Bernd; Sans, Nathalie; Montcouquiol, Mireille

2017-04-01

Mutations in GPSM2 cause Chudley-McCullough syndrome (CMCS), an autosomal recessive neurological disorder characterized by early-onset sensorineural deafness and brain anomalies. Here, we show that mutation of the mouse orthologue of GPSM2 affects actin-rich stereocilia elongation in auditory and vestibular hair cells, causing deafness and balance defects. The G-protein subunit Gαi3, a well-documented partner of Gpsm2, participates in the elongation process, and its absence also causes hearing deficits. We show that Gpsm2 defines an ~200 nm nanodomain at the tips of stereocilia and this localization requires the presence of Gαi3, myosin 15 and whirlin. Using single-molecule tracking, we report that loss of Gpsm2 leads to decreased outgrowth and a disruption of actin dynamics in neuronal growth cones. Our results elucidate the aetiology of CMCS and highlight a new molecular role for Gpsm2/Gαi3 in the regulation of actin dynamics in epithelial and neuronal tissues.

Gravisensing in single-celled systems - update on characean rhizoids and protonemata

NASA Astrophysics Data System (ADS)

Braun, M.; Limbach, C.

Single-celled and tip-growing rhizoids and protonemata of the characean algae have been intensively studied and there is considerable progress in the understanding of the molecular and cellular mechanisms underlying gravisensing and gravity-dependent growth. In higher plant statocytes, the role of actin in both processes is still a matter of intense debate, but there is clear evidence that actin coordinates both processes in characean rhizoids and protonemata. The multiple functions and dynamic nature of the actin cytoskeleton in these cells are based on the concerted action of a variety of actin-binding proteins. Profilin, actin-depolymerizing factor, a spectrin-like protein, villin and fimbrin have been detected which control apical actin polymerization and regulate the dynamic remodeling of the actin arrangement. An actomyosin-based system was shown to (i) mediate the transport of secretory vesicles to the growing tip, (ii) establish the incorporation of cell wall material and (iii) coordinate the tip-focussed distribution of calcium channels which establish the tip-high calcium gradient for local exocytosis. Experiments performed in microgravity have shown that the actomyosin system precisely coordinates the position of statoliths in rhizoids and protonemata and, upon a change in orientation, directs sedimenting statoliths to specific areas at the plasma membrane where physical contact with gravisensor molecules initiates growth reorientation. The upward growth response of protonemata was shown to be preceded by a statolith-induced and actin-dependent relocalization of the Ca2+-gradient to the upper flank that does not occur in positively gravitropic rhizoids, in which sedimented statoliths cause differential growth of the opposite subapical cell flank. Thus, constant actin polymerization in the growing tip and the spatiotemporal control of actin remodeling by numerous actin-binding proteins are essential for gravity sensing and polarized growth of characean rhizoids and protonemata.

Fimbrin phosphorylation by metaphase Cdk1 regulates actin cable dynamics in budding yeast

PubMed Central

Miao, Yansong; Han, Xuemei; Zheng, Liangzhen; Xie, Ying; Mu, Yuguang; Yates, John R.; Drubin, David G.

2016-01-01

Actin cables, composed of actin filament bundles nucleated by formins, mediate intracellular transport for cell polarity establishment and maintenance. We previously observed that metaphase cells preferentially promote actin cable assembly through cyclin-dependent kinase 1 (Cdk1) activity. However, the relevant metaphase Cdk1 targets were not known. Here we show that the highly conserved actin filament crosslinking protein fimbrin is a critical Cdk1 target for actin cable assembly regulation in budding yeast. Fimbrin is specifically phosphorylated on threonine 103 by the metaphase cyclin–Cdk1 complex, in vivo and in vitro. On the basis of conformational simulations, we suggest that this phosphorylation stabilizes fimbrin's N-terminal domain, and modulates actin filament binding to regulate actin cable assembly and stability in cells. Overall, this work identifies fimbrin as a key target for cell cycle regulation of actin cable assembly in budding yeast, and suggests an underlying mechanism. PMID:27068241

Tropomyosin Promotes Lamellipodial Persistence by Collaborating with Arp2/3 at the Leading Edge.

PubMed

Brayford, Simon; Bryce, Nicole S; Schevzov, Galina; Haynes, Elizabeth M; Bear, James E; Hardeman, Edna C; Gunning, Peter W

2016-05-23

At the leading edge of migrating cells, protrusion of the lamellipodium is driven by Arp2/3-mediated polymerization of actin filaments [1]. This dense, branched actin network is promoted and stabilized by cortactin [2, 3]. In order to drive filament turnover, Arp2/3 networks are remodeled by proteins such as GMF, which blocks the actin-Arp2/3 interaction [4, 5], and coronin 1B, which acts by directing SSH1L to the lamellipodium where it activates the actin-severing protein cofilin [6, 7]. It has been shown in vitro that cofilin-mediated severing of Arp2/3 actin networks results in the generation of new pointed ends to which the actin-stabilizing protein tropomyosin (Tpm) can bind [8]. The presence of Tpm in lamellipodia, however, is disputed in the literature [9-19]. Here, we report that the Tpm isoforms 1.8/9 are enriched in the lamellipodium of fibroblasts as detected with a novel isoform-specific monoclonal antibody. RNAi-mediated silencing of Tpm1.8/9 led to an increase of Arp2/3 accumulation at the cell periphery and a decrease in the persistence of lamellipodia and cell motility, a phenotype consistent with cortactin- and coronin 1B-deficient cells [2, 7]. In the absence of coronin 1B or cofilin, Tpm1.8/9 protein levels are reduced while, conversely, inhibition of Arp2/3 with CK666 leads to an increase in Tpm1.8/9 protein. These findings establish a novel regulatory mechanism within the lamellipodium whereby Tpm collaborates with Arp2/3 to promote lamellipodial-based cell migration. Copyright © 2016 Elsevier Ltd. All rights reserved.

The Membrane Skeleton Controls Diffusion Dynamics and Signaling through the B Cell Receptor

PubMed Central

Treanor, Bebhinn; Depoil, David; Gonzalez-Granja, Aitor; Barral, Patricia; Weber, Michele; Dushek, Omer; Bruckbauer, Andreas; Batista, Facundo D.

2010-01-01

Summary Early events of B cell activation after B cell receptor (BCR) triggering have been well characterized. However, little is known about the steady state of the BCR on the cell surface. Here, we simultaneously visualize single BCR particles and components of the membrane skeleton. We show that an ezrin- and actin-defined network influenced steady-state BCR diffusion by creating boundaries that restrict BCR diffusion. We identified the intracellular domain of Igβ as important in mediating this restriction in diffusion. Importantly, alteration of this network was sufficient to induce robust intracellular signaling and concomitant increase in BCR mobility. Moreover, by using B cells deficient in key signaling molecules, we show that this signaling was most probably initiated by the BCR. Thus, our results suggest the membrane skeleton plays a crucial function in controlling BCR dynamics and thereby signaling, in a way that could be important for understanding tonic signaling necessary for B cell development and survival. PMID:20171124

NanoDLSay: a new platform technology for biomolecular detection and analysis using gold nanoparticle probes coupled with dynamic light scattering

NASA Astrophysics Data System (ADS)

Bogdanovic, Jelena; Huo, Qun

2010-04-01

Most analytical techniques that are routinely used in biomedical research for detection and quantification of biomolecules are time-consuming, expensive and labor-intensive, and there is always a need for rapid, affordable and convenient methods. Recently we have developed a new platform technology for biomolecular detection and analysis: NanoDLSay. NanoDLSay employs antibody-coated gold nanoparticles (GNPs) and dynamic light scattering, and correlates the specific increase in particle size after antigen-antibody interaction to the target antigen concentration. We applied this technology to develop an assay for rapid detection of actin, a protein widely used as a loading control in Western Blot analysis. GNPs were coated with two types of polyclonal anti-actin antibodies, and used in the assay to detect two types of actin: β- and bovine skeletal muscle actin in RIPA buffer. The results of our study revealed some complex aspects of actin binding characteristics, which depended on the type of actin reagent and anti-actin antibody used. A surprising finding was a reverse dose-response relationship between the actin concentration and the average particle size in the assay solution, which we attributed to the effect of RIPA buffer. Our results indicate that RIPA may also interfere in other types of nanoparticle-based assays, and that this interference deserves further study.

Rac-WAVE-mediated actin reorganization is required for organization and maintenance of cell-cell adhesion.

PubMed

Yamazaki, Daisuke; Oikawa, Tsukasa; Takenawa, Tadaomi

2007-01-01

During cadherin-dependent cell-cell adhesion, the actin cytoskeleton undergoes dynamic reorganization in epithelial cells. Rho-family small GTPases, which regulate actin dynamics, play pivotal roles in cadherin-dependent cell-cell adhesion; however, the precise molecular mechanisms that underlie cell-cell adhesion formation remain unclear. Here we show that Wiskott-Aldrich syndrome protein family verprolin-homologous protein (WAVE)-mediated reorganization of actin, downstream of Rac plays an important role in normal development of cadherin-dependent cell-cell adhesions in MDCK cells. Rac-induced development of cadherin-dependent adhesions required WAVE2-dependent actin reorganization. The process of cell-cell adhesion is divided into three steps: formation of new cell-cell contacts, stabilization of these new contacts and junction maturation. WAVE1 and WAVE2 were expressed in MDCK cells. The functions of WAVE1 and WAVE2 were redundant in this system but WAVE2 appeared to play a more significant role. During the first step, WAVE2-dependent lamellipodial protrusions facilitated formation of cell-cell contacts. During the second step, WAVE2 recruited actin filaments to new cell-cell contacts and stabilized newly formed cadherin clusters. During the third step, WAVE2-dependent actin reorganization was required for organization and maintenance of mature cell-cell adhesions. Thus, Rac-WAVE-dependent actin reorganization is not only involved in formation of cell-cell adhesions but is also required for their maintenance.

Membrane tension regulates clathrin-coated pit dynamics

NASA Astrophysics Data System (ADS)

Liu, Allen

2014-03-01

Intracellular organization depends on close communication between the extracellular environment and a network of cytoskeleton filaments. The interactions between cytoskeletal filaments and the plasma membrane lead to changes in membrane tension that in turns help regulate biological processes. Endocytosis is thought to be stimulated by low membrane tension and the removal of membrane increases membrane tension. While it is appreciated that the opposing effects of exocytosis and endocytosis have on keeping plasma membrane tension to a set point, it is not clear how membrane tension affects the dynamics of clathrin-coated pits (CCPs), the individual functional units of clathrin-mediated endocytosis. Furthermore, although it was recently shown that actin dynamics counteracts membrane tension during CCP formation, it is not clear what roles plasma membrane tension plays during CCP initiation. Based on the notion that plasma membrane tension is increased when the membrane area increases during cell spreading, we designed micro-patterned surfaces of different sizes to control the cell spreading sizes. Total internal reflection fluorescence microscopy of living cells and high content image analysis were used to quantify the dynamics of CCPs. We found that there is an increased proportion of CCPs with short (<20s) lifetime for cells on larger patterns. Interestingly, cells on larger patterns have higher CCP initiation density, an effect unexpected based on the conventional view of decreasing endocytosis with increasing membrane tension. Furthermore, by analyzing the intensity profiles of CCPs that were longer-lived, we found CCP intensity decreases with increasing cell size, indicating that the CCPs are smaller with increasing membrane tension. Finally, disruption of actin dynamics significantly increased the number of short-lived CCPs, but also decreased CCP initiation rate. Together, our study reveals new mechanistic insights into how plasma membrane tension regulates the dynamics of CCPs.

Involvement of the Rac1-IRSp53-Wave2-Arp2/3 Signaling Pathway in HIV-1 Gag Particle Release in CD4 T Cells.

PubMed

Thomas, Audrey; Mariani-Floderer, Charlotte; López-Huertas, Maria Rosa; Gros, Nathalie; Hamard-Péron, Elise; Favard, Cyril; Ohlmann, Theophile; Alcamí, José; Muriaux, Delphine

2015-08-01

During HIV-1 assembly, the Gag viral proteins are targeted and assemble at the inner leaflet of the cell plasma membrane. This process could modulate the cortical actin cytoskeleton, located underneath the plasma membrane, since actin dynamics are able to promote localized membrane reorganization. In addition, activated small Rho GTPases are known for regulating actin dynamics and membrane remodeling. Therefore, the modulation of such Rho GTPase activity and of F-actin by the Gag protein during virus particle formation was considered. Here, we studied the implication of the main Rac1, Cdc42, and RhoA small GTPases, and some of their effectors, in this process. The effect of small interfering RNA (siRNA)-mediated Rho GTPases and silencing of their effectors on Gag localization, Gag membrane attachment, and virus-like particle production was analyzed by immunofluorescence coupled to confocal microscopy, membrane flotation assays, and immunoblot assays, respectively. In parallel, the effect of Gag expression on the Rac1 activation level was monitored by G-LISA, and the intracellular F-actin content in T cells was monitored by flow cytometry and fluorescence microscopy. Our results revealed the involvement of activated Rac1 and of the IRSp53-Wave2-Arp2/3 signaling pathway in HIV-1 Gag membrane localization and particle release in T cells as well as a role for actin branching and polymerization, and this was solely dependent on the Gag viral protein. In conclusion, our results highlight a new role for the Rac1-IRSp53-Wave2-Arp2/3 signaling pathway in the late steps of HIV-1 replication in CD4 T lymphocytes. During HIV-1 assembly, the Gag proteins are targeted and assembled at the inner leaflet of the host cell plasma membrane. Gag interacts with specific membrane phospholipids that can also modulate the regulation of cortical actin cytoskeleton dynamics. Actin dynamics can promote localized membrane reorganization and thus can be involved in facilitating Gag assembly and particle formation. Activated small Rho GTPases and effectors are regulators of actin dynamics and membrane remodeling. We thus studied the effects of the Rac1, Cdc42, and RhoA GTPases and their specific effectors on HIV-1 Gag membrane localization and viral particle release in T cells. Our results show that activated Rac1 and the IRSp53-Wave2-Arp2/3 signaling pathway are involved in Gag plasma membrane localization and viral particle production. This work uncovers a role for cortical actin through the activation of Rac1 and the IRSp53/Wave2 signaling pathway in HIV-1 particle formation in CD4 T lymphocytes. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Involvement of the Rac1-IRSp53-Wave2-Arp2/3 Signaling Pathway in HIV-1 Gag Particle Release in CD4 T Cells

PubMed Central

Thomas, Audrey; Mariani-Floderer, Charlotte; López-Huertas, Maria Rosa; Gros, Nathalie; Hamard-Péron, Elise; Favard, Cyril; Ohlmann, Theophile; Alcamí, José

2015-01-01

ABSTRACT During HIV-1 assembly, the Gag viral proteins are targeted and assemble at the inner leaflet of the cell plasma membrane. This process could modulate the cortical actin cytoskeleton, located underneath the plasma membrane, since actin dynamics are able to promote localized membrane reorganization. In addition, activated small Rho GTPases are known for regulating actin dynamics and membrane remodeling. Therefore, the modulation of such Rho GTPase activity and of F-actin by the Gag protein during virus particle formation was considered. Here, we studied the implication of the main Rac1, Cdc42, and RhoA small GTPases, and some of their effectors, in this process. The effect of small interfering RNA (siRNA)-mediated Rho GTPases and silencing of their effectors on Gag localization, Gag membrane attachment, and virus-like particle production was analyzed by immunofluorescence coupled to confocal microscopy, membrane flotation assays, and immunoblot assays, respectively. In parallel, the effect of Gag expression on the Rac1 activation level was monitored by G-LISA, and the intracellular F-actin content in T cells was monitored by flow cytometry and fluorescence microscopy. Our results revealed the involvement of activated Rac1 and of the IRSp53-Wave2-Arp2/3 signaling pathway in HIV-1 Gag membrane localization and particle release in T cells as well as a role for actin branching and polymerization, and this was solely dependent on the Gag viral protein. In conclusion, our results highlight a new role for the Rac1-IRSp53-Wave2-Arp2/3 signaling pathway in the late steps of HIV-1 replication in CD4 T lymphocytes. IMPORTANCE During HIV-1 assembly, the Gag proteins are targeted and assembled at the inner leaflet of the host cell plasma membrane. Gag interacts with specific membrane phospholipids that can also modulate the regulation of cortical actin cytoskeleton dynamics. Actin dynamics can promote localized membrane reorganization and thus can be involved in facilitating Gag assembly and particle formation. Activated small Rho GTPases and effectors are regulators of actin dynamics and membrane remodeling. We thus studied the effects of the Rac1, Cdc42, and RhoA GTPases and their specific effectors on HIV-1 Gag membrane localization and viral particle release in T cells. Our results show that activated Rac1 and the IRSp53-Wave2-Arp2/3 signaling pathway are involved in Gag plasma membrane localization and viral particle production. This work uncovers a role for cortical actin through the activation of Rac1 and the IRSp53/Wave2 signaling pathway in HIV-1 particle formation in CD4 T lymphocytes. PMID:26018170

Three-color single molecule imaging shows WASP detachment from Arp2/3 complex triggers actin filament branch formation.

PubMed

Smith, Benjamin A; Padrick, Shae B; Doolittle, Lynda K; Daugherty-Clarke, Karen; Corrêa, Ivan R; Xu, Ming-Qun; Goode, Bruce L; Rosen, Michael K; Gelles, Jeff

2013-09-03

During cell locomotion and endocytosis, membrane-tethered WASP proteins stimulate actin filament nucleation by the Arp2/3 complex. This process generates highly branched arrays of filaments that grow toward the membrane to which they are tethered, a conflict that seemingly would restrict filament growth. Using three-color single-molecule imaging in vitro we revealed how the dynamic associations of Arp2/3 complex with mother filament and WASP are temporally coordinated with initiation of daughter filament growth. We found that WASP proteins dissociated from filament-bound Arp2/3 complex prior to new filament growth. Further, mutations that accelerated release of WASP from filament-bound Arp2/3 complex proportionally accelerated branch formation. These data suggest that while WASP promotes formation of pre-nucleation complexes, filament growth cannot occur until it is triggered by WASP release. This provides a mechanism by which membrane-bound WASP proteins can stimulate network growth without restraining it. DOI:http://dx.doi.org/10.7554/eLife.01008.001.

Mutations in KPTN cause macrocephaly, neurodevelopmental delay, and seizures.

PubMed

Baple, Emma L; Maroofian, Reza; Chioza, Barry A; Izadi, Maryam; Cross, Harold E; Al-Turki, Saeed; Barwick, Katy; Skrzypiec, Anna; Pawlak, Robert; Wagner, Karin; Coblentz, Roselyn; Zainy, Tala; Patton, Michael A; Mansour, Sahar; Rich, Phillip; Qualmann, Britta; Hurles, Matt E; Kessels, Michael M; Crosby, Andrew H

2014-01-02

The proper development of neuronal circuits during neuromorphogenesis and neuronal-network formation is critically dependent on a coordinated and intricate series of molecular and cellular cues and responses. Although the cortical actin cytoskeleton is known to play a key role in neuromorphogenesis, relatively little is known about the specific molecules important for this process. Using linkage analysis and whole-exome sequencing on samples from families from the Amish community of Ohio, we have demonstrated that mutations in KPTN, encoding kaptin, cause a syndrome typified by macrocephaly, neurodevelopmental delay, and seizures. Our immunofluorescence analyses in primary neuronal cell cultures showed that endogenous and GFP-tagged kaptin associates with dynamic actin cytoskeletal structures and that this association is lost upon introduction of the identified mutations. Taken together, our studies have identified kaptin alterations responsible for macrocephaly and neurodevelopmental delay and define kaptin as a molecule crucial for normal human neuromorphogenesis. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Structural Determination of Biomolecules in Microfluidic Systems

NASA Astrophysics Data System (ADS)

Butler, John C.; Menard, Etienne; Rogers, John A.; Wong, Gerard C. L.

2004-03-01

Supramolecular biological complexes are often too large to be crystallized for structural studies. Here, we explore the use of microfluidic arrays to order a model self-assembled cytoskeletal system. Filamentous actin (F-actin) is a negatively charged protein rod and is a key structural component in the eukaryotic cytoskeleton. In this context, F-actin can self-assemble with actin binding proteins (ABP) in a highly regulated manner to dynamically form structures for a wide range of biomechanical functions. In this work, we will systematically study the action of 3 types of actin binding proteins (a-actinin, fimbrin, cofilin) on the self-assembled structures of F-actin that have been aligned in microfluidic arrays.

Toll-like receptor ligands sensitize B-cell receptor signalling by reducing actin-dependent spatial confinement of the receptor.

PubMed

Freeman, Spencer A; Jaumouillé, Valentin; Choi, Kate; Hsu, Brian E; Wong, Harikesh S; Abraham, Libin; Graves, Marcia L; Coombs, Daniel; Roskelley, Calvin D; Das, Raibatak; Grinstein, Sergio; Gold, Michael R

2015-02-03

Integrating signals from multiple receptors allows cells to interpret the physiological context in which a signal is received. Here we describe a mechanism for receptor crosstalk in which receptor-induced increases in actin dynamics lower the threshold for signalling by another receptor. We show that the Toll-like receptor ligands lipopolysaccharide and CpG DNA, which are conserved microbial molecules, enhance signalling by the B-cell antigen receptor (BCR) by activating the actin-severing protein cofilin. Single-particle tracking reveals that increased severing of actin filaments reduces the spatial confinement of the BCR within the plasma membrane and increases BCR mobility. This allows more frequent collisions between BCRs and greater signalling in response to low densities of membrane-bound antigen. These findings implicate actin dynamics as a means of tuning receptor signalling and as a mechanism by which B cells distinguish inert antigens from those that are accompanied by indicators of microbial infection.

Toll-like receptor ligands sensitize B-cell receptor signalling by reducing actin-dependent spatial confinement of the receptor

PubMed Central

Freeman, Spencer A.; Jaumouillé, Valentin; Choi, Kate; Hsu, Brian E.; Wong, Harikesh S.; Abraham, Libin; Graves, Marcia L.; Coombs, Daniel; Roskelley, Calvin D.; Das, Raibatak; Grinstein, Sergio; Gold, Michael R.

2015-01-01

Integrating signals from multiple receptors allows cells to interpret the physiological context in which a signal is received. Here we describe a mechanism for receptor crosstalk in which receptor-induced increases in actin dynamics lower the threshold for signalling by another receptor. We show that the Toll-like receptor ligands lipopolysaccharide and CpG DNA, which are conserved microbial molecules, enhance signalling by the B-cell antigen receptor (BCR) by activating the actin-severing protein cofilin. Single-particle tracking reveals that increased severing of actin filaments reduces the spatial confinement of the BCR within the plasma membrane and increases BCR mobility. This allows more frequent collisions between BCRs and greater signalling in response to low densities of membrane-bound antigen. These findings implicate actin dynamics as a means of tuning receptor signalling and as a mechanism by which B cells distinguish inert antigens from those that are accompanied by indicators of microbial infection. PMID:25644899

Novel Regulation of Ski Protein Stability and Endosomal Sorting by Actin Cytoskeleton Dynamics in Hepatocytes*

PubMed Central

Vázquez-Victorio, Genaro; Caligaris, Cassandre; Del Valle-Espinosa, Eugenio; Sosa-Garrocho, Marcela; González-Arenas, Nelly R.; Reyes-Cruz, Guadalupe; Briones-Orta, Marco A.; Macías-Silva, Marina

2015-01-01

TGF-β-induced antimitotic signals are highly regulated during cell proliferation under normal and pathological conditions, such as liver regeneration and cancer. Up-regulation of the transcriptional cofactors Ski and SnoN during liver regeneration may favor hepatocyte proliferation by inhibiting TGF-β signals. In this study, we found a novel mechanism that regulates Ski protein stability through TGF-β and G protein-coupled receptor (GPCR) signaling. Ski protein is distributed between the nucleus and cytoplasm of normal hepatocytes, and the molecular mechanisms controlling Ski protein stability involve the participation of actin cytoskeleton dynamics. Cytoplasmic Ski is partially associated with actin and localized in cholesterol-rich vesicles. Ski protein stability is decreased by TGF-β/Smads, GPCR/Rho signals, and actin polymerization, whereas GPCR/cAMP signals and actin depolymerization promote Ski protein stability. In conclusion, TGF-β and GPCR signals differentially regulate Ski protein stability and sorting in hepatocytes, and this cross-talk may occur during liver regeneration. PMID:25561741

Technical advance: identification of plant actin-binding proteins by F-actin affinity chromatography

NASA Technical Reports Server (NTRS)

Hu, S.; Brady, S. R.; Kovar, D. R.; Staiger, C. J.; Clark, G. B.; Roux, S. J.; Muday, G. K.

2000-01-01

Proteins that interact with the actin cytoskeleton often modulate the dynamics or organization of the cytoskeleton or use the cytoskeleton to control their localization. In plants, very few actin-binding proteins have been identified and most are thought to modulate cytoskeleton function. To identify actin-binding proteins that are unique to plants, the development of new biochemical procedures will be critical. Affinity columns using actin monomers (globular actin, G-actin) or actin filaments (filamentous actin, F-actin) have been used to identify actin-binding proteins from a wide variety of organisms. Monomeric actin from zucchini (Cucurbita pepo L.) hypocotyl tissue was purified to electrophoretic homogeneity and shown to be native and competent for polymerization to actin filaments. G-actin, F-actin and bovine serum albumin affinity columns were prepared and used to separate samples enriched in either soluble or membrane-associated actin-binding proteins. Extracts of soluble actin-binding proteins yield distinct patterns when eluted from the G-actin and F-actin columns, respectively, leading to the identification of a putative F-actin-binding protein of approximately 40 kDa. When plasma membrane-associated proteins were applied to these columns, two abundant polypeptides eluted selectively from the F-actin column and cross-reacted with antiserum against pea annexins. Additionally, a protein that binds auxin transport inhibitors, the naphthylphthalamic acid binding protein, which has been previously suggested to associate with the actin cytoskeleton, was eluted in a single peak from the F-actin column. These experiments provide a new approach that may help to identify novel actin-binding proteins from plants.

Technical advance: identification of plant actin-binding proteins by F-actin affinity chromatography.

PubMed

Hu, S; Brady, S R; Kovar, D R; Staiger, C J; Clark, G B; Roux, S J; Muday, G K

2000-10-01

Proteins that interact with the actin cytoskeleton often modulate the dynamics or organization of the cytoskeleton or use the cytoskeleton to control their localization. In plants, very few actin-binding proteins have been identified and most are thought to modulate cytoskeleton function. To identify actin-binding proteins that are unique to plants, the development of new biochemical procedures will be critical. Affinity columns using actin monomers (globular actin, G-actin) or actin filaments (filamentous actin, F-actin) have been used to identify actin-binding proteins from a wide variety of organisms. Monomeric actin from zucchini (Cucurbita pepo L.) hypocotyl tissue was purified to electrophoretic homogeneity and shown to be native and competent for polymerization to actin filaments. G-actin, F-actin and bovine serum albumin affinity columns were prepared and used to separate samples enriched in either soluble or membrane-associated actin-binding proteins. Extracts of soluble actin-binding proteins yield distinct patterns when eluted from the G-actin and F-actin columns, respectively, leading to the identification of a putative F-actin-binding protein of approximately 40 kDa. When plasma membrane-associated proteins were applied to these columns, two abundant polypeptides eluted selectively from the F-actin column and cross-reacted with antiserum against pea annexins. Additionally, a protein that binds auxin transport inhibitors, the naphthylphthalamic acid binding protein, which has been previously suggested to associate with the actin cytoskeleton, was eluted in a single peak from the F-actin column. These experiments provide a new approach that may help to identify novel actin-binding proteins from plants.

Monoubiquitination Inhibits the Actin Bundling Activity of Fascin*

PubMed Central

Lin, Shengchen; Lu, Shuang; Mulaj, Mentor; Fang, Bin; Keeley, Tyler; Wan, Lixin; Hao, Jihui; Muschol, Martin; Sun, Jianwei; Yang, Shengyu

2016-01-01

Fascin is an actin bundling protein that cross-links individual actin filaments into straight, compact, and stiff bundles, which are crucial for the formation of filopodia, stereocillia, and other finger-like membrane protrusions. The dysregulation of fascin has been implicated in cancer metastasis, hearing loss, and blindness. Here we identified monoubiquitination as a novel mechanism that regulates fascin bundling activity and dynamics. The monoubiquitination sites were identified to be Lys247 and Lys250, two residues located in a positive charge patch at the actin binding site 2 of fascin. Using a chemical ubiquitination method, we synthesized chemically monoubiquitinated fascin and determined the effects of monoubiquitination on fascin bundling activity and dynamics. Our data demonstrated that monoubiquitination decreased the fascin bundling EC50, delayed the initiation of bundle assembly, and accelerated the disassembly of existing bundles. By analyzing the electrostatic properties on the solvent-accessible surface of fascin, we proposed that monoubiquitination introduced steric hindrance to interfere with the interaction between actin filaments and the positively charged patch at actin binding site 2. We also identified Smurf1 as a E3 ligase regulating the monoubiquitination of fascin. Our findings revealed a previously unidentified regulatory mechanism for fascin, which will have important implications for the understanding of actin bundle regulation under physiological and pathological conditions. PMID:27879315

Determinants of fluidlike behavior and effective viscosity in cross-linked actin networks.

PubMed

Kim, Taeyoon; Gardel, Margaret L; Munro, Ed

2014-02-04

The actin cortex has a well-documented ability to rapidly remodel and flow while maintaining long-range connectivity, but how this is achieved remains poorly understood. Here, we use computer simulations to explore how stress relaxation in cross-linked actin networks subjected to extensional stress depends on the interplay between network architecture and turnover. We characterize a regime in which a network response is nonaffine and stress relaxation is governed by the continuous dissipation of elastic energy via cyclic formation, elongation, and turnover of tension-bearing elements. Within this regime, for a wide range of network parameters, we observe a constant deformation (creep) rate that is linearly proportional to the rate of filament turnover, leading to a constant effective viscosity that is inversely proportional to turnover rate. Significantly, we observe a biphasic dependence of the creep rate on applied stress: below a critical stress threshold, the creep rate increases linearly with applied stress; above that threshold, the creep rate becomes independent of applied stress. We show that this biphasic stress dependence can be understood in terms of the nonlinear force-extension behavior of individual force-transmitting network elements. These results have important implications for understanding the origins and control of viscous flows both in the cortex of living cells and in other polymer networks. Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Birefringence imaging directly reveals architectural dynamics of filamentous actin in living growth cones.

PubMed

Katoh, K; Hammar, K; Smith, P J; Oldenbourg, R

1999-01-01

We have investigated the dynamic behavior of cytoskeletal fine structure in the lamellipodium of nerve growth cones using a new type of polarized light microscope (the Pol-Scope). Pol-Scope images display with exquisite resolution and definition birefringent fine structures, such as filaments and membranes, without having to treat the cell with exogenous dyes or fluorescent labels. Furthermore, the measured birefringence of protein fibers in the thin lamellipodial region can be interpreted in terms of the number of filaments in the bundles. We confirmed that birefringent fibers are actin-based using conventional fluorescence-labeling methods. By recording movies of time-lapsed Pol-Scope images, we analyzed the creation and dynamic composition of radial fibers, filopodia, and intrapodia in advancing growth cones. The strictly quantitative information available in time-lapsed Pol-Scope images confirms previously deduced behavior and provides new insight into the architectural dynamics of filamentous actin.

Dynamin1 is a novel target for IRSp53 protein and works with mammalian enabled (Mena) protein and Eps8 to regulate filopodial dynamics.

PubMed

Chou, Ai Mei; Sem, Kai Ping; Wright, Graham Daniel; Sudhaharan, Thankiah; Ahmed, Sohail

2014-08-29

Filopodia are dynamic actin-based structures that play roles in processes such as cell migration, wound healing, and axonal guidance. Cdc42 induces filopodial formation through IRSp53, an Inverse-Bin-Amphiphysins-Rvs (I-BAR) domain protein. Previous work from a number of laboratories has shown that IRSp53 generates filopodia by coupling membrane protrusion with actin dynamics through its Src homology 3 domain binding partners. Here, we show that dynamin1 (Dyn1), the large guanosine triphosphatase, is an interacting partner of IRSp53 through pulldown and Förster resonance energy transfer analysis, and we explore its role in filopodial formation. In neuroblastoma cells, Dyn1 localizes to filopodia, associated tip complexes, and the leading edge just behind the anti-capping protein mammalian enabled (Mena). Dyn1 knockdown reduces filopodial formation, which can be rescued by overexpressing wild-type Dyn1 but not the GTPase mutant Dyn1-K44A and the loss-of-function actin binding domain mutant Dyn1-K/E. Interestingly, dynasore, an inhibitor of Dyn GTPase, also reduced filopodial number and increased their lifetime. Using rapid time-lapse total internal reflection fluorescence microscopy, we show that Dyn1 and Mena localize to filopodia only during initiation and assembly. Dyn1 actin binding domain mutant inhibits filopodial formation, suggesting a role in actin elongation. In contrast, Eps8, an actin capping protein, is seen most strongly at filopodial tips during disassembly. Taken together, the results suggest IRSp53 partners with Dyn1, Mena, and Eps8 to regulate filopodial dynamics. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Actin and microtubule networks contribute differently to cell response for small and large strains

NASA Astrophysics Data System (ADS)

Kubitschke, H.; Schnauss, J.; Nnetu, K. D.; Warmt, E.; Stange, R.; Kaes, J.

2017-09-01

Cytoskeletal filaments provide cells with mechanical stability and organization. The main key players are actin filaments and microtubules governing a cell’s response to mechanical stimuli. We investigated the specific influences of these crucial components by deforming MCF-7 epithelial cells at small (≤5% deformation) and large strains (>5% deformation). To understand specific contributions of actin filaments and microtubules, we systematically studied cellular responses after treatment with cytoskeleton influencing drugs. Quantification with the microfluidic optical stretcher allowed capturing the relative deformation and relaxation of cells under different conditions. We separated distinctive deformational and relaxational contributions to cell mechanics for actin and microtubule networks for two orders of magnitude of drug dosages. Disrupting actin filaments via latrunculin A, for instance, revealed a strain-independent softening. Stabilizing these filaments by treatment with jasplakinolide yielded cell softening for small strains but showed no significant change at large strains. In contrast, cells treated with nocodazole to disrupt microtubules displayed a softening at large strains but remained unchanged at small strains. Stabilizing microtubules within the cells via paclitaxel revealed no significant changes for deformations at small strains, but concentration-dependent impact at large strains. This suggests that for suspended cells, the actin cortex is probed at small strains, while at larger strains; the whole cell is probed with a significant contribution from the microtubules.

Mechanical Response of Single Filamin A (ABP-280) Molecules and Its Role in the Actin/Filamin A Gel

NASA Astrophysics Data System (ADS)

Sano, Ryoko; Furuike, Shou; Ito, Tadanao; Ohashi, Kazuyo; Yamazaki, Masahito

2004-04-01

Actin/filamin A gel plays important roles in mechanical response of cells. We found a force (50 to 220 pN)-induced unfolding of single filamin A molecules using AFM, and have proposed a hypothesis on the role of single filamin A in the novel property of viscoelasticity of actin/filamin A gel. We also investigated structure and its dynamics of actin/filamin A gel formed in a giant liposome using fluorescence microscopy.

Every day I'm rufflin': Calcium sensing and actin dynamics in the growth factor-independent membrane ruffling of professional phagocytes.

PubMed

Schlam, Daniel; Canton, Johnathan

2017-04-03

Professional phagocytes continuously extend dynamic, actin-driven membrane protrusions. These protrusions, often referred to as membrane ruffles, serve a critical role in the essential phagocyte processes of macropinocytosis and phagocytosis. Small GTPases, such as RAC1/2, spatially and temporally regulate membrane ruffle formation. We have recently shown that extracellular calcium regulates the elaboration of membrane ruffles primarily through the synthesis of phosphatidic acid (PtdOH) at the plasma membrane. RAC1/2 guanine nucleotide exchange factors harbouring polybasic stretches are recruited by PtdOH to sites of ruffle formation. Here we discuss our findings and offer perspectives on how the regulation of dynamic actin structures at the plasma membrane by small GTPases is a critical component of phagocyte function.

Computational model of polarized actin cables and cytokinetic actin ring formation in budding yeast

PubMed Central

Tang, Haosu; Bidone, Tamara C.

2015-01-01

The budding yeast actin cables and contractile ring are important for polarized growth and division, revealing basic aspects of cytoskeletal function. To study these formin-nucleated structures, we built a 3D computational model with actin filaments represented as beads connected by springs. Polymerization by formins at the bud tip and bud neck, crosslinking, severing, and myosin pulling, are included. Parameter values were estimated from prior experiments. The model generates actin cable structures and dynamics similar to those of wild type and formin deletion mutant cells. Simulations with increased polymerization rate result in long, wavy cables. Simulated pulling by type V myosin stretches actin cables. Increasing the affinity of actin filaments for the bud neck together with reduced myosin V pulling promotes the formation of a bundle of antiparallel filaments at the bud neck, which we suggest as a model for the assembly of actin filaments to the contractile ring. PMID:26538307

Formaldehyde fixation is detrimental to actin cables in glucose-depleted S. cerevisiae cells

PubMed Central

Vasicova, Pavla; Rinnerthaler, Mark; Haskova, Danusa; Novakova, Lenka; Malcova, Ivana; Breitenbach, Michael; Hasek, Jiri

2016-01-01

Actin filaments form cortical patches and emanating cables in fermenting cells of Saccharomyces cerevisiae. This pattern has been shown to be depolarized in glucose-depleted cells after formaldehyde fixation and staining with rhodamine-tagged phalloidin. Loss of actin cables in mother cells was remarkable. Here we extend our knowledge on actin in live glucose-depleted cells co-expressing the marker of actin patches (Abp1-RFP) with the marker of actin cables (Abp140-GFP). Glucose depletion resulted in appearance of actin patches also in mother cells. However, even after 80 min of glucose deprivation these cells showed a clear network of actin cables labeled with Abp140-GFP in contrast to previously published data. In live cells with a mitochondrial dysfunction (rho0 cells), glucose depletion resulted in almost immediate appearance of Abp140-GFP foci partially overlapping with Abp1-RFP patches in mother cells. Residual actin cables were clustered in patch-associated bundles. A similar overlapping “patchy” pattern of both actin markers was observed upon treatment of glucose-deprived rho+ cells with FCCP (the inhibitor of oxidative phosphorylation) and upon treatment with formaldehyde. While the formaldehyde-targeted process stays unknown, our results indicate that published data on yeast actin cytoskeleton obtained from glucose-depleted cells after fixation should be considered with caution. PMID:28357356

Neonatal isolation augments social dominance by altering actin dynamics in the medial prefrontal cortex.

PubMed

Tada, Hirobumi; Miyazaki, Tomoyuki; Takemoto, Kiwamu; Takase, Kenkichi; Jitsuki, Susumu; Nakajima, Waki; Koide, Mayu; Yamamoto, Naoko; Komiya, Kasane; Suyama, Kumiko; Sano, Akane; Taguchi, Akiko; Takahashi, Takuya

2016-10-25

Social separation early in life can lead to the development of impaired interpersonal relationships and profound social disorders. However, the underlying cellular and molecular mechanisms involved are largely unknown. Here, we found that isolation of neonatal rats induced glucocorticoid-dependent social dominance over nonisolated control rats in juveniles from the same litter. Furthermore, neonatal isolation inactivated the actin-depolymerizing factor (ADF)/cofilin in the juvenile medial prefrontal cortex (mPFC). Isolation-induced inactivation of ADF/cofilin increased stable actin fractions at dendritic spines in the juvenile mPFC, decreasing glutamate synaptic AMPA receptors. Expression of constitutively active ADF/cofilin in the mPFC rescued the effect of isolation on social dominance. Thus, neonatal isolation affects spines in the mPFC by reducing actin dynamics, leading to altered social behavior later in life.

Neonatal isolation augments social dominance by altering actin dynamics in the medial prefrontal cortex

PubMed Central

Tada, Hirobumi; Miyazaki, Tomoyuki; Takemoto, Kiwamu; Takase, Kenkichi; Jitsuki, Susumu; Nakajima, Waki; Koide, Mayu; Yamamoto, Naoko; Komiya, Kasane; Suyama, Kumiko; Sano, Akane; Taguchi, Akiko; Takahashi, Takuya

2016-01-01

Social separation early in life can lead to the development of impaired interpersonal relationships and profound social disorders. However, the underlying cellular and molecular mechanisms involved are largely unknown. Here, we found that isolation of neonatal rats induced glucocorticoid-dependent social dominance over nonisolated control rats in juveniles from the same litter. Furthermore, neonatal isolation inactivated the actin-depolymerizing factor (ADF)/cofilin in the juvenile medial prefrontal cortex (mPFC). Isolation-induced inactivation of ADF/cofilin increased stable actin fractions at dendritic spines in the juvenile mPFC, decreasing glutamate synaptic AMPA receptors. Expression of constitutively active ADF/cofilin in the mPFC rescued the effect of isolation on social dominance. Thus, neonatal isolation affects spines in the mPFC by reducing actin dynamics, leading to altered social behavior later in life. PMID:27791080

The Stochastic Dynamics of Filopodial Growth

NASA Astrophysics Data System (ADS)

Papoian, Garegin A.; Lan, Yueheng; Zhuravlev, Pavel

2008-03-01

A filopodium is a cytoplasmic projection, exquisitely built and regulated, which extends from the leading edge of the migrating cell, exploring the cell's neighborhood. Commonly, filopodia grow and retract after their initiation, exhibiting rich dynamical behaviors. We model the growth of a filopodium based on a stochastic description which incorporates mechanical, physical and biochemical components. Our model provides a full stochastic treatment of the actin monomer diffusion and polymerization of each individual actin filament under stress of the fluctuating membrane. We have investigated the length distribution of individual filaments in a growing filopodium and studied how it depends on various physical parameters. The distribution of filament lengths turned out to be narrow, which we explained by the negative feedback created by the membrane load and monomeric G-actin gradient. We also discovered that filopodial growth is strongly diminished upon increasing retrograde flow, suggesting that regulating the retrograde flow rate would be a highly efficient way to control filopodial extension dynamics. The filopodial length increases as the membrane fluctuations decrease, which we attributed to the unequal loading of the mem- brane force among individual filaments, which, in turn, results in larger average polymerization rates. We also observed significant diffusional noise of G-actin monomers, which leads to smaller G-actin flux along the filopodial tube compared with the prediction using the diffusion equation.

Cofilin is a pH sensor for actin free barbed end formation: role of phosphoinositide binding.

PubMed

Frantz, Christian; Barreiro, Gabriela; Dominguez, Laura; Chen, Xiaoming; Eddy, Robert; Condeelis, John; Kelly, Mark J S; Jacobson, Matthew P; Barber, Diane L

2008-12-01

Newly generated actin free barbed ends at the front of motile cells provide sites for actin filament assembly driving membrane protrusion. Growth factors induce a rapid biphasic increase in actin free barbed ends, and we found both phases absent in fibroblasts lacking H(+) efflux by the Na-H exchanger NHE1. The first phase is restored by expression of mutant cofilin-H133A but not unphosphorylated cofilin-S3A. Constant pH molecular dynamics simulations and nuclear magnetic resonance (NMR) reveal pH-sensitive structural changes in the cofilin C-terminal filamentous actin binding site dependent on His133. However, cofilin-H133A retains pH-sensitive changes in NMR spectra and severing activity in vitro, which suggests that it has a more complex behavior in cells. Cofilin activity is inhibited by phosphoinositide binding, and we found that phosphoinositide binding is pH-dependent for wild-type cofilin, with decreased binding at a higher pH. In contrast, phosphoinositide binding by cofilin-H133A is attenuated and pH insensitive. These data suggest a molecular mechanism whereby cofilin acts as a pH sensor to mediate a pH-dependent actin filament dynamics.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Krauss, Sharon Wald; Chen, Cynthia; Penman, Sheldon

Structural protein 4.1, which has crucial interactions within the spectin-actin lattice of the human red cell membrane skeleton, also is widely distributed at diverse intracellular sites in nucleated cells. We previously showed that 4.1 is essential for assembly of functional nuclei in vitro and that the capacity of 4.1 to bind actin is required. Here we report that 4.1 and actin colocalize in mammalian cell nuclei using fluorescence microscopy and, by higher resolution cell whole mount electron microscopy, are associated on nuclear filaments. We also devised a cell-free assay using Xenopus egg extract containing fluorescent actin to follow actin duringmore » nuclear assembly. By directly imaging actin under non-perturbing conditions, the total nuclear actin population is retained and is visualized in situ relative to intact chromatin. We detected actin initially when chromatin and nuclear pores began assembling. As the nuclear lamina assembled, but preceding DNA synthesis, a discrete actin network formed throughout the nucleus. Protein 4.1 epitopes also were detected when actin began to accumulate in nuclei, producing a diffuse coincident pattern. As nuclei matured, actin was detected both coincident with and also independent of 4.1 epitopes. To test whether acquisition of nuclear actin is required for nuclear assembly, the actin inhibitor latrunculin A was added to Xenopus egg extracts during nuclear assembly. Latrunculin A strongly perturbed nuclear assembly and produced distorted nuclear structures containing neither actin nor protein 4.1. Our results suggest that actin as well as 4.1 is necessary for nuclear assembly and that 4.1-actin interactions may be critical.« less

Demonstration of prominent actin filaments in the root columella

NASA Technical Reports Server (NTRS)

Collings, D. A.; Zsuppan, G.; Allen, N. S.; Blancaflor, E. B.; Brown, C. S. (Principal Investigator)

2001-01-01

The distribution of actin filaments within the gravity-sensing columella cells of plant roots remains poorly understood, with studies over numerous years providing inconsistent descriptions of actin organization in these cells. This uncertainty in actin organization, and thus in actin's role in graviperception and gravisignaling, has led us to investigate actin arrangements in the columella cells of Zea mays L., Medicago truncatula Gaertn., Linum usitatissiilium L. and Nicotianla benthamiana Domin. Actin organization was examined using a combination of optimized immunofluorescence techniques, and an improved fluorochrome-conjugated phalloidin labeling method reliant on 3-maleimidobenzoyl-N-hydroxy-succinimide ester (MBS) cross-linking combined with glycerol permeabilization. Confocal microscopy of root sections labeled with anti-actin antibodies revealed patterns suggestive of actin throughout the columella region. These patterns included short and fragmented actin bundles, fluorescent rings around amyloplasts and intense fluorescence originating from the nucleus. Additionally, confocal microscopy of MBS-stabilized and Alexa Fluor-phalloidin-labeled root sections revealed a previously undetected state of actin organization in the columella. Discrete actin structures surrounded the amyloplasts and prominent actin cables radiated from the nuclear surface toward the cell periphery. Furthermore, the cortex of the columella cells contained fine actin bundles (or single filaments) that had a predominant transverse orientation. We also used confocal microscopy of plant roots expressing endoplasmic reticulum (ER)-targeted green fluorescent protein to demonstrate rapid ER movements within the columella cells, suggesting that the imaged actin network is functional. The successful identification of discrete actin structures in the root columella cells forms the perception and signaling.

The plant host pathogen interface: cell wall and membrane dynamics of pathogen-induced responses.

PubMed

Day, Brad; Graham, Terry

2007-10-01

Perception of pathogens by their hosts is the outcome of a highly coordinated and sophisticated surveillance network, tightly regulated by both host and pathogen elicitors, effectors, and signaling processes. In this article, we focus on two relatively well-studied host-pathogens systems, one involving a bacterial-plant interaction (Pseudomonas syringae-Arabidopsis) and the other involving an oomycete-plant interaction (Phytophthora sojae-soybean). We discuss the status of current research related to events occurring at the host-pathogen interface in these two systems, and how these events influence the organization and activation of resistance responses in the respective hosts. This recent research has revealed that in addition to the previously identified resistance machinery (R-proteins, molecular chaperones, etc.), the dynamics of the cell wall, membrane trafficking, and the actin cytoskeleton are intimately associated with the activation of resistance in plants. Specifically, in Arabidopsis, a possible connection between the actin machinery and R-protein- mediated induction of disease resistance is described. In the case of the P. sojae-soybean interaction, we describe the fact that a classical basal resistance elicitor, the cell wall glucan elicitor from the pathogen, can directly activate host hypersensitive cell death, which is apparently modulated in a race-specific manner by the presence of R genes in the host.

Stomatal Complex Development and F-Actin Organization in Maize Leaf Epidermis Depend on Cellulose Synthesis.

PubMed

Panteris, Emmanuel; Achlati, Theonymphi; Daras, Gerasimos; Rigas, Stamatis

2018-06-06

Cellulose microfibrils reinforce the cell wall for morphogenesis in plants. Herein, we provide evidence on a series of defects regarding stomatal complex development and F-actin organization in Zea mays leaf epidermis, due to inhibition of cellulose synthesis. Formative cell divisions of stomatal complex ontogenesis were delayed or inhibited, resulting in lack of subsidiary cells and frequently in unicellular stomata, with an atypical stomatal pore. Guard cells failed to acquire a dumbbell shape, becoming rounded, while subsidiary cells, whenever present, exhibited aberrant morphogenesis. F-actin organization was also affected, since the stomatal complex-specific arrays were scarcely observed. At late developmental stages, the overall F-actin network was diminished in all epidermal cells, although thick actin bundles persisted. Taken together, stomatal complex development strongly depends on cell wall mechanical properties. Moreover, F-actin organization exhibits a tight relationship with the cell wall.

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

PubMed Central

Hocking, Kyle M.; Baudenbacher, Franz J.; Putumbaka, Gowthami; Venkatraman, Sneha; Cheung-Flynn, Joyce; Brophy, Colleen M.; Komalavilas, Padmini

2013-01-01

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. PMID:23593369

Role of cyclic nucleotide-dependent actin cytoskeletal dynamics:Ca(2+)](i) and force suppression in forskolin-pretreated porcine coronary arteries.

PubMed

Hocking, Kyle M; Baudenbacher, Franz J; Putumbaka, Gowthami; Venkatraman, Sneha; Cheung-Flynn, Joyce; Brophy, Colleen M; Komalavilas, Padmini

2013-01-01

Initiation of force generation during vascular smooth muscle contraction involves a rise in intracellular calcium ([Ca(2+)]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 [Ca(2+)]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.

Dynamics of actin-based movement by Rickettsia rickettsii in vero cells.

PubMed

Heinzen, R A; Grieshaber, S S; Van Kirk, L S; Devin, C J

1999-08-01

Actin-based motility (ABM) is a virulence mechanism exploited by invasive bacterial pathogens in the genera Listeria, Shigella, and Rickettsia. Due to experimental constraints imposed by the lack of genetic tools and their obligate intracellular nature, little is known about rickettsial ABM relative to Listeria and Shigella ABM systems. In this study, we directly compared the dynamics and behavior of ABM of Rickettsia rickettsii and Listeria monocytogenes. A time-lapse video of moving intracellular bacteria was obtained by laser-scanning confocal microscopy of infected Vero cells synthesizing beta-actin coupled to green fluorescent protein (GFP). Analysis of time-lapse images demonstrated that R. rickettsii organisms move through the cell cytoplasm at an average rate of 4.8 +/- 0.6 micrometer/min (mean +/- standard deviation). This speed was 2.5 times slower than that of L. monocytogenes, which moved at an average rate of 12.0 +/- 3.1 micrometers/min. Although rickettsiae moved more slowly, the actin filaments comprising the actin comet tail were significantly more stable, with an average half-life approximately three times that of L. monocytogenes (100.6 +/- 19.2 s versus 33.0 +/- 7.6 s, respectively). The actin tail associated with intracytoplasmic rickettsiae remained stationary in the cytoplasm as the organism moved forward. In contrast, actin tails of rickettsiae trapped within the nucleus displayed dramatic movements. The observed phenotypic differences between the ABM of Listeria and Rickettsia may indicate fundamental differences in the mechanisms of actin recruitment and polymerization.

Nonmedially assembled F-actin cables incorporate into the actomyosin ring in fission yeast

PubMed Central

Huang, Junqi; Huang, Yinyi; Yu, Haochen; Subramanian, Dhivya; Padmanabhan, Anup; Thadani, Rahul; Tao, Yaqiong; Tang, Xie; Wedlich-Soldner, Roland

2012-01-01

In many eukaryotes, cytokinesis requires the assembly and constriction of an actomyosin-based contractile ring. Despite the central role of this ring in cytokinesis, the mechanism of F-actin assembly and accumulation in the ring is not fully understood. In this paper, we investigate the mechanism of F-actin assembly during cytokinesis in Schizosaccharomyces pombe using lifeact as a probe to monitor actin dynamics. Previous work has shown that F-actin in the actomyosin ring is assembled de novo at the division site. Surprisingly, we find that a significant fraction of F-actin in the ring was recruited from formin-Cdc12p nucleated long actin cables that were generated at multiple nonmedial locations and incorporated into the ring by a combination of myosin II and myosin V activities. Our results, together with findings in animal cells, suggest that de novo F-actin assembly at the division site and directed transport of F-actin cables assembled elsewhere can contribute to ring assembly. PMID:23185032

A nucleator arms race: cellular control of actin assembly.

PubMed

Campellone, Kenneth G; Welch, Matthew D

2010-04-01

For over a decade, the actin-related protein 2/3 (ARP2/3) complex, a handful of nucleation-promoting factors and formins were the only molecules known to directly nucleate actin filament formation de novo. However, the past several years have seen a surge in the discovery of mammalian proteins with roles in actin nucleation and dynamics. Newly recognized nucleation-promoting factors, such as WASP and SCAR homologue (WASH), WASP homologue associated with actin, membranes and microtubules (WHAMM), and junction-mediating regulatory protein (JMY), stimulate ARP2/3 activity at distinct cellular locations. Formin nucleators with additional biochemical and cellular activities have also been uncovered. Finally, the Spire, cordon-bleu and leiomodin nucleators have revealed new ways of overcoming the kinetic barriers to actin polymerization.

Biphasic interactions between a cationic dendrimer and actin.

PubMed

Ruenraroengsak, Pakatip; Florence, Alexander T

2010-12-01

Gene delivery systems face the problem not only of the route toward the cell and tissues in question, but also of the molecularly crowded environment of both the cytoplasm and the nucleus itself. One of the physical barriers in the cytoplasm for diffusing nanoparticles is an actin network. Here, we describe the finding that a self-fluorescent sixth generation cationic dendrimer (6 nm in diameter) interacts reversibly and possibly electrostatically with actin filaments in vitro. Not only does this interaction slow the diffusion of the dendrimer but it also affects actin polymerization in a biphasic manner. At low concentrations the dendrimer behaves like a G-binding actin protein, retarding actin polymerization, whereas at high concentrations the dendrimer acts as a nucleating protein accelerating the polymerization. Thus in vivo the diffusion of a dendrimer carrier such as this has both physical and chemical elements: by decreasing polymerization it might accelerate its own transport, and by enhancing actin polymerization retard it. This finding suggests that such a dendrimer may have a role as an anticancer agent through its inhibitory effect on actin polymerization.

WAVE binds Ena/VASP for enhanced Arp2/3 complex–based actin assembly

PubMed Central

Havrylenko, Svitlana; Noguera, Philippe; Abou-Ghali, Majdouline; Manzi, John; Faqir, Fahima; Lamora, Audrey; Guérin, Christophe; Blanchoin, Laurent; Plastino, Julie

2015-01-01

The WAVE complex is the main activator of the Arp2/3 complex for actin filament nucleation and assembly in the lamellipodia of moving cells. Other important players in lamellipodial protrusion are Ena/VASP proteins, which enhance actin filament elongation. Here we examine the molecular coordination between the nucleating activity of the Arp2/3 complex and the elongating activity of Ena/VASP proteins for the formation of actin networks. Using an in vitro bead motility assay, we show that WAVE directly binds VASP, resulting in an increase in Arp2/3 complex–based actin assembly. We show that this interaction is important in vivo as well, for the formation of lamellipodia during the ventral enclosure event of Caenorhabditis elegans embryogenesis. Ena/VASP's ability to bind F-actin and profilin-complexed G-actin are important for its effect, whereas Ena/VASP tetramerization is not necessary. Our data are consistent with the idea that binding of Ena/VASP to WAVE potentiates Arp2/3 complex activity and lamellipodial actin assembly. PMID:25355952

Structural alterations of thin actin filaments in muscle contraction by synchrotron X-ray fiber diffraction.

PubMed

Wakabayashi, Katsuzo; Sugimoto, Yasunobu; Takezawa, Yasunori; Ueno, Yutaka; Minakata, Shiho; Oshima, Kanji; Matsuo, Tatsuhito; Kobayashi, Takakazu

2007-01-01

Strong evidence has been accumulated that the conformational changes of the thin actin filaments are occurring and playing an important role in the entire process of muscle contraction. The conformational changes and the mechanical properties of the thin actin filaments we have found by X-ray fiber diffraction on skeletal muscle contraction are explored. Recent studies on the conformational changes of regulatory proteins bound to actin filaments upon activation and in the force generation process are also described. Finally, the roles of structural alterations and dynamics of the actin filaments are discussed in conjunction with the regulation mechanism and the force generation mechanism.

Actin cytoskeletal rearrangement and dysfunction due to activation of the receptor for advanced glycation end products is inhibited by thymosin beta 4

PubMed Central

Kim, Sokho; Kwon, Jungkee

2015-01-01

The receptor of advanced glycation end products (RAGE) is a cell-surface receptor that is a key factor in the pathogenesis of diabetic complications, including vascular disorders. Dysfunction of the actin cytoskeleton contributes to disruption of cell membrane repair in response to various type of endothelial cell damage. However, mechanism underlying RAGE remodelling of the actin cytoskeleton, by which globular actin (G-actin) forms to filamentous actin (F-actin), remains unclear. In this study we examined the role of thymosin beta 4 (Tβ4) – which binds to actin, blocks actin polymerization, and maintains the dynamic equilibrium between G-actin and F-actin in human umbilical vein endothelial cells (HUVECs) – in the response to RAGE. Tβ4 increased cell viability and decreased levels of reactive oxygen species in HUVECs incubated with AGEs. Tβ4 reduced the expression of RAGE, consistent with a down-regulation of the F-actin to G-actin ratio. The effect of remodelling of the actin cytoskeleton on RAGE expression was clarified by adding Phalloidin, which stabilizes F-actin. Moreover, small interfering RNA was used to determine whether intrinsic Tβ4 regulates RAGE expression in the actin cytoskeleton. The absence of intrinsic Tβ4 in HUVECs evoked actin cytoskeleton disorder and increased RAGE expression. These findings suggest that regulation of the actin cytoskeleton by Tβ4 plays a pivotal role in the RAGE response to AGEs. PMID:25640761

Tight coupling between nucleus and cell migration through the perinuclear actin cap

PubMed Central

Kim, Dong-Hwee; Cho, Sangkyun; Wirtz, Denis

2014-01-01

ABSTRACT Although eukaryotic cells are known to alternate between ‘advancing’ episodes of fast and persistent movement and ‘hesitation’ episodes of low speed and low persistence, the molecular mechanism that controls the dynamic changes in morphology, speed and persistence of eukaryotic migratory cells remains unclear. Here, we show that the movement of the interphase nucleus during random cell migration switches intermittently between two distinct modes – rotation and translocation – that follow with high fidelity the sequential rounded and elongated morphologies of the nucleus and cell body, respectively. Nuclear rotation and translocation mediate the stop-and-go motion of the cell through the dynamic formation and dissolution, respectively, of the contractile perinuclear actin cap, which is dynamically coupled to the nuclear lamina and the nuclear envelope through LINC complexes. A persistent cell movement and nuclear translocation driven by the actin cap are halted following the disruption of the actin cap, which in turn allows the cell to repolarize for its next persistent move owing to nuclear rotation mediated by cytoplasmic dynein light intermediate chain 2. PMID:24639463

Myosin IIb-dependent Regulation of Actin Dynamics Is Required for N-Methyl-D-aspartate Receptor Trafficking during Synaptic Plasticity.

PubMed

Bu, Yunfei; Wang, Ning; Wang, Shaoli; Sheng, Tao; Tian, Tian; Chen, Linlin; Pan, Weiwei; Zhu, Minsheng; Luo, Jianhong; Lu, Wei

2015-10-16

N-Methyl-d-aspartate receptor (NMDAR) synaptic incorporation changes the number of NMDARs at synapses and is thus critical to various NMDAR-dependent brain functions. To date, the molecules involved in NMDAR trafficking and the underlying mechanisms are poorly understood. Here, we report that myosin IIb is an essential molecule in NMDAR synaptic incorporation during PKC- or θ burst stimulation-induced synaptic plasticity. Moreover, we demonstrate that myosin light chain kinase (MLCK)-dependent actin reorganization contributes to NMDAR trafficking. The findings from additional mutual occlusion experiments demonstrate that PKC and MLCK share a common signaling pathway in NMDAR-mediated synaptic regulation. Because myosin IIb is the primary substrate of MLCK and can regulate actin dynamics during synaptic plasticity, we propose that the MLCK- and myosin IIb-dependent regulation of actin dynamics is required for NMDAR trafficking during synaptic plasticity. This study provides important insights into a mechanical framework for understanding NMDAR trafficking associated with synaptic plasticity. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Capping protein regulatory cycle driven by CARMIL and V-1 may promote actin network assembly at protruding edges

PubMed Central

Fujiwara, Ikuko; Remmert, Kirsten; Piszczek, Grzegorz; Hammer, John A.

2014-01-01

Although capping protein (CP) terminates actin filament elongation, it promotes Arp2/3-dependent actin network assembly and accelerates actin-based motility both in vitro and in vivo. In vitro, capping protein Arp2/3 myosin I linker (CARMIL) antagonizes CP by reducing its affinity for the barbed end and by uncapping CP-capped filaments, whereas the protein V-1/myotrophin sequesters CP in an inactive complex. Previous work showed that CARMIL can readily retrieve CP from the CP:V-1 complex, thereby converting inactive CP into a version with moderate affinity for the barbed end. Here we further clarify the mechanism of this exchange reaction, and we demonstrate that the CP:CARMIL complex created by complex exchange slows the rate of barbed-end elongation by rapidly associating with, and dissociating from, the barbed end. Importantly, the cellular concentrations of V-1 and CP determined here argue that most CP is sequestered by V-1 at steady state in vivo. Finally, we show that CARMIL is recruited to the plasma membrane and only at cell edges undergoing active protrusion. Assuming that CARMIL is active only at this location, our data argue that a large pool of freely diffusing, inactive CP (CP:V-1) feeds, via CARMIL-driven complex exchange, the formation of weak-capping complexes (CP:CARMIL) at the plasma membrane of protruding edges. In vivo, therefore, CARMIL should promote Arp2/3-dependent actin network assembly at the leading edge by promoting barbed-end capping there. PMID:24778263

The actin cytoskeleton in whole mount preparations and sections.

PubMed

Resch, Guenter P; Urban, Edit; Jacob, Sonja

2010-01-01

In non-muscle cells, the actin cytoskeleton plays a key role by providing a scaffold contributing to the definition of cell shape, force for driving cell motility, cytokinesis, endocytosis, and propulsion of pathogens, as well as tracks for intracellular transport. A thorough understanding of these processes requires insight into the spatial and temporal organisation of actin filaments into diverse higher-order structures, such as networks, parallel bundles, and contractile arrays. Transmission and scanning electron microscopy can be used to visualise the actin cytoskeleton, but due to the delicate nature of actin filaments, they are easily affected by standard preparation protocols, yielding variable degrees of ultrastructural preservation. In this chapter, we describe different conventional and cryo-approaches to visualise the actin cytoskeleton using transmission electron microscopy and discuss their specific advantages and drawbacks. In the first part, we present three different whole mount techniques, which allow visualisation of actin in the peripheral, thinly spread parts of cells grown in monolayers. In the second part, we describe specific issues concerning the visualisation of actin in thin sections. Techniques for three-dimensional visualisation of actin, protein localisation, and correlative light and electron microscopy are also included. Copyright © 2010 Elsevier Inc. All rights reserved.

Correlative nanoscale imaging of actin filaments and their complexes

NASA Astrophysics Data System (ADS)

Sharma, Shivani; Zhu, Huanqi; Grintsevich, Elena E.; Reisler, Emil; Gimzewski, James K.

2013-06-01

Actin remodeling is an area of interest in biology in which correlative microscopy can bring a new way to analyze protein complexes at the nanoscale. Advances in EM, X-ray diffraction, fluorescence, and single molecule techniques have provided a wealth of information about the modulation of the F-actin structure and its regulation by actin binding proteins (ABPs). Yet, there are technological limitations of these approaches to achieving quantitative molecular level information on the structural and biophysical changes resulting from ABPs interaction with F-actin. Fundamental questions about the actin structure and dynamics and how these determine the function of ABPs remain unanswered. Specifically, how local and long-range structural and conformational changes result in ABPs induced remodeling of F-actin needs to be addressed at the single filament level. Advanced, sensitive and accurate experimental tools for detailed understanding of ABP-actin interactions are much needed. This article discusses the current understanding of nanoscale structural and mechanical modulation of F-actin by ABPs at the single filament level using several correlative microscopic techniques, focusing mainly on results obtained by Atomic Force Microscopy (AFM) analysis of ABP-actin complexes.

Monoubiquitination Inhibits the Actin Bundling Activity of Fascin.

PubMed

Lin, Shengchen; Lu, Shuang; Mulaj, Mentor; Fang, Bin; Keeley, Tyler; Wan, Lixin; Hao, Jihui; Muschol, Martin; Sun, Jianwei; Yang, Shengyu

2016-12-30

Fascin is an actin bundling protein that cross-links individual actin filaments into straight, compact, and stiff bundles, which are crucial for the formation of filopodia, stereocillia, and other finger-like membrane protrusions. The dysregulation of fascin has been implicated in cancer metastasis, hearing loss, and blindness. Here we identified monoubiquitination as a novel mechanism that regulates fascin bundling activity and dynamics. The monoubiquitination sites were identified to be Lys 247 and Lys 250 , two residues located in a positive charge patch at the actin binding site 2 of fascin. Using a chemical ubiquitination method, we synthesized chemically monoubiquitinated fascin and determined the effects of monoubiquitination on fascin bundling activity and dynamics. Our data demonstrated that monoubiquitination decreased the fascin bundling EC 50 , delayed the initiation of bundle assembly, and accelerated the disassembly of existing bundles. By analyzing the electrostatic properties on the solvent-accessible surface of fascin, we proposed that monoubiquitination introduced steric hindrance to interfere with the interaction between actin filaments and the positively charged patch at actin binding site 2. We also identified Smurf1 as a E3 ligase regulating the monoubiquitination of fascin. Our findings revealed a previously unidentified regulatory mechanism for fascin, which will have important implications for the understanding of actin bundle regulation under physiological and pathological conditions. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Affimer proteins for F-actin: novel affinity reagents that label F-actin in live and fixed cells.

PubMed

Lopata, Anna; Hughes, Ruth; Tiede, Christian; Heissler, Sarah M; Sellers, James R; Knight, Peter J; Tomlinson, Darren; Peckham, Michelle

2018-04-26

Imaging the actin cytoskeleton in cells uses a wide range of approaches. Typically, a fluorescent derivative of the small cyclic peptide phalloidin is used to image F-actin in fixed cells. Lifeact and F-tractin are popular for imaging the cytoskeleton in live cells. Here we characterised novel affinity reagents called Affimers that specifically bind to F-actin in vitro to determine if they are suitable alternatives as eGFP-fusion proteins, to label actin in live cells, or for labeling F-actin in fixed cells. In vitro experiments showed that 3 out of the 4 Affimers (Affimers 6, 14 and 24) tested bind tightly to purified F-actin, and appear to have overlapping binding sites. As eGFP-fusion proteins, the same 3 Affimers label F-actin in live cells. FRAP experiments suggest that eGFP-Affimer 6 behaves most similarly to F-tractin and Lifeact. However, it does not colocalise with mCherry-actin in dynamic ruffles, and may preferentially bind stable actin filaments. All 4 Affimers label F-actin in methanol fixed cells, while only Affimer 14 labels F-actin after paraformaldehyde fixation. eGFP-Affimer 6 has potential for use in selectively imaging the stable actin cytoskeleton in live cells, while all 4 Affimers are strong alternatives to phalloidin for labelling F-actin in fixed cells.

Phosphoinositide 3-Kinase Regulates Glycolysis through Mobilization of Aldolase from the Actin Cytoskeleton.

PubMed

Hu, Hai; Juvekar, Ashish; Lyssiotis, Costas A; Lien, Evan C; Albeck, John G; Oh, Doogie; Varma, Gopal; Hung, Yin Pun; Ullas, Soumya; Lauring, Josh; Seth, Pankaj; Lundquist, Mark R; Tolan, Dean R; Grant, Aaron K; Needleman, Daniel J; Asara, John M; Cantley, Lewis C; Wulf, Gerburg M

2016-01-28

The phosphoinositide 3-kinase (PI3K) pathway regulates multiple steps in glucose metabolism and also cytoskeletal functions, such as cell movement and attachment. Here, we show that PI3K directly coordinates glycolysis with cytoskeletal dynamics in an AKT-independent manner. Growth factors or insulin stimulate the PI3K-dependent activation of Rac, leading to disruption of the actin cytoskeleton, release of filamentous actin-bound aldolase A, and an increase in aldolase activity. Consistently, PI3K inhibitors, but not AKT, SGK, or mTOR inhibitors, cause a significant decrease in glycolysis at the step catalyzed by aldolase, while activating PIK3CA mutations have the opposite effect. These results point toward a master regulatory function of PI3K that integrates an epithelial cell's metabolism and its form, shape, and function, coordinating glycolysis with the energy-intensive dynamics of actin remodeling. Copyright © 2016 Elsevier Inc. All rights reserved.

Phosphoinositide 3-Kinase Regulates Glycolysis through Mobilization of Aldolase from the Actin cytoskeleton

PubMed Central

Hu, Hai; Juvekar, Ashish; Lyssiotis, Costas A.; Lien, Evan C.; Albeck, John G.; Oh, Doogie; Varma, Gopal; Hung, Yin Pun; Ullas, Soumya; Lauring, Josh; Seth, Pankaj; Lundquist, Mark R.; Tolan, Dean R.; Grant, Aaron K.; Needleman, Daniel J.; Asara, John M.; Cantley, Lewis C.

2016-01-01

Summary The Phosphoinositide 3-Kinase (PI3K) pathway regulates multiple steps in glucose metabolism but also cytoskeletal functions, such as cell movement and attachment. Here we show that PI3K directly coordinates glycolysis with cytoskeletal dynamics in an AKT-independent manner. Growth factors or insulin stimulate the PI3K-dependent activation of Rac, leading to disruption of the actin cytoskeleton, release of filamentous actin-bound aldolase A and an increase in aldolase activity. Consistently, PI3K-, but not AKT-, SGK- or mTOR-inhibitors, cause a significant decrease in glycolysis at the step catalyzed by aldolase, while activating PIK3CA mutations have the opposite effect. These results point towards a master regulatory function of PI3K that integrates an epithelial cell’s metabolism and its form, shape and function, coordinating glycolysis with the energy-intensive dynamics of actin remodeling. PMID:26824656

The Cytoskeleton-Autophagy Connection.

PubMed

Kast, David J; Dominguez, Roberto

2017-04-24

Actin cytoskeleton dynamics play vital roles in most forms of intracellular trafficking by promoting the biogenesis and transport of vesicular cargoes. Mounting evidence indicates that actin dynamics and membrane-cytoskeleton scaffolds also have essential roles in macroautophagy, the process by which cellular waste is isolated inside specialized vesicles called autophagosomes for recycling and degradation. Branched actin polymerization is necessary for the biogenesis of autophagosomes from the endoplasmic reticulum (ER) membrane. Actomyosin-based transport is then used to feed the growing phagophore with pre-selected cargoes and debris derived from different membranous organelles inside the cell. Finally, mature autophagosomes detach from the ER membrane by an as yet unknown mechanism, undergo intracellular transport and then fuse with lysosomes, endosomes and multivesicular bodies through mechanisms that involve actin- and microtubule-mediated motility, cytoskeleton-membrane scaffolds and signaling proteins. In this review, we highlight the considerable progress made recently towards understanding the diverse roles of the cytoskeleton in autophagy. Published by Elsevier Ltd.

The Cytoskeleton-Autophagy Connection

PubMed Central

Kast, David J.; Dominguez, Roberto

2017-01-01

Summary Actin cytoskeleton dynamics plays vital roles in most forms of intracellular trafficking by promoting the biogenesis and transport of vesicular cargoes. Mounting evidence indicates that actin dynamics and membrane-cytoskeleton scaffolds also play essential roles in macroautophagy, the process by which cellular waste is isolated inside specialized vesicles called autophagosomes for recycling and degradation. Thus, branched-actin polymerization is necessary for the biogenesis of autophagosomes from the endoplasmic reticulum (ER) membrane. Actomyosin-based transport is then used to feed the growing phagophore with pre-selected cargoes and debris derived from different membranous organelles inside the cell. Mature autophagosomes then detach from the ER membrane by an unknown mechanism, and are transported and fused with lysosomes, endosomes and multi-vesicular bodies through mechanisms that involve actin- and microtubule-based motility, cytoskeleton-membrane scaffolds and signaling proteins. In this minireview, we highlight the considerable progress made recently towards understanding the diverse roles of the cytoskeleton in autophagy. PMID:28441569

Inter-dependent apical microtubule and actin dynamics orchestrate centrosome retention and neuronal delamination

PubMed Central

Kasioulis, Ioannis

2017-01-01

Detachment of newborn neurons from the neuroepithelium is required for correct neuronal architecture and functional circuitry. This process, also known as delamination, involves adherens-junction disassembly and acto-myosin-mediated abscission, during which the centrosome is retained while apical/ciliary membranes are shed. Cell-biological mechanisms mediating delamination are, however, poorly understood. Using live-tissue and super-resolution imaging, we uncover a centrosome-nucleated wheel-like microtubule configuration, aligned with the apical actin cable and adherens-junctions within chick and mouse neuroepithelial cells. These microtubules maintain adherens-junctions while actin maintains microtubules, adherens-junctions and apical end-foot dimensions. During neuronal delamination, acto-myosin constriction generates a tunnel-like actin-microtubule configuration through which the centrosome translocates. This movement requires inter-dependent actin and microtubule activity, and we identify drebrin as a potential coordinator of these cytoskeletal dynamics. Furthermore, centrosome compromise revealed that this organelle is required for delamination. These findings identify new cytoskeletal configurations and regulatory relationships that orchestrate neuronal delamination and may inform mechanisms underlying pathological epithelial cell detachment. PMID:29058679

Dia-Interacting Protein (DIP) Imposes Migratory Plasticity in mDia2-Dependent Tumor Cells in Three-Dimensional Matrices

PubMed Central

Wyse, Meghan M.; Lei, Jun; Nestor-Kalinoski, Andrea L.; Eisenmann, Kathryn M.

2012-01-01

Tumor cells rely upon membrane pliancy to escape primary lesions and invade secondary metastatic sites. This process relies upon localized assembly and disassembly cycles of F-actin that support and underlie the plasma membrane. Dynamic actin generates both spear-like and bleb structures respectively characterizing mesenchymal and amoeboid motility programs utilized by metastatic cells in three-dimensional matrices. The molecular mechanism and physiological trigger(s) driving membrane plasticity are poorly understood. mDia formins are F-actin assembly factors directing membrane pliancy in motile cells. mDia2 is functionally coupled with its binding partner DIP, regulating cortical actin and inducing membrane blebbing in amoeboid cells. Here we show that mDia2 and DIP co-tether to nascent blebs and this linkage is required for bleb formation. DIP controls mesenchymal/amoeboid cell interconvertability, while CXCL12 induces assembly of mDia2:DIP complexes to bleb cortices in 3D matrices. These results demonstrate how DIP-directed mDia2-dependent F-actin dynamics regulate morphological plasticity in motile cancer cells. PMID:23024796

Long non-coding RNA CRYBG3 blocks cytokinesis by directly binding G-actin.

PubMed

Pei, Hailong; Hu, Wentao; Guo, Ziyang; Chen, Huaiyuan; Ma, Ji; Mao, Weidong; Li, Bingyan; Wang, Aiqing; Wan, Jianmei; Zhang, Jian; Nie, Jing; Zhou, Guangming; Hei, Tom K

2018-06-22

The dynamic interchange between monomeric globular actin (G-actin) and polymeric filamentous actin filaments (F-actin) is fundamental and essential to many cellular processes including cytokinesis and maintenance of genomic stability. Here we report that the long non-coding RNA LNC CRYBG3 directly binds G-actin to inhibit its polymerization and formation of contractile rings, resulting in M-Phase cell arrest. Knockdown of LNC CRYBG3 in tumor cells enhanced their malignant phenotypes. Nucleotide sequence 228-237 of the full-length LNC CRYBG3 and the ser14 domain of beta-actin are essential for their interaction, and mutation of either of these sites abrogated binding of LNC CRYBG3 to G-actin. Binding of LNC CRYBG3 to G-actin blocked nuclear localization of MAL, which consequently kept serum response factor (SRF) away from the promoter region of several immediate early genes, including JUNB and Arp3, which are necessary for cellular proliferation, tumor growth, adhesion, movement, and metastasis. These findings reveal a novel lncRNA-actin-MAL-SRF pathway and highlight LNC CRYBG3 as a means to block cytokinesis and treat cancer by targeting the actin cytoskeleton. Copyright ©2018, American Association for Cancer Research.

Actin cable dynamics in budding yeast

PubMed Central

Yang, Hyeong-Cheol; Pon, Liza A.

2002-01-01

Actin cables, bundles of actin filaments that align along the long axis of budding yeast, are crucial for establishment of cell polarity. We fused green fluorescent protein (GFP) to actin binding protein 140 (Abp140p) and visualized actin cable dynamics in living yeast. We detected two populations of actin cables: (i) bud-associated cables, which extend from the bud along the mother-bud axis, and (ii) randomly oriented cables, which are relatively short. Time-lapse imaging of Abp140p–GFP revealed an apparent increase in the length of bud-associated actin cables. Analysis of movement of Abp140p–GFP fiduciary marks on bud-associated cables and fluorescence loss in photobleaching experiments revealed that this apparent elongation occurs by assembly of new material at the end of the cable within the bud and movement of the opposite end of the cable toward the tip of the mother cell distal to the bud. The rate of extension of the tip of an elongating actin cable is 0.29 ± 0.08 μm/s. Latrunculin A (Lat-A) treatment completely blocked this process. We also observed movement of randomly oriented cables around the cortex of cells at a rate of 0.59 ± 0.14 μm/s. Mild treatment with Lat-A did not affect the velocity of movement of randomly oriented cables. However, Lat-A treatment did increase the number of randomly oriented, motile cables per cell. Our observations suggest that establishment of bud-associated actin cables during the cell cycle is accomplished not by realignment of existing cables but by assembly of new cables within the bud or bud neck, followed by elongation. PMID:11805329

Sensory role of actin in auxin-dependent responses of tobacco BY-2.

PubMed

Huang, Xiang; Maisch, Jan; Nick, Peter

2017-11-01

Polar auxin transport depends on the polar localization of auxin-efflux carriers. The cycling of these carriers between cell interior and plasma membrane depends on actin. The dynamic of actin not only affects auxin transport, but also changes the auxin-responsiveness. To study the potential link between auxin responsiveness and actin dynamics, we investigated developmental responses of the non-transformed BY-2 (Nicotiana tabacum L. cv Bright Yellow 2) cell line and the transgenic BY-2 strain GF11 (stably transformed BY-2 cells with a GFP-fimbrin actin-binding domain 2 construct). The developmental process was divided into three distinct stages: cell cycling, cell elongation and file disintegration. Several phenotypes were measured to monitor the cellular responses to different concentrations of exogenous natural auxin (Indole-3-acetic acid, IAA). We found that auxin stimulated and prolonged the mitotic activity, and delayed the exit from the proliferation phase. However, both responses were suppressed in the GF11 line. At the stationary phase of the cultivation cycle, auxin strongly accelerated the cell file disintegration. Interestingly, it was not suppressed but progressed to a more complete disintegration in the GF11 line. During the cultivation cycle, we also followed the organization of actin in the GF11 line and did not detect any significant difference in actin organization from untreated control or exogenous IAA treatment. Therefore, our findings indicate that the specific differences observed in the GF11 line must be linked with a function of actin that is not structural. It means that there is a sensory role of actin for auxin signaling. Copyright © 2017 Elsevier GmbH. All rights reserved.

Actin dynamics at the living cell submembrane imaged by total internal reflection fluorescence photobleaching.

PubMed Central

Sund, S E; Axelrod, D

2000-01-01

Although reversible chemistry is crucial to dynamical processes in living cells, relatively little is known about relevant chemical kinetic rates in vivo. Total internal reflection/fluorescence recovery after photobleaching (TIR/FRAP), an established technique previously demonstrated to measure reversible biomolecular kinetic rates at surfaces in vitro, is extended here to measure reversible biomolecular kinetic rates of actin at the cytofacial (subplasma membrane) surface of living cells. For the first time, spatial imaging (with a charge-coupled device camera) is used in conjunction with TIR/FRAP. TIR/FRAP imaging produces both spatial maps of kinetic parameters (off-rates and mobile fractions) and estimates of kinetic correlation distances, cell-wide kinetic gradients, and dependences of kinetic parameters on initial fluorescence intensity. For microinjected rhodamine actin in living cultured smooth muscle (BC3H1) cells, the unbinding rate at or near the cytofacial surface of the plasma membrane (averaged over the entire cell) is measured at 0.032 +/- 0.007 s(-1). The corresponding rate for actin marked by microinjected rhodamine phalloidin is very similar, 0.033 +/- 0.013 s(-1), suggesting that TIR/FRAP is reporting the dynamics of entire filaments or protofilaments. For submembrane fluorescence-marked actin, the intensity, off-rate, and mobile fraction show a positive correlation over a characteristic distance of 1-3 microm and a negative correlation over larger distances greater than approximately 7-14 microm. Furthermore, the kinetic parameters display a statistically significant cell-wide gradient, with the cell having a "fast" and "slow" end with respect to actin kinetics. PMID:10969025

Morphogenesis of the mouse neural plate depends on distinct roles of cofilin 1 in apical and basal epithelial domains

PubMed Central

Grego-Bessa, Joaquim; Hildebrand, Jeffrey; Anderson, Kathryn V.

2015-01-01

The genetic control of mammalian epithelial polarity and dynamics can be studied in vivo at cellular resolution during morphogenesis of the mouse neural tube. The mouse neural plate is a simple epithelium that is transformed into a columnar pseudostratified tube over the course of ∼24 h. Apical F-actin is known to be important for neural tube closure, but the precise roles of actin dynamics in the neural epithelium are not known. To determine how the organization of the neural epithelium and neural tube closure are affected when actin dynamics are blocked, we examined the cellular basis of the neural tube closure defect in mouse mutants that lack the actin-severing protein cofilin 1 (CFL1). Although apical localization of the adherens junctions, the Par complex, the Crumbs complex and SHROOM3 is normal in the mutants, CFL1 has at least two distinct functions in the apical and basal domains of the neural plate. Apically, in the absence of CFL1 myosin light chain does not become phosphorylated, indicating that CFL1 is required for the activation of apical actomyosin required for neural tube closure. On the basal side of the neural plate, loss of CFL1 has the opposite effect on myosin: excess F-actin and myosin accumulate and the ectopic myosin light chain is phosphorylated. The basal accumulation of F-actin is associated with the assembly of ectopic basal tight junctions and focal disruptions of the basement membrane, which eventually lead to a breakdown of epithelial organization. PMID:25742799

Profilin as a regulator of the membrane-actin cytoskeleton interface in plant cells

PubMed Central

Sun, Tiantian; Li, Shanwei; Ren, Haiyun

2013-01-01

Membrane structures and cytoskeleton dynamics are intimately inter-connected in the eukaryotic cell. Recently, the molecular mechanisms operating at this interface have been progressively addressed. Many experiments have revealed that the actin cytoskeleton can interact with membranes through various discrete membrane domains. The actin-binding protein, profilin has been proven to inhibit actin polymerization and to promote F-actin elongation. This is dependent on many factors, such as the profilin/G-actin ratio and the ionic environment of the cell. Additionally, profilin has specific domains that interact with phosphoinositides and poly-L-proline rich proteins; theoretically, this gives profilin the opportunity to interact with membranes, and a large number of experiments have confirmed this possibility. In this article, we summarize recent findings in plant cells, and discuss the evidence of the connections among actin cytoskeleton, profilin and biomembranes through direct or indirect relationships. PMID:24391654

Spatial control of actin polymerization during neutrophil chemotaxis

PubMed Central

Weiner, Orion D.; Servant, Guy; Welch, Matthew D.; Mitchison, Timothy J.; Sedat, John W.; Bourne, Henry R.

2010-01-01

Neutrophils respond to chemotactic stimuli by increasing the nucleation and polymerization of actin filaments, but the location and regulation of these processes are not well understood. Here, using a permeabilized-cell assay, we show that chemotactic stimuli cause neutrophils to organize many discrete sites of actin polymerization, the distribution of which is biased by external chemotactic gradients. Furthermore, the Arp2/3 complex, which can nucleate actin polymerization, dynamically redistributes to the region of living neutrophils that receives maximal chemotactic stimulation, and the least-extractable pool of the Arp2/3 complex co-localizes with sites of actin polymerization. Our observations indicate that chemoattractant-stimulated neutrophils may establish discrete foci of actin polymerization that are similar to those generated at the posterior surface of the intracellular bacterium Listeria monocytogenes. We propose that asymmetrical establishment and/or maintenance of sites of actin polymerization produces directional migration of neutrophils in response to chemotactic gradients. PMID:10559877

Spatial control of actin polymerization during neutrophil chemotaxis.

PubMed

Weiner, O D; Servant, G; Welch, M D; Mitchison, T J; Sedat, J W; Bourne, H R

1999-06-01

Neutrophils respond to chemotactic stimuli by increasing the nucleation and polymerization of actin filaments, but the location and regulation of these processes are not well understood. Here, using a permeabilized-cell assay, we show that chemotactic stimuli cause neutrophils to organize many discrete sites of actin polymerization, the distribution of which is biased by external chemotactic gradients. Furthermore, the Arp2/3 complex, which can nucleate actin polymerization, dynamically redistributes to the region of living neutrophils that receives maximal chemotactic stimulation, and the least-extractable pool of the Arp2/3 complex co-localizes with sites of actin polymerization. Our observations indicate that chemoattractant-stimulated neutrophils may establish discrete foci of actin polymerization that are similar to those generated at the posterior surface of the intracellular bacterium Listeria monocytogenes. We propose that asymmetrical establishment and/or maintenance of sites of actin polymerization produces directional migration of neutrophils in response to chemotactic gradients.

Calcium-mediated actin reset (CaAR) mediates acute cell adaptations.

PubMed

Wales, Pauline; Schuberth, Christian E; Aufschnaiter, Roland; Fels, Johannes; García-Aguilar, Ireth; Janning, Annette; Dlugos, Christopher P; Schäfer-Herte, Marco; Klingner, Christoph; Wälte, Mike; Kuhlmann, Julian; Menis, Ekaterina; Hockaday Kang, Laura; Maier, Kerstin C; Hou, Wenya; Russo, Antonella; Higgs, Henry N; Pavenstädt, Hermann; Vogl, Thomas; Roth, Johannes; Qualmann, Britta; Kessels, Michael M; Martin, Dietmar E; Mulder, Bela; Wedlich-Söldner, Roland

2016-12-06

Actin has well established functions in cellular morphogenesis. However, it is not well understood how the various actin assemblies in a cell are kept in a dynamic equilibrium, in particular when cells have to respond to acute signals. Here, we characterize a rapid and transient actin reset in response to increased intracellular calcium levels. Within seconds of calcium influx, the formin INF2 stimulates filament polymerization at the endoplasmic reticulum (ER), while cortical actin is disassembled. The reaction is then reversed within a few minutes. This Calcium-mediated actin reset (CaAR) occurs in a wide range of mammalian cell types and in response to many physiological cues. CaAR leads to transient immobilization of organelles, drives reorganization of actin during cell cortex repair, cell spreading and wound healing, and induces long-lasting changes in gene expression. Our findings suggest that CaAR acts as fundamental facilitator of cellular adaptations in response to acute signals and stress.

Analysis of Actin-Based Intracellular Trafficking in Pollen Tubes.

PubMed

Jiang, Yuxiang; Zhang, Meng; Huang, Shanjin

2017-01-01

Underlying rapid and directional pollen tube growth is the active intracellular trafficking system that carries materials necessary for cell wall synthesis and membrane expansion to the expanding point of the pollen tube. The actin cytoskeleton has been shown to control various intracellular trafficking events in the pollen tube, but the underlying cellular and molecular mechanisms remain poorly understood. To better understand how the actin cytoskeleton is involved in the regulation of intracellular trafficking events, we need to establish assays to visualize and quantify the distribution and dynamics of organelles, vesicles, or secreted proteins. In this chapter, we introduce methods regarding the visualization and quantification of the distribution and dynamics of organelles or vesicles in pollen tubes.

Model of turnover kinetics in the lamellipodium: implications of slow- and fast- diffusing capping protein and Arp2/3 complex

NASA Astrophysics Data System (ADS)

McMillen, Laura M.; Vavylonis, Dimitrios

2016-12-01

Cell protrusion through polymerization of actin filaments at the leading edge of motile cells may be influenced by spatial gradients of diffuse actin and regulators. Here we study the distribution of two of the most important regulators, capping protein and Arp2/3 complex, which regulate actin polymerization in the lamellipodium through capping and nucleation of free barbed ends. We modeled their kinetics using data from prior single molecule microscopy experiments on XTC cells. These experiments have provided evidence for a broad distribution of diffusion coefficients of both capping protein and Arp2/3 complex. The slowly diffusing proteins appear as extended ‘clouds’ while proteins bound to the actin filament network appear as speckles that undergo retrograde flow. Speckle appearance and disappearance events correspond to assembly and dissociation from the actin filament network and speckle lifetimes correspond to the dissociation rate. The slowly diffusing capping protein could represent severed capped actin filament fragments or membrane-bound capping protein. Prior evidence suggests that slowly diffusing Apr2/3 complex associates with the membrane. We use the measured rates and estimates of diffusion coefficients of capping protein and Arp2/3 complex in a Monte Carlo simulation that includes particles in association with a filament network and diffuse in the cytoplasm. We consider two separate pools of diffuse proteins, representing fast and slowly diffusing species. We find a steady state with concentration gradients involving a balance of diffusive flow of fast and slow species with retrograde flow. We show that simulations of FRAP are consistent with prior experiments performed on different cell types. We provide estimates for the ratio of bound to diffuse complexes and calculate conditions where Arp2/3 complex recycling by diffusion may become limiting. We discuss the implications of slowly diffusing populations and suggest experiments to distinguish among mechanisms that influence long range transport.

Curved tails in polymerization-based bacterial motility

NASA Astrophysics Data System (ADS)

Rutenberg, Andrew D.; Grant, Martin

2001-08-01

The curved actin ``comet-tail'' of the bacterium Listeria monocytogenes is a visually striking signature of actin polymerization-based motility. Similar actin tails are associated with Shigella flexneri, spotted-fever Rickettsiae, the Vaccinia virus, and vesicles and microspheres in related in vitro systems. We show that the torque required to produce the curvature in the tail can arise from randomly placed actin filaments pushing the bacterium or particle. We find that the curvature magnitude determines the number of actively pushing filaments, independent of viscosity and of the molecular details of force generation. The variation of the curvature with time can be used to infer the dynamics of actin filaments at the bacterial surface.

Endocytosis-dependent coordination of multiple actin regulators is required for wound healing

PubMed Central

Matsubayashi, Yutaka; Coulson-Gilmer, Camilla

2015-01-01

The ability to heal wounds efficiently is essential for life. After wounding of an epithelium, the cells bordering the wound form dynamic actin protrusions and/or a contractile actomyosin cable, and these actin structures drive wound closure. Despite their importance in wound healing, the molecular mechanisms that regulate the assembly of these actin structures at wound edges are not well understood. In this paper, using Drosophila melanogaster embryos, we demonstrate that Diaphanous, SCAR, and WASp play distinct but overlapping roles in regulating actin assembly during wound healing. Moreover, we show that endocytosis is essential for wound edge actin assembly and wound closure. We identify adherens junctions (AJs) as a key target of endocytosis during wound healing and propose that endocytic remodeling of AJs is required to form “signaling centers” along the wound edge that control actin assembly. We conclude that coordination of actin assembly, AJ remodeling, and membrane traffic is required for the construction of a motile leading edge during wound healing. PMID:26216900

Actin–microtubule coordination at growing microtubule ends

PubMed Central

López, Magdalena Preciado; Huber, Florian; Grigoriev, Ilya; Steinmetz, Michel O.; Akhmanova, Anna; Koenderink, Gijsje H.; Dogterom, Marileen

2014-01-01

To power dynamic processes in cells, the actin and microtubule cytoskeletons organize into complex structures. Although it is known that cytoskeletal coordination is vital for cell function, the mechanisms by which cross-linking proteins coordinate actin and microtubule activities remain poorly understood. In particular, it is unknown how the distinct mechanical properties of different actin architectures modulate the outcome of actin–microtubule interactions. To address this question, we engineered the protein TipAct, which links growing microtubule ends via end-binding proteins to actin filaments. We show that growing microtubules can be captured and guided by stiff actin bundles, leading to global actin–microtubule alignment. Conversely, growing microtubule ends can transport, stretch and bundle individual actin filaments, thereby globally defining actin filament organization. Our results provide a physical basis to understand actin–microtubule cross-talk, and reveal that a simple cross-linker can enable a mechanical feedback between actin and microtubule organization that is relevant to diverse biological contexts. PMID:25159196

Tracking single Kv2.1 channels in live cells reveals anomalous subdiffusion and ergodicity breaking

NASA Astrophysics Data System (ADS)

Weigel, Aubrey; Simon, Blair; Tamkun, Michael; Krapf, Diego

2011-03-01

The dynamic organization of the plasma membrane is responsible for essential cellular processes, such as receptor trafficking and signaling. By studying the dynamics of transmembrane proteins a greater understanding of these processes as a whole can be achieved. It is broadly observed that the diffusion pattern of membrane protein displays anomalous subdiffusion. However, the mechanisms responsible for this behavior are not yet established. We explore the dynamics of the voltage gated potassium channel Kv2.1 by using single-particle tracking. We analyze Kv2.1 channel trajectories in terms of the time and ensemble distributions of square displacements. Our results reveal that all Kv2.1 channels experience anomalous subdiffusion and we observe that the Kv2.1 diffusion pattern is non-ergodic. We further investigated the role of the actin cytoskeleton in these channel dynamics by applying actin depolymerizing drugs. It is seen that with the breakdown of the actin cytoskeleton the Kv2.1 channel trajectories recover ergodicity.

Action and Traction: Cytoskeletal Control of Receptor Triggering at the Immunological Synapse

PubMed Central

Comrie, William A.; Burkhardt, Janis K.

2016-01-01

It is well known that F-actin dynamics drive the micron-scale cell shape changes required for migration and immunological synapse (IS) formation. In addition, recent evidence points to a more intimate role for the actin cytoskeleton in promoting T cell activation. Mechanotransduction, the conversion of mechanical input into intracellular biochemical changes, is thought to play a critical role in several aspects of immunoreceptor triggering and downstream signal transduction. Multiple molecules associated with signaling events at the IS have been shown to respond to physical force, including the TCR, costimulatory molecules, adhesion molecules, and several downstream adapters. In at least some cases, it is clear that the relevant forces are exerted by dynamics of the T cell actomyosin cytoskeleton. Interestingly, there is evidence that the cytoskeleton of the antigen-presenting cell also plays an active role in T cell activation, by countering the molecular forces exerted by the T cell at the IS. Since actin polymerization is itself driven by TCR and costimulatory signaling pathways, a complex relationship exists between actin dynamics and receptor activation. This review will focus on recent advances in our understanding of the mechanosensitive aspects of T cell activation, paying specific attention to how F-actin-directed forces applied from both sides of the IS fit into current models of receptor triggering and activation. PMID:27014258

CD94/NKG2A inhibits NK cell activation by disrupting the actin network at the immunological synapse.

PubMed

Masilamani, Madhan; Nguyen, Connie; Kabat, Juraj; Borrego, Francisco; Coligan, John E

2006-09-15

An adequate immune response is the result of the fine balance between activation and inhibitory signals. The exact means by which inhibitory signals obviate activation signals in immune cells are not totally elucidated. Human CD94/NKG2A is an ITIM-containing inhibitory receptor expressed by NK cells and some CD8+ T cells that recognize HLA-E. We show that the engagement of this receptor prevents NK cell activation by disruption of the actin network and exclusion of lipid rafts at the point of contact with its ligand (inhibitory NK cell immunological synapse, iNKIS). CD94/NKG2A engagement leads to recruitment and activation of src homology 2 domain-bearing tyrosine phosphatase 1. This likely explains the observed dephosphorylation of guanine nucleotide exchange factor and regulator of actin, Vav1, as well as ezrin-radixin-moesin proteins that connect actin filaments to membrane structures. In contrast, NK cell activation by NKG2D induced Vav1 and ezrin-radixin-moesin phosphorylation. Thus, CD94/NKG2A prevents actin-dependent recruitment of raft-associated activation receptors complexes to the activating synapse. This was further substantiated by showing that inhibition of actin polymerization abolished lipid rafts exclusion at the iNKIS, whereas cholesterol depletion had no effect on actin disruption at the iNKIS. These data indicate that the lipid rafts exclusion at the iNKIS is an active process which requires an intact cytoskeleton to maintain lipid rafts outside the inhibitory synapse. The net effect is to maintain an inhibitory state in the proximity of the iNKIS, while allowing the formation of activation synapse at distal points within the same NK cell.

Arabidopsis ACTIN-DEPOLYMERIZING FACTOR3 Is Required for Controlling Aphid Feeding from the Phloem1[OPEN

PubMed Central

Mondal, Hossain A.; Louis, Joe; Archer, Lani; Patel, Monika; Nalam, Vamsi J.; Sarowar, Sujon; Sivapalan, Vishala

2018-01-01

The actin cytoskeleton network has an important role in plant cell growth, division, and stress response. Actin-depolymerizing factors (ADFs) are a group of actin-binding proteins that contribute to reorganization of the actin network. Here, we show that the Arabidopsis (Arabidopsis thaliana) ADF3 is required in the phloem for controlling infestation by Myzus persicae Sülzer, commonly known as the green peach aphid (GPA), which is an important phloem sap-consuming pest of more than fifty plant families. In agreement with a role for the actin-depolymerizing function of ADF3 in defense against the GPA, we show that resistance in adf3 was restored by overexpression of the related ADF4 and the actin cytoskeleton destabilizers, cytochalasin D and latrunculin B. Electrical monitoring of the GPA feeding behavior indicates that the GPA stylets found sieve elements faster when feeding on the adf3 mutant compared to the wild-type plant. In addition, once they found the sieve elements, the GPA fed for a more prolonged period from sieve elements of adf3 compared to the wild-type plant. The longer feeding period correlated with an increase in fecundity and population size of the GPA and a parallel reduction in callose deposition in the adf3 mutant. The adf3-conferred susceptibility to GPA was overcome by expression of the ADF3 coding sequence from the phloem-specific SUC2 promoter, thus confirming the importance of ADF3 function in the phloem. We further demonstrate that the ADF3-dependent defense mechanism is linked to the transcriptional up-regulation of PHYTOALEXIN-DEFICIENT4, which is an important regulator of defenses against the GPA. PMID:29133373

F-actin distribution at nodes of Ranvier and Schmidt-Lanterman incisures in mammalian sciatic nerves.

PubMed

Kun, Alejandra; Canclini, Lucía; Rosso, Gonzalo; Bresque, Mariana; Romeo, Carlos; Hanusz, Alicia; Cal, Karina; Calliari, Aldo; Sotelo Silveira, José; Sotelo, José R

2012-07-01

Very little is known about the function of the F-actin cytoskeleton in the regeneration and pathology of peripheral nerve fibers. The actin cytoskeleton has been associated with maintenance of tissue structure, transmission of traction and contraction forces, and an involvement in cell motility. Therefore, the state of the actin cytoskeleton strongly influences the mechanical properties of cells and intracellular transport therein. In this work, we analyze the distribution of F-actin at Schmidt-Lanterman Incisures (SLI) and nodes of Ranvier (NR) domains in normal, regenerating and pathologic Trembler J (TrJ/+) sciatic nerve fibers, of rats and mice. F-actin was quantified and it was found increased in TrJ/+, both in SLI and NR. However, SLI and NR of regenerating rat sciatic nerve did not show significant differences in F-actin, as compared with normal nerves. Cytochalasin-D and Latrunculin-A were used to disrupt the F-actin network in normal and regenerating rat sciatic nerve fibers. Both drugs disrupt F-actin, but in different ways. Cytochalasin-D did not disrupt Schwann cell (SC) F-actin at the NR. Latrunculin-A did not disrupt F-actin at the boundary region between SC and axon at the NR domain. We surmise that the rearrangement of F-actin in neurological disorders, as presented here, is an important feature of TrJ/+ pathology as a Charcot-Marie-Tooth (CMT) model. Copyright © 2012 Wiley Periodicals, Inc.

Spatiotemporal Patterns of Noise-Driven Confined Actin Waves in Living Cells.

PubMed

Bernitt, Erik; Döbereiner, Hans-Günther

2017-01-27

Cells utilize waves of polymerizing actin to reshape their morphologies, which is central to physiological and pathological processes alike. Here, we force dorsal actin waves to propagate on one-dimensional domains with periodic boundary conditions, which results in striking spatiotemporal patterns with a clear signature of noise-driven dynamics. We show that these patterns can be very closely reproduced with a noise-driven active medium at coherence resonance.

Hypergravity Loading the Cultured Osteoblasts: Modeling and Experimental Analysis of Cellular Morphology and the Cytoskeleton

NASA Technical Reports Server (NTRS)

Searby, N. D.; Steele, C. R.; Globus, R. K.; Dalton, Bonnie P. (Technical Monitor)

2001-01-01

Bone forming cells, osteoblasts, respond to various mechanical forces, including mechanical strain and fluid-induced shear stress. This study examined whether osteoblasts detect changes in gravity as a mechanical force, as assessed by cellular morphology and dimensions of the cytoskeletal network. We used modeling to evaluate how gravity influences cell morphology given theoretical differences in densities between the surrounding medium, cytoplasm, and nucleus. A mechanical model was built based on analysis of axisymmetric shell structures (Fast4 software) to study the effects of 10 times gravity (10G) on cell height. The model indicated 0.02% decrease in overall cell height when the medium was 10% denser than the nucleus or cytoplasm, 5.9 x 10(exp-5)% decrease when the nucleus was 10% denser than the cytoplasm or medium, and 1.3 x 10(exp-5)% decrease when the cell cytoplasm was 10% denser than the nucleus or medium. To experimentally evaluate the influence of gravity, cultured primary fetal rat osteoblasts were grown to near confluence and centrifuged at 10G for 3 hours. Actin, microtubules, and nuclei were fluorescently labeled and analyzed by confocal microscopy to determine overall microtubule and actin network height. Centrifugation led to an apparent reduction in height of both the microtubule (-16%) and the actin (-20%) networks relative to stationary controls. Thus, both modeling and experiments indicate that hypergravity reduces the height of the osteoblast cell layer and their microtubule and actin networks. This combination of modeling and experimental analyses will help us to better understand the mechanical loading of osteoblasts.

WASP and SCAR are evolutionarily conserved in actin-filled pseudopod-based motility

PubMed Central

2017-01-01

Diverse eukaryotic cells crawl through complex environments using distinct modes of migration. To understand the underlying mechanisms and their evolutionary relationships, we must define each mode and identify its phenotypic and molecular markers. In this study, we focus on a widely dispersed migration mode characterized by dynamic actin-filled pseudopods that we call “α-motility.” Mining genomic data reveals a clear trend: only organisms with both WASP and SCAR/WAVE—activators of branched actin assembly—make actin-filled pseudopods. Although SCAR has been shown to drive pseudopod formation, WASP’s role in this process is controversial. We hypothesize that these genes collectively represent a genetic signature of α-motility because both are used for pseudopod formation. WASP depletion from human neutrophils confirms that both proteins are involved in explosive actin polymerization, pseudopod formation, and cell migration. WASP and WAVE also colocalize to dynamic signaling structures. Moreover, retention of WASP together with SCAR correctly predicts α-motility in disease-causing chytrid fungi, which we show crawl at >30 µm/min with actin-filled pseudopods. By focusing on one migration mode in many eukaryotes, we identify a genetic marker of pseudopod formation, the morphological feature of α-motility, providing evidence for a widely distributed mode of cell crawling with a single evolutionary origin. PMID:28473602

Xenopus extract approaches to studying microtubule organization and signaling in cytokinesis

PubMed Central

Field, Christine M.; Pelletier, James F.; Mitchison, Timothy J.

2017-01-01

We report optimized methods for preparing actin-intact Xenopus egg extract. This extract is minimally perturbed, undiluted egg cytoplasm where the cell cycle can be experimentally controlled. It contains abundant organelles and glycogen, and supports active metabolism and cytoskeletal dynamics that closely mimic egg physiology. The concentration of the most abundant ~11,000 proteins is known from mass spectrometry. Actin-intact egg extract can be used for analysis of actin dynamics and interaction of actin with other cytoplasmic systems, as well as microtubule organization. It can be spread as thin layers, and naturally depletes oxygen though mitochondrial metabolism, which makes it ideal for fluorescence imaging. When combined with artificial lipid bilayers, it allows reconstitution and analysis of the spatially controlled signaling that positions the cleavage furrow during early cytokinesis. Actin-intact extract is generally useful for probing the biochemistry and biophysics of the large Xenopus egg. Protocols are provided for preparation of actin-intact egg extract, control of the cell cycle, fluorescent probes for cytoskeleton and cytoskeleton-dependent signaling, preparation of glass surfaces for imaging experiments, and immunodepletion to probe the role of specific proteins and protein complexes. We also describe methods for adding supported lipid bilayers to mimic the plasma membrane and for confining in microfluidic droplets to explore size scaling issues. PMID:28065319