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.

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

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.

RAI14 (retinoic acid induced protein 14) is an F-actin regulator

PubMed Central

Qian, Xiaojing; Mruk, Dolores D.; Cheng, Yan-ho; Cheng, C. Yan

2013-01-01

RAI14 (retinoic acid induced protein 14) is an actin-binding protein first identified in the liver. In the testis, RAI14 is expressed by both Sertoli and germ cells in the seminiferous epithelium. Besides binding to actin in the testis, RAI14 is also a binding protein for palladin, an actin cross-linking and bundling protein. A recent report has shown that RAI14 displays stage-specific and spatiotemporal expression at the ES [ectoplasmic specialization, a testis-specific filamentous (F)-actin-rich adherens junction] in the seminiferous epithelium of adult rat testes during the epithelial cycle of spermatogenesis, illustrating its likely involvement in F-actin organization at the ES. Functional studies in which RAI14 was knocked down by RNAi in Sertoli cells in vitro and also in testicular cells in vivo have illustrated its role in conferring the integrity of actin filament bundles at the ES, perturbing the Sertoli cell tight junction (TJ)-pemeability barrier function in vitro, and also spermatid polarity and adhesion in vivo, thereby regulating spermatid transport at spermiation. Herein, we critically evaluate these earlier findings and also provide a likely hypothetic model based on the functional role of RAI14 at the ES, and how RAI14 is working with palladin and other actin regulatory proteins in the testis to regulate the transport of (1) spermatids and (2) preleptotene spermatocytes across the seminiferous epithelium and the blood-testis barrier (BTB), respectively, during spermatogenesis. This model should serve as a framework upon which functional experiments can be designed to better understand the biology of RAI14 and other actin-binding and regulatory proteins in the testis. PMID:23885305

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.

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.

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

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.

ER sheet persistence is coupled to myosin 1c-regulated dynamic actin filament arrays.

PubMed

Joensuu, Merja; Belevich, Ilya; Rämö, Olli; Nevzorov, Ilya; Vihinen, Helena; Puhka, Maija; Witkos, Tomasz M; Lowe, Martin; Vartiainen, Maria K; Jokitalo, Eija

2014-04-01

The endoplasmic reticulum (ER) comprises a dynamic three-dimensional (3D) network with diverse structural and functional domains. Proper ER operation requires an intricate balance within and between dynamics, morphology, and functions, but how these processes are coupled in cells has been unclear. Using live-cell imaging and 3D electron microscopy, we identify a specific subset of actin filaments localizing to polygons defined by ER sheets and tubules and describe a role for these actin arrays in ER sheet persistence and, thereby, in maintenance of the characteristic network architecture by showing that actin depolymerization leads to increased sheet fluctuation and transformations and results in small and less abundant sheet remnants and a defective ER network distribution. Furthermore, we identify myosin 1c localizing to the ER-associated actin filament arrays and reveal a novel role for myosin 1c in regulating these actin structures, as myosin 1c manipulations lead to loss of the actin filaments and to similar ER phenotype as observed after actin depolymerization. We propose that ER-associated actin filaments have a role in ER sheet persistence regulation and thus support the maintenance of sheets as a stationary subdomain of the dynamic ER network.

An atomic model of the tropomyosin cable on F-actin.

PubMed

Orzechowski, Marek; Li, Xiaochuan Edward; Fischer, Stefan; Lehman, William

2014-08-05

Tropomyosin regulates a wide variety of actin filament functions and is best known for the role that it plays together with troponin in controlling muscle activity. For effective performance on actin filaments, adjacent 42-nm-long tropomyosin molecules are joined together by a 9- to 10-residue head-to-tail overlapping domain to form a continuous cable that wraps around the F-actin helix. Yet, despite the apparent simplicity of tropomyosin's coiled-coil structure and its well-known periodic association with successive actin subunits along F-actin, the structure of the tropomyosin cable on actin is uncertain. This is because the conformation of the overlap region that joins neighboring molecules is poorly understood, thus leaving a significant gap in our understanding of thin-filament structure and regulation. However, recent molecular-dynamics simulations of overlap segments defined their overall shape and provided unique and sufficient cues to model the whole actin-tropomyosin filament assembly in atomic detail. In this study, we show that these MD structures merge seamlessly onto the ends of tropomyosin coiled-coils. Adjacent tropomyosin molecules can then be joined together to provide a comprehensive model of the tropomyosin cable running continuously on F-actin. The resulting complete model presented here describes for the first time (to our knowledge) an atomic-level structure of αα-striated muscle tropomyosin bound to an actin filament that includes the critical overlap domain. Thus, the model provides a structural correlate to evaluate thin-filament mechanics, self-assembly mechanisms, and the effect of disease-causing mutations. Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.

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.

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

ER sheet persistence is coupled to myosin 1c–regulated dynamic actin filament arrays

PubMed Central

Joensuu, Merja; Belevich, Ilya; Rämö, Olli; Nevzorov, Ilya; Vihinen, Helena; Puhka, Maija; Witkos, Tomasz M.; Lowe, Martin; Vartiainen, Maria K.; Jokitalo, Eija

2014-01-01

The endoplasmic reticulum (ER) comprises a dynamic three-dimensional (3D) network with diverse structural and functional domains. Proper ER operation requires an intricate balance within and between dynamics, morphology, and functions, but how these processes are coupled in cells has been unclear. Using live-cell imaging and 3D electron microscopy, we identify a specific subset of actin filaments localizing to polygons defined by ER sheets and tubules and describe a role for these actin arrays in ER sheet persistence and, thereby, in maintenance of the characteristic network architecture by showing that actin depolymerization leads to increased sheet fluctuation and transformations and results in small and less abundant sheet remnants and a defective ER network distribution. Furthermore, we identify myosin 1c localizing to the ER-associated actin filament arrays and reveal a novel role for myosin 1c in regulating these actin structures, as myosin 1c manipulations lead to loss of the actin filaments and to similar ER phenotype as observed after actin depolymerization. We propose that ER-associated actin filaments have a role in ER sheet persistence regulation and thus support the maintenance of sheets as a stationary subdomain of the dynamic ER network. PMID:24523293

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

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.

Structural dynamics of F-actin: I. Changes in the C terminus.

PubMed

Orlova, A; Egelman, E H

1995-02-03

The biochemical properties of G-actin, and the kinetics of polymerization of G-actin into F-actin, are dependent upon whether Mg2+ or Ca2+ is bound at the high-affinity metal-binding site in actin. Three-dimensional reconstructions from electron micrographs show that a bridge of density, that we interpret as arising from a major shift of the C terminus, exists between the two strands of the filament in Ca(2+)-actin that is absent in Mg(2+)-actin. This bridge is also absent in models of F-actin built from an atomic structure of G-Ca(2+)-actin. The cleavage of the DNase I-binding loop in actin between residues 42 and 43, with the non-covalent association of the 42 cleaved residues with the remainder of the actin, induces an even larger bridge of density between the two strands. When the bridge is absent, the two C-terminal residues in F-actin are easily cleaved by trypsin, while these residues become increasingly resistant to tryptic cleavage as the bridge becomes more prominent. Conversely, cleavage of the two C-terminal residues leads to a conformational change in the DNase I-binding loop. Since both the DNase I-binding loop and the metal-binding site are quite distant from the C terminus, large allosteric effects must exist in F-actin. The conformational change in F-actin that results from the creation of this bridge may be induced by myosin binding, since this movement generates changes in actin's diffraction that are very similar to the changes in the muscle X-ray pattern during activation that are associated with the binding of myosin to the thin filament.

Cell Elasticity Is Regulated by the Tropomyosin Isoform Composition of the Actin Cytoskeleton

PubMed Central

Jalilian, Iman; Heu, Celine; Cheng, Hong; Freittag, Hannah; Desouza, Melissa; Stehn, Justine R.; Bryce, Nicole S.; Whan, Renee M.; Hardeman, Edna C.

2015-01-01

The actin cytoskeleton is the primary polymer system within cells responsible for regulating cellular stiffness. While various actin binding proteins regulate the organization and dynamics of the actin cytoskeleton, the proteins responsible for regulating the mechanical properties of cells are still not fully understood. In the present study, we have addressed the significance of the actin associated protein, tropomyosin (Tpm), in influencing the mechanical properties of cells. Tpms belong to a multi-gene family that form a co-polymer with actin filaments and differentially regulate actin filament stability, function and organization. Tpm isoform expression is highly regulated and together with the ability to sort to specific intracellular sites, result in the generation of distinct Tpm isoform-containing actin filament populations. Nanomechanical measurements conducted with an Atomic Force Microscope using indentation in Peak Force Tapping in indentation/ramping mode, demonstrated that Tpm impacts on cell stiffness and the observed effect occurred in a Tpm isoform-specific manner. Quantitative analysis of the cellular filamentous actin (F-actin) pool conducted both biochemically and with the use of a linear detection algorithm to evaluate actin structures revealed that an altered F-actin pool does not absolutely predict changes in cell stiffness. Inhibition of non-muscle myosin II revealed that intracellular tension generated by myosin II is required for the observed increase in cell stiffness. Lastly, we show that the observed increase in cell stiffness is partially recapitulated in vivo as detected in epididymal fat pads isolated from a Tpm3.1 transgenic mouse line. Together these data are consistent with a role for Tpm in regulating cell stiffness via the generation of specific populations of Tpm isoform-containing actin filaments. PMID:25978408

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

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

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.

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

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 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

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

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

Leishmania infection inhibits macrophage motility by altering F-actin dynamics and the expression of adhesion complex proteins

PubMed Central

de Menezes, Juliana Perrone Bezerra; Koushik, Amrita; Das, Satarupa; Guven, Can; Siegel, Ariel; Laranjeira-Silva, Maria Fernanda; Losert, Wolfgang; Andrews, Norma W.

2016-01-01

Leishmania is an intracellular protozoan parasite that causes a broad spectrum of clinical manifestations, ranging from self-healing skin lesions to fatal visceralizing disease. As the host cells of choice for all species of Leishmania, macrophages are critical for the establishment of infections. How macrophages contribute to parasite homing to specific tissues and how parasites modulate macrophage function is still poorly understood. In this study we show that L. amazonensis infection inhibits macrophage roaming motility. The reduction in macrophage speed is not dependent on particle load or on factors released by infected macrophages. L. amazonensis-infected macrophages also show reduced directional migration in response to the chemokine MCP-1. We found that infected macrophages have lower levels of total paxillin, phosphorylated paxillin and phosphorylated FAK when compared to non-infected macrophages, indicating abnormalities in the formation of signaling adhesion complexes that regulate motility. Analysis of the dynamics of actin polymerization at peripheral sites also revealed a markedly enhanced F-actin turnover frequency in L. amazonensis-infected macrophages. Thus, Leishmania infection inhibits macrophage motility by altering actin dynamics and impairing the expression of proteins that function in plasma membrane-extracellular matrix interactions. PMID:27641840

Role of gelsolin interaction with actin in regulation and creation of actin nuclei in chemotactic peptide activated polymorphonuclear neutrophils.

PubMed Central

Deaton, J D; Guerrero, T; Howard, T H

1992-01-01

In vitro Ca++ activates gelsolin to sever F-actin and form a gelsolin-actin (GA) complex at the+end of F-actin that is not dissociated by ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) but is separated by EGTA+PIP/PIP2. The gelsolin blocks the+end on the actin filament, but the-end of the filament can still initiate actin polymerization. In thrombin activated platelets, evidence suggests that severing of F-actin by gelsolin increases GA complex, creates one-end actin nucleus and one cryptic+end actin nucleus per cut, and then dissociates to yield free+ends to nucleate rapid actin assembly. We examined the role of F-actin severing in creation and regulation of nuclei and polymerization in polymorphonuclear neutrophils (PMNs). At 2-s intervals after formyl peptide (FMLP) activation of endotoxin free (ETF) PMNs, change in GA complex was correlated with change in+end actin nuclei,-end actin nuclei, and F-actin content. GA complex was quantitated by electrophoretograms of proteins absorbed by antigelsolin from cells lysed in 10 mM EGTA,+end actin nuclei as cytochalasin (CD) sensitive and-end actin nuclei as CD insensitive increases in G-pyrenyl actin polymerization rates induced by the same PMNs, and F-actin content by NBDphallacidin binding to fixed cells. Thirty three percent of gelsolin was in GA complex in basal ETF PMNs; from 2-6 s, GA complexes dissociate (low = 15% at 10 s) and sequentially+end nuclei and F-actin content and then-end nuclei increase to a maximum at 10 s. At > s GA complex increase toward basal and + end nuclei and F-actin content returned toward basal. These kinetic data show gelsolin regulates availability of + end nuclei and actin polymerization in FMLP. However, absence of an initial increase in GA complex or - end nucleating activity shows FMLP activation does not cause gelsolin to sever F- or to bind G-actin to create cryptic + end nuclei in PMNs; the results suggest the + nucleus formation is gelsolin

Role of gelsolin interaction with actin in regulation and creation of actin nuclei in chemotactic peptide activated polymorphonuclear neutrophils.

PubMed

Deaton, J D; Guerrero, T; Howard, T H

1992-12-01

In vitro Ca++ activates gelsolin to sever F-actin and form a gelsolin-actin (GA) complex at the+end of F-actin that is not dissociated by ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) but is separated by EGTA+PIP/PIP2. The gelsolin blocks the+end on the actin filament, but the-end of the filament can still initiate actin polymerization. In thrombin activated platelets, evidence suggests that severing of F-actin by gelsolin increases GA complex, creates one-end actin nucleus and one cryptic+end actin nucleus per cut, and then dissociates to yield free+ends to nucleate rapid actin assembly. We examined the role of F-actin severing in creation and regulation of nuclei and polymerization in polymorphonuclear neutrophils (PMNs). At 2-s intervals after formyl peptide (FMLP) activation of endotoxin free (ETF) PMNs, change in GA complex was correlated with change in+end actin nuclei,-end actin nuclei, and F-actin content. GA complex was quantitated by electrophoretograms of proteins absorbed by antigelsolin from cells lysed in 10 mM EGTA,+end actin nuclei as cytochalasin (CD) sensitive and-end actin nuclei as CD insensitive increases in G-pyrenyl actin polymerization rates induced by the same PMNs, and F-actin content by NBDphallacidin binding to fixed cells. Thirty three percent of gelsolin was in GA complex in basal ETF PMNs; from 2-6 s, GA complexes dissociate (low = 15% at 10 s) and sequentially+end nuclei and F-actin content and then-end nuclei increase to a maximum at 10 s. At > s GA complex increase toward basal and + end nuclei and F-actin content returned toward basal. These kinetic data show gelsolin regulates availability of + end nuclei and actin polymerization in FMLP. However, absence of an initial increase in GA complex or - end nucleating activity shows FMLP activation does not cause gelsolin to sever F- or to bind G-actin to create cryptic + end nuclei in PMNs; the results suggest the + nucleus formation is gelsolin

Actin-binding proteins sensitively mediate F-actin bundle stiffness

NASA Astrophysics Data System (ADS)

Claessens, Mireille M. A. E.; Bathe, Mark; Frey, Erwin; Bausch, Andreas R.

2006-09-01

Bundles of filamentous actin (F-actin) form primary structural components of a broad range of cytoskeletal processes including filopodia, sensory hair cell bristles and microvilli. Actin-binding proteins (ABPs) allow the cell to tailor the dimensions and mechanical properties of the bundles to suit specific biological functions. Therefore, it is important to obtain quantitative knowledge on the effect of ABPs on the mechanical properties of F-actin bundles. Here we measure the bending stiffness of F-actin bundles crosslinked by three ABPs that are ubiquitous in eukaryotes. We observe distinct regimes of bundle bending stiffness that differ by orders of magnitude depending on ABP type, concentration and bundle size. The behaviour observed experimentally is reproduced quantitatively by a molecular-based mechanical model in which ABP shearing competes with F-actin extension/compression. Our results shed new light on the biomechanical function of ABPs and demonstrate how single-molecule properties determine mesoscopic behaviour. The bending mechanics of F-actin fibre bundles are general and have implications for cytoskeletal mechanics and for the rational design of functional materials.

The nuclear F-actin interactome of Xenopus oocytes reveals an actin-bundling kinesin that is essential for meiotic cytokinesis

PubMed Central

Samwer, Matthias; Dehne, Heinz-Jürgen; Spira, Felix; Kollmar, Martin; Gerlich, Daniel W; Urlaub, Henning; Görlich, Dirk

2013-01-01

Nuclei of Xenopus laevis oocytes grow 100 000-fold larger in volume than a typical somatic nucleus and require an unusual intranuclear F-actin scaffold for mechanical stability. We now developed a method for mapping F-actin interactomes and identified a comprehensive set of F-actin binders from the oocyte nuclei. Unexpectedly, the most prominent interactor was a novel kinesin termed NabKin (Nuclear and meiotic actin-bundling Kinesin). NabKin not only binds microtubules but also F-actin structures, such as the intranuclear actin bundles in prophase and the contractile actomyosin ring during cytokinesis. The interaction between NabKin and F-actin is negatively regulated by Importin-β and is responsive to spatial information provided by RanGTP. Disconnecting NabKin from F-actin during meiosis caused cytokinesis failure and egg polyploidy. We also found actin-bundling activity in Nabkin's somatic paralogue KIF14, which was previously shown to be essential for somatic cell division. Our data are consistent with the notion that NabKin/KIF14 directly link microtubules with F-actin and that such link is essential for cytokinesis. PMID:23727888

Triggering signaling pathways using F-actin self-organization.

PubMed

Colin, A; Bonnemay, L; Gayrard, C; Gautier, J; Gueroui, Z

2016-10-04

The spatiotemporal organization of proteins within cells is essential for cell fate behavior. Although it is known that the cytoskeleton is vital for numerous cellular functions, it remains unclear how cytoskeletal activity can shape and control signaling pathways in space and time throughout the cell cytoplasm. Here we show that F-actin self-organization can trigger signaling pathways by engineering two novel properties of the microfilament self-organization: (1) the confinement of signaling proteins and (2) their scaffolding along actin polymers. Using in vitro reconstitutions of cellular functions, we found that both the confinement of nanoparticle-based signaling platforms powered by F-actin contractility and the scaffolding of engineered signaling proteins along actin microfilaments can drive a signaling switch. Using Ran-dependent microtubule nucleation, we found that F-actin dynamics promotes the robust assembly of microtubules. Our in vitro assay is a first step towards the development of novel bottom-up strategies to decipher the interplay between cytoskeleton spatial organization and signaling pathway activity.

Triggering signaling pathways using F-actin self-organization

PubMed Central

Colin, A.; Bonnemay, L.; Gayrard, C.; Gautier, J.; Gueroui, Z.

2016-01-01

The spatiotemporal organization of proteins within cells is essential for cell fate behavior. Although it is known that the cytoskeleton is vital for numerous cellular functions, it remains unclear how cytoskeletal activity can shape and control signaling pathways in space and time throughout the cell cytoplasm. Here we show that F-actin self-organization can trigger signaling pathways by engineering two novel properties of the microfilament self-organization: (1) the confinement of signaling proteins and (2) their scaffolding along actin polymers. Using in vitro reconstitutions of cellular functions, we found that both the confinement of nanoparticle-based signaling platforms powered by F-actin contractility and the scaffolding of engineered signaling proteins along actin microfilaments can drive a signaling switch. Using Ran-dependent microtubule nucleation, we found that F-actin dynamics promotes the robust assembly of microtubules. Our in vitro assay is a first step towards the development of novel bottom-up strategies to decipher the interplay between cytoskeleton spatial organization and signaling pathway activity. PMID:27698406

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.

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

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.

Leishmania infection inhibits macrophage motility by altering F-actin dynamics and the expression of adhesion complex proteins.

PubMed

de Menezes, Juliana Perrone Bezerra; Koushik, Amrita; Das, Satarupa; Guven, Can; Siegel, Ariel; Laranjeira-Silva, Maria Fernanda; Losert, Wolfgang; Andrews, Norma W

2017-03-01

Leishmania is an intracellular protozoan parasite that causes a broad spectrum of clinical manifestations, ranging from self-healing skin lesions to fatal visceralizing disease. As the host cells of choice for all species of Leishmania, macrophages are critical for the establishment of infections. How macrophages contribute to parasite homing to specific tissues and how parasites modulate macrophage function are still poorly understood. In this study, we show that Leishmania amazonensis infection inhibits macrophage roaming motility. The reduction in macrophage speed is not dependent on particle load or on factors released by infected macrophages. L. amazonensis-infected macrophages also show reduced directional migration in response to the chemokine MCP-1. We found that infected macrophages have lower levels of total paxillin, phosphorylated paxillin, and phosphorylated focal adhesion kinase when compared to noninfected macrophages, indicating abnormalities in the formation of signaling adhesion complexes that regulate motility. Analysis of the dynamics of actin polymerization at peripheral sites also revealed a markedly enhanced F-actin turnover frequency in L. amazonensis-infected macrophages. Thus, Leishmania infection inhibits macrophage motility by altering actin dynamics and impairing the expression of proteins that function in plasma membrane-extracellular matrix interactions. © 2016 John Wiley & Sons Ltd.

An F-actin-depleted zone is present at the hyphal tip of invasive hyphae of Neurospora crassa.

PubMed

Suei, S; Garrill, A

2008-01-01

The distribution of filamentous actin (F-actin) in invasive and noninvasive hyphae of the ascomycete Neurospora crassa was investigated. Eighty six percent of noninvasive hyphae had F-actin in the tip region compared to only 9% of invasive hyphae. The remaining 91% of the invasive hyphae had no obvious tip high concentration of F-actin staining; instead they had an F-actin-depleted zone in this region, although some F-actin, possibly associated with the Spitzenkörper, remained at the tip. The size of the F-actin-depleted zone in invasive hyphae increased with an increase in agar concentration. The membrane stain FM 4-64 reveals a slightly larger accumulation of vesicles at the tips of invasive hyphae relative to noninvasive hyphae, although this difference is unlikely to be sufficient to account for the exclusion of F-actin from the depleted zone. Antibodies raised against the actin filament-severing protein cofilin from both yeast and human cells localize to the tips of invasive hyphae. The human cofilin antibody shows a more random distribution in noninvasive hyphae locating primarily at the hyphal periphery but with some diffuse cytoplasmic staining. This antibody also identifies a single band at 21 kDa in immunoblots of whole hyphal fractions. These data suggest that a protein with epitopic similarity to cofilin may function in F-actin dynamics that underlie invasive growth. The F-actin-depleted zone may play a role in the regulation of tip yielding to turgor pressure, thus increasing the protrusive force necessary for invasive growth.

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

The role of F-actin in the transport and secretion of chromaffin granules: an historic perspective.

PubMed

Gutiérrez, Luis M; Villanueva, José

2018-01-01

Actin is one of the most ubiquitous protein playing fundamental roles in a variety of cellular processes. Since early in the 1980s, it was evident that filamentous actin (F-actin) formed a peripheral cortical barrier that prevented vesicles to access secretory sites in chromaffin cells in culture. Later, around 2000, it was described that the F-actin structure accomplishes a dual role serving both vesicle transport and retentive purposes and undergoing dynamic transient changes during cell stimulation. The complex role of the F-actin cytoskeleton in neuroendocrine secretion was further evidenced when it has been proved to participate in the scaffold structure holding together the secretory machinery at active sites and participate in the generation of mechanical forces that drive the opening of the fusion pore, during the first decade of the present century. The complex vision of the multiple roles of F-actin in secretion we have acquired to date comes largely from studies performed on traditional 2D cultures of primary cells; however, recent evidences suggest that these may not accurately mimic the 3D in vivo environment, and thus, more work is now needed on adrenomedullary cells kept in a more "native" configuration to fully understand the role of F-actin in regulating chromaffin granule transport and secretion under physiological conditions.

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.

MAP18 regulates the direction of pollen tube growth in Arabidopsis by modulating F-actin organization.

PubMed

Zhu, Lei; Zhang, Yan; Kang, Erfang; Xu, Qiangyi; Wang, Miaoying; Rui, Yue; Liu, Baoquan; Yuan, Ming; Fu, Ying

2013-03-01

For fertilization to occur in plants, the pollen tube must be guided to enter the ovule via the micropyle. Previous reports have implicated actin filaments, actin binding proteins, and the tip-focused calcium gradient as key contributors to polar growth of pollen tubes; however, the regulation of directional pollen tube growth is largely unknown. We reported previously that Arabidopsis thaliana MICROTUBULE-ASSOCIATED PROTEIN18 (MAP18) contributes to directional cell growth and cortical microtubule organization. The preferential expression of MAP18 in pollen and in pollen tubes suggests that MAP18 also may function in pollen tube growth. In this study, we demonstrate that MAP18 functions in pollen tubes by influencing actin organization, rather than microtubule assembly. In vitro biochemical results indicate that MAP18 exhibits Ca(2+)-dependent filamentous (F)-actin-severing activity. Abnormal expression of MAP18 in map18 and MAP18 OX plants was associated with disorganization of the actin cytoskeleton in the tube apex, resulting in aberrant pollen tube growth patterns and morphologies, inaccurate micropyle targeting, and fewer fertilization events. Experiments with MAP18 mutants created by site-directed mutagenesis suggest that F-actin-severing activity is essential to the effects of MAP18 on pollen tube growth direction. Our study demonstrates that in Arabidopsis, MAP18 guides the direction of pollen tube growth by modulating actin filaments.

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.

Regulation of Retinoschisin Secretion in Weri-Rb1 Cells by the F-Actin and Microtubule Cytoskeleton

PubMed Central

Kitamura, Eiko; Gribanova, Yekaterina E.; Farber, Debora B.

2011-01-01

Retinoschisin is encoded by the gene responsible for X-linked retinoschisis (XLRS), an early onset macular degeneration that results in a splitting of the inner layers of the retina and severe loss in vision. Retinoschisin is predominantly expressed and secreted from photoreceptor cells as a homo-oligomer protein; it then associates with the surface of retinal cells and maintains the retina cellular architecture. Many missense mutations in the XLRS1 gene are known to cause intracellular retention of retinoschisin, indicating that the secretion process of the protein is a critical step for its normal function in the retina. However, the molecular mechanisms underlying retinoschisin's secretion remain to be fully elucidated. In this study, we investigated the role of the F-actin cytoskeleton in the secretion of retinoschisin by treating Weri-Rb1 cells, which are known to secrete retinoschisin, with cytochalasin D, jasplakinolide, Y-27632, and dibutyryl cGMP. Our results show that cytochalasin D and jasplakinolide inhibit retinoschisin secretion, whereas Y-27632 and dibutyryl cGMP enhance secretion causing F-actin alterations. We also demonstrate that high concentrations of taxol, which hyperpolymerizes microtubules, inhibit retinoschisin secretion. Our data suggest that retinoschisin secretion is regulated by the F-actin cytoskeleton, that cGMP or inhibition of ROCK alters F-actin structure enhancing the secretion, and that the microtubule cytoskeleton is also involved in this process. PMID:21738583

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

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

PubMed Central

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

2017-01-01

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. DOI: http://dx.doi.org/10.7554/eLife.24119.001 PMID:28322189

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.

Plasma levels of F-actin and F:G-actin ratio as potential new biomarkers in patients with septic shock.

PubMed

Belsky, Justin B; Morris, Daniel C; Bouchebl, Ralph; Filbin, Michael R; Bobbitt, Kevin R; Jaehne, Anja K; Rivers, Emanuel P

2016-01-01

To compare plasma levels of F-actin, G-actin and thymosin beta 4 (TB4) in humans with septic shock, noninfectious systemic inflammatory response syndrome (SIRS) and healthy controls. F-actin was significantly elevated in septic shock as compared with noninfectious SIRS and healthy controls. G-actin levels were greatest in the noninfectious SIRS group but significantly elevated in septic shock as compared with healthy controls. TB4 was not detectable in the septic shock or noninfectious SIRS group above the assay's lowest detection range (78 ng/ml). F-actin is significantly elevated in patients with septic shock as compared with noninfectious SIRS. F-actin and the F:G-actin ratio are potential biomarkers for the diagnosis of septic shock.

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.

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

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.

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

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

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

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.

Kindlin-2 directly binds actin and regulates integrin outside-in signaling

PubMed Central

Bledzka, Kamila; Bialkowska, Katarzyna; Sossey-Alaoui, Khalid; Vaynberg, Julia; Pluskota, Elzbieta

2016-01-01

Reduced levels of kindlin-2 (K2) in endothelial cells derived from K2+/− mice or C2C12 myoblastoid cells treated with K2 siRNA showed disorganization of their actin cytoskeleton and decreased spreading. These marked changes led us to examine direct binding between K2 and actin. Purified K2 interacts with F-actin in cosedimentation and surface plasmon resonance analyses and induces actin aggregation. We further find that the F0 domain of K2 binds actin. A mutation, LK47/AA, within a predicted actin binding site (ABS) of F0 diminishes its interaction with actin by approximately fivefold. Wild-type K2 and K2 bearing the LK47/AA mutation were equivalent in their ability to coactivate integrin αIIbβ3 in a CHO cell system when coexpressed with talin. However, K2-LK47/AA exhibited a diminished ability to support cell spreading and actin organization compared with wild-type K2. The presence of an ABS in F0 of K2 that influences outside-in signaling across integrins establishes a new foundation for considering how kindlins might regulate cellular responses. PMID:27044892

Regulation of myosin IIA and filamentous actin during insulin-stimulated glucose uptake in 3T3-L1 adipocytes

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

Stall, Richard; Ramos, Joseph; Kent Fulcher, F.

Insulin stimulated glucose uptake requires the colocalization of myosin IIA (MyoIIA) and the insulin-responsive glucose transporter 4 (GLUT4) at the plasma membrane for proper GLUT4 fusion. MyoIIA facilitates filamentous actin (F-actin) reorganization in various cell types. In adipocytes F-actin reorganization is required for insulin-stimulated glucose uptake. What is not known is whether MyoIIA interacts with F-actin to regulate insulin-induced GLUT4 fusion at the plasma membrane. To elucidate the relationship between MyoIIA and F-actin, we examined the colocalization of MyoIIA and F-actin at the plasma membrane upon insulin stimulation as well as the regulation of this interaction. Our findings demonstrated thatmore » MyoIIA and F-actin colocalized at the site of GLUT4 fusion with the plasma membrane upon insulin stimulation. Furthermore, inhibition of MyoII with blebbistatin impaired F-actin localization at the plasma membrane. Next we examined the regulatory role of calcium in MyoIIA-F-actin colocalization. Reduced calcium or calmodulin levels decreased colocalization of MyoIIA and F-actin at the plasma membrane. While calcium alone can translocate MyoIIA it did not stimulate F-actin accumulation at the plasma membrane. Taken together, we established that while MyoIIA activity is required for F-actin localization at the plasma membrane, it alone is insufficient to localize F-actin to the plasma membrane. - Highlights: • Insulin induces colocalization of MyoIIA and F-actin at the cortex in adipocytes. • MyoIIA is necessary but not sufficient to localize F-actin at the cell cortex. • MyoIIA-F-actin colocalization is regulated by calcium and calmodulin.« less

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

ADF Proteins Are Involved in the Control of Flowering and Regulate F-Actin Organization, Cell Expansion, and Organ Growth in Arabidopsis

PubMed Central

Dong, Chun-Hai; Xia, Gui-Xian; Hong, Yan; Ramachandran, Srinivasan; Kost, Benedikt; Chua, Nam-Hai

2001-01-01

Based mostly on the results of in vitro experiments, ADF (actin-depolymerizing factor) proteins are thought to be key modulators of the dynamic organization of the actin cytoskeleton. The few studies concerned with the in vivo function of ADF proteins that have been reported to date were performed almost exclusively using single-cell systems and have failed to produce consistent results. To investigate ADF functions in vivo and during the development of multicellular organs, we generated transgenic Arabidopsis plants that express a cDNA encoding an ADF protein (AtADF1) in the sense or the antisense orientation under the control of a strong constitutively active promoter. Selected lines with significantly altered levels of AtADF protein expression were characterized phenotypically. Overexpression of AtADF1 resulted in the disappearance of thick actin cables in different cell types, caused irregular cellular and tissue morphogenesis, and reduced the growth of cells and organs. In contrast, reduced AtADF expression promoted the formation of actin cables, resulted in a delay in flowering, and stimulated cell expansion as well as organ growth. These results are consistent with the molecular functions of ADF as predicted by in vitro studies, support the global roles of ADF proteins during the development of a multicellular organism, and demonstrate that these proteins are key regulators of F-actin organization, flowering, and cell and organ expansion in Arabidopsis. PMID:11402164

Rho Kinase (ROCK) collaborates with Pak to Regulate Actin Polymerization and Contraction in Airway Smooth Muscle.

PubMed

Zhang, Wenwu; Bhetwal, Bhupal P; Gunst, Susan J

2018-05-10

The mechanisms by which Rho kinase (ROCK) regulates airway smooth muscle contraction were determined in tracheal smooth muscle tissues. ROCK may mediate smooth muscle contraction by inhibiting myosin regulatory light chain (RLC) phosphatase. ROCK can also regulate F-actin dynamics during cell migration, and actin polymerization is critical for airway smooth muscle contraction. Our results show that ROCK does not regulate airway smooth muscle contraction by inhibiting myosin RLC phosphatase or by stimulating myosin RLC phosphorylation. We find that ROCK regulates airway smooth muscle contraction by activating the serine-threonine kinase Pak, which mediates the activation of Cdc42 and Neuronal-Wiskott-Aldrich Syndrome protein (N-WASp). N-WASP transmits signals from cdc42 to the Arp2/3 complex for the nucleation of actin filaments. These results demonstrate a novel molecular function for ROCK in the regulation of Pak and cdc42 activation that is critical for the processes of actin polymerization and contractility in airway smooth muscle. Rho kinase (ROCK), a RhoA GTPase effector, can regulate the contraction of airway and other smooth muscle tissues. In some tissues, ROCK can inhibit myosin regulatory light chain (RLC) phosphatase, which increases the phosphorylation of myosin RLC and promotes smooth muscle contraction. ROCK can also regulate cell motility and migration by affecting F-actin dynamics. Actin polymerization is stimulated by contractile agonists in airway smooth muscle tissues and is required for contractile tension development in addition to myosin RLC phosphorylation. We investigated the mechanisms by which ROCK regulates the contractility of tracheal smooth muscle tissues by expressing a kinase inactive mutant of ROCK, ROCK-K121G, in the tissues or by treating them with the ROCK inhibitor, H-1152P. Our results show no role for ROCK in the regulation of non-muscle or smooth muscle myosin RLC phosphorylation during contractile stimulation in this tissue

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.

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.

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

A new F-actin structure in fungi: actin ring formation around the cell nucleus of Cryptococcus neoformans.

PubMed

Kopecká, Marie; Kawamoto, Susumu; Yamaguchi, Masashi

2013-04-01

The F-actin cytoskeleton of Cryptococcus neoformans is known to comprise actin cables, cortical patches and cytokinetic ring. Here, we describe a new F-actin structure in fungi, a perinuclear F-actin collar ring around the cell nucleus, by fluorescent microscopic imaging of rhodamine phalloidin-stained F-actin. Perinuclear F-actin rings form in Cryptococcus neoformans treated with the microtubule inhibitor Nocodazole or with the drug solvent dimethyl sulfoxide (DMSO) or grown in yeast extract peptone dextrose (YEPD) medium, but they are absent in cells treated with Latrunculin A. Perinuclear F-actin rings may function as 'funicular cabin' for the cell nucleus, and actin cables as intracellular 'funicular' suspending nucleus in the central position in the cell and moving nucleus along the polarity axis along actin cables.

Subunits of the Drosophila Actin-Capping Protein Heterodimer Regulate Each Other at Multiple Levels

PubMed Central

Amândio, Ana Rita; Gaspar, Pedro; Whited, Jessica L.; Janody, Florence

2014-01-01

The actin-Capping Protein heterodimer, composed of the α and β subunits, is a master F-actin regulator. In addition to its role in many cellular processes, Capping Protein acts as a main tumor suppressor module in Drosophila and in humans, in part, by restricting the activity of Yorkie/YAP/TAZ oncogenes. We aimed in this report to understand how both subunits regulate each other in vivo. We show that the levels and capping activities of both subunits must be tightly regulated to control F-actin levels and consequently growth of the Drosophila wing. Overexpressing capping protein α and β decreases both F-actin levels and tissue growth, while expressing forms of Capping Protein that have dominant negative effects on F-actin promote tissue growth. Both subunits regulate each other's protein levels. In addition, overexpressing one of the subunit in tissues knocked-down for the other increases the mRNA and protein levels of the subunit knocked-down and compensates for its loss. We propose that the ability of the α and β subunits to control each other's levels assures that a pool of functional heterodimer is produced in sufficient quantities to restrict the development of tumor but not in excess to sustain normal tissue growth. PMID:24788460

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.

Platelet rich plasma promotes skeletal muscle cell migration in association with up-regulation of FAK, paxillin, and F-Actin formation.

PubMed

Tsai, Wen-Chung; Yu, Tung-Yang; Lin, Li-Ping; Lin, Mioa-Sui; Tsai, Ting-Ta; Pang, Jong-Hwei S

2017-11-01

Platelet rich plasma (PRP) contains various cytokines and growth factors which may be beneficial to the healing process of injured muscle. The aim of this study was to investigate the effect and molecular mechanism of PRP on migration of skeletal muscle cells. Skeletal muscle cells intrinsic to Sprague-Dawley rats were treated with PRP. The cell migration was evaluated by transwell filter migration assay and electric cell-substrate impedance sensing. The spreading of cells was evaluated microscopically. The formation of filamentous actin (F-actin) cytoskeleton was assessed by immunofluorescence staining. The protein expressions of paxillin and focal adhesion kinase (FAK) were assessed by Western blot analysis. Transfection of paxillin small-interfering RNA (siRNAs) to muscle cells was performed to validate the role of paxillin in PRP-mediated promotion of cell migration. Dose-dependently PRP promotes migration of and spreading and muscle cells. Protein expressions of paxillin and FAK were up-regulated dose-dependently. F-actin formation was also enhanced by PRP treatment. Furthermore, the knockdown of paxillin expression impaired the effect of PRP to promote cell migration. It was concluded that PRP promoting migration of muscle cells is associated with up-regulation of proteins expression of paxillin and FAK as well as increasing F-actin formation. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2506-2512, 2017. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

Assembly kinetics determine the architecture of α-actinin crosslinked F-actin networks.

PubMed

Falzone, Tobias T; Lenz, Martin; Kovar, David R; Gardel, Margaret L

2012-05-29

The actin cytoskeleton is organized into diverse meshworks and bundles that support many aspects of cell physiology. Understanding the self-assembly of these actin-based structures is essential for developing predictive models of cytoskeletal organization. Here we show that the competing kinetics of bundle formation with the onset of dynamic arrest arising from filament entanglements and crosslinking determine the architecture of reconstituted actin networks formed with α-actinin crosslinks. Crosslink-mediated bundle formation only occurs in dilute solutions of highly mobile actin filaments. As actin polymerization proceeds, filament mobility and bundle formation are arrested concomitantly. By controlling the onset of dynamic arrest, perturbations to actin assembly kinetics dramatically alter the architecture of biochemically identical samples. Thus, the morphology of reconstituted F-actin networks is a kinetically determined structure similar to those formed by physical gels and glasses. These results establish mechanisms controlling the structure and mechanics in diverse semiflexible biopolymer networks.

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

Tropomyosin movement on F-actin during muscle activation explained by energy landscapes

PubMed Central

Orzechowski, Marek; Moore, Jeffrey R.; Fischer, Stefan; Lehman, William

2014-01-01

Muscle contraction is regulated by tropomyosin movement across the thin filament surface, which exposes or blocks myosin-binding sites on actin. Recent atomic structures of F-actin-tropomyosin have yielded the positions of tropomyosin on myosin-free and myosin-decorated actin. Here, the repositioning of α-tropomyosin between these locations on F-actin was systematically examined by optimizing the energy of the complex for a wide range of tropomyosin positions on F-actin. The resulting energy landscape provides a full-map of the F-actin surface preferred by tropomyosin, revealing a broad energy basin associated with the tropomyosin position that blocks myosin-binding. This is consistent with previously proposed low-energy oscillations of semi-rigid tropomyosin, necessary for shifting of tropomyosin following troponin-binding. In contrast, the landscape shows much less favorable energies when tropomyosin locates near its myosin-induced “open-state” position. This indicates that spontaneous movement of tropomyosin away from its energetic “ground-state” to the open-state is unlikely in absence of myosin. Instead, myosin-binding must drive tropomyosin toward the open-state to activate the thin filament. Additional energy landscapes were computed for disease-causing actin mutants that distort the topology of the actin-tropomyosin energy landscape, explaining their phenotypes. Thus, the computation of such energy landscapes offers a sensitive way to estimate the impact of mutations. PMID:24412204

Tropomyosin movement on F-actin during muscle activation explained by energy landscapes.

PubMed

Orzechowski, Marek; Moore, Jeffrey R; Fischer, Stefan; Lehman, William

2014-03-01

Muscle contraction is regulated by tropomyosin movement across the thin filament surface, which exposes or blocks myosin-binding sites on actin. Recent atomic structures of F-actin-tropomyosin have yielded the positions of tropomyosin on myosin-free and myosin-decorated actin. Here, the repositioning of α-tropomyosin between these locations on F-actin was systematically examined by optimizing the energy of the complex for a wide range of tropomyosin positions on F-actin. The resulting energy landscape provides a full-map of the F-actin surface preferred by tropomyosin, revealing a broad energy basin associated with the tropomyosin position that blocks myosin-binding. This is consistent with previously proposed low-energy oscillations of semi-rigid tropomyosin, necessary for shifting of tropomyosin following troponin-binding. In contrast, the landscape shows much less favorable energies when tropomyosin locates near its myosin-induced "open-state" position. This indicates that spontaneous movement of tropomyosin away from its energetic "ground-state" to the open-state is unlikely in absence of myosin. Instead, myosin-binding must drive tropomyosin toward the open-state to activate the thin filament. Additional energy landscapes were computed for disease-causing actin mutants that distort the topology of the actin-tropomyosin energy landscape, explaining their phenotypes. Thus, the computation of such energy landscapes offers a sensitive way to estimate the impact of mutations. Copyright © 2014 Elsevier Inc. All rights reserved.

Dynamics of F-actin prefigure the structure of butterfly wing scales.

PubMed

Dinwiddie, April; Null, Ryan; Pizzano, Maria; Chuong, Lisa; Leigh Krup, Alexis; Ee Tan, Hwei; Patel, Nipam H

2014-08-15

The wings of butterflies and moths consist of dorsal and ventral epidermal surfaces that give rise to overlapping layers of scales and hairs (Lepidoptera, "scale wing"). Wing scales (average length ~200 µm) are homologous to insect bristles (macrochaetes), and their colors create the patterns that characterize lepidopteran wings. The topology and surface sculpture of wing scales vary widely, and this architectural complexity arises from variations in the developmental program of the individual scale cells of the wing epithelium. One of the more striking features of lepidopteran wing scales are the longitudinal ridges that run the length of the mature (dead) cell, gathering the cuticularized scale cell surface into pleats on the sides of each scale. While also present around the periphery of other insect bristles and hairs, longitudinal ridges in lepidopteran wing scales gain new significance for their creation of iridescent color through microribs and lamellae. Here we show the dynamics of the highly organized F-actin filaments during scale cell development, and present experimental manipulations of actin polymerization that reveal the essential role of this cytoskeletal component in wing scale elongation and the positioning of longitudinal ribs. Copyright © 2014 Elsevier Inc. All rights reserved.

Assembly Kinetics Determine the Architecture of α-actinin Crosslinked F-actin Networks

PubMed Central

Falzone, Tobias T.; Lenz, Martin; Kovar, David R.; Gardel, Margaret L.

2013-01-01

The actin cytoskeleton is organized into diverse meshworks and bundles that support many aspects of cell physiology. Understanding the self-assembly of these actin-based structures is essential for developing predictive models of cytoskeletal organization. Here we show that the competing kinetics of bundle formation with the onset of dynamic arrest arising from filament entanglements and cross-linking determine the architecture of reconstituted actin networks formed with α-actinin cross-links. Cross-link mediated bundle formation only occurs in dilute solutions of highly mobile actin filaments. As actin polymerization proceeds, filament mobility and bundle formation are arrested concomitantly. By controlling the onset of dynamic arrest, perturbations to actin assembly kinetics dramatically alter the architecture of biochemically identical samples. Thus, the morphology of reconstituted F-actin networks is a kinetically determined structure similar to those formed by physical gels and glasses. These results establish mechanisms controlling the structure and mechanics in diverse semi-flexible biopolymer networks. PMID:22643888

F-actin and G-actin binding are uncoupled by mutation of conserved tyrosine residues in maize actin depolymerizing factor (ZmADF)

PubMed Central

Jiang, Chang-Jie; Weeds, Alan G.; Khan, Safina; Hussey, Patrick J.

1997-01-01

Actin depolymerizing factors (ADF) are stimulus responsive actin cytoskeleton modulating proteins. They bind both monomeric actin (G-actin) and filamentous actin (F-actin) and, under certain conditions, F-actin binding is followed by filament severing. In this paper, using mutant maize ADF3 proteins, we demonstrate that the maize ADF3 binding of F-actin can be spatially distinguished from that of G-actin. One mutant, zmadf3–1, in which Tyr-103 and Ala-104 (equivalent to destrin Tyr-117 and Ala-118) have been replaced by phenylalanine and glycine, respectively, binds more weakly to both G-actin and F-actin compared with maize ADF3. A second mutant, zmadf3–2, in which both Tyr-67 and Tyr-70 are replaced by phenylalanine, shows an affinity for G-actin similar to maize ADF3, but F-actin binding is abolished. The two tyrosines, Tyr-67 and Tyr-70, are in the equivalent position to Tyr-82 and Tyr-85 of destrin, respectively. Using the tertiary structure of destrin, yeast cofilin, and Acanthamoeba actophorin, we discuss the implications of removing the aromatic hydroxyls of Tyr-82 and Tyr-85 (i.e., the effect of substituting phenylalanine for tyrosine) and conclude that Tyr-82 plays a critical role in stabilizing the tertiary structure that is essential for F-actin binding. We propose that this tertiary structure is maintained as a result of a hydrogen bond between the hydroxyl of Tyr-82 and the carbonyl of Tyr-117, which is located in the long α-helix; amino acid components of this helix (Leu-111 to Phe-128) have been implicated in G-actin and F-actin binding. The structures of human destrin and yeast cofilin indicate a hydrogen distance of 2.61 and 2.77 Å, respectively, with corresponding bond angles of 99.5° and 113°, close to the optimum for a strong hydrogen bond. PMID:9275236

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

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.

Yap-Hippo pathway regulates cerebral hypoxia-reoxygenation injury in neuroblastoma N2a cells via inhibiting ROCK1/F-actin/mitochondrial fission pathways.

PubMed

Geng, Chizi; Wei, Jianchao; Wu, Chengsi

2018-05-23

Yes-associated protein (Yap), a regulator of cellular apoptosis, has been demonstrated to be involved in cerebral ischemia-reperfusion (IR) injury through poorly defined mechanisms. The present study aimed to explore the role of Yap in regulating cerebral IR injury in vitro, with a focus on mitochondrial fission and ROCK1/F-actin pathways. Our data demonstrated that Yap was actually downregulated in N2a cells after cerebral hypoxia-reoxygenation (HR) injury, and that lower expression of Yap was closely associated with increased cell death. However, the reintroduction of Yap was able to suppress the HR-mediated N2a cells death via blocking the mitochondria-related apoptotic signal. At the molecular levels, Yap overexpression sustained mitochondrial potential, normalized the mitochondrial respiratory function, reduced ROS overproduction, limited HtrA2/Omi release from mitochondria into the nucleus, and suppressed pro-apoptotic proteins activation. Subsequently, functional studies have further illustrated that HR-mediated mitochondrial apoptosis was highly regulated by mitochondrial fission, whereas Yap overexpression was able to attenuate HR-mediated mitochondrial fission and, thus, promote N2a cell survival in the context of HR injury. At last, we demonstrated that Yap handled mitochondrial fission via closing ROCK1/F-actin signaling pathways. Activation of ROCK1/F-actin pathways abrogated the protective role of Yap overexpression on mitochondrial homeostasis and N2a cell survival in the setting of HR injury. Altogether, our data identified Yap as the endogenous defender to relieve HR-mediated nerve damage via antagonizing ROCK1/F-actin/mitochondrial fission pathways.

Fascin regulates nuclear actin during Drosophila oogenesis

PubMed Central

Kelpsch, Daniel J.; Groen, Christopher M.; Fagan, Tiffany N.; Sudhir, Sweta; Tootle, Tina L.

2016-01-01

Drosophila oogenesis provides a developmental system with which to study nuclear actin. During Stages 5–9, nuclear actin levels are high in the oocyte and exhibit variation within the nurse cells. Cofilin and Profilin, which regulate the nuclear import and export of actin, also localize to the nuclei. Expression of GFP-tagged Actin results in nuclear actin rod formation. These findings indicate that nuclear actin must be tightly regulated during oogenesis. One factor mediating this regulation is Fascin. Overexpression of Fascin enhances nuclear GFP-Actin rod formation, and Fascin colocalizes with the rods. Loss of Fascin reduces, whereas overexpression of Fascin increases, the frequency of nurse cells with high levels of nuclear actin, but neither alters the overall nuclear level of actin within the ovary. These data suggest that Fascin regulates the ability of specific cells to accumulate nuclear actin. Evidence indicates that Fascin positively regulates nuclear actin through Cofilin. Loss of Fascin results in decreased nuclear Cofilin. In addition, Fascin and Cofilin genetically interact, as double heterozygotes exhibit a reduction in the number of nurse cells with high nuclear actin levels. These findings are likely applicable beyond Drosophila follicle development, as the localization and functions of Fascin and the mechanisms regulating nuclear actin are widely conserved. PMID:27535426

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.

Live-Cell Imaging of F-Actin Dynamics During Fertilization in Arabidopsis thaliana.

PubMed

Susaki, Daichi; Maruyama, Daisuke; Yelagandula, Ramesh; Berger, Frederic; Kawashima, Tomokazu

2017-01-01

Fertilization comprises a complex series of cellular processes leading to the fusion of a male and female gamete. Many studies have been carried out to investigate each step of fertilization in plants; however, our comprehensive understanding of all the sequential events during fertilization is still limited. This is largely due to difficulty in investigating events in the female gametophyte, which is deeply embedded in the maternal tissue. Recent advances in confocal microcopy assisted by fluorescent marker lines have contributed to visualizing subcellular dynamics in real time during fertilization in vivo. In this chapter, we describe a method focusing on the investigation of F-actin dynamics in the central cell during male gamete nuclear migration. This method also allows the study of a wide range of early sexual reproduction events, from pollen tube guidance to the early stage of seed development.

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

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.

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

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 Temporal Model of Cofilin Regulation and the Early Peak of Actin Barbed Ends in Invasive Tumor Cells

PubMed Central

Tania, Nessy; Prosk, Erin; Condeelis, John; Edelstein-Keshet, Leah

2011-01-01

Cofilin is an important regulator of actin polymerization, cell migration, and chemotaxis. Recent experimental data on mammary carcinoma cells reveal that stimulation by epidermal growth factor (EGF) generates a pool of active cofilin that results in a peak of actin filament barbed ends on the timescale of 1 min. Here, we present results of a mathematical model for the dynamics of cofilin and its transition between several pools in response to EGF stimulation. We describe the interactions of phospholipase C, membrane lipids (PIP2), and cofilin bound to PIP2 and to F-actin, as well as diffusible cofilin in active G-actin-monomer-bound or phosphorylated states. We consider a simplified representation in which the thin cell edge (lamellipod) and the cell interior are represented by two compartments that are linked by diffusion. We demonstrate that a high basal level of active cofilin stored by binding to PIP2, as well as the highly enriched local milieu of F-actin at the cell edge, is essential to capture the EGF-induced barbed-end amplification observed experimentally. PMID:21504724

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 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

Myopodin is an F-actin bundling protein with multiple independent actin-binding regions.

PubMed

Linnemann, Anja; Vakeel, Padmanabhan; Bezerra, Eduardo; Orfanos, Zacharias; Djinović-Carugo, Kristina; van der Ven, Peter F M; Kirfel, Gregor; Fürst, Dieter O

2013-02-01

The assembly of striated muscle myofibrils is a multistep process in which a variety of proteins is involved. One of the first and most important steps in myofibrillogenesis is the arrangement of thin myofilaments into ordered I-Z-I brushes, requiring the coordinated activity of numerous actin binding proteins. The early expression of myopodin prior to sarcomeric α-actinin, as well as its binding to actin, α-actinin and filamin indicate an important role for this protein in actin cytoskeleton remodelling with the precise function of myopodin in this process yet remaining to be resolved. While myopodin was previously described as a protein capable of cross-linking actin filaments into thick bundles upon transient transfections, it has remained unclear whether myopodin alone is capable of bundling actin, or if additional proteins are involved. We have therefore investigated the in vitro actin binding properties of myopodin. High speed cosedimentation assays with skeletal muscle actin confirmed direct binding of myopodin to F-actin and showed that this interaction is mediated by at least two independent actin binding sites, found in all myopodin isoforms identified to date. Furthermore, low-speed cosedimentation assays revealed that not only full length myopodin, but also the fragment containing only the second binding site, bundles microfilaments in the absence of accessory proteins. Ultrastructural analysis demonstrated that this bundling activity resembled that of α-actinin. Biochemical experiments revealed that bundling was not achieved by myopodin's ability to dimerize, indicating the presence of two individual F-actin binding sites within the second binding segment. Thus full length myopodin contains at least three F-actin binding sites. These data provide further understanding of the mechanisms by which myopodin contributes to actin reorganization during myofibril assembly.

Cdc42 regulates junctional actin but not cell polarization in the Caenorhabditis elegans epidermis

PubMed Central

Zilberman, Yuliya; Abrams, Joshua; Anderson, Dorian C.

2017-01-01

During morphogenesis, adherens junctions (AJs) remodel to allow changes in cell shape and position while preserving adhesion. Here, we examine the function of Rho guanosine triphosphatase CDC-42 in AJ formation and regulation during Caenorhabditis elegans embryo elongation, a process driven by asymmetric epidermal cell shape changes. cdc-42 mutant embryos arrest during elongation with epidermal ruptures. Unexpectedly, we find using time-lapse fluorescence imaging that cdc-42 is not required for epidermal cell polarization or junction assembly, but rather is needed for proper junctional actin regulation during elongation. We show that the RhoGAP PAC-1/ARHGAP21 inhibits CDC-42 activity at AJs, and loss of PAC-1 or the interacting linker protein PICC-1/CCDC85A-C blocks elongation in embryos with compromised AJ function. pac-1 embryos exhibit dynamic accumulations of junctional F-actin and an increase in AJ protein levels. Our findings identify a previously unrecognized molecular mechanism for inhibiting junctional CDC-42 to control actin organization and AJ protein levels during epithelial morphogenesis. PMID:28903999

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

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.

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

F-actin mechanics control spindle centring in the mouse zygote

NASA Astrophysics Data System (ADS)

Chaigne, Agathe; Campillo, Clément; Voituriez, Raphaël; Gov, Nir S.; Sykes, Cécile; Verlhac, Marie-Hélène; Terret, Marie-Emilie

2016-01-01

Mitotic spindle position relies on interactions between astral microtubules nucleated by centrosomes and a rigid cortex. Some cells, such as mouse oocytes, do not possess centrosomes and astral microtubules. These cells rely only on actin and on a soft cortex to position their spindle off-centre and undergo asymmetric divisions. While the first mouse embryonic division also occurs in the absence of centrosomes, it is symmetric and not much is known on how the spindle is positioned at the exact cell centre. Using interdisciplinary approaches, we demonstrate that zygotic spindle positioning follows a three-step process: (1) coarse centring of pronuclei relying on the dynamics of an F-actin/Myosin-Vb meshwork; (2) fine centring of the metaphase plate depending on a high cortical tension; (3) passive maintenance at the cell centre. Altogether, we show that F-actin-dependent mechanics operate the switch between asymmetric to symmetric division required at the oocyte to embryo transition.

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

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.

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.

Investigating the effects of tropomyosin mutations on its flexibility and interactions with filamentous actin using molecular dynamics simulation.

PubMed

Zheng, Wenjun; Hitchcock-DeGregori, Sarah E; Barua, Bipasha

2016-10-01

Tropomyosin (Tpm) is a two-chained α-helical coiled-coil protein that binds to filamentous actin (F-actin), and regulates its interactions with myosin by occupying three average positions on F-actin (blocked, closed, and open). Mutations in the Tpm are linked to heart diseases including hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM). To elucidate the molecular mechanisms of Tpm mutations (including DCM mutation E54K, HCM mutations E62Q, A63V, K70T, V95A, D175N, E180G, L185R, E192K, and a designed synthetic mutation D137L) in terms of their effects on Tpm flexibility and its interactions with F-actin, we conducted extensive molecular dynamics simulations for the wild-type and mutant Tpm in complex with F-actin (total simulation time 160 ns per mutant). The mutants exhibited distinct changes (i.e., increase or decrease) in the overall and local flexibility of the Tpm coiled-coil, with each mutation causing both local and long-range modifications of the Tpm flexibility. In addition, our binding calculations revealed weakened Tpm-F-actin interactions (except for L185R, D137L and A63V) involving five periods of Tpm, which correlate with elevated fluctuation of Tpm relative to the blocked position on F-actin that may lead to easier activation and increased Ca 2+ -sensitivity. We also simulated the αβ/βα-Tpm heterodimer in comparison with the αα-Tpm homodimer, which revealed greater flexibility and weaker actin binding in the heterodimer. Our findings are consistent with a complex mechanism underlying how different Tpm mutations perturb the Tpm function in distinct ways (e.g., by affecting specific sites of Tpm), which bear no simple links to the disease phenotypes (e.g., HCM vs. DCM).

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

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

A temporal model of cofilin regulation and the early peak of actin barbed ends in invasive tumor cells.

PubMed

Tania, Nessy; Prosk, Erin; Condeelis, John; Edelstein-Keshet, Leah

2011-04-20

Cofilin is an important regulator of actin polymerization, cell migration, and chemotaxis. Recent experimental data on mammary carcinoma cells reveal that stimulation by epidermal growth factor (EGF) generates a pool of active cofilin that results in a peak of actin filament barbed ends on the timescale of 1 min. Here, we present results of a mathematical model for the dynamics of cofilin and its transition between several pools in response to EGF stimulation. We describe the interactions of phospholipase C, membrane lipids (PIP(2)), and cofilin bound to PIP(2) and to F-actin, as well as diffusible cofilin in active G-actin-monomer-bound or phosphorylated states. We consider a simplified representation in which the thin cell edge (lamellipod) and the cell interior are represented by two compartments that are linked by diffusion. We demonstrate that a high basal level of active cofilin stored by binding to PIP(2), as well as the highly enriched local milieu of F-actin at the cell edge, is essential to capture the EGF-induced barbed-end amplification observed experimentally. Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Myosin Va Bound to Phagosomes Binds to F-Actin and Delays Microtubule-dependent Motility

PubMed Central

Al-Haddad, Ahmed; Shonn, Marion A.; Redlich, Bärbel; Blocker, Ariel; Burkhardt, Janis K.; Yu, Hanry; Hammer, John A.; Weiss, Dieter G.; Steffen, Walter; Griffiths, Gareth; Kuznetsov, Sergei A.

2001-01-01

We established a light microscopy-based assay that reconstitutes the binding of phagosomes purified from mouse macrophages to preassembled F-actin in vitro. Both endogenous myosin Va from mouse macrophages and exogenous myosin Va from chicken brain stimulated the phagosome–F-actin interaction. Myosin Va association with phagosomes correlated with their ability to bind F-actin in an ATP-regulated manner and antibodies to myosin Va specifically blocked the ATP-sensitive phagosome binding to F-actin. The uptake and retrograde transport of phagosomes from the periphery to the center of cells in bone marrow macrophages was observed in both normal mice and mice homozygous for the dilute-lethal spontaneous mutation (myosin Va null). However, in dilute-lethal macrophages the accumulation of phagosomes in the perinuclear region occurred twofold faster than in normal macrophages. Motion analysis revealed saltatory phagosome movement with temporarily reversed direction in normal macrophages, whereas almost no reversals in direction were observed in dilute-lethal macrophages. These observations demonstrate that myosin Va mediates phagosome binding to F-actin, resulting in a delay in microtubule-dependent retrograde phagosome movement toward the cell center. We propose an “antagonistic/cooperative mechanism” to explain the saltatory phagosome movement toward the cell center in normal macrophages. PMID:11553713

Multiple roles for the actin cytoskeleton during regulated exocytosis

PubMed Central

Porat-Shliom, Natalie; Milberg, Oleg; Masedunskas, Andrius; Weigert, Roberto

2014-01-01

Regulated exocytosis is the main mechanism utilized by specialized secretory cells to deliver molecules to the cell surface by virtue of membranous containers (i.e. secretory vesicles). The process involves a series of highly coordinated and sequential steps, which include the biogenesis of the vesicles, their delivery to the cell periphery, their fusion with the plasma membrane and the release of their content into the extracellular space. Each of these steps is regulated by the actin cytoskeleton. In this review, we summarize the current knowledge regarding the involvement of actin and its associated molecules during each of the exocytic steps in vertebrates, and suggest that the overall role of the actin cytoskeleton during regulated exocytosis is linked to the architecture and the physiology of the secretory cells under examination. Specifically, in neurons, neuroendocrine, endocrine, and hematopoietic cells, which contain small secretory vesicles that undergo rapid exocytosis (on the order of milliseconds), the actin cytoskeleton plays a role in pre-fusion events, where it acts primarily as a functional barrier and facilitates docking. In exocrine and other secretory cells, which contain large secretory vesicles that undergo slow exocytosis (seconds to minutes), the actin cytoskeleton plays a role in post-fusion events, where it regulates the dynamics of the fusion pore, facilitates the integration of the vesicles into the plasma membrane, provides structural support, and promotes the expulsion of large cargo molecules. PMID:22986507

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.

Investigating Molecular Level Stress-Strain Relationships in Entangled F-Actin Networks by Combined Force-Measuring Optical Tweezers and Fluorescence Microscopy

NASA Astrophysics Data System (ADS)

Lee, Kent; Henze, Dean; Robertson-Anderson, Rae

2013-03-01

Actin is an important cytoskeletal protein involved in cell structure and motility, cancer invasion and metastasis, and muscle contraction. The intricate viscoelastic properties of filamentous actin (F-actin) networks allow for the many dynamic roles of actin, thus warranting investigation. Exploration of this unique stress-strain/strain-rate relationship in complex F-actin networks can also improve biomimetic materials engineering. Here, we use optical tweezers with fluorescence microscopy to study the viscoelastic properties of F-actin networks on the microscopic level. Optically trapped microspheres embedded in various F-actin networks are moved through the network using a nanoprecision piezoelectric stage. The force exerted on the microspheres by the F-actin network and subsequent force relaxation are measured, while a fraction of the filaments in the network are fluorescent-labeled to observe filament deformation in real-time. The dependence of the viscoelastic properties of the network on strain rates and amplitudes as well as F-actin concentration is quantified. This approach provides the much-needed link between induced force and deformation over localized regimes (tens of microns) and down to the single molecule level.

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 Out: Regulation of the Synaptic Cytoskeleton

PubMed Central

Spence, Erin F.; Soderling, Scott H.

2015-01-01

The small size of dendritic spines belies the elaborate role they play in excitatory synaptic transmission and ultimately complex behaviors. The cytoskeletal architecture of the spine is predominately composed of actin filaments. These filaments, which at first glance might appear simple, are also surprisingly complex. They dynamically assemble into different structures and serve as a platform for orchestrating the elaborate responses of the spine during spinogenesis and experience-dependent plasticity. Multiple mutations associated with human neurodevelopmental and psychiatric disorders involve genes that encode regulators of the synaptic cytoskeleton. A major, unresolved question is how the disruption of specific actin filament structures leads to the onset and progression of complex synaptic and behavioral phenotypes. This review will cover established and emerging mechanisms of actin cytoskeletal remodeling and how this influences specific aspects of spine biology that are implicated in disease. PMID:26453304

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 Age Orchestrates Myosin-5 and Myosin-6 Runlengths

PubMed Central

Zimmermann, Dennis; Santos, Alicja; Kovar, David R.; Rock, Ronald S.

2015-01-01

Summary Unlike a static and immobile skeleton, the actin cytoskeleton is a highly dynamic network of filamentous actin (F-actin) polymers that continuously turn over. In addition to generating mechanical forces and sensing mechanical deformation, dynamic F-actin networks serve as cellular tracks for myosin motor traffic. However, much of our mechanistic understanding of processive myosins comes from in vitro studies where motility was studied on pre-assembled and artificially stabilized, static F-actin tracks. In this work, we examine the role of actin dynamics in single-molecule myosin motility using assembling F-actin and the two highly processive motors, myosin-5 and myosin-6. These two myosins have distinct functions in the cell and travel in opposite directions along actin filaments [1–3]. Myosin-5 walks towards the barbed ends of F-actin, traveling to sites of actin polymerization at the cell periphery [4]. Myosin-6 walks towards the pointed end of F-actin [5], traveling towards the cell center along older segments of the actin filament. We find that myosin-5 takes 1.3 to 1.5-fold longer runs on ADP•Pi (young) F-actin, while myosin-6 takes 1.7 to 3.6-fold longer runs along ADP (old) F-actin. These results suggest that conformational differences between ADP•Pi and ADP F-actin tailor these myosins to walk farther toward their preferred actin filament end. Taken together, these experiments define a new mechanism by which myosin traffic may sort to different F-actin networks depending on filament age. PMID:26190073

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.

Apical and basal epitheliomuscular F-actin dynamics during Hydra bud evagination

PubMed Central

Aufschnaiter, Roland; Wedlich-Söldner, Roland; Zhang, Xiaoming

2017-01-01

ABSTRACT Bending of 2D cell sheets is a fundamental morphogenetic mechanism during animal development and reproduction. A critical player driving cell shape during tissue bending is the actin cytoskeleton. Much of our current knowledge about actin dynamics in whole organisms stems from studies of embryonic development in bilaterian model organisms. Here, we have analyzed actin-based processes during asexual bud evagination in the simple metazoan Hydra. We created transgenic Hydra strains stably expressing the actin marker Lifeact-GFP in either ectodermal or endodermal epitheliomuscular cells. We then combined live imaging with conventional phalloidin staining to directly follow actin reorganization. Bending of the Hydra epithelial double layer is initiated by a group of epitheliomuscular cells in the endodermal layer. These cells shorten their apical-basal axis and arrange their basal muscle processes in a circular configuration. We propose that this rearrangement generates the initial forces to bend the endoderm towards the ectoderm. Convergent tissue movement in both epithelial layers towards the centre of evagination then leads to elongation and extension of the bud along its new body axis. Tissue movement into the bud is associated with lateral intercalation of epithelial cells, remodelling of apical septate junctions, and rearrangement of basal muscle processes. The work presented here extends the analysis of morphogenetic mechanisms beyond embryonic tissues of model bilaterians. PMID:28630355

Latrunculin B-induced plant dwarfism: Plant cell elongation is F-actin-dependent.

PubMed

Baluska, F; Jasik, J; Edelmann, H G; Salajová, T; Volkmann, D

2001-03-01

Marine macrolides latrunculins are highly specific toxins which effectively depolymerize actin filaments (generally F-actin) in all eukaryotic cells. We show that latrunculin B is effective on diverse cell types in higher plants and describe the use of this drug in probing F-actin-dependent growth and in plant development-related processes. In contrast to other eukaryotic organisms, cell divisions occurs in plant cells devoid of all actin filaments. However, the alignment of the division planes is often distorted. In addition to cell division, postembryonic development and morphogenesis also continue in the absence of F-actin. These experimental data suggest that F-actin is of little importance in the morphogenesis of higher plants, and that plants can develop more or less normally without F-actin. In contrast, F-actin turns out to be essential for cell elongation. When latrunculin B was added during germination, morphologically normal Arabidopsis and rye seedlings developed but, as a result of the absence of cell elongation, these were stunted, resembling either genetic dwarfs or environmental bonsai plants. In conclusion, F-actin is essential for the plant cell elongation, while this F-actin-dependent cell elongation is not an essential feature of plant-specific developmental programs.

Agonist-induced PIP(2) hydrolysis inhibits cortical actin dynamics: regulation at a global but not at a micrometer scale.

PubMed

van Rheenen, Jacco; Jalink, Kees

2002-09-01

Phosphatidylinositol 4, 5-bisphosphate (PIP(2)) at the inner leaflet of the plasma membrane has been proposed to locally regulate the actin cytoskeleton. Indeed, recent studies that use GFP-tagged pleckstrin homology domains (GFP-PH) as fluorescent PIP(2) sensors suggest that this lipid is enriched in membrane microdomains. Here we report that this concept needs revision. Using three distinct fluorescent GFP-tagged pleckstrin homology domains, we show that highly mobile GFP-PH patches colocalize perfectly with various lipophilic membrane dyes and, hence, represent increased lipid content rather than PIP(2)-enriched microdomains. We show that bright patches are caused by submicroscopical folds and ruffles in the membrane that can be directly visualized at approximately 15 nm axial resolution with a novel numerically enhanced imaging method. F-actin motility is inhibited significantly by agonist-induced PIP(2) breakdown, and it resumes as soon as PIP(2) levels are back to normal. Thus, our data support a role for PIP(2) in the regulation of cortical actin, but they challenge a model in which spatial differences in PIP(2) regulation of the cytoskeleton exist at a micrometer scale.

How actin network dynamics control the onset of actin-based motility

PubMed Central

Kawska, Agnieszka; Carvalho, Kévin; Manzi, John; Boujemaa-Paterski, Rajaa; Blanchoin, Laurent; Martiel, Jean-Louis; Sykes, Cécile

2012-01-01

Cells use their dynamic actin network to control their mechanics and motility. These networks are made of branched actin filaments generated by the Arp2/3 complex. Here we study under which conditions the microscopic organization of branched actin networks builds up a sufficient stress to trigger sustained motility. In our experimental setup, dynamic actin networks or “gels” are grown on a hard bead in a controlled minimal protein system containing actin monomers, profilin, the Arp2/3 complex and capping protein. We vary protein concentrations and follow experimentally and through simulations the shape and mechanical properties of the actin gel growing around beads. Actin gel morphology is controlled by elementary steps including “primer” contact, growth of the network, entanglement, mechanical interaction and force production. We show that varying the biochemical orchestration of these steps can lead to the loss of network cohesion and the lack of effective force production. We propose a predictive phase diagram of actin gel fate as a function of protein concentrations. This work unveils how, in growing actin networks, a tight biochemical and physical coupling smoothens initial primer-caused heterogeneities and governs force buildup and cell motility. PMID:22908255

Boron nitride nanotube-mediated stimulation modulates F/G-actin ratio and mechanical properties of human dermal fibroblasts

NASA Astrophysics Data System (ADS)

Ricotti, Leonardo; das Neves, Ricardo Pires; Ciofani, Gianni; Canale, Claudio; Nitti, Simone; Mattoli, Virgilio; Mazzolai, Barbara; Ferreira, Lino; Menciassi, Arianna

2014-02-01

F/G-actin ratio modulation is known to have an important role in many cell functions and in the regulation of specific cell behaviors. Several attempts have been made in the latest decades to finely control actin production and polymerization, in order to promote certain cell responses. In this paper we demonstrate the possibility of modulating F/G-actin ratio and mechanical properties of normal human dermal fibroblasts by using boron nitride nanotubes dispersed in the culture medium and by stimulating them with ultrasound transducers. Increasing concentrations of nanotubes were tested with the cells, without any evidence of cytotoxicity up to 10 μg/ml concentration of nanoparticles. Cells treated with nanoparticles and ultrasound stimulation showed a significantly higher F/G-actin ratio in comparison with the controls, as well as a higher Young's modulus. Assessment of Cdc42 activity revealed that actin nucleation/polymerization pathways, involving Rho GTPases, are probably influenced by nanotube-mediated stimulation, but they do not play a primary role in the significant increase of F/G-actin ratio of treated cells, such effect being mainly due to actin overexpression.

Coupled excitable Ras and F-actin activation mediates spontaneous pseudopod formation and directed cell movement

PubMed Central

van Haastert, Peter J. M.; Keizer-Gunnink, Ineke; Kortholt, Arjan

2017-01-01

Many eukaryotic cells regulate their mobility by external cues. Genetic studies have identified >100 components that participate in chemotaxis, which hinders the identification of the conceptual framework of how cells sense and respond to shallow chemical gradients. The activation of Ras occurs during basal locomotion and is an essential connector between receptor and cytoskeleton during chemotaxis. Using a sensitive assay for activated Ras, we show here that activation of Ras and F-actin forms two excitable systems that are coupled through mutual positive feedback and memory. This coupled excitable system leads to short-lived patches of activated Ras and associated F-actin that precede the extension of protrusions. In buffer, excitability starts frequently with Ras activation in the back/side of the cell or with F-actin in the front of the cell. In a shallow gradient of chemoattractant, local Ras activation triggers full excitation of Ras and subsequently F-actin at the side of the cell facing the chemoattractant, leading to directed pseudopod extension and chemotaxis. A computational model shows that the coupled excitable Ras/F-actin system forms the driving heart for the ordered-stochastic extension of pseudopods in buffer and for efficient directional extension of pseudopods in chemotactic gradients. PMID:28148648

F-actin-based Ca signaling-a critical comparison with the current concept of Ca signaling.

PubMed

Lange, Klaus; Gartzke, Joachim

2006-11-01

A short comparative survey on the current idea of Ca signaling and the alternative concept of F-actin-based Ca signaling is given. The two hypotheses differ in one central aspect, the mechanism of Ca storage. The current theory rests on the assumption of Ca-accumulating endoplasmic/sarcoplasmic reticulum-derived vesicles equipped with an ATP-dependent Ca pump and IP3- or ryanodine-sensitive channel-receptors for Ca-release. The alternative hypothesis proceeds from the idea of Ca storage at the high-affinity binding sites of actin filaments. Cellular sites of F-actin-based Ca storage are microvilli and the submembrane cytoskeleton. Several specific features of Ca signaling such as store-channel coupling, quantal Ca release, spiking and oscillations, biphasic and "phasic" uptake kinetics, and Ca-induced Ca release (CICR), which are not adequately described by the current concept, are inherent properties of the F-actin system and its dynamic state of treadmilling. Copyright 2006 Wiley-Liss, Inc.

A critical comparison of the current view of Ca signaling with the novel concept of F-actin-based Ca signaling.

PubMed

Lange, Klaus; Gartzke, Joachim

2006-01-01

A detailed comparative survey on the current idea of Ca signaling and the alternative concept of F-actin-based Ca signaling is given. The two hypotheses differ in one central aspect - the mechanism of Ca storage. The current theory rests on the assumption of Ca-accumulating vesicles derived from the endoplasmic/ sarcoplasmic reticulum, which are equipped with an ATP-dependent Ca pump and IP3- or ryanodine-sensitive Ca-release channels/receptors. The alternative hypothesis proceeds from the idea of Ca storage at the high-affinity binding sites of F-actin subunits. Several prominent features of Ca signaling, which are not adequately described by the current concept, are inherent properties of the F-actin system and its dynamic state of treadmilling. F-actin is the only known biological Ca-binding system that has been proven by in vitro experiments to work within the physiological range of Ca concentrations and the only system that meets all necessary conditions to function as receptor-operated Ca store and as a coupling device between the Ca store and the store-operated Ca influx pathway. The most important properties of Ca signaling, such as store-channel coupling, quantal Ca release, spiking and oscillations, biphasic and "phasic" uptake kinetics, and Ca-induced Ca release, turn out to be systematic features of the new concept but remain unexplained by the classical vesicle storage hypothesis. A number of novel findings, specifically recent reports about direct effects of actin-specific toxins on Ca stores, have strengthened the new concept. The concept of F-actin-based Ca signaling combined with the notion of microvillar regulation of ion and substrate fluxes opens new aspects and far-reaching consequences, not only for cellular Ca signaling but also for various other cell functions, and represents an opportunity to connect several fields of cell physiology on the basis of a common mechanism.

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.

The minus-end actin capping protein, UNC-94/tropomodulin, regulates development of the Caenorhabditis elegans intestine

PubMed Central

Cox-Paulson, Elisabeth; Cannataro, Vincent; Gallagher, Thomas; Hoffman, Corey; Mantione, Gary; McIntosh, Matthew; Silva, Malan; Vissichelli, Nicole; Walker, Rachel; Simske, Jeffrey; Ono, Shoichiro; Hoops, Harold

2014-01-01

Background Tropomodulins are actin capping proteins that regulate the stability of the slow growing, minus-ends of actin filaments. The C. elegans tropomodulin homolog, UNC-94 has sequence and functional similarity to vertebrate tropomodulins. We investigated the role of UNC-94 in C. elegans intestinal morphogenesis. Results In the embryonic C. elegans intestine, UNC-94 localizes to the terminal web, an actin and intermediate filament rich structure that underlies the apical membrane. Loss of UNC-94 function results in areas of flattened intestinal lumen. In worms homozygous for the strong loss-of-function allele, unc-94(tm724), the terminal web is thinner and the amount of F-actin is reduced, pointing to a role for UNC-94 in regulating the structure of the terminal web. The non-muscle myosin, NMY-1, also localizes to the terminal web; and we present evidence that increasing actomyosin contractility by depleting the myosin phosphatase regulatory subunit, mel-11, can rescue the flattened lumen phenotype of unc-94 mutants. Conclusions The data support a model in which minus-end actin capping by UNC-94 promotes proper F-actin structure and contraction in the terminal web, yielding proper shape of the intestinal lumen. This establishes a new role for a tropomodulin in regulating lumen shape during tubulogenesis. PMID:24677443

Actin polymerization drives polar growth in Arabidopsis root hair cells.

PubMed

Vazquez, Luis Alfredo Bañuelos; Sanchez, Rosana; Hernandez-Barrera, Alejandra; Zepeda-Jazo, Isaac; Sánchez, Federico; Quinto, Carmen; Torres, Luis Cárdenas

2014-01-01

In plants, the actin cytoskeleton is a prime regulator of cell polarity, growth, and cytoplasmic streaming. Tip growth, as observed in root hairs, caulonema, and pollen tubes, is governed by many factors, including calcium gradients, exocytosis and endocytosis, reactive oxygen species, and the cytoskeleton. Several studies indicate that the polymerization of G-actin into F-actin also contributes to tip growth. The structure and function of F-actin within the apical dome is variable, ranging from a dense meshwork to sparse single filaments. The presence of multiple F-actin structures in the elongating apices of tip-growing cells suggests that this cytoskeletal array is tightly regulated. We recently reported that sublethal concentrations of fluorescently labeled cytochalasin could be used to visualize the distribution of microfilament plus ends using fluorescence microscopy, and found that the tip region of the growing root hair cells of a legume plant exhibits a clear response to the nodulation factors secreted by Rhizobium. (1) In this current work, we expanded our analysis using confocal microscopy and demonstrated the existence of highly dynamic fluorescent foci along Arabidopsis root hair cells. Furthermore, we show that the strongest fluorescence signal accumulates in the tip dome of the growing root hair and seems to be in close proximity to the apical plasma membrane. Based on these findings, we propose that actin polymerization within the dome of growing root hair cells regulates polar growth.

Effects of basic calponin on the flexural mechanics and stability of F-actin.

PubMed

Jensen, Mikkel Herholdt; Watt, James; Hodgkinson, Julie L; Gallant, Cynthia; Appel, Sarah; El-Mezgueldi, Mohammed; Angelini, Thomas E; Morgan, Kathleen G; Lehman, William; Moore, Jeffrey R

2012-01-01

The cellular actin cytoskeleton plays a central role in the ability of cells to properly sense, propagate, and respond to external stresses and other mechanical stimuli. Calponin, an actin-binding protein found both in muscle and non-muscle cells, has been implicated in actin cytoskeletal organization and regulation. In this work, we studied the mechanical and structural interaction of actin with basic calponin, a differentiation marker in smooth muscle cells, on a single filament level. We imaged fluorescently labeled thermally fluctuating actin filaments and found that at moderate calponin binding densities, actin filaments were more flexible, evident as a reduction in persistence length from 8.0 to 5.8 μm. When calponin-decorated actin filaments were subjected to shear, we observed a marked reduction of filament lengths after decoration with calponin, which we argue was due to shear-induced filament rupture rather than depolymerization. This increased shear susceptibility was exacerbated with calponin concentration. Cryo-electron microscopy results confirmed previously published negative stain electron microscopy results and suggested alterations in actin involving actin subdomain 2. A weakening of F-actin intermolecular association is discussed as the underlying cause of the observed mechanical perturbations. Copyright © 2011 Wiley Periodicals, Inc.

Secretagogin affects insulin secretion in pancreatic β-cells by regulating actin dynamics and focal adhesion.

PubMed

Yang, Seo-Yun; Lee, Jae-Jin; Lee, Jin-Hee; Lee, Kyungeun; Oh, Seung Hoon; Lim, Yu-Mi; Lee, Myung-Shik; Lee, Kong-Joo

2016-06-15

Secretagogin (SCGN), a Ca(2+)-binding protein having six EF-hands, is selectively expressed in pancreatic β-cells and neuroendocrine cells. Previous studies suggested that SCGN enhances insulin secretion by functioning as a Ca(2+)-sensor protein, but the underlying mechanism has not been elucidated. The present study explored the mechanism by which SCGN enhances glucose-induced insulin secretion in NIT-1 insulinoma cells. To determine whether SCGN influences the first or second phase of insulin secretion, we examined how SCGN affects the kinetics of insulin secretion in NIT-1 cells. We found that silencing SCGN suppressed the second phase of insulin secretion induced by glucose and H2O2, but not the first phase induced by KCl stimulation. Recruitment of insulin granules in the second phase of insulin secretion was significantly impaired by knocking down SCGN in NIT-1 cells. In addition, we found that SCGN interacts with the actin cytoskeleton in the plasma membrane and regulates actin remodelling in a glucose-dependent manner. Since actin dynamics are known to regulate focal adhesion, a critical step in the second phase of insulin secretion, we examined the effect of silencing SCGN on focal adhesion molecules, including FAK (focal adhesion kinase) and paxillin, and the cell survival molecules ERK1/2 (extracellular-signal-regulated kinase 1/2) and Akt. We found that glucose- and H2O2-induced activation of FAK, paxillin, ERK1/2 and Akt was significantly blocked by silencing SCGN. We conclude that SCGN controls glucose-stimulated insulin secretion and thus may be useful in the therapy of Type 2 diabetes. © 2016 The Author(s).

MoCAP proteins regulated by MoArk1-mediated phosphorylation coordinate endocytosis and actin dynamics to govern development and virulence of Magnaporthe oryzae

PubMed Central

Yang, Jun; Chen, Deng; Liu, Muxing; Zhang, Haifeng; Zheng, Xiaobo; Wang, Ping; Peng, Youliang; Zhang, Zhengguang

2017-01-01

Actin organization is a conserved cellular process that regulates the growth and development of eukaryotic cells. It also governs the virulence process of pathogenic fungi, such as the rice blast fungus Magnaporthe oryzae, with mechanisms not yet fully understood. In a previous study, we found that actin-regulating kinase MoArk1 displays conserved functions important in endocytosis and actin organization, and MoArk1 is required for maintaining the growth and full virulence of M. oryzae. To understand how MoArk1 might function, we identified capping protein homologs from M. oryzae (MoCAP) that interact with MoArk1 in vivo. MoCAP is heterodimer consisting of α and β subunits MoCapA and MoCapB. Single and double deletions of MoCAP subunits resulted in abnormal mycelial growth and conidia formation. The ΔMocap mutants also exhibited reduced appressorium penetration and invasive hyphal growth within host cells. Furthermore, the ΔMocap mutants exhibited delayed endocytosis and abnormal cytoskeleton assembly. Consistent with above findings, MoCAP proteins interacted with MoAct1, co-localized with actin during mycelial development, and participated in appressorial actin ring formation. Further analysis revealed that the S85 residue of MoCapA and the S285 residue of MoCapB were subject to phosphorylation by MoArk1 that negatively regulates MoCAP functions. Finally, the addition of exogenous phosphatidylinositol 4,5-bisphosphate (PIP2) failed to modulate actin ring formation in ΔMocap mutants, in contrast to the wild-type strain, suggesting that MoCAP may also mediate phospholipid signaling in the regulation of the actin organization. These results together demonstrate that MoCAP proteins whose functions are regulated by MoArk1 and PIP2 are important for endocytosis and actin dynamics that are directly linked to growth, conidiation and pathogenicity of M. oryzae. PMID:28542408

F-actin distribution and function during sexual development in Eimeria maxima.

PubMed

Frölich, Sonja; Wallach, Michael

2015-06-01

To determine the involvement of the actin cytoskeleton in macrogametocyte growth and oocyst wall formation, freshly purified macrogametocytes and oocysts were stained with Oregon Green 514 conjugated phalloidin to visualize F-actin microfilaments, while Evans blue staining was used to detect type 1 wall forming bodies (WFB1s) and the outer oocyst wall. The double-labelled parasites were then analysed at various stages of sexual development using three-dimensional confocal microscopy. The results showed F-actin filaments were distributed throughout the entire cytoplasm of mature Eimeria maxima macrogametocytes forming a web-like meshwork of actin filaments linking the type 1 WFBs together into structures resembling 'beads on a string'. At the early stages of oocyst wall formation, F-actin localization changed in alignment with the egg-shaped morphology of the forming oocysts with F-actin microfilaments making direct contact with the WFB1s. In tissue oocysts, the labelled actin cytoskeleton was situated underneath the forming outer layer of the oocyst wall. Treatment of macrogametocytes in vitro with the actin depolymerizing agents, Cytochalasin D and Latrunculin, led to a reduction in the numbers of mature WFB1s in the cytoplasm of the developing macrogametocytes, indicating that the actin plays an important role in WFB1 transport and oocyst wall formation in E. maxima.

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

Cell-cycle regulation of formin-mediated actin cable assembly

PubMed Central

Miao, Yansong; Wong, Catherine C. L.; Mennella, Vito; Michelot, Alphée; Agard, David A.; Holt, Liam J.; Yates, John R.; Drubin, David G.

2013-01-01

Assembly of appropriately oriented actin cables nucleated by formin proteins is necessary for many biological processes in diverse eukaryotes. However, compared with knowledge of how nucleation of dendritic actin filament arrays by the actin-related protein-2/3 complex is regulated, the in vivo regulatory mechanisms for actin cable formation are less clear. To gain insights into mechanisms for regulating actin cable assembly, we reconstituted the assembly process in vitro by introducing microspheres functionalized with the C terminus of the budding yeast formin Bni1 into extracts prepared from yeast cells at different cell-cycle stages. EM studies showed that unbranched actin filament bundles were reconstituted successfully in the yeast extracts. Only extracts enriched in the mitotic cyclin Clb2 were competent for actin cable assembly, and cyclin-dependent kinase 1 activity was indispensible. Cyclin-dependent kinase 1 activity also was found to regulate cable assembly in vivo. Here we present evidence that formin cell-cycle regulation is conserved in vertebrates. The use of the cable-reconstitution system to test roles for the key actin-binding proteins tropomyosin, capping protein, and cofilin provided important insights into assembly regulation. Furthermore, using mass spectrometry, we identified components of the actin cables formed in yeast extracts, providing the basis for comprehensive understanding of cable assembly and regulation. PMID:24133141

Fascin 1 is an actin filament-bundling protein that regulates ectoplasmic specialization dynamics in the rat testis

PubMed Central

Gungor-Ordueri, N. Ece; Celik-Ozenci, Ciler

2014-01-01

In the testis, spermatids are polarized cells, with their heads pointing toward the basement membrane during maturation. This polarity is crucial to pack the maximal number of spermatids in the seminiferous epithelium so that millions of sperms can be produced daily. A loss of spermatid polarity is detected after rodents are exposed to toxicants (e.g., cadmium) or nonhormonal male contraceptives (e.g., adjudin), which is associated with a disruption on the expression and/or localization of polarity proteins. In the rat testis, fascin 1, an actin-bundling protein found in mammalian cells, was expressed by Sertoli and germ cells. Fascin 1 was a component of the ectoplasmic specialization (ES), a testis-specific anchoring junction known to confer spermatid adhesion and polarity. Its expression in the seminiferous epithelium was stage specific. Fascin 1 was localized to the basal ES at the Sertoli cell-cell interface of the blood-testis barrier in all stages of the epithelial cycle, except it diminished considerably at late stage VIII. Fascin 1 was highly expressed at the apical ES at stage VII–early stage VIII and restricted to the step 19 spermatids. Its knockdown by RNAi that silenced fascin 1 by ∼70% in Sertoli cells cultured in vitro was found to perturb the tight junction-permeability barrier via a disruption of F-actin organization. Knockdown of fascin 1 in vivo by ∼60–70% induced defects in spermatid polarity, which was mediated by a mislocalization and/or downregulation of actin-bundling proteins Eps8 and palladin, thereby impeding F-actin organization and disrupting spermatid polarity. In summary, these findings provide insightful information on spermatid polarity regulation. PMID:25159326

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

Quantitative Kinetic Study of the Actin-Bundling Protein L-Plastin and of Its Impact on Actin Turn-Over

PubMed Central

Al Tanoury, Ziad; Schaffner-Reckinger, Elisabeth; Halavatyi, Aliaksandr; Hoffmann, Céline; Moes, Michèle; Hadzic, Ermin; Catillon, Marie; Yatskou, Mikalai; Friederich, Evelyne

2010-01-01

Background Initially detected in leukocytes and cancer cells derived from solid tissues, L-plastin/fimbrin belongs to a large family of actin crosslinkers and is considered as a marker for many cancers. Phosphorylation of L-plastin on residue Ser5 increases its F-actin binding activity and is required for L-plastin-mediated cell invasion. Methodology/Principal Findings To study the kinetics of L-plastin and the impact of L-plastin Ser5 phosphorylation on L-plastin dynamics and actin turn-over in live cells, simian Vero cells were transfected with GFP-coupled WT-L-plastin, Ser5 substitution variants (S5/A, S5/E) or actin and analyzed by fluorescence recovery after photobleaching (FRAP). FRAP data were explored by mathematical modeling to estimate steady-state reaction parameters. We demonstrate that in Vero cell focal adhesions L-plastin undergoes rapid cycles of association/dissociation following a two-binding-state model. Phosphorylation of L-plastin increased its association rates by two-fold, whereas dissociation rates were unaffected. Importantly, L-plastin affected actin turn-over by decreasing the actin dissociation rate by four-fold, increasing thereby the amount of F-actin in the focal adhesions, all these effects being promoted by Ser5 phosphorylation. In MCF-7 breast carcinoma cells, phorbol 12-myristate 13-acetate (PMA) treatment induced L-plastin translocation to de novo actin polymerization sites in ruffling membranes and spike-like structures and highly increased its Ser5 phosphorylation. Both inhibition studies and siRNA knock-down of PKC isozymes pointed to the involvement of the novel PKC-δ isozyme in the PMA-elicited signaling pathway leading to L-plastin Ser5 phosphorylation. Furthermore, the L-plastin contribution to actin dynamics regulation was substantiated by its association with a protein complex comprising cortactin, which is known to be involved in this process. Conclusions/Significance Altogether these findings quantitatively

Barrier role of actin filaments in regulated mucin secretion from airway goblet cells.

PubMed

Ehre, Camille; Rossi, Andrea H; Abdullah, Lubna H; De Pestel, Kathleen; Hill, Sandra; Olsen, John C; Davis, C William

2005-01-01

Airway goblet cells secrete mucin onto mucosal surfaces under the regulation of an apical, phospholipase C/G(q)-coupled P2Y(2) receptor. We tested whether cortical actin filaments negatively regulate exocytosis in goblet cells by forming a barrier between secretory granules and plasma membrane docking sites as postulated for other secretory cells. Immunostaining of human lung tissues and SPOC1 cells (an epithelial, mucin-secreting cell line) revealed an apical distribution of beta- and gamma-actin in ciliated and goblet cells. In goblet cells, actin appeared as a prominent subplasmalemmal sheet lying between granules and the apical membrane, and it disappeared from SPOC1 cells activated by purinergic agonist. Disruption of actin filaments with latrunculin A stimulated SPOC1 cell mucin secretion under basal and agonist-activated conditions, whereas stabilization with jasplakinolide or overexpression of beta- or gamma-actin conjugated to yellow fluorescent protein (YFP) inhibited secretion. Myristoylated alanine-rich C kinase substrate, a PKC-activated actin-plasma membrane tethering protein, was phosphorylated after agonist stimulation, suggesting a translocation to the cytosol. Scinderin (or adseverin), a Ca(2+)-activated actin filament severing and capping protein was cloned from human airway and SPOC1 cells, and synthetic peptides corresponding to its actin-binding domains inhibited mucin secretion. We conclude that actin filaments negatively regulate mucin secretion basally in airway goblet cells and are dynamically remodeled in agonist-stimulated cells to promote exocytosis.

Visualization of highly dynamic F-actin plus ends in growing phaseolus vulgaris root hair cells and their responses to Rhizobium etli nod factors.

PubMed

Zepeda, Isaac; Sánchez-López, Rosana; Kunkel, Joseph G; Bañuelos, Luis A; Hernández-Barrera, Alejandra; Sánchez, Federico; Quinto, Carmen; Cárdenas, Luis

2014-03-01

Legume plants secrete signaling molecules called flavonoids into the rhizosphere. These molecules activate the transcription of rhizobial nod genes, which encode proteins involved in the synthesis of signaling compounds named Nod factors (NFs). NFs, in turn, trigger changes in plant gene expression, cortical cell dedifferentiation and mitosis, depolarization of the root hair cell membrane potential and rearrangement of the actin cytoskeleton. Actin polymerization plays an important role in apical growth in hyphae and pollen tubes. Using sublethal concentrations of fluorescently labeled cytochalasin D (Cyt-Fl), we visualized the distribution of filamentous actin (F-actin) plus ends in living Phaseolus vulgaris and Arabidopsis root hairs during apical growth. We demonstrated that Cyt-Fl specifically labeled the newly available plus ends of actin microfilaments, which probably represent sites of polymerization. The addition of unlabeled competing cytochalasin reduced the signal, suggesting that the labeled and unlabeled forms of the drug bind to the same site on F-actin. Exposure to Rhizobium etli NFs resulted in a rapid increase in the number of F-actin plus ends in P. vulgaris root hairs and in the re-localization of F-actin plus ends to infection thread initiation sites. These data suggest that NFs promote the formation of F-actin plus ends, which results in actin cytoskeleton rearrangements that facilitate infection thread formation.

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

Bulkiness or aromatic nature of tyrosine-143 of actin is important for the weak binding between F-actin and myosin-ADP-phosphate

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

Gomibuchi, Yuki; Uyeda, Taro Q.P.; Wakabayashi, Takeyuki, E-mail: tw007@nasu.bio.teikyo-u.ac.jp

2013-11-29

Highlights: •The effect of mutation of Tyr143 that becomes more exposed on assembly was examined. •Mutation of tyrosine-143 of Dictyostelium actin changed actin polymerizability. •The bulkiness or aromatic nature of Tyr143 is important for the weak binding. •The weak interaction between myosin and actin strengthened by Tyr143Trp mutation. -- Abstract: Actin filaments (F-actin) interact with myosin and activate its ATPase to support force generation. By comparing crystal structures of G-actin and the quasi-atomic model of F-actin based on high-resolution cryo-electron microscopy, the tyrosine-143 was found to be exposed more than 60 Å{sup 2} to the solvent in F-actin. Because tyrosine-143more » flanks the hydrophobic cleft near the hydrophobic helix that binds to myosin, the mutant actins, of which the tyrosine-143 was replaced with tryptophan, phenylalanine, or isoleucine, were generated using the Dictyostelium expression system. It polymerized significantly poorly when induced by NaCl, but almost normally by KCl. In the presence of phalloidin and KCl, the extents of the polymerization of all the mutant actins were comparable to that of the wild-type actin so that the actin-activated myosin ATPase activity could be reliably compared. The affinity of skeletal heavy meromyosin to F-actin and the maximum ATPase activity (V{sub max}) were estimated by a double reciprocal plot. The Tyr143Trp-actin showed the higher affinity (smaller K{sub app}) than that of the wild-type actin, with the V{sub max} being almost unchanged. The K{sub app} and V{sub max} of the Tyr143Phe-actin were similar to those of the wild-type actin. However, the activation by Tyr143Ile-actin was much smaller than the wild-type actin and the accurate determination of K{sub app} was difficult. Comparison of the myosin ATPase activated by the various mutant actins at the same concentration of F-actin showed that the extent of activation correlates well with the solvent-accessible surface areas

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

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

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

Interactions between globular proteins and F-actin in isotonic saline solution.

PubMed

Lakatos, S; Minton, A P

1991-10-05

Solutions of each of three different globular proteins (cytochrome c, chromophorically labeled serum albumin, and chromophorically labeled aldolase), mixed with another unlabeled globular protein or with fibrous actin, were prepared in pH 8.0 Tris-HCl buffer containing 0.15 M NaCl. Each solution was centrifuged at low speed, at 5 degrees C, until unassociated globular protein in solution achieved sedimentation equilibrium. Individual absorbance gradients of both macrosolutes in the mixtures subsequent to centrifugation were obtained via optical scans of the centrifuge tubes at two wavelengths. The gradients of each macrosolute in mixtures of two globular proteins revealed no association of globular proteins under the conditions of these experiments, but perturbation of the gradients of serum albumin, aldolase, and cytochrome c in the presence of F-actin indicated association of all three globular proteins with F-actin. Perturbation of actin gradients in the presence of serum albumin and aldolase suggested partial depolymerization of the F-actin by the globular protein. Analysis of the data with a simple phenomenological model relating free globular protein, bound globular protein, and total actin concentration provided estimates of the respective equilibrium constants for association of serum albumin and aldolase with F-actin, under the conditions of these experiments, of the order of 0.1 microM-1.

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

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.

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

Actin Polymerization: An Event Regulated by Tyrosine Phosphorylation During Buffalo Sperm Capacitation.

PubMed

Naresh, S; Atreja, S K

2015-12-01

In the female reproductive tract, the spermatozoa undergo a series of physiological and biochemical changes, prior to gaining the ability to fertilize, that result to capacitation. However, the actin polymerization and protein tyrosine phosphorylation are the two necessary steps for capacitation. In this study, we have demonstrated the actin polymerization and established the correlation between protein tyrosine phosphorylation and actin reorganization during in vitro capacitation in buffalo (Bubalus bubalis) spermatozoa. Indirect immunofluorescence and Western blot techniques were used to detect actin polymerization and tyrosine phosphorylation. The time-dependent fluorimetric studies revealed that the actin polymerization starts from the tail region and progressed towards the head region of spermatozoa during capacitation. The lysophosphatidyl choline (LPC)-induced acrosome reaction (AR) stimulated quick actin depolymerization. The inhibitor cytochalasin D (CD) blocked the in vitro capacitation by inhibiting the actin polymerization. In addition, we also performed different inhibitor (Genistein, H-89, PD9809 and GF-109) and enhancer (dbcAMP, H(2)O(2) and vanadate) studies on actin tyrosine phosphorylation and actin polymerization. The inhibitors of tyrosine phosphorylation inhibit actin tyrosine phosphorylation and polymerization, whereas enhancers of tyrosine phosphorylation stimulate F-actin formation and tyrosine phosphorylation. These observations suggest that the tyrosine phosphorylation regulates the actin polymerization, and both are coupled processes during capacitation of buffalo spermatozoa. © 2015 Blackwell Verlag GmbH.

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.

Aβ mediates F-actin disassembly in dendritic spines leading to cognitive deficits in Alzheimer's disease.

PubMed

Kommaddi, Reddy Peera; Das, Debajyoti; Karunakaran, Smitha; Nanguneri, Siddharth; Bapat, Deepti; Ray, Ajit; Shaw, Eisha; Bennett, David A; Nair, Deepak; Ravindranath, Vijayalakshmi

2018-01-31

Dendritic spine loss is recognized as an early feature of Alzheimer's disease (AD), but the underlying mechanisms are poorly understood. Dendritic spine structure is defined by filamentous actin (F-actin) and we observed depolymerization of synaptosomal F-actin accompanied by increased globular-actin (G-actin) at as early as 1 month of age in a mouse model of AD (APPswe/PS1ΔE9, male mice). This led to recall deficit after contextual fear conditioning (cFC) at 2 months of age in APPswe/PS1ΔE9 male mice, which could be reversed by the actin-polymerizing agent jasplakinolide. Further, the F-actin-depolymerizing agent latrunculin induced recall deficit after cFC in WT mice, indicating the importance of maintaining F-/G-actin equilibrium for optimal behavioral response. Using direct stochastic optical reconstruction microscopy (dSTORM), we show that F-actin depolymerization in spines leads to a breakdown of the nano-organization of outwardly radiating F-actin rods in cortical neurons from APPswe/PS1ΔE9 mice. Our results demonstrate that synaptic dysfunction seen as F-actin disassembly occurs very early, before onset of pathological hallmarks in AD mice, and contributes to behavioral dysfunction, indicating that depolymerization of F-actin is causal and not consequent to decreased spine density. Further, we observed decreased synaptosomal F-actin levels in postmortem brain from mild cognitive impairment and AD patients compared with subjects with normal cognition. F-actin decrease correlated inversely with increasing AD pathology (Braak score, Aβ load, and tangle density) and directly with performance in episodic and working memory tasks, suggesting its role in human disease pathogenesis and progression. SIGNIFICANCE STATEMENT Synaptic dysfunction underlies cognitive deficits in Alzheimer's disease (AD). The cytoskeletal protein actin plays a critical role in maintaining structure and function of synapses. Using cultured neurons and an AD mouse model, we show for the

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.

[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

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.

Circadian actin dynamics drive rhythmic fibroblast mobilisation during wound healing

PubMed Central

Hoyle, Nathaniel P.; Seinkmane, Estere; Putker, Marrit; Feeney, Kevin A.; Krogager, Toke P.; Chesham, Johanna E.; Bray, Liam K.; Thomas, Justyn M.; Dunn, Ken; Blaikley, John; O’Neill, John S.

2017-01-01

Fibroblasts are primary cellular protagonists of wound healing. They also exhibit circadian timekeeping which imparts a ~24-hour rhythm to their biological function. We interrogated the functional consequences of the cell-autonomous clockwork in fibroblasts using a proteome-wide screen for rhythmically expressed proteins. We observed temporal coordination of actin regulators that drives cell-intrinsic rhythms in actin dynamics. In consequence the cellular clock modulates the efficiency of actin-dependent processes such as cell migration and adhesion, which ultimately impact the efficacy of wound healing. Accordingly, skin wounds incurred during a mouse’s active phase exhibited increased fibroblast invasion in vivo and ex vivo, as well as in cultured fibroblasts and keratinocytes. Our experimental results correlate with the observation that the time of injury significantly affects healing after burns in humans, with daytime wounds healing ~60% faster than night-time wounds. We suggest that circadian regulation of the cytoskeleton influences wound healing efficacy from the cellular to the organismal scale. PMID:29118260

IFT88 influences chondrocyte actin organization and biomechanics.

PubMed

Wang, Z; Wann, A K T; Thompson, C L; Hassen, A; Wang, W; Knight, M M

2016-03-01

Primary cilia are microtubule based organelles which control a variety of signalling pathways important in cartilage development, health and disease. This study examines the role of the intraflagellar transport (IFT) protein, IFT88, in regulating fundamental actin organisation and mechanics in articular chondrocytes. The study used an established chondrocyte cell line with and without hypomorphic mutation of IFT88 (IFT88(orpk)). Confocal microscopy was used to quantify F-actin and myosin IIB organisation. Viscoelastic cell and actin cortex mechanics were determined using micropipette aspiration with actin dynamics visualised in live cells transfected with LifeACT-GFP. IFT88(orpk) cells exhibited a significant increase in acto-myosin stress fibre organisation relative to wild-type (WT) cells in monolayer and an altered response to cytochalasin D. Rounded IFT88(orpk) cells cultured in suspension exhibited reduced cortical actin expression with reduced cellular equilibrium modulus. Micropipette aspiration resulted in reduced membrane bleb formation in IFT88(orpk) cells. Following membrane blebbing, IFT88(orpk) cells exhibited slower reformation of the actin cortex. IFT88(orpk) cells showed increased actin deformability and reduced cortical tension confirming that IFT regulates actin cortex mechanics. The reduced cortical tension is also consistent with the reduced bleb formation. This study demonstrates for the first time that the ciliary protein IFT88 regulates fundamental actin organisation and the stiffness of the actin cortex leading to alterations in cell deformation, mechanical properties and blebbing in an IFT88 chondrocyte cell line. This adds to the growing understanding of the role of primary cilia and IFT in regulating cartilage biology. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Regulation of actin dynamics and protein trafficking during spermatogenesis – insights into a complex process*

PubMed Central

Su, Wenhui; Mruk, Dolores; Cheng, C Yan

2013-01-01

In the mammalian testis, extensive restructuring takes place across the seminiferous epithelium at the Sertoli-Sertoli and Sertoli-germ cell interface during the epithelial cycle of spermatogenesis, which is important to facilitate changes in the cell shape and morphology of developing germ cells. However, precise communications also take place at the cell junctions to coordinate the discrete events pertinent to spermatogenesis, namely spermatogonial renewal via mitosis, cell cycle progression and meiosis, spermiogenesis, and spermiation. It is obvious that these cellular events are intimately related to the underlying actin-based cytoskeleton which is being used by different cell junctions for their attachment. However, little is known on the biology and regulation of this cytoskeleton, in particular its possible involvement in endocytic vesicle-mediated trafficking during spermatogenesis, which in turn affects cell adhesive function and communication at the cell-cell interface. Studies in other epithelia in recent years have shed insightful information on the intimate involvement of actin dynamics and protein trafficking in regulating cell adhesion and communications. The goal of this critical review is to provide an updated assessment of the latest findings in the field on how these complex processes regulate spermatogenesis. We also provide a working model based on the latest findings in the field to provide our thoughts on an apparent complicated subject, which also serves as the framework for investigators in the field. It is obvious that this model will be rapidly updated when more data are available in future years. PMID:23339542

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.

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.

Cortactin binding to F-actin revealed by electron microscopy and 3D reconstruction.

PubMed

Pant, Kiran; Chereau, David; Hatch, Victoria; Dominguez, Roberto; Lehman, William

2006-06-16

Cortactin and WASP activate Arp2/3-mediated actin filament nucleation and branching. However, different mechanisms underlie activation by the two proteins, which rely on distinct actin-binding modules and modes of binding to actin filaments. It is generally thought that cortactin binds to "mother" actin filaments, while WASP donates actin monomers to Arp2/3-generated "daughter" filament branches. Interestingly, cortactin also binds WASP in addition to F-actin and the Arp2/3 complex. However, the structural basis for the role of cortactin in filament branching remains unknown, making interpretation difficult. Here, electron microscopy and 3D reconstruction were carried out on F-actin decorated with the actin-binding repeating domain of cortactin, revealing conspicuous density on F-actin attributable to cortactin that is located on a consensus-binding site on subdomain-1 of actin subunits. Strikingly, the binding of cortactin widens the gap between the two long-pitch filament strands. Although other proteins have been found to alter the structure of the filament, the cortactin-induced conformational change appears unique. The results are consistent with a mechanism whereby alterations of the F-actin structure may facilitate recruitment of the Arp2/3 complex to the "mother" filament in the cortex of cells. In addition, cortactin may act as a structural adapter protein, stabilizing nascent filament branches while mediating the simultaneous recruitment of Arp2/3 and WASP.

Stromal cell-derived factor 1 regulates the actin organization of chondrocytes and chondrocyte hypertrophy.

PubMed

Murata, Koichi; Kitaori, Toshiyuki; Oishi, Shinya; Watanabe, Naoki; Yoshitomi, Hiroyuki; Tanida, Shimei; Ishikawa, Masahiro; Kasahara, Takashi; Shibuya, Hideyuki; Fujii, Nobutaka; Nagasawa, Takashi; Nakamura, Takashi; Ito, Hiromu

2012-01-01

Stromal cell-derived factor 1 (SDF-1/CXCL12/PBSF) plays important roles in the biological and physiological functions of haematopoietic and mesenchymal stem cells. This chemokine regulates the formation of multiple organ systems during embryogenesis. However, its roles in skeletal development remain unclear. Here we investigated the roles of SDF-1 in chondrocyte differentiation. We demonstrated that SDF-1 protein was expressed at pre-hypertrophic and hypertrophic chondrocytes in the newly formed endochondral callus of rib fracture as well as in the growth plate of normal mouse tibia by immunohistochemical analysis. Using SDF-1(-/-) mouse embryo, we histologically showed that the total length of the whole humeri of SDF-1(-/-) mice was significantly shorter than that of wild-type mice, which was contributed mainly by shorter hypertrophic and calcified zones in SDF-1(-/-) mice. Actin cytoskeleton of hypertrophic chondrocytes in SDF-1(-/-) mouse humeri showed less F-actin and rounder shape than that of wild-type mice. Primary chondrocytes from SDF-1(-/-) mice showed the enhanced formation of philopodia and loss of F-actin. The administration of SDF-1 to primary chondrocytes of wild-type mice and SDF-1(-/-) mice promoted the formation of actin stress fibers. Organ culture of embryonic metatarsals from SDF-1(-/-) mice showed the growth delay, which was recovered by an exogenous administration of SDF-1. mRNA expression of type X collagen in metatarsals and in primary chondrocytes of SDF-1(-/-) mouse embryo was down-regulated while the administration of SDF-1 to metatarsals recovered. These data suggests that SDF-1 regulates the actin organization and stimulates bone growth by mediating chondrocyte hypertrophy.

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.

PI(4,5)P2 regulates myoblast fusion through Arp2/3 regulator localization at the fusion site

PubMed Central

Bothe, Ingo; Deng, Su; Baylies, Mary

2014-01-01

Cell-cell fusion is a regulated process that requires merging of the opposing membranes and underlying cytoskeletons. However, the integration between membrane and cytoskeleton signaling during fusion is not known. Using Drosophila, we demonstrate that the membrane phosphoinositide PI(4,5)P2 is a crucial regulator of F-actin dynamics during myoblast fusion. PI(4,5)P2 is locally enriched and colocalizes spatially and temporally with the F-actin focus that defines the fusion site. PI(4,5)P2 enrichment depends on receptor engagement but is upstream or parallel to actin remodeling. Regulators of actin branching via Arp2/3 colocalize with PI(4,5)P2 in vivo and bind PI(4,5)P2 in vitro. Manipulation of PI(4,5)P2 availability leads to impaired fusion, with a reduction in the F-actin focus size and altered focus morphology. Mechanistically, the changes in the actin focus are due to a failure in the enrichment of actin regulators at the fusion site. Moreover, improper localization of these regulators hinders expansion of the fusion interface. Thus, PI(4,5)P2 enrichment at the fusion site encodes spatial and temporal information that regulates fusion progression through the localization of activators of actin polymerization. PMID:24821989

A glycolytic metabolon in Saccharomyces cerevisiae is stabilized by F-actin.

PubMed

Araiza-Olivera, Daniela; Chiquete-Felix, Natalia; Rosas-Lemus, Mónica; Sampedro, José G; Peña, Antonio; Mujica, Adela; Uribe-Carvajal, Salvador

2013-08-01

In the Saccharomyces cerevisiae glycolytic pathway, 11 enzymes catalyze the stepwise conversion of glucose to two molecules of ethanol plus two CO₂ molecules. In the highly crowded cytoplasm, this pathway would be very inefficient if it were dependent on substrate/enzyme diffusion. Therefore, the existence of a multi-enzymatic glycolytic complex has been suggested. This complex probably uses the cytoskeleton to stabilize the interaction of the various enzymes. Here, the role of filamentous actin (F-actin) in stabilization of a putative glycolytic metabolon is reported. Experiments were performed in isolated enzyme/actin mixtures, cytoplasmic extracts and permeabilized yeast cells. Polymerization of actin was promoted using phalloidin or inhibited using cytochalasin D or latrunculin. The polymeric filamentous F-actin, but not the monomeric globular G-actin, stabilized both the interaction of isolated glycolytic pathway enzyme mixtures and the whole fermentation pathway, leading to higher fermentation activity. The associated complexes were resistant against inhibition as a result of viscosity (promoted by the disaccharide trehalose) or inactivation (using specific enzyme antibodies). In S. cerevisiae, a glycolytic metabolon appear to assemble in association with F-actin. In this complex, fermentation activity is enhanced and enzymes are partially protected against inhibition by trehalose or by antibodies. © 2013 FEBS.

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

Environmental toxicants perturb human Sertoli cell adhesive function via changes in F-actin organization mediated by actin regulatory proteins

PubMed Central

Xiao, Xiang; Mruk, Dolores D.; Tang, Elizabeth I.; Wong, Chris K.C.; Lee, Will M.; John, Constance M.; Turek, Paul J.; Silvestrini, Bruno; Cheng, C. Yan

2014-01-01

STUDY QUESTION Can human Sertoli cells cultured in vitro and that have formed an epithelium be used as a model to monitor toxicant-induced junction disruption and to better understand the mechanism(s) by which toxicants disrupt cell adhesion at the Sertoli cell blood–testis barrier (BTB)? SUMMARY ANSWER Our findings illustrate that human Sertoli cells cultured in vitro serve as a reliable system to monitor the impact of environmental toxicants on the BTB function. WHAT IS KNOWN ALREADY Suspicions of a declining trend in semen quality and a concomitant increase in exposures to environmental toxicants over the past decades reveal the need of an in vitro system that efficiently and reliably monitors the impact of toxicants on male reproductive function. Furthermore, studies in rodents have confirmed that environmental toxicants impede Sertoli cell BTB function in vitro and in vivo. STUDY DESIGN, SIZE AND DURATION We examined the effects of two environmental toxicants: cadmium chloride (0.5–20 µM) and bisphenol A (0.4–200 µM) on human Sertoli cell function. Cultured Sertoli cells from three men were used in this study, which spanned an 18-month period. PARTICIPANTS/MATERIALS, SETTING, METHODS Human Sertoli cells from three subjects were cultured in F12/DMEM containing 5% fetal bovine serum. Changes in protein expression were monitored by immunoblotting using specific antibodies. Immunofluorescence analyses were used to assess changes in the distribution of adhesion proteins, F-actin and actin regulatory proteins following exposure to two toxicants: cadmium chloride and bisphenol A (BPA). MAIN RESULTS AND THE ROLE OF CHANCE Human Sertoli cells were sensitive to cadmium and BPA toxicity. Changes in the localization of cell adhesion proteins were mediated by an alteration of the actin-based cytoskeleton. This alteration of F-actin network in Sertoli cells as manifested by truncation and depolymerization of actin microfilaments at the Sertoli cell BTB was caused by

Kinetic analysis of F-actin depolymerization in polymorphonuclear leukocyte lysates indicates that chemoattractant stimulation increases actin filament number without altering the filament length distribution

PubMed Central

1991-01-01

The rate of filamentous actin (F-actin) depolymerization is proportional to the number of filaments depolarizing and changes in the rate are proportional to changes in filament number. To determine the number and length of actin filaments in polymorphonuclear leukocytes and the change in filament number and length that occurs during the increase in F-actin upon chemoattractant stimulation, the time course of cellular F-actin depolymerization in lysates of control and peptide- stimulated cells was examined. F-actin was quantified by the TRITC- labeled phalloidin staining of pelletable actin. Lysis in 1.2 M KCl and 10 microM DNase I minimized the effects of F-actin binding proteins and G-actin, respectively, on the kinetics of depolymerization. To determine filament number and length from a depolymerization time course, depolymerization kinetics must be limited by the actin monomer dissociation rate. Comparison of time courses of depolymerization in the presence (pointed ends free) or absence (barbed and pointed ends free) of cytochalasin suggested depolymerization occurred from both ends of the filament and that monomer dissociation was rate limiting. Control cells had 1.7 +/- 0.4 x 10(5) filaments with an average length of 0.29 +/- 0.09 microns. Chemo-attractant stimulation for 90 s at room temperature with 0.02 microM N-formylnorleucylleucylphenylalanine caused a twofold increase in F-actin and about a two-fold increase in the total number of actin filaments to 4.0 +/- 0.5 x 10(5) filaments with an average length of 0.27 +/- 0.07 microns. In both cases, most (approximately 80%) of the filaments were quite short (less than or equal to 0.18 micron). The length distributions of actin filaments in stimulated and control cells were similar. PMID:1918158

A WASp–VASP complex regulates actin polymerization at the plasma membrane

PubMed Central

Castellano, Flavia; Le Clainche, Christophe; Patin, Delphine; Carlier, Marie-France; Chavrier, Philippe

2001-01-01

Proteins of the Wiskott–Aldrich syndrome and Ena/VASP families both play essential functions in the regulation of actin dynamics at the cell leading edge. However, possibilities of functional interplay between members of these two families have not been addressed. Here we show that, in hemopoietic cells, recruitment of the C-terminal VCA (Verprolin homology, Cofilin homology, Acidic) domain of WASp at the plasma membrane by a ligand technique using rapamycin as an intermediate is not sufficient to elicit efficient Arp2/3 complex-mediated actin polymerization. Other domains of WASp, in particular the proline-rich domain, are required for the formation of actin-rich structures. An in vitro analysis demonstrates that the proline-rich domain of WASp binds VASP with an affinity of ∼106 M–1. In addition, WASp and VASP both accumulate in actin-rich phagocytic cups. Finally, in a reconstituted motility medium, VASP enhances actin-based propulsion of WASp-coated beads in a fashion reminiscent of its effect on Listeria movement. We propose that VASP and WASp cooperation is essential in stimulating actin assembly and membrane protrusion at the leading edge. PMID:11598004

Oxidation of F-actin controls the terminal steps of cytokinesis

PubMed Central

Frémont, Stéphane; Hammich, Hussein; Bai, Jian; Wioland, Hugo; Klinkert, Kerstin; Rocancourt, Murielle; Kikuti, Carlos; Stroebel, David; Romet-Lemonne, Guillaume; Pylypenko, Olena; Houdusse, Anne; Echard, Arnaud

2017-01-01

Cytokinetic abscission, the terminal step of cell division, crucially depends on the local constriction of ESCRT-III helices after cytoskeleton disassembly. While the microtubules of the intercellular bridge are cut by the ESCRT-associated enzyme Spastin, the mechanism that clears F-actin at the abscission site is unknown. Here we show that oxidation-mediated depolymerization of actin by the redox enzyme MICAL1 is key for ESCRT-III recruitment and successful abscission. MICAL1 is recruited to the abscission site by the Rab35 GTPase through a direct interaction with a flat three-helix domain found in MICAL1 C terminus. Mechanistically, in vitro assays on single actin filaments demonstrate that MICAL1 is activated by Rab35. Moreover, in our experimental conditions, MICAL1 does not act as a severing enzyme, as initially thought, but instead induces F-actin depolymerization from both ends. Our work reveals an unexpected role for oxidoreduction in triggering local actin depolymerization to control a fundamental step of cell division. PMID:28230050

Cytoskeletal actin dynamics shape a ramifying actin network underpinning immunological synapse formation

PubMed Central

Fritzsche, Marco; Fernandes, Ricardo A.; Chang, Veronica T.; Colin-York, Huw; Clausen, Mathias P.; Felce, James H.; Galiani, Silvia; Erlenkämper, Christoph; Santos, Ana M.; Heddleston, John M.; Pedroza-Pacheco, Isabela; Waithe, Dominic; de la Serna, Jorge Bernardino; Lagerholm, B. Christoffer; Liu, Tsung-li; Chew, Teng-Leong; Betzig, Eric; Davis, Simon J.; Eggeling, Christian

2017-01-01

T cell activation and especially trafficking of T cell receptor microclusters during immunological synapse formation are widely thought to rely on cytoskeletal remodeling. However, important details on the involvement of actin in the latter transport processes are missing. Using a suite of advanced optical microscopes to analyze resting and activated T cells, we show that, following contact formation with activating surfaces, these cells sequentially rearrange their cortical actin across the entire cell, creating a previously unreported ramifying actin network above the immunological synapse. This network shows all the characteristics of an inward-growing transportation network and its dynamics correlating with T cell receptor rearrangements. This actin reorganization is accompanied by an increase in the nanoscale actin meshwork size and the dynamic adjustment of the turnover times and filament lengths of two differently sized filamentous actin populations, wherein formin-mediated long actin filaments support a very flat and stiff contact at the immunological synapse interface. The initiation of immunological synapse formation, as highlighted by calcium release, requires markedly little contact with activating surfaces and no cytoskeletal rearrangements. Our work suggests that incipient signaling in T cells initiates global cytoskeletal rearrangements across the whole cell, including a stiffening process for possibly mechanically supporting contact formation at the immunological synapse interface as well as a central ramified transportation network apparently directed at the consolidation of the contact and the delivery of effector functions. PMID:28691087

Ezrin is down-regulated in diabetic kidney glomeruli and regulates actin reorganization and glucose uptake via GLUT1 in cultured podocytes.

PubMed

Wasik, Anita A; Koskelainen, Susanna; Hyvönen, Mervi E; Musante, Luca; Lehtonen, Eero; Koskenniemi, Kerttu; Tienari, Jukka; Vaheri, Antti; Kerjaschki, Dontscho; Szalay, Csaba; Révész, Csaba; Varmanen, Pekka; Nyman, Tuula A; Hamar, Peter; Holthöfer, Harry; Lehtonen, Sanna

2014-06-01

Diabetic nephropathy is a complication of diabetes and a major cause of end-stage renal disease. To characterize the early pathophysiological mechanisms leading to glomerular podocyte injury in diabetic nephropathy, we performed quantitative proteomic profiling of glomeruli isolated from rats with streptozotocin-induced diabetes and controls. Fluorescence-based two-dimensional difference gel electrophoresis, coupled with mass spectrometry, identified 29 differentially expressed spots, including actin-binding protein ezrin and its interaction partner, NHERF2, which were down-regulated in the streptozotocin group. Knockdown of ezrin by siRNA in cultured podocytes increased glucose uptake compared with control siRNA-transfected cells, apparently by increasing translocation of glucose transporter GLUT1 to the plasma membrane. Knockdown of ezrin also induced actin remodeling under basal conditions, but reduced insulin-stimulated actin reorganization. Ezrin-dependent actin remodeling involved cofilin-1 that is essential for the turnover and reorganization of actin filaments. Phosphorylated, inactive cofilin-1 was up-regulated in diabetic glomeruli, suggesting altered actin dynamics. Furthermore, IHC analysis revealed reduced expression of ezrin in the podocytes of patients with diabetes. Our findings suggest that ezrin may play a role in the development of the renal complication in diabetes by regulating transport of glucose and organization of the actin cytoskeleton in podocytes. Copyright © 2014 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Strong Dependence of Hydration State of F-Actin on the Bound Mg(2+)/Ca(2+) Ions.

PubMed

Suzuki, Makoto; Imao, Asato; Mogami, George; Chishima, Ryotaro; Watanabe, Takahiro; Yamaguchi, Takaya; Morimoto, Nobuyuki; Wazawa, Tetsuichi

2016-07-21

Understanding of the hydration state is an important issue in the chemomechanical energetics of versatile biological functions of polymerized actin (F-actin). In this study, hydration-state differences of F-actin by the bound divalent cations are revealed through precision microwave dielectric relaxation (DR) spectroscopy. G- and F-actin in Ca- and Mg-containing buffer solutions exhibit dual hydration components comprising restrained water with DR frequency f2 (f1 (>fw). The hydration state of F-actin is strongly dependent on the ionic composition. In every buffer tested, the HMW signal Dhyme (≡ (f1 - fw)δ1/(fwδw)) of F-actin is stronger than that of G-actin, where δw is DR-amplitude of bulk solvent and δ1 is that of HMW in a fixed-volume ellipsoid containing an F-actin and surrounding water in solution. Dhyme value of F-actin in Ca2.0-buffer (containing 2 mM Ca(2+)) is markedly higher than in Mg2.0-buffer (containing 2 mM Mg(2+)). Moreover, in the presence of 2 mM Mg(2+), the hydration state of F-actin is changed by adding a small fraction of Ca(2+) (∼0.1 mM) and becomes closer to that of the Ca-bound form in Ca2.0-buffer. This is consistent with the results of the partial specific volume and the Cotton effect around 290 nm in the CD spectra, indicating a change in the tertiary structure and less apparent change in the secondary structure of actin. The number of restrained water molecules per actin (N2) is estimated to be 1600-2100 for Ca2.0- and F-buffer and ∼2500 for Mg2.0-buffer at 10-15 °C. These numbers are comparable to those estimated from the available F-actin atomic structures as in the first water layer. The number of HMW molecules is roughly explained by the volume between the equipotential surface of -kT/2e and the first water layer of the actin surface by solving the Poisson-Boltzmann equation using UCSF Chimera.

Activator-inhibitor coupling between Rho signaling and actin assembly make the cell cortex an excitable medium

PubMed Central

Bement, William M.; Leda, Marcin; Moe, Alison M.; Kita, Angela M.; Larson, Matthew E.; Golding, Adriana E.; Pfeuti, Courtney; Su, Kuan-Chung; Miller, Ann L.; Goryachev, Andrew B.; von Dassow, George

2016-01-01

Animal cell cytokinesis results from patterned activation of the small GTPase Rho, which directs assembly of actomyosin in the equatorial cortex. Cytokinesis is restricted to a portion of the cell cycle following anaphase onset in which the cortex is responsive to signals from the spindle. We show that shortly after anaphase onset oocytes and embryonic cells of frogs and echinoderms exhibit cortical waves of Rho activity and F-actin polymerization. The waves are modulated by cyclin-dependent kinase 1 (Cdk1) activity and require the Rho GEF (guanine nucleotide exchange factor), Ect2. Surprisingly, during wave propagation, while Rho activity elicits F-actin assembly, F-actin subsequently inactivates Rho. Experimental and modeling results show that waves represent excitable dynamics of a reaction diffusion system with Rho as the activator and F-actin the inhibitor. We propose that cortical excitability explains fundamental features of cytokinesis including its cell cycle regulation. PMID:26479320

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

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

The actin cytoskeleton inhibits pore expansion during PIV5 fusion protein-promoted cell-cell fusion

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

Wurth, Mark A.; Schowalter, Rachel M.; Smith, Everett Clinton

2010-08-15

Paramyxovirus fusion (F) proteins promote both virus-cell fusion, required for viral entry, and cell-cell fusion, resulting in syncytia formation. We used the F-actin stabilizing drug, jasplakinolide, and the G-actin sequestrant, latrunculin A, to examine the role of actin dynamics in cell-cell fusion mediated by the parainfluenza virus 5 (PIV5) F protein. Jasplakinolide treatment caused a dose-dependent increase in cell-cell fusion as measured by both syncytia and reporter gene assays, and latrunculin A treatment also resulted in fusion stimulation. Treatment with jasplakinolide or latrunculin A partially rescued a fusion pore opening defect caused by deletion of the PIV5 F protein cytoplasmicmore » tail, but these drugs had no effect on fusion inhibited at earlier stages by either temperature arrest or by a PIV5 heptad repeat peptide. These data suggest that the cortical actin cytoskeleton is an important regulator of fusion pore enlargement, an energetically costly stage of viral fusion protein-mediated membrane merger.« less

The actin cytoskeleton inhibits pore expansion during PIV5 fusion protein-promoted cell-cell fusion

PubMed Central

Wurth, Mark A.; Schowalter, Rachel M.; Smith, Everett Clinton; Moncman, Carole L.; Dutch, Rebecca Ellis; McCann, Richard O.

2010-01-01

Paramyxovirus fusion (F) proteins promote both virus-cell fusion, required for viral entry, and cell-cell fusion, resulting in syncytia formation. We used the F-actin stabilizing drug, jasplakinolide, and the G-actin sequestrant, latrunculin A, to examine the role of actin dynamics in cell-cell fusion mediated by the parainfluenza virus 5 (PIV5) F protein. Jasplakinolide treatment caused a dose-dependent increase in cell-cell fusion as measured by both syncytia and reporter gene assays, and latrunculin A treatment also resulted in fusion stimulation. Treatment with jasplakinolide or latrunculin A partially rescued a fusion pore opening defect caused by deletion of the PIV5 F protein cytoplasmic tail, but these drugs had no effect on fusion inhibited at earlier stages by either temperature arrest or by a PIV5 heptad repeat peptide. These data suggest that the cortical actin cytoskeleton is an important regulator of fusion pore enlargement, an energetically costly stage of viral fusion protein-mediated membrane merger. PMID:20537366

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

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

Polarized Growth in the Absence of F-Actin in Saccharomyces cerevisiae Exiting Quiescence

PubMed Central

Sahin, Annelise; Daignan-Fornier, Bertrand; Sagot, Isabelle

2008-01-01

Background Polarity establishment and maintenance are crucial for morphogenesis and development. In budding yeast, these two intricate processes involve the superposition of regulatory loops between polarity landmarks, RHO GTPases, actin-mediated vesicles transport and endocytosis. Deciphering the chronology and the significance of each molecular step of polarized growth is therefore very challenging. Principal Findings We have taken advantage of the fact that yeast quiescent cells display actin bodies, a non polarized actin structure, to evaluate the role of F-actin in bud emergence. Here we show that upon exit from quiescence, actin cables are not required for the first steps of polarized growth. We further show that polarized growth can occur in the absence of actin patch-mediated endocytosis. We finally establish, using latrunculin-A, that the first steps of polarized growth do not require any F-actin containing structures. Yet, these structures are required for the formation of a bona fide daughter cell and cell cycle completion. We propose that upon exit from quiescence in the absence of F-actin, secretory vesicles randomly reach the plasma membrane but preferentially dock and fuse where polarity cues are localized, this being sufficient to trigger polarized growth. PMID:18596916

Rab8 Regulates the Actin-based Movement of MelanosomesV⃞

PubMed Central

Chabrillat, Marion L.; Wilhelm, Claire; Wasmeier, Christina; Sviderskaya, Elena V.; Louvard, Daniel; Coudrier, Evelyne

2005-01-01

Rab GTPases have been implicated in the regulation of specific microtubule- and actin-based motor proteins. We devised an in vitro motility assay reconstituting the movement of melanosomes on actin bundles in the presence of ATP to investigate the role of Rab proteins in the actin-dependent movement of melanosomes. Using this assay, we confirmed that Rab27 is required for the actin-dependent movement of melanosomes, and we showed that a second Rab protein, Rab8, also regulates this movement. Rab8 was partially associated with mature melanosomes. Expression of Rab8Q67L perturbed the cellular distribution and increased the frequency of microtubule-independent movement of melanosomes in vivo. Furthermore, anti-Rab8 antibodies decreased the number of melanosomes moving in vitro on actin bundles, whereas melanosomes isolated from cells expressing Rab8Q67L exhibited 70% more movements than wild-type melanosomes. Together, our observations suggest that Rab8 is involved in regulating the actin-dependent movement of melanosomes. PMID:15673612

Dynamics between actin and the VE-cadherin/catenin complex

PubMed Central

Abu Taha, Abdallah; Schnittler, Hans-J

2014-01-01

Endothelial adherens junctions are critical for physiological and pathological processes such as differentiation, maintenance of entire monolayer integrity, and the remodeling. The endothelial-specific VE-cadherin/catenin complex provides the backbone of adherens junctions and acts in close interaction with actin filaments and actin/myosin-mediated contractility to fulfill the junction demands. The functional connection between the cadherin/catenin complex and actin filaments might be either directly through α-catenins, or indirectly e.g., via linker proteins such as vinculin, p120ctn, α-actinin, or EPLIN. However, both junction integrity and dynamic remodeling have to be contemporarily coordinated. The actin-related protein complex ARP2/3 and its activating molecules, such as N-WASP and WAVE, have been shown to regulate the lammellipodia-mediated formation of cell junctions in both epithelium and endothelium. Recent reports now demonstrate a novel aspect of the ARP2/3 complex and the nucleating-promoting factors in the maintenance of endothelial barrier function and junction remodeling of established endothelial cell junctions. Those mechanisms open novel possibilities; not only in fulfilling physiological demands but obtained information may be of critical importance in pathologies such as wound healing, angiogenesis, inflammation, and cell diapedesis. PMID:24621569

Actin retrograde flow controls natural killer cell response by regulating the conformation state of SHP-1.

PubMed

Matalon, Omri; Ben-Shmuel, Aviad; Kivelevitz, Jessica; Sabag, Batel; Fried, Sophia; Joseph, Noah; Noy, Elad; Biber, Guy; Barda-Saad, Mira

2018-03-01

Natural killer (NK) cells are a powerful weapon against viral infections and tumor growth. Although the actin-myosin (actomyosin) cytoskeleton is crucial for a variety of cellular processes, the role of mechanotransduction, the conversion of actomyosin mechanical forces into signaling cascades, was never explored in NK cells. Here, we demonstrate that actomyosin retrograde flow (ARF) controls the immune response of primary human NK cells through a novel interaction between β-actin and the SH2-domain-containing protein tyrosine phosphatase-1 (SHP-1), converting its conformation state, and thereby regulating NK cell cytotoxicity. Our results identify ARF as a master regulator of the NK cell immune response. Since actin dynamics occur in multiple cellular processes, this mechanism might also regulate the activity of SHP-1 in additional cellular systems. © 2018 The Authors.

Ligand-mediated Galectin-1 endocytosis prevents intraneural H2O2 production promoting F-actin dynamics reactivation and axonal re-growth.

PubMed

Quintá, Héctor R; Wilson, Carlos; Blidner, Ada G; González-Billault, Christian; Pasquini, Laura A; Rabinovich, Gabriel A; Pasquini, Juana M

2016-09-01

Axonal growth cone collapse following spinal cord injury (SCI) is promoted by semaphorin3A (Sema3A) signaling via PlexinA4 surface receptor. This interaction triggers intracellular signaling events leading to increased hydrogen peroxide levels which in turn promote filamentous actin (F-actin) destabilization and subsequent inhibition of axonal re-growth. In the current study, we demonstrated that treatment with galectin-1 (Gal-1), in its dimeric form, promotes a decrease in hydrogen peroxide (H2O2) levels and F-actin repolimerization in the growth cone and in the filopodium of neuron surfaces. This effect was dependent on the carbohydrate recognition activity of Gal-1, as it was prevented using a Gal-1 mutant lacking carbohydrate-binding activity. Furthermore, Gal-1 promoted its own active ligand-mediated endocytosis together with the PlexinA4 receptor, through mechanisms involving complex branched N-glycans. In summary, our results suggest that Gal-1, mainly in its dimeric form, promotes re-activation of actin cytoskeleton dynamics via internalization of the PlexinA4/Gal-1 complex. This mechanism could explain, at least in part, critical events in axonal regeneration including the full axonal re-growth process, de novo formation of synapse clustering, axonal re-myelination and functional recovery of coordinated locomotor activities in an in vivo acute and chronic SCI model. Axonal regeneration is a response of injured nerve cells critical for nerve repair in human spinal cord injury. Understanding the molecular mechanisms controlling nerve repair by Galectin-1, may be critical for therapeutic intervention. Our results show that Galectin-1; in its dimeric form, interferes with hydrogen peroxide production triggered by Semaphorin3A. The high levels of this reactive oxygen species (ROS) seem to be the main factor preventing axonal regeneration due to promotion of actin depolymerization at the axonal growth cone. Thus, Galectin-1 administration emerges as a novel

The Differential Organization of F-Actin Alters the Distribution of Organelles in Cultured When Compared to Native Chromaffin Cells

PubMed Central

Gimenez-Molina, Yolanda; Villanueva, José; Nanclares, Carmen; Lopez-Font, Inmaculada; Viniegra, Salvador; Francés, Maria del Mar; Gandia, Luis; Gil, Amparo; Gutiérrez, Luis M.

2017-01-01

Cultured bovine chromaffin cells have been used extensively as a neuroendocrine model to study regulated secretion. In order to extend such experimental findings to the physiological situation, it is necessary to study mayor cellular structures affecting secretion in cultured cells with their counterparts present in the adrenomedullary tissue. F-actin concentrates in a peripheral ring in cultured cells, as witnessed by phalloidin–rodhamine labeling, while extends throughout the cytoplasm in native cells. This result is also confirmed when studying the localization of α-fodrin, a F-actin-associated protein. Furthermore, as a consequence of this redistribution of F-actin, we observed that chromaffin granules and mitochondria located into two different cortical and internal populations in cultured cells, whereas they are homogeneously distributed throughout the cytoplasm in the adrenomedullary tissue. Nevertheless, secretion from isolated cells and adrenal gland pieces is remarkably similar when measured by amperometry. Finally, we generate mathematical models to consider how the distribution of organelles affects the secretory kinetics of intact and cultured cells. Our results imply that we have to consider F-actin structural changes to interpret functional data obtained in cultured neuroendocrine cells. PMID:28522964

eNOS S-nitrosylates β-actin on Cys374 and regulates PKC-θ at the immune synapse by impairing actin binding to profilin-1

PubMed Central

García-Ortiz, Almudena; Martín-Cofreces, Noa B.; Ibiza, Sales; Ortega, Ángel; Izquierdo-Álvarez, Alicia; Trullo, Antonio; Victor, Víctor M.; Calvo, Enrique; Sot, Begoña; Martínez-Ruiz, Antonio; Vázquez, Jesús; Sánchez-Madrid, Francisco

2017-01-01

The actin cytoskeleton coordinates the organization of signaling microclusters at the immune synapse (IS); however, the mechanisms involved remain poorly understood. We show here that nitric oxide (NO) generated by endothelial nitric oxide synthase (eNOS) controls the coalescence of protein kinase C-θ (PKC-θ) at the central supramolecular activation cluster (c-SMAC) of the IS. eNOS translocated with the Golgi to the IS and partially colocalized with F-actin around the c-SMAC. This resulted in reduced actin polymerization and centripetal retrograde flow of β-actin and PKC-θ from the lamellipodium-like distal (d)-SMAC, promoting PKC-θ activation. Furthermore, eNOS-derived NO S-nitrosylated β-actin on Cys374 and impaired actin binding to profilin-1 (PFN1), as confirmed with the transnitrosylating agent S-nitroso-L-cysteine (Cys-NO). The importance of NO and the formation of PFN1-actin complexes on the regulation of PKC-θ was corroborated by overexpression of PFN1- and actin-binding defective mutants of β-actin (C374S) and PFN1 (H119E), respectively, which reduced the coalescence of PKC-θ at the c-SMAC. These findings unveil a novel NO-dependent mechanism by which the actin cytoskeleton controls the organization and activation of signaling microclusters at the IS. PMID:28394935

p21-Activated kinase (Pak) regulates airway smooth muscle contraction by regulating paxillin complexes that mediate actin polymerization.

PubMed

Zhang, Wenwu; Huang, Youliang; Gunst, Susan J

2016-09-01

tissue extracts and proximity ligation assays in dissociated cells showed that Pak activation and paxillin Ser273 phosphorylation triggered the formation of an adhesion junction signalling complex with paxillin that included G-protein-coupled receptor kinase-interacting protein (GIT1) and the cdc42 guanine exchange factor, βPIX (Pak interactive exchange factor). Assembly of the Pak-GIT1-βPIX-paxillin complex was necessary for cdc42 and neuronal Wiskott-Aldrich syndrome protein (N-WASP) activation, actin polymerization and contraction in response to ACh. RhoA activation was also required for the recruitment of Pak to adhesion junctions, Pak activation, paxillin Ser273 phosphorylation and paxillin complex assembly. These studies demonstrate a novel role for Pak in the regulation of N-WASP activation, actin dynamics and cell contractility. © 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.

Dendrite architecture organized by transcriptional control of the F-actin nucleator Spire.

PubMed

Ferreira, Tiago; Ou, Yimiao; Li, Sally; Giniger, Edward; van Meyel, Donald J

2014-02-01

The architectures of dendritic trees are crucial for the wiring and function of neuronal circuits because they determine coverage of receptive territories, as well as the nature and strength of sensory or synaptic inputs. Here, we describe a cell-intrinsic pathway sculpting dendritic arborization (da) neurons in Drosophila that requires Longitudinals Lacking (Lola), a BTB/POZ transcription factor, and its control of the F-actin cytoskeleton through Spire (Spir), an actin nucleation protein. Loss of Lola from da neurons reduced the overall length of dendritic arbors, increased the expression of Spir, and produced inappropriate F-actin-rich dendrites at positions too near the cell soma. Selective removal of Lola from only class IV da neurons decreased the evasive responses of larvae to nociception. The increased Spir expression contributed to the abnormal F-actin-rich dendrites and the decreased nocifensive responses because both were suppressed by reduced dose of Spir. Thus, an important role of Lola is to limit expression of Spir to appropriate levels within da neurons. We found Spir to be expressed in dendritic arbors and to be important for their development. Removal of Spir from class IV da neurons reduced F-actin levels and total branch number, shifted the position of greatest branch density away from the cell soma, and compromised nocifensive behavior. We conclude that the Lola-Spir pathway is crucial for the spatial arrangement of branches within dendritic trees and for neural circuit function because it provides balanced control of the F-actin cytoskeleton.

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.

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

Binding mode of cytochalasin B to F-actin is altered by lateral binding of regulatory proteins.

PubMed

Suzuki, N; Mihashi, K

1991-01-01

The binding of cytochalasin B (CB) to F-actin was studied using a trace amount of [3H]-cytochalasin B. F-Actin-bound CB was separated from free CB by ultracentrifugation and the amount of F-actin-bound CB was determined by comparing the radioactivity both in the supernatant and in the precipitate. A filament of pure F-actin possessed one high-affinity binding site for CB (Kd = 5.0 nM) at the B-end. When the filament was bound to native tropomyosin (complex of tropomyosin and troponin), two low-affinity binding sites for CB (Kd = 230 nM) were created, while the high-affinity binding site was reserved (Kd = 3.4 nM). It was concluded that the creation of low-affinity binding sites was primarily due to binding of tropomyosin to F-actin, as judged from the following two observations: (1) a filament of F-actin/tropomyosin complex possessed one high-affinity binding site (Kd = 3.9 nM) plus two low-affinity binding sites (Kd = 550 nM); (2) the Ca2(+)-receptive state of troponin C in F-actin/native tropomyosin complex did not affect CB binding.

The interplay between viscoelastic and thermodynamic properties determines the birefringence of F-actin gels.

PubMed

Helfer, Emmanuèle; Panine, Pierre; Carlier, Marie-France; Davidson, Patrick

2005-07-01

F-actin gels of increasing concentrations (25-300 microM) display in vitro a progressive onset of birefringence due to orientational ordering of actin filaments. At F-actin concentrations <100 microM, this birefringence can be erased and restored at will by sonication and gentle flow, respectively. Hence, the orientational ordering does not result from a thermodynamic transition to a nematic phase but instead is due to mechanical stresses stored in the gels. In contrast, at F-actin concentrations > or =100 microM, gels display spontaneous birefringence recovery, at rest, which is the sign of true nematic ordering, in good agreement with statistical physics models of the isotropic/nematic transition. Well-aligned samples of F-actin gels could be produced and their small-angle x-ray scattering patterns are quite anisotropic. These patterns show no sign of filament positional short-range order and could be modeled by averaging the form factor with the Maier-Saupe nematic distribution function. The derived nematic order parameter S of the gels ranged from S = 0.7 at 300 microM to S = 0.4 at 25 microM. Both birefringence and small-angle x-ray scattering data indicate that, even in absence of cross-linking proteins, spontaneous cooperative alignment of actin filaments may arise in motile regions of living cells where F-actin concentrations can reach values of a few 100 microM.

Strong fascin expression promotes metastasis independent of its F-actin bundling activity.

PubMed

Heinz, Lisa S; Muhs, Stefanie; Schiewek, Johanna; Grüb, Saskia; Nalaskowski, Marcus; Lin, Yuan-Na; Wikman, Harriet; Oliveira-Ferrer, Leticia; Lange, Tobias; Wellbrock, Jasmin; Konietzny, Anja; Mikhaylova, Marina; Windhorst, Sabine

2017-12-15

High expression of the actin bundling protein Fascin increases the malignancy of tumor cells. Here we show that fascin expression is up-regulated in more malignant sub-cell lines of MDA-MB-231 cells as compared to parental cells. Since also parental MDA-MB-231 cells exhibit high fascin levels, increased fascin expression was termed as "hyperexpression". To examine the effect of fascin hyperexpression, fascin was hyperexpressed in parental MDA-MB-231 cells and metastasis was analyzed in NOD scid gamma (NSG) mice. In addition, the effect of fascin mutants with inactive or constitutively active actin bundling activity was examined. Unexpectedly, we found that hyperexpression of both, wildtype (wt) and mutant fascin strongly increased metastasis in vivo , showing that the effect of fascin hyperexpression did not depend on its actin bundling activity. Cellular assays revealed that hyperexpression of wt and mutant fascin increased adhesion of MDA-MB-231 cells while transmigration and proliferation were not affected. Since it has been shown that fascin controls adhesion by directly interacting with microtubules ( MTs), we analyzed if fascin hyperexpression affects MT dynamics. We found that at high concentrations fascin significantly increased MT dynamics in cells and in cell-free approaches. In summary our data show that strong expression of fascin in breast cancer cells increases metastasis independent of its actin bundling activity. Thus, it seems that the mechanism of fascin-stimulated metastasis depends on its concentration.

A p21-activated kinase (PAK1) signaling cascade coordinately regulates F-actin remodeling and insulin granule exocytosis in pancreatic β cells

PubMed Central

Kalwat, Michael A.; Yoder, Stephanie M.; Wang, Zhanxiang; Thurmond, Debbie C.

2012-01-01

Human islet studies implicate an important signaling role for the Cdc42 effector protein p21-activated kinase (PAK1) in the sustained/second-phase of insulin secretion. Because human islets from type 2 diabetic donors lack ~80% of normal PAK1 protein levels, the mechanistic requirement for PAK1 signaling in islet function was interrogated. Similar to MIN6 β cells, human islets elicited glucose-stimulated PAK1 activation that was sensitive to the PAK1 inhibitor, IPA3. Given that sustained insulin secretion has been correlated with glucose-induced filamentous actin (F-actin) remodeling, we tested the hypothesis that a Cdc42-activated PAK1 signaling cascade is required to elicit F-actin remodeling to mobilize granules to the cell surface. Live-cell imaging captured the glucose-induced cortical F-actin remodeling in MIN6 β cells; IPA3-mediated inhibition of PAK1 abolished this remodeling. IPA3 also ablated glucose-stimulated insulin granule accumulation at the plasma membrane, consistent with its role in sustained/second-phase insulin release. Both IPA3 and a selective inhibitor of the Cdc42 GTPase, ML-141, blunted the glucose-stimulated activation of Raf-1, suggesting Raf-1 to be downstream of Cdc42→PAK1. IPA3 also inhibited MEK1/2 activation, implicating the MEK1/2→ERK1/2 cascade to occur downstream of PAK1. Importantly, PD0325901, a new selective inhibitor of MEK1/2→ERK1/2 activation, impaired F-actin remodeling and the sustained/amplification pathway of insulin release. Taken together, these data suggest that glucose-mediated activation of Cdc42 leads to activation of PAK1 and prompts activation of its downstream targets Raf-1, MEK1/2 and ERK1/2 to elicit F-actin remodeling and recruitment of insulin granules to the plasma membrane to support the sustained phase of insulin release. PMID:23246867

CaMKIIβ Association with the Actin Cytoskeleton Is Regulated by Alternative Splicing

PubMed Central

O'Leary, Heather; Lasda, Erika

2006-01-01

The Ca2+/calmodulin (CaM)-dependent protein kinase II (CaMKII)β has morphogenic functions in neurons not shared by the α isoform. CaMKIIβ contains three exons (v1, v3, and v4) not present in the CaMKIIα gene, and two of these exons (v1 and v4) are subject to differential alternative splicing. We show here that CaMKIIβ, but not α, mediated bundling of F-actin filaments in vitro. Most importantly, inclusion of exon v1 was required for CaMKIIβ association with the F-actin cytoskeleton within cells. CaMKIIβe, which is the dominant variant around birth and lacks exon v1 sequences, failed to associate with F-actin. By contrast, CaMKIIβ′, which instead lacks exon v4, associated with F-actin as full-length CaMKIIβ. Previous studies with CaMKIIβ mutants have indicated a role of nonstimulated kinase activity in enhancing dendritic arborization. Here, we show that F-actin–targeted CaMKIIβ, but not α, was able to phosphorylate actin in vitro even by nonstimulated basal activity in absence of Ca2+/CaM. In rat pancreatic islets and in skeletal muscle, the actin-associated CaMKIIβ′ and βM were the predominant variants, respectively. Thus, cytoskeletal targeting may mediate functions of CaMKIIβ variants also outside the nervous system. PMID:16928958

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

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

p21‐Activated kinase (Pak) regulates airway smooth muscle contraction by regulating paxillin complexes that mediate actin polymerization

PubMed Central

Zhang, Wenwu; Huang, Youliang

2016-01-01

. Immunoprecipitation analysis of tissue extracts and proximity ligation assays in dissociated cells showed that Pak activation and paxillin Ser273 phosphorylation triggered the formation of an adhesion junction signalling complex with paxillin that included G‐protein‐coupled receptor kinase‐interacting protein (GIT1) and the cdc42 guanine exchange factor, βPIX (Pak interactive exchange factor). Assembly of the Pak–GIT1–βPIX–paxillin complex was necessary for cdc42 and neuronal Wiskott–Aldrich syndrome protein (N‐WASP) activation, actin polymerization and contraction in response to ACh. RhoA activation was also required for the recruitment of Pak to adhesion junctions, Pak activation, paxillin Ser273 phosphorylation and paxillin complex assembly. These studies demonstrate a novel role for Pak in the regulation of N‐WASP activation, actin dynamics and cell contractility. PMID:27038336

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

Nck adaptor proteins link Tks5 to invadopodia actin regulation and ECM degradation.

PubMed

Stylli, Stanley S; Stacey, T T I; Verhagen, Anne M; Xu, San San; Pass, Ian; Courtneidge, Sara A; Lock, Peter

2009-08-01

Invadopodia are actin-based projections enriched with proteases, which invasive cancer cells use to degrade the extracellular matrix (ECM). The Phox homology (PX)-Src homology (SH)3 domain adaptor protein Tks5 (also known as SH3PXD2A) cooperates with Src tyrosine kinase to promote invadopodia formation but the underlying pathway is not clear. Here we show that Src phosphorylates Tks5 at Y557, inducing it to associate directly with the SH3-SH2 domain adaptor proteins Nck1 and Nck2 in invadopodia. Tks5 mutants unable to bind Nck show reduced matrix degradation-promoting activity and recruit actin to invadopodia inefficiently. Conversely, Src- and Tks5-driven matrix proteolysis and actin assembly in invadopodia are enhanced by Nck1 or Nck2 overexpression and inhibited by Nck1 depletion. We show that clustering at the plasma membrane of the Tks5 inter-SH3 region containing Y557 triggers phosphorylation at this site, facilitating Nck recruitment and F-actin assembly. These results identify a Src-Tks5-Nck pathway in ECM-degrading invadopodia that shows parallels with pathways linking several mammalian and pathogen-derived proteins to local actin regulation.

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.

The carboxyl terminus of the alpha-subunit of the amiloride-sensitive epithelial sodium channel binds to F-actin.

PubMed

Mazzochi, Christopher; Bubien, James K; Smith, Peter R; Benos, Dale J

2006-03-10

The activity of the amiloride-sensitive epithelial sodium channel (ENaC) is modulated by F-actin. However, it is unknown if there is a direct interaction between alpha-ENaC and actin. We have investigated the hypothesis that the actin cytoskeleton directly binds to the carboxyl terminus of alpha-ENaC using a combination of confocal microscopy, co-immunoprecipitation, and protein binding studies. Confocal microscopy of Madin-Darby canine kidney cell monolayers stably transfected with wild type, rat isoforms of alpha-, beta-, and gamma-ENaC revealed co-localization of alpha-ENaC with the cortical F-actin cytoskeleton both at the apical membrane and within the subapical cytoplasm. F-actin was found to co-immunoprecipitate with alpha-ENaC from whole cell lysates of this cell line. Gel overlay assays demonstrated that F-actin specifically binds to the carboxyl terminus of alpha-ENaC. A direct interaction between F-actin and the COOH terminus of alpha-ENaC was further corroborated by F-actin co-sedimentation studies. This is the first study to report a direct and specific biochemical interaction between F-actin and ENaC.

Total synthesis of (-)-doliculide, structure-activity relationship studies and its binding to F-actin.

PubMed

Matcha, Kiran; Madduri, Ashoka V R; Roy, Sayantani; Ziegler, Slava; Waldmann, Herbert; Hirsch, Anna K H; Minnaard, Adriaan J

2012-11-26

Actin, an abundant protein in most eukaryotic cells, is one of the targets in cancer research. Recently, a great deal of attention has been paid to the synthesis and function of actin-targeting compounds and their use as effective molecular probes in chemical biology. In this study, we have developed an efficient synthesis of (-)-doliculide, a very potent actin binder with a higher cell-membrane permeability than phalloidin. Actin polymerization assays with (-)-doliculide and two analogues on HeLa and BSC-1 cells, together with a prediction of their binding mode to F-actin by unbiased computational docking, show that doliculide stabilizes F-actin in a similar way to jasplakinolide and chondramide C. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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.

A WAVE2-Abi1 complex mediates CSF-1-induced F-actin-rich membrane protrusions and migration in macrophages.

PubMed

Kheir, Wassim Abou; Gevrey, Jean-Claude; Yamaguchi, Hideki; Isaac, Beth; Cox, Dianne

2005-11-15

Colony-stimulating factor 1 (CSF-1) is an important physiological chemoattractant for macrophages. The mechanisms by which CSF-1 elicits the formation of filamentous actin (F-actin)-rich membrane protrusions and induces macrophage migration are not fully understood. In particular, very little is known regarding the contribution of the different members of the Wiskott-Aldrich Syndrome protein (WASP) family of actin regulators in response to CSF-1. Although a role for WASP itself in macrophage chemotaxis has been previously identified, no data was available regarding the function of WASP family verprolin-homologous (WAVE) proteins in this cell type. We found that WAVE2 was the predominant isoform to be expressed in primary macrophages and in cells derived from the murine monocyte/macrophage RAW264.7 cell line (RAW/LR5). CSF-1 treatment of macrophages resulted in WAVE2 accumulation in F-actin-rich protrusions induced by CSF-1. Inhibition of WAVE2 function by expressing a dominant-negative mutant or introducing anti-WAVE2 antibodies in RAW/LR5 cells, as well as reduction of endogenous WAVE2 expression by RNA-mediated interference (RNAi), resulted in a significant reduction of CSF-1-elicited F-actin protrusions. WAVE2 was found in a protein complex together with Abelson kinase interactor 1 (Abi1) in resting or stimulated cells. Both WAVE2 and Abi1 were recruited to and necessary for the formation of F-actin protrusions in response to CSF-1. Reducing the levels of WAVE2, directly or by targeting Abi1, resulted in an impaired cell migration to CSF-1. Altogether these data identify a WAVE2-Abi1 complex crucial for the normal actin cytoskeleton reorganization and migration of macrophages in response to CSF-1.

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.

Role of gelsolin in the formation and organization of triton-soluble F-actin during myeloid differentiation of HL-60 cells.

PubMed

Watts, R G

1995-04-15

Structurally and functionally distinct F-actin pools coexist with globular (G)-actin in a variety of eukaryotic cells, including polymorphonuclear leukocytes (PMNs). In PMNs, a Triton-soluble F-actin pool (TSF) exists as short cytoplasmic filaments capped with gelsolin, while Triton-insoluble F-actin (TIF) is a three-dimensional meshwork of F-actin associated with actin-binding protein 280 (ABP-280), alpha-actinin, and tropomyosin. The unique association of gelsolin with the TSF suggests a role for gelsolin in creation or regulation of TSF. To evaluate gelsolin's role in TSF formation, the quantities of actin and gelsolin were determined by quantitative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblots in uninduced HL-60 cells (U-HL-60) and in HL-60 cells induced to myeloid differentiation with 1.25% dimethyl sulfoxide for 4 to 5 days (I-HL-60). U-HL-60 cells contain 17.76 +/- 6.01 pmol actin per 10(6) cells (TIF, 5.3 +/- 1.5; TSF, 2.17 +/- 0.37; G, 10.3 +/- 5.7; n = 5) and 0.073 pmol gelsolin per 10(6) cells (TIF, 0; TSF, 0.002 +/- 0.005; G, 0.07 +/- 0.01; n = 3), representing molar actin to gelsolin (A:G) ratios of 1,085:1 for TSF and 147:1 for G. After myeloid differentiation, the actin content increases 1.80-fold (31.94 +/- 6.14 pmol/10(6) cells) equally in each actin pool (TIF, 9.36 +/- 2.35; TSF, 3.29 +/- 0.62; G, 19.29 +/- 4.83). Gelsolin increases 2.4-fold overall (0.178 +/- 0.02 pmol/10(6) cells) but 19-fold in TSF (0.038 +/- 0.009) and only 1.9-fold in G pool (0.139 +/- 0.006), resulting in A:G ratios of 87:1 in TSF and 139:1 in G. The findings of an increase in TSF gelsolin with decreased A:G ratios (1,085:1 v 87:1) with myeloid differentiation suggest shortening of TSF filaments, while the A:G ratios of unbound gelsolin are unchanged (147:1 v 139:1). Measurement of EGTA-resistant gelsolin/actin complexes in HL-60 cells shows that 95% to 100% of complexes exist in the TSF-actin pool only. These findings are consistent

Capillary pericytes express α-smooth muscle actin, which requires prevention of filamentous-actin depolymerization for detection.

PubMed

Alarcon-Martinez, Luis; Yilmaz-Ozcan, Sinem; Yemisci, Muge; Schallek, Jesse; Kılıç, Kıvılcım; Can, Alp; Di Polo, Adriana; Dalkara, Turgay

2018-03-21

Recent evidence suggests that capillary pericytes are contractile and play a crucial role in the regulation of microcirculation. However, failure to detect components of the contractile apparatus in capillary pericytes, most notably α-smooth muscle actin (α-SMA), has questioned these findings. Using strategies that allow rapid filamentous-actin (F-actin) fixation (i.e. snap freeze fixation with methanol at -20°C) or prevent F-actin depolymerization (i.e. with F-actin stabilizing agents), we demonstrate that pericytes on mouse retinal capillaries, including those in intermediate and deeper plexus, express α-SMA. Junctional pericytes were more frequently α-SMA-positive relative to pericytes on linear capillary segments. Intravitreal administration of short interfering RNA (α-SMA-siRNA) suppressed α-SMA expression preferentially in high order branch capillary pericytes, confirming the existence of a smaller pool of α-SMA in distal capillary pericytes that is quickly lost by depolymerization. We conclude that capillary pericytes do express α-SMA, which rapidly depolymerizes during tissue fixation thus evading detection by immunolabeling. © 2018, Alarcon-Martinez et al.

14-3-3 Regulates Actin Filament Formation in the Deep-Branching Eukaryote Giardia lamblia

PubMed Central

Xu, Jennifer; Steele-Ogus, Melissa; Alas, Germain C. M.

2017-01-01

ABSTRACT The phosphoserine/phosphothreonine-binding protein 14-3-3 is known to regulate actin; this function has been previously attributed to sequestration of phosphorylated cofilin. 14-3-3 was identified as an actin-associated protein in the deep-branching eukaryote Giardia lamblia; however, Giardia lacks cofilin and all other canonical actin-binding proteins (ABPs). Thus, the role of G. lamblia 14-3-3 (Gl-14-3-3) in actin regulation was unknown. Gl-14-3-3 depletion resulted in an overall disruption of actin organization characterized by ectopically distributed short actin filaments. Using phosphatase and kinase inhibitors, we demonstrated that actin phosphorylation correlated with destabilization of the actin network and increased complex formation with 14-3-3, while blocking actin phosphorylation stabilized actin filaments and attenuated complex formation. Giardia’s sole Rho family GTPase, Gl-Rac, modulates Gl-14-3-3’s association with actin, providing the first connection between Gl-Rac and the actin cytoskeleton in Giardia. Giardia actin (Gl-actin) contains two putative 14-3-3 binding motifs, one of which (S330) is conserved in mammalian actin. Mutation of these sites reduced, but did not completely disrupt, the association with 14-3-3. Native gels and overlay assays indicate that intermediate proteins are required to support complex formation between 14-3-3 and actin. Overall, our results support a role for 14-3-3 as a regulator of actin; however, the presence of multiple 14-3-3–actin complexes suggests a more complex regulatory relationship than might be expected for a minimalistic parasite. IMPORTANCE Giardia lacks canonical actin-binding proteins. Gl-14-3-3 was identified as an actin interactor, but the significance of this interaction was unknown. Loss of Gl-14-3-3 results in ectopic short actin filaments, indicating that Gl-14-3-3 is an important regulator of the actin cytoskeleton in Giardia. Drug studies indicate that Gl-14-3-3 complex

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

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

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.

Myosin II Motors and F-Actin Dynamics Drive the Coordinated Movement of the Centrosome and Soma during CNS Glial-Guided Neuronal Migration

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

Solecki, Dr. David; Trivedi, Dr. Niraj; Govek, Eve-Ellen

2009-01-01

Lamination of cortical regions of the vertebrate brain depends on glial-guided neuronal migration. The conserved polarity protein Par6{alpha} localizes to the centrosome and coordinates forward movement of the centrosome and soma in migrating neurons. The cytoskeletal components that produce this unique form of cell polarity and their relationship to polarity signaling cascades are unknown. We show that F-actin and Myosin II motors are enriched in the neuronal leading process and that Myosin II activity is necessary for leading process actin dynamics. Inhibition of Myosin II decreased the speed of centrosome and somal movement, whereas Myosin II activation increased coordinated movement.more » Ectopic expression or silencing of Par6{alpha} inhibited Myosin II motors by decreasing Myosin light-chain phosphorylation. These findings suggest leading-process Myosin II may function to 'pull' the centrosome and soma forward during glial-guided migration by a mechanism involving the conserved polarity protein Par6{alpha}.« less

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.

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

Cortactin promotes exosome secretion by controlling branched actin dynamics

PubMed Central

Sinha, Seema; Hoshino, Daisuke; Hong, Nan Hyung; Seiki, Motoharu; Tyska, Matthew J.

2016-01-01

Exosomes are extracellular vesicles that influence cellular behavior and enhance cancer aggressiveness by carrying bioactive molecules. The mechanisms that regulate exosome secretion are poorly understood. Here, we show that the actin cytoskeletal regulatory protein cortactin promotes exosome secretion. Knockdown or overexpression of cortactin in cancer cells leads to a respective decrease or increase in exosome secretion, without altering exosome cargo content. Live-cell imaging revealed that cortactin controls both trafficking and plasma membrane docking of multivesicular late endosomes (MVEs). Regulation of exosome secretion by cortactin requires binding to the branched actin nucleating Arp2/3 complex and to actin filaments. Furthermore, cortactin, Rab27a, and coronin 1b coordinately control stability of cortical actin MVE docking sites and exosome secretion. Functionally, the addition of purified exosomes to cortactin-knockdown cells rescued defects of those cells in serum-independent growth and invasion. These data suggest a model in which cortactin promotes exosome secretion by stabilizing cortical actin-rich MVE docking sites. PMID:27402952

Cortactin promotes exosome secretion by controlling branched actin dynamics.

PubMed

Sinha, Seema; Hoshino, Daisuke; Hong, Nan Hyung; Kirkbride, Kellye C; Grega-Larson, Nathan E; Seiki, Motoharu; Tyska, Matthew J; Weaver, Alissa M

2016-07-18

Exosomes are extracellular vesicles that influence cellular behavior and enhance cancer aggressiveness by carrying bioactive molecules. The mechanisms that regulate exosome secretion are poorly understood. Here, we show that the actin cytoskeletal regulatory protein cortactin promotes exosome secretion. Knockdown or overexpression of cortactin in cancer cells leads to a respective decrease or increase in exosome secretion, without altering exosome cargo content. Live-cell imaging revealed that cortactin controls both trafficking and plasma membrane docking of multivesicular late endosomes (MVEs). Regulation of exosome secretion by cortactin requires binding to the branched actin nucleating Arp2/3 complex and to actin filaments. Furthermore, cortactin, Rab27a, and coronin 1b coordinately control stability of cortical actin MVE docking sites and exosome secretion. Functionally, the addition of purified exosomes to cortactin-knockdown cells rescued defects of those cells in serum-independent growth and invasion. These data suggest a model in which cortactin promotes exosome secretion by stabilizing cortical actin-rich MVE docking sites. © 2016 Sinha et al.

Theoretical kinetic studies of models for binding myosin subfragment-1 to regulated actin: Hill model versus Geeves model.

PubMed Central

Chen , Y; Yan, B; Chalovich, J M; Brenner, B

2001-01-01

It was previously shown that a one-dimensional Ising model could successfully simulate the equilibrium binding of myosin S1 to regulated actin filaments (T. L. Hill, E. Eisenberg and L. Greene, Proc. Natl. Acad. Sci. U.S.A. 77:3186-3190, 1980). However, the time course of myosin S1 binding to regulated actin was thought to be incompatible with this model, and a three-state model was subsequently developed (D. F. McKillop and M. A. Geeves, Biophys. J. 65:693-701, 1993). A quantitative analysis of the predicted time course of myosin S1 binding to regulated actin, however, was never done for either model. Here we present the procedure for the theoretical evaluation of the time course of myosin S1 binding for both models and then show that 1) the Hill model can predict the "lag" in the binding of myosin S1 to regulated actin that is observed in the absence of Ca++ when S1 is in excess of actin, and 2) both models generate very similar families of binding curves when [S1]/[actin] is varied. This result shows that, just based on the equilibrium and pre-steady-state kinetic binding data alone, it is not possible to differentiate between the two models. Thus, the model of Hill et al. cannot be ruled out on the basis of existing pre-steady-state and equilibrium binding data. Physical mechanisms underlying the generation of the lag in the Hill model are discussed. PMID:11325734

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

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

The accessibility of etheno-nucleotides to collisional quenchers and the nucleotide cleft in G- and F-actin.

PubMed Central

Root, D. D.; Reisler, E.

1992-01-01

Recent publication of the atomic structure of G-actin (Kabsch, W., Mannherz, H. G., Suck, D., Pai, E. F., & Holmes, K. C., 1990, Nature 347, 37-44) raises questions about how the conformation of actin changes upon its polymerization. In this work, the effects of various quenchers of etheno-nucleotides bound to G- and F-actin were examined in order to assess polymerization-related changes in the nucleotide phosphate site. The Mg(2+)-induced polymerization of actin quenched the fluorescence of the etheno-nucleotides by approximately 20% simultaneously with the increase in light scattering by actin. A conformational change at the nucleotide binding site was also indicated by greater accessibility of F-actin than G-actin to positively, negatively, and neutrally charged collisional quenchers. The difference in accessibility between G- and F-actin was greatest for I-, indicating that the environment of the etheno group is more positively charged in the polymerized form of actin. Based on calculations of the change in electric potential of the environment of the etheno group, specific polymerization-related movements of charged residues in the atomic structure of G-actin are suggested. The binding of S-1 to epsilon-ATP-G-actin increased the accessibility of the etheno group to I- even over that in Mg(2+)-polymerized actin. The quenching of the etheno group by nitromethane was, however, unaffected by the binding of S-1 to actin. Thus, the binding of S-1 induces conformational changes in the cleft region of actin that are different from those caused by Mg2+ polymerization of actin.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1304380

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

PubMed Central

Deshpande, Siddharth; Pfohl, Thomas

2015-01-01

Understanding the cytoskeletal functionality and its relation to other cellular components and properties is a prominent question in biophysics. The dynamics of actin cytoskeleton and its polymorphic nature are indispensable for the proper functioning of living cells. Actin bundles are involved in cell motility, environmental exploration, intracellular transport and mechanical stability. Though the viscoelastic properties of actin-based structures have been extensively probed, the underlying microstructure dynamics, especially their disassembly, is not fully understood. In this article, we explore the rich dynamics and emergent properties exhibited by actin bundles within flow-free confinements using a microfluidic set-up and epifluorescence microscopy. After forming entangled actin filaments within cell-sized quasi two-dimensional confinements, we induce their bundling using three different fundamental mechanisms: counterion condensation, depletion interactions and specific protein-protein interactions. Intriguingly, long actin filaments form emerging networks of actin bundles via percolation leading to remarkable properties such as stress generation and spindle-like intermediate structures. Simultaneous sharing of filaments in different links of the network is an important parameter, as short filaments do not form networks but segregated clusters of bundles instead. We encounter a hierarchical process of bundling and its subsequent disassembly. Additionally, our study suggests that such percolated networks are likely to exist within living cells in a dynamic fashion. These observations render a perspective about differential cytoskeletal responses towards numerous stimuli. PMID:25785606

Stability of actin-lysozyme complexes formed in cystic fibrosis disease.

PubMed

Mohammadinejad, Sarah; Ghamkhari, Behnoush; Abdolmaleki, Sarah

2016-08-21

Finding the conditions for destabilizing actin-lysozyme complexes is of biomedical importance in preventing infections in cystic fibrosis. In this manuscript, the effects of different charge-mutants of lysozyme and salt concentration on the stability of actin-lysozyme complexes are studied using Langevin dynamics simulation. A coarse-grained model of F-actin is used in which both its twist and bending rigidities are considered. We observe that the attraction between F-actins is stronger in the presence of wild-type lysozymes relative to the mutated lysozymes of lower charges. By calculating the potential of mean force between F-actins, we conclude that the stability of actin-lysozyme complexes is decreased by reducing the charge of lysozyme mutants. The distributions of different lysozyme charge-mutants show that wild-type (+9e) lysozymes are mostly accumulated in the center of triangles formed by three adjacent F-actins, while lysozyme mutants of charges +7e and +5e occupy the bridging regions between F-actins. Low-charge mutants of lysozyme (+3e) distribute uniformly around F-actins. A rough estimate of the electrostatic energy for these different distributions proves that the distribution in which lysozymes reside in the center of triangles leads to more stable complexes. Also our results in the presence of a salt suggest that at physiological salt concentration of airway, F-actin complexes are not formed by charge-reduced mutants of lysozyme. The findings are interesting because if we can design charge-reduced lysozyme mutants with considerable antibacterial activity, they are not sequestered inside F-actin aggregates and can play their role as antibacterial agents against airway infection.

Electrostatic interaction map reveals a new binding position for tropomyosin on F-actin.

PubMed

Rynkiewicz, Michael J; Schott, Veronika; Orzechowski, Marek; Lehman, William; Fischer, Stefan

2015-12-01

Azimuthal movement of tropomyosin around the F-actin thin filament is responsible for muscle activation and relaxation. Recently a model of αα-tropomyosin, derived from molecular-mechanics and electron microscopy of different contractile states, showed that tropomyosin is rather stiff and pre-bent to present one specific face to F-actin during azimuthal transitions. However, a new model based on cryo-EM of troponin- and myosin-free filaments proposes that the interacting-face of tropomyosin can differ significantly from that in the original model. Because resolution was insufficient to assign tropomyosin side-chains, the interacting-face could not be unambiguously determined. Here, we use structural analysis and energy landscapes to further examine the proposed models. The observed bend in seven crystal structures of tropomyosin is much closer in direction and extent to the original model than to the new model. Additionally, we computed the interaction map for repositioning tropomyosin over the F-actin surface, but now extended over a much larger surface than previously (using the original interacting-face). This map shows two energy minima-one corresponding to the "blocked-state" as in the original model, and the other related by a simple 24 Å translation of tropomyosin parallel to the F-actin axis. The tropomyosin-actin complex defined by the second minimum fits perfectly into the recent cryo-EM density, without requiring any change in the interacting-face. Together, these data suggest that movement of tropomyosin between regulatory states does not require interacting-face rotation. Further, they imply that thin filament assembly may involve an interplay between initially seeded tropomyosin molecules growing from distinct binding-site regions on actin.

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.

Distinct roles for Arp2/3 regulators in actin assembly and endocytosis.

PubMed

Galletta, Brian J; Chuang, Dennis Y; Cooper, John A

2008-01-01

The Arp2/3 complex is essential for actin assembly and motility in many cell processes, and a large number of proteins have been found to bind and regulate it in vitro. A critical challenge is to understand the actions of these proteins in cells, especially in settings where multiple regulators are present. In a systematic study of the sequential multicomponent actin assembly processes that accompany endocytosis in yeast, we examined and compared the roles of WASp, two type-I myosins, and two other Arp2/3 activators, along with that of coronin, which is a proposed inhibitor of Arp2/3. Quantitative analysis of high-speed fluorescence imaging revealed individual functions for the regulators, manifested in part by novel phenotypes. We conclude that Arp2/3 regulators have distinct and overlapping roles in the processes of actin assembly that drive endocytosis in yeast. The formation of the endocytic actin patch, the creation of the endocytic vesicle, and the movement of the vesicle into the cytoplasm display distinct dependencies on different Arp2/3 regulators. Knowledge of these roles provides insight into the in vivo relevance of the dendritic nucleation model for actin assembly.

The Association of Myosin IB with Actin Waves in Dictyostelium Requires Both the Plasma Membrane-Binding Site and Actin-Binding Region in the Myosin Tail

PubMed Central

Brzeska, Hanna; Pridham, Kevin; Chery, Godefroy; Titus, Margaret A.; Korn, Edward D.

2014-01-01

F-actin structures and their distribution are important determinants of the dynamic shapes and functions of eukaryotic cells. Actin waves are F-actin formations that move along the ventral cell membrane driven by actin polymerization. Dictyostelium myosin IB is associated with actin waves but its role in the wave is unknown. Myosin IB is a monomeric, non-filamentous myosin with a globular head that binds to F-actin and has motor activity, and a non-helical tail comprising a basic region, a glycine-proline-glutamine-rich region and an SH3-domain. The basic region binds to acidic phospholipids in the plasma membrane through a short basic-hydrophobic site and the Gly-Pro-Gln region binds F-actin. In the current work we found that both the basic-hydrophobic site in the basic region and the Gly-Pro-Gln region of the tail are required for the association of myosin IB with actin waves. This is the first evidence that the Gly-Pro-Gln region is required for localization of myosin IB to a specific actin structure in situ. The head is not required for myosin IB association with actin waves but binding of the head to F-actin strengthens the association of myosin IB with waves and stabilizes waves. Neither the SH3-domain nor motor activity is required for association of myosin IB with actin waves. We conclude that myosin IB contributes to anchoring actin waves to the plasma membranes by binding of the basic-hydrophobic site to acidic phospholipids in the plasma membrane and binding of the Gly-Pro-Gln region to F-actin in the wave. PMID:24747353

Regulation of blood-testis barrier by actin binding proteins and protein kinases

PubMed Central

Li, Nan; Tang, Elizabeth I.; Cheng, C. Yan

2016-01-01

The blood-testis barrier (BTB) is an important ultrastructure in the testis since the onset of spermatogenesis coincides with the establishment of a functional barrier in rodents and humans. It is also noted that a delay in the assembly of a functional BTB following treatment of neonatal rats with drugs such as diethylstilbestrol or adjudin also delays the first wave of spermiation. While the BTB is one of the tightest blood-tissue barriers, it undergoes extensive remodeling, in particular at stage VIII of the epithelial cycle to facilitate the transport of preleptotene spermatocytes connected in clones across the immunological barrier. Without this timely transport of preleptotene spermatocytes derived from type B spermatogonia, meiosis will be arrested, causing aspermatogenesis. Yet the biology and regulation of the BTB remains largely unexplored since the morphological studies in the 1970s. Recent studies, however, have shed new light on the biology of the BTB. Herein, we critically evaluate some of these findings, illustrating that the Sertoli cell BTB is regulated by actin binding proteins (ABPs), likely supported by non-receptor protein kinases, to modulate the organization of actin microfilament bundles at the site. Furthermore, microtubule (MT)-based cytoskeleton is also working in concert with the actin-based cytoskeleton to confer BTB dynamics. This timely review provides an update on the unique biology and regulation of the BTB based on the latest findings in the field, focusing on the role of ABPs and non-receptor protein kinases. PMID:26628556

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

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.

WASH and WAVE actin regulators of the Wiskott-Aldrich syndrome protein (WASP) family are controlled by analogous structurally related complexes.

PubMed

Jia, Da; Gomez, Timothy S; Metlagel, Zoltan; Umetani, Junko; Otwinowski, Zbyszek; Rosen, Michael K; Billadeau, Daniel D

2010-06-08

We recently showed that the Wiskott-Aldrich syndrome protein (WASP) family member, WASH, localizes to endosomal subdomains and regulates endocytic vesicle scission in an Arp2/3-dependent manner. Mechanisms regulating WASH activity are unknown. Here we show that WASH functions in cells within a 500 kDa core complex containing Strumpellin, FAM21, KIAA1033 (SWIP), and CCDC53. Although recombinant WASH is constitutively active toward the Arp2/3 complex, the reconstituted core assembly is inhibited, suggesting that it functions in cells to regulate actin dynamics through WASH. FAM21 interacts directly with CAPZ and inhibits its actin-capping activity. Four of the five core components show distant (approximately 15% amino acid sequence identify) but significant structural homology to components of a complex that negatively regulates the WASP family member, WAVE. Moreover, biochemical and electron microscopic analyses show that the WASH and WAVE complexes are structurally similar. Thus, these two distantly related WASP family members are controlled by analogous structurally related mechanisms. Strumpellin is mutated in the human disease hereditary spastic paraplegia, and its link to WASH suggests that misregulation of actin dynamics on endosomes may play a role in this disorder.

A Requirement for Mena, an Actin Regulator, in Local mRNA Translation in Developing Neurons.

PubMed

Vidaki, Marina; Drees, Frauke; Saxena, Tanvi; Lanslots, Erwin; Taliaferro, Matthew J; Tatarakis, Antonios; Burge, Christopher B; Wang, Eric T; Gertler, Frank B

2017-08-02

During neuronal development, local mRNA translation is required for axon guidance and synaptogenesis, and dysregulation of this process contributes to multiple neurodevelopmental and cognitive disorders. However, regulation of local protein synthesis in developing axons remains poorly understood. Here, we uncover a novel role for the actin-regulatory protein Mena in the formation of a ribonucleoprotein complex that involves the RNA-binding proteins HnrnpK and PCBP1 and regulates local translation of specific mRNAs in developing axons. We find that translation of dyrk1a, a Down syndrome- and autism spectrum disorders-related gene, is dependent on Mena, both in steady-state conditions and upon BDNF stimulation. We identify hundreds of additional mRNAs that associate with the Mena complex, suggesting that it plays broader role(s) in post-transcriptional gene regulation. Our work establishes a dual role for Mena in neurons, providing a potential link between regulation of actin dynamics and local translation. Copyright © 2017 Elsevier Inc. All rights reserved.

Hematopoietic Protein-1 Regulates the Actin Membrane Skeleton and Membrane Stability in Murine Erythrocytes

PubMed Central

Chan, Maia M.; Wooden, Jason M.; Tsang, Mark; Gilligan, Diana M.; Hirenallur-S, Dinesh K.; Finney, Greg L.; Rynes, Eric; MacCoss, Michael; Ramirez, Julita A.; Park, Heon; Iritani, Brian M.

2013-01-01

Hematopoietic protein-1 (Hem-1) is a hematopoietic cell specific member of the WAVE (Wiskott-Aldrich syndrome verprolin-homologous protein) complex, which regulates filamentous actin (F-actin) polymerization in many cell types including immune cells. However, the roles of Hem-1 and the WAVE complex in erythrocyte biology are not known. In this study, we utilized mice lacking Hem-1 expression due to a non-coding point mutation in the Hem1 gene to show that absence of Hem-1 results in microcytic, hypochromic anemia characterized by abnormally shaped erythrocytes with aberrant F-actin foci and decreased lifespan. We find that Hem-1 and members of the associated WAVE complex are normally expressed in wildtype erythrocyte progenitors and mature erythrocytes. Using mass spectrometry and global proteomics, Coomassie staining, and immunoblotting, we find that the absence of Hem-1 results in decreased representation of essential erythrocyte membrane skeletal proteins including α- and β- spectrin, dematin, p55, adducin, ankyrin, tropomodulin 1, band 3, and band 4.1. Hem1−/− erythrocytes exhibit increased protein kinase C-dependent phosphorylation of adducin at Ser724, which targets adducin family members for dissociation from spectrin and actin, and subsequent proteolysis. Increased adducin Ser724 phosphorylation in Hem1−/− erythrocytes correlates with decreased protein expression of the regulatory subunit of protein phosphatase 2A (PP2A), which is required for PP2A-dependent dephosphorylation of PKC targets. These results reveal a novel, critical role for Hem-1 in the homeostasis of structural proteins required for formation and stability of the actin membrane skeleton in erythrocytes. PMID:23424621

Allele-specific Effects of Human Deafness γ-Actin Mutations (DFNA20/26) on the Actin/Cofilin Interaction*

PubMed Central

Bryan, Keith E.; Rubenstein, Peter A.

2009-01-01

Auditory hair cell function requires proper assembly and regulation of the nonmuscle gamma isoactin-rich cytoskeleton, and six point mutations in this isoactin cause a type of delayed onset autosomal dominant nonsyndromic progressive hearing loss, DFNA20/26. The molecular basis underlying this actin-dependent hearing loss is unknown. To address this problem, the mutations have been introduced into yeast actin, and their effects on actin function were assessed in vivo and in vitro. Because we previously showed that polymerization was unaffected in five of the six mutants, we have focused on proteins that regulate actin, in particular cofilin, which severs F-actin and sequesters actin monomers. The mutations do not affect the interaction of cofilin with G-actin. However, T89I and V370A mutant F-actins are much more susceptible to cofilin disassembly than WT filaments in vitro. Conversely, P332A filaments demonstrate enhanced resistance. Wild type actin solutions containing T89I, K118M, or P332A mutant actins at mole fractions similar to those found in the hair cell respond in vitro toward cofilin in a manner proportional to the level of the mutant present. Finally, depression of cofilin action in vivo by elimination of the cofilin-activating protein, Aip1p, rescues the inability to grow on glycerol caused by K118M, T278I, P332A, and V370A. These results suggest that a filament instability caused by these mutations can be balanced by decreasing a system in vivo that promotes increased filament turnover. Such mutant-dependent filament destabilization could easily result in hair cell malfunction leading to the late-onset hearing loss observed in these patients. PMID:19419963

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

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.

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.

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

Rice actin-binding protein RMD is a key link in the auxin-actin regulatory loop that controls cell growth.

PubMed

Li, Gang; Liang, Wanqi; Zhang, Xiaoqing; Ren, Haiyun; Hu, Jianping; Bennett, Malcolm J; Zhang, Dabing

2014-07-15

The plant hormone auxin plays a central role in plant growth and development. Auxin transport and signaling depend on actin organization. Despite its functional importance, the mechanistic link between actin filaments (F-actin) and auxin intracellular signaling remains unclear. Here, we report that the actin-organizing protein Rice Morphology Determinant (RMD), a type II formin from rice (Oryza sativa), provides a key link. Mutants lacking RMD display abnormal cell growth and altered configuration of F-actin array direction. The rmd mutants also exhibit an inhibition of auxin-mediated cell elongation, decreased polar auxin transport, altered auxin distribution gradients in root tips, and suppression of plasma membrane localization of auxin transporters O. sativa PIN-FORMED 1b (OsPIN1b) and OsPIN2 in root cells. We demonstrate that RMD is required for endocytosis, exocytosis, and auxin-mediated OsPIN2 recycling to the plasma membrane. Moreover, RMD expression is directly regulated by heterodimerized O. sativa auxin response factor 23 (OsARF23) and OsARF24, providing evidence that auxin modulates the orientation of F-actin arrays through RMD. In support of this regulatory loop, osarf23 and lines with reduced expression of both OsARF23 and OsARF24 display reduced RMD expression, disrupted F-actin organization and cell growth, less sensitivity to auxin response, and altered auxin distribution and OsPIN localization. Our findings establish RMD as a crucial component of the auxin-actin self-organizing regulatory loop from the nucleus to cytoplasm that controls rice cell growth and morphogenesis.

Microscale Mechanics of Actin Networks During Dynamic Assembly and Dissociation

NASA Astrophysics Data System (ADS)

Gurmessa, Bekele; Robertson-Anderson, Rae; Ross, Jennifer; Nguyen, Dan; Saleh, Omar

Actin is one of the key components of the cytoskeleton, enabling cells to move and divide while maintaining shape by dynamic polymerization, dissociation and crosslinking. Actin polymerization and network formation is driven by ATP hydrolysis and varies depending on the concentrations of actin monomers and crosslinking proteins. The viscoelastic properties of steady-state actin networks have been well-characterized, yet the mechanical properties of these non-equilibrium systems during dynamic assembly and disassembly remain to be understood. We use semipermeable microfluidic devices to induce in situ dissolution and re-polymerization of entangled and crosslinked actin networks, by varying ATP concentrations in real-time, while measuring the mechanical properties during disassembly and re-assembly. We use optical tweezers to sinusoidally oscillate embedded microspheres and measure the resulting force at set time-intervals and in different regions of the network during cyclic assembly/disassembly. We determine the time-dependent viscoelastic properties of non-equilibrium network intermediates and the reproducibility and homogeneity of network formation and dissolution. Results inform the role that cytoskeleton reorganization plays in the dynamic multifunctional mechanics of cells. NSF CAREER Award (DMR-1255446) and a Scialog Collaborative Innovation Award funded by Research Corporation for Scientific Advancement (Grant No. 24192).

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.

Modulation of actin dynamics as potential macrophage subtype-targeting anti-tumour strategy.

PubMed

Pergola, Carlo; Schubert, Katrin; Pace, Simona; Ziereisen, Jana; Nikels, Felix; Scherer, Olga; Hüttel, Stephan; Zahler, Stefan; Vollmar, Angelika M; Weinigel, Christina; Rummler, Silke; Müller, Rolf; Raasch, Martin; Mosig, Alexander; Koeberle, Andreas; Werz, Oliver

2017-01-30

Tumour-associated macrophages mainly comprise immunosuppressive M2 phenotypes that promote tumour progression besides anti-tumoural M1 subsets. Selective depletion or reprogramming of M2 may represent an innovative anti-cancer strategy. The actin cytoskeleton is central for cellular homeostasis and is targeted for anti-cancer chemotherapy. Here, we show that targeting G-actin nucleation using chondramide A (ChA) predominantly depletes human M2 while promoting the tumour-suppressive M1 phenotype. ChA reduced the viability of M2, with minor effects on M1, but increased tumour necrosis factor (TNF)α release from M1. Interestingly, ChA caused rapid disruption of dynamic F-actin filaments and polymerization of G-actin, followed by reduction of cell size, binucleation and cell division, without cellular collapse. In M1, but not in M2, ChA caused marked activation of SAPK/JNK and NFκB, with slight or no effects on Akt, STAT-1/-3, ERK-1/2, and p38 MAPK, seemingly accounting for the better survival of M1 and TNFα secretion. In a microfluidically-supported human tumour biochip model, circulating ChA-treated M1 markedly reduced tumour cell viability through enhanced release of TNFα. Together, ChA may cause an anti-tumoural microenvironment by depletion of M2 and activation of M1, suggesting induction of G-actin nucleation as potential strategy to target tumour-associated macrophages in addition to neoplastic cells.

Modulation of actin dynamics as potential macrophage subtype-targeting anti-tumour strategy

PubMed Central

Pergola, Carlo; Schubert, Katrin; Pace, Simona; Ziereisen, Jana; Nikels, Felix; Scherer, Olga; Hüttel, Stephan; Zahler, Stefan; Vollmar, Angelika M.; Weinigel, Christina; Rummler, Silke; Müller, Rolf; Raasch, Martin; Mosig, Alexander; Koeberle, Andreas; Werz, Oliver

2017-01-01

Tumour-associated macrophages mainly comprise immunosuppressive M2 phenotypes that promote tumour progression besides anti-tumoural M1 subsets. Selective depletion or reprogramming of M2 may represent an innovative anti-cancer strategy. The actin cytoskeleton is central for cellular homeostasis and is targeted for anti-cancer chemotherapy. Here, we show that targeting G-actin nucleation using chondramide A (ChA) predominantly depletes human M2 while promoting the tumour-suppressive M1 phenotype. ChA reduced the viability of M2, with minor effects on M1, but increased tumour necrosis factor (TNF)α release from M1. Interestingly, ChA caused rapid disruption of dynamic F-actin filaments and polymerization of G-actin, followed by reduction of cell size, binucleation and cell division, without cellular collapse. In M1, but not in M2, ChA caused marked activation of SAPK/JNK and NFκB, with slight or no effects on Akt, STAT-1/-3, ERK-1/2, and p38 MAPK, seemingly accounting for the better survival of M1 and TNFα secretion. In a microfluidically-supported human tumour biochip model, circulating ChA-treated M1 markedly reduced tumour cell viability through enhanced release of TNFα. Together, ChA may cause an anti-tumoural microenvironment by depletion of M2 and activation of M1, suggesting induction of G-actin nucleation as potential strategy to target tumour-associated macrophages in addition to neoplastic cells. PMID:28134280

A mitochondria-anchored isoform of the actin-nucleating spire protein regulates mitochondrial division.

PubMed

Manor, Uri; Bartholomew, Sadie; Golani, Gonen; Christenson, Eric; Kozlov, Michael; Higgs, Henry; Spudich, James; Lippincott-Schwartz, Jennifer

2015-08-25

Mitochondrial division, essential for survival in mammals, is enhanced by an inter-organellar process involving ER tubules encircling and constricting mitochondria. The force for constriction is thought to involve actin polymerization by the ER-anchored isoform of the formin protein inverted formin 2 (INF2). Unknown is the mechanism triggering INF2-mediated actin polymerization at ER-mitochondria intersections. We show that a novel isoform of the formin-binding, actin-nucleating protein Spire, Spire1C, localizes to mitochondria and directly links mitochondria to the actin cytoskeleton and the ER. Spire1C binds INF2 and promotes actin assembly on mitochondrial surfaces. Disrupting either Spire1C actin- or formin-binding activities reduces mitochondrial constriction and division. We propose Spire1C cooperates with INF2 to regulate actin assembly at ER-mitochondrial contacts. Simulations support this model's feasibility and demonstrate polymerizing actin filaments can induce mitochondrial constriction. Thus, Spire1C is optimally positioned to serve as a molecular hub that links mitochondria to actin and the ER for regulation of mitochondrial division.

The cell wall of Arabidopsis thaliana influences actin network dynamics.

PubMed

Tolmie, Frances; Poulet, Axel; McKenna, Joseph; Sassmann, Stefan; Graumann, Katja; Deeks, Michael; Runions, John

2017-07-20

In plant cells, molecular connections link the cell wall-plasma membrane-actin cytoskeleton to form a continuum. It is hypothesized that the cell wall provides stable anchor points around which the actin cytoskeleton remodels. Here we use live cell imaging of fluorescently labelled marker proteins to quantify the organization and dynamics of the actin cytoskeleton and to determine the impact of disrupting connections within the continuum. Labelling of the actin cytoskeleton with green fluorescent protein (GFP)-fimbrin actin-binding domain 2 (FABD2) resulted in a network composed of fine filaments and thicker bundles that appeared as a highly dynamic remodelling meshwork. This differed substantially from the GFP-Lifeact-labelled network that appeared much more sparse with thick bundles that underwent 'simple movement', in which the bundles slightly change position, but in such a manner that the structure of the network was not substantially altered during the time of observation. Label-dependent differences in actin network morphology and remodelling necessitated development of two new image analysis techniques. The first of these, 'pairwise image subtraction', was applied to measurement of the more rapidly remodelling actin network labelled with GFP-FABD2, while the second, 'cumulative fluorescence intensity', was used to measure bulk remodelling of the actin cytoskeleton when labelled with GFP-Lifeact. In each case, these analysis techniques show that the actin cytoskeleton has a decreased rate of bulk remodelling when the cell wall-plasma membrane-actin continuum is disrupted either by plasmolysis or with isoxaben, a drug that specifically inhibits cellulose deposition. Changes in the rate of actin remodelling also affect its functionality, as observed by alteration in Golgi body motility. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

The formin DIAPH1 (mDia1) regulates megakaryocyte proplatelet formation by remodeling the actin and microtubule cytoskeletons.

PubMed

Pan, Jiajia; Lordier, Larissa; Meyran, Deborah; Rameau, Philippe; Lecluse, Yann; Kitchen-Goosen, Susan; Badirou, Idinath; Mokrani, Hayat; Narumiya, Shuh; Alberts, Arthur S; Vainchenker, William; Chang, Yunhua

2014-12-18

Megakaryocytes are highly specialized precursor cells that produce platelets via cytoplasmic extensions called proplatelets. Proplatelet formation (PPF) requires profound changes in microtubule and actin organization. In this work, we demonstrated that DIAPH1 (mDia1), a mammalian homolog of Drosophila diaphanous that works as an effector of the small GTPase Rho, negatively regulates PPF by controlling the dynamics of the actin and microtubule cytoskeletons. Moreover, we showed that inhibition of both DIAPH1 and the Rho-associated protein kinase (Rock)/myosin pathway increased PPF via coordination of both cytoskeletons. We provide evidence that 2 major effectors of the Rho GTPase pathway (DIAPH1 and Rock/myosin II) are involved not only in Rho-mediated stress fibers assembly, but also in the regulation of microtubule stability and dynamics during PPF. © 2014 by The American Society of Hematology.

Actin-interacting protein 1 controls assembly and permeability of intestinal epithelial apical junctions

PubMed Central

Baranwal, Somesh

2015-01-01

Adherens junctions (AJs) and tight junctions (TJs) are crucial regulators of the integrity and restitution of the intestinal epithelial barrier. The structure and function of epithelial junctions depend on their association with the cortical actin cytoskeleton that, in polarized epithelial cells, is represented by a prominent perijunctional actomyosin belt. The assembly and stability of the perijunctional cytoskeleton is controlled by constant turnover (disassembly and reassembly) of actin filaments. Actin-interacting protein (Aip) 1 is an emerging regulator of the actin cytoskeleton, playing a critical role in filament disassembly. In this study, we examined the roles of Aip1 in regulating the structure and remodeling of AJs and TJs in human intestinal epithelium. Aip1 was enriched at apical junctions in polarized human intestinal epithelial cells and normal mouse colonic mucosa. Knockdown of Aip1 by RNA interference increased the paracellular permeability of epithelial cell monolayers, decreased recruitment of AJ/TJ proteins to steady-state intercellular contacts, and attenuated junctional reassembly in a calcium-switch model. The observed defects of AJ/TJ structure and functions were accompanied by abnormal organization and dynamics of the perijunctional F-actin cytoskeleton. Moreover, loss of Aip1 impaired the apico-basal polarity of intestinal epithelial cell monolayers and inhibited formation of polarized epithelial cysts in 3-D Matrigel. Our findings demonstrate a previously unanticipated role of Aip1 in regulating the structure and remodeling of intestinal epithelial junctions and early steps of epithelial morphogenesis. PMID:25792565

Actin-interacting protein 1 controls assembly and permeability of intestinal epithelial apical junctions.

PubMed

Lechuga, Susana; Baranwal, Somesh; Ivanov, Andrei I

2015-05-01

Adherens junctions (AJs) and tight junctions (TJs) are crucial regulators of the integrity and restitution of the intestinal epithelial barrier. The structure and function of epithelial junctions depend on their association with the cortical actin cytoskeleton that, in polarized epithelial cells, is represented by a prominent perijunctional actomyosin belt. The assembly and stability of the perijunctional cytoskeleton is controlled by constant turnover (disassembly and reassembly) of actin filaments. Actin-interacting protein (Aip) 1 is an emerging regulator of the actin cytoskeleton, playing a critical role in filament disassembly. In this study, we examined the roles of Aip1 in regulating the structure and remodeling of AJs and TJs in human intestinal epithelium. Aip1 was enriched at apical junctions in polarized human intestinal epithelial cells and normal mouse colonic mucosa. Knockdown of Aip1 by RNA interference increased the paracellular permeability of epithelial cell monolayers, decreased recruitment of AJ/TJ proteins to steady-state intercellular contacts, and attenuated junctional reassembly in a calcium-switch model. The observed defects of AJ/TJ structure and functions were accompanied by abnormal organization and dynamics of the perijunctional F-actin cytoskeleton. Moreover, loss of Aip1 impaired the apico-basal polarity of intestinal epithelial cell monolayers and inhibited formation of polarized epithelial cysts in 3-D Matrigel. Our findings demonstrate a previously unanticipated role of Aip1 in regulating the structure and remodeling of intestinal epithelial junctions and early steps of epithelial morphogenesis. Copyright © 2015 the American Physiological Society.

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.

Direct Transmembrane Interaction between Actin and the Pore-Competent, Cholesterol-Dependent Cytolysin Pneumolysin

PubMed Central

Hupp, Sabrina; Förtsch, Christina; Wippel, Carolin; Ma, Jiangtao; Mitchell, Timothy J.; Iliev, Asparouh I.

2013-01-01

The eukaryotic actin cytoskeleton is an evolutionarily well-established pathogen target, as a large number of bacterial factors disturb its dynamics to alter the function of the host cells. These pathogenic factors modulate or mimic actin effector proteins or they modify actin directly, leading to an imbalance of the precisely regulated actin turnover. Here, we show that the pore-forming, cholesterol-dependent cytolysin pneumolysin (PLY), a major neurotoxin of Streptococcus pneumoniae, has the capacity to bind actin directly and to enhance actin polymerisation in vitro. In cells, the toxin co-localised with F-actin shortly after exposure, and this direct interaction was verified by Förster resonance energy transfer. PLY was capable of exerting its effect on actin through the lipid bilayer of giant unilamellar vesicles, but only when its pore competence was preserved. The dissociation constant of G-actin binding to PLY in a biochemical environment was 170–190 nM, which is indicative of a high-affinity interaction, comparable to the affinity of other intracellular actin-binding factors. Our results demonstrate the first example of a direct interaction of a pore-forming toxin with cytoskeletal components, suggesting that the cross talk between pore-forming cytolysins and cells is more complex than previously thought. PMID:23219469

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.

BAR Proteins PSTPIP1/2 Regulate Podosome Dynamics and the Resorption Activity of Osteoclasts

PubMed Central

Sztacho, Martin; Segeletz, Sandra; Sanchez-Fernandez, Maria Arantzazu; Czupalla, Cornelia; Niehage, Christian; Hoflack, Bernard

2016-01-01

Bone resorption in vertebrates relies on the ability of osteoclasts to assemble F-actin-rich podosomes that condense into podosomal belts, forming sealing zones. Sealing zones segregate bone-facing ruffled membranes from other membrane domains, and disassemble when osteoclasts migrate to new areas. How podosome/sealing zone dynamics is regulated remains unknown. We illustrate the essential role of the membrane scaffolding F-BAR-Proline-Serine-Threonine Phosphatase Interacting Proteins (PSTPIP) 1 and 2 in this process. Whereas PSTPIP2 regulates podosome assembly, PSTPIP1 regulates their disassembly. PSTPIP1 recruits, through its F-BAR domain, the protein tyrosine phosphatase non-receptor type 6 (PTPN6) that de-phosphophorylates the phosphatidylinositol 5-phosphatases SHIP1/2 bound to the SH3 domain of PSTPIP1. Depletion of any component of this complex prevents sealing zone disassembly and increases osteoclast activity. Thus, our results illustrate the importance of BAR domain proteins in podosome structure and dynamics, and identify a new PSTPIP1/PTPN6/SHIP1/2-dependent negative feedback mechanism that counterbalances Src and PI(3,4,5)P3 signalling to control osteoclast cell polarity and activity during bone resorption. PMID:27760174

Evidence Coupling Increased Hexosamine Biosynthesis Pathway Activity to Membrane Cholesterol Toxicity and Cortical Filamentous Actin Derangement Contributing to Cellular Insulin Resistance†

PubMed Central

Bhonagiri, Padma; Pattar, Guruprasad R.; Habegger, Kirk M.; McCarthy, Alicia M.; Tackett, Lixuan

2011-01-01

Hyperinsulinemia is known to promote the progression/worsening of insulin resistance. Evidence reveals a hidden cost of hyperinsulinemia on plasma membrane (PM) phosphatidylinositol 4,5-bisphosphate (PIP2)-regulated filamentous actin (F-actin) structure, components critical to the normal operation of the insulin-regulated glucose transport system. Here we delineated whether increased glucose flux through the hexosamine biosynthesis pathway (HBP) causes PIP2/F-actin dysregulation and subsequent insulin resistance. Increased glycosylation events were detected in 3T3-L1 adipocytes cultured under conditions closely resembling physiological hyperinsulinemia (5 nm insulin; 12 h) and in cells in which HBP activity was amplified by 2 mm glucosamine (GlcN). Both the physiological hyperinsulinemia and experimental GlcN challenge induced comparable losses of PIP2 and F-actin. In addition to protecting against the insulin-induced membrane/cytoskeletal abnormality and insulin-resistant state, exogenous PIP2 corrected the GlcN-induced insult on these parameters. Moreover, in accordance with HBP flux directly weakening PIP2/F-actin structure, pharmacological inhibition of the rate-limiting HBP enzyme [glutamine-fructose-6-phosphate amidotransferase (GFAT)] restored PIP2-regulated F-actin structure and insulin responsiveness. Conversely, overexpression of GFAT was associated with a loss of detectable PM PIP2 and insulin sensitivity. Even less invasive challenges with glucose, in the absence of insulin, also led to PIP2/F-actin dysregulation. Mechanistically we found that increased HBP activity increased PM cholesterol, the removal of which normalized PIP2/F-actin levels. Accordingly, these data suggest that glucose transporter-4 functionality, dependent on PIP2 and/or F-actin status, can be critically compromised by inappropriate HBP activity. Furthermore, these data are consistent with the PM cholesterol accrual/toxicity as a mechanistic basis of the HBP-induced defects in PIP2/F-actin

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

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.

Identification of Actin-Binding Proteins from Maize Pollen

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

Staiger, C.J.

Specific Aims--The goal of this project was to gain an understanding of how actin filament organization and dynamics are controlled in flowering plants. Specifically, we proposed to identify unique proteins with novel functions by investigating biochemical strategies for the isolation and characterization of actin-binding proteins (ABPs). In particular, our hunt was designed to identify capping proteins and nucleation factors. The specific aims included: (1) to use F-actin affinity chromatography (FAAC) as a general strategy to isolate pollen ABPs (2) to produce polyclonal antisera and perform subcellular localization in pollen tubes (3) to isolate cDNA clones for the most promising ABPsmore » (4) to further purify and characterize ABP interactions with actin in vitro. Summary of Progress By employing affinity chromatography on F-actin or DNase I columns, we have identified at least two novel ABPs from pollen, PrABP80 (gelsolin-like) and ZmABP30, We have also cloned and expressed recombinant protein, as well as generated polyclonal antisera, for 6 interesting ABPs from Arabidopsis (fimbrin AtFIM1, capping protein a/b (AtCP), adenylyl cyclase-associated protein (AtCAP), AtCapG & AtVLN1). We performed quantitative analyses of the biochemical properties for two of these previously uncharacterized ABPs (fimbrin and capping protein). Our studies provide the first evidence for fimbrin activity in plants, demonstrate the existence of barbed-end capping factors and a gelsolin-like severing activity, and provide the quantitative data necessary to establish and test models of F-actin organization and dynamics in plant cells.« less

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

Synthetic peptides that cause F-actin bundling and block actin depolymerization

DOEpatents

Sederoff, Heike [Raleigh, NC; Huber, Steven C [Savoy, IL; Larabell, Carolyn A [Berkeley, CA

2011-10-18

Synthetic peptides derived from sucrose synthase, and having homology to actin and actin-related proteins, sharing a common motif, useful for causing acting bundling and preventing actin depolymerization. Peptides exhibiting the common motif are described, as well as specific synthetic peptides which caused bundled actin and inhibit actin depolymerization. These peptides can be useful for treating a subject suffering from a disease characterized by cells having neoplastic growth, for anti-cancer therapeutics, delivered to subjects solely, or concomitantly or sequentially with other known cancer therapeutics. These peptides can also be used for stabilizing microfilaments in living cells and inhibiting growth of cells.

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

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.

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

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.

A mitochondria-anchored isoform of the actin-nucleating spire protein regulates mitochondrial division

PubMed Central

Manor, Uri; Bartholomew, Sadie; Golani, Gonen; Christenson, Eric; Kozlov, Michael; Higgs, Henry; Spudich, James; Lippincott-Schwartz, Jennifer

2015-01-01

Mitochondrial division, essential for survival in mammals, is enhanced by an inter-organellar process involving ER tubules encircling and constricting mitochondria. The force for constriction is thought to involve actin polymerization by the ER-anchored isoform of the formin protein inverted formin 2 (INF2). Unknown is the mechanism triggering INF2-mediated actin polymerization at ER-mitochondria intersections. We show that a novel isoform of the formin-binding, actin-nucleating protein Spire, Spire1C, localizes to mitochondria and directly links mitochondria to the actin cytoskeleton and the ER. Spire1C binds INF2 and promotes actin assembly on mitochondrial surfaces. Disrupting either Spire1C actin- or formin-binding activities reduces mitochondrial constriction and division. We propose Spire1C cooperates with INF2 to regulate actin assembly at ER-mitochondrial contacts. Simulations support this model's feasibility and demonstrate polymerizing actin filaments can induce mitochondrial constriction. Thus, Spire1C is optimally positioned to serve as a molecular hub that links mitochondria to actin and the ER for regulation of mitochondrial division. DOI: http://dx.doi.org/10.7554/eLife.08828.001 PMID:26305500

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.

Single molecules of the bacterial actin MreB undergo directed treadmilling motion in Caulobacter crescentus.

PubMed

Kim, So Yeon; Gitai, Zemer; Kinkhabwala, Anika; Shapiro, Lucy; Moerner, W E

2006-07-18

The actin cytoskeleton represents a key regulator of multiple essential cellular functions in both eukaryotes and prokaryotes. In eukaryotes, these functions depend on the orchestrated dynamics of actin filament assembly and disassembly. However, the dynamics of the bacterial actin homolog MreB have yet to be examined in vivo. In this study, we observed the motion of single fluorescent MreB-yellow fluorescent protein fusions in living Caulobacter cells in a background of unlabeled MreB. With time-lapse imaging, polymerized MreB [filamentous MreB (fMreB)] and unpolymerized MreB [globular MreB (gMreB)] monomers could be distinguished: gMreB showed fast motion that was characteristic of Brownian diffusion, whereas the labeled molecules in fMreB displayed slow, directed motion. This directional movement of labeled MreB in the growing polymer provides an indication that, like actin, MreB monomers treadmill through MreB filaments by preferential polymerization at one filament end and depolymerization at the other filament end. From these data, we extract several characteristics of single MreB filaments, including that they are, on average, much shorter than the cell length and that the direction of their polarized assembly seems to be independent of the overall cellular polarity. Thus, MreB, like actin, exhibits treadmilling behavior in vivo, and the long MreB structures that have been visualized in multiple bacterial species seem to represent bundles of short filaments that lack a uniform global polarity.

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

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

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).

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

The Actin Nucleator Cobl Is Controlled by Calcium and Calmodulin

PubMed Central

Haag, Natja; Kessels, Michael M.; Qualmann, Britta

2015-01-01

Actin nucleation triggers the formation of new actin filaments and has the power to shape cells but requires tight control in order to bring about proper morphologies. The regulation of the members of the novel class of WASP Homology 2 (WH2) domain-based actin nucleators, however, thus far has largely remained elusive. Our study reveals signal cascades and mechanisms regulating Cordon-Bleu (Cobl). Cobl plays some, albeit not fully understood, role in early arborization of neurons and nucleates actin by a mechanism that requires a combination of all three of its actin monomer–binding WH2 domains. Our experiments reveal that Cobl is regulated by Ca2+ and multiple, direct associations of the Ca2+ sensor Calmodulin (CaM). Overexpression analyses and rescue experiments of Cobl loss-of-function phenotypes with Cobl mutants in primary neurons and in tissue slices demonstrated the importance of CaM binding for Cobl’s functions. Cobl-induced dendritic branch initiation was preceded by Ca2+ signals and coincided with local F-actin and CaM accumulations. CaM inhibitor studies showed that Cobl-mediated branching is strictly dependent on CaM activity. Mechanistic studies revealed that Ca2+/CaM modulates Cobl’s actin binding properties and furthermore promotes Cobl’s previously identified interactions with the membrane-shaping F-BAR protein syndapin I, which accumulated with Cobl at nascent dendritic protrusion sites. The findings of our study demonstrate a direct regulation of an actin nucleator by Ca2+/CaM and reveal that the Ca2+/CaM-controlled molecular mechanisms we discovered are crucial for Cobl’s cellular functions. By unveiling the means of Cobl regulation and the mechanisms, by which Ca2+/CaM signals directly converge on a cellular effector promoting actin filament formation, our work furthermore sheds light on how local Ca2+ signals steer and power branch initiation during early arborization of nerve cells—a key process in neuronal network formation. PMID

In Vivo, Villin Is Required for Ca2+-Dependent F-Actin Disruption in Intestinal Brush Borders

PubMed Central

Ferrary, Evelyne; Cohen-Tannoudji, Michel; Pehau-Arnaudet, Gérard; Lapillonne, Alexandre; Athman, Rafika; Ruiz, Tereza; Boulouha, Lilia; El Marjou, Fatima; Doye, Anne; Fontaine, Jean-Jacques; Antony, Claude; Babinet, Charles; Louvard, Daniel; Jaisser, Frédéric; Robine, Sylvie

1999-01-01

Villin is an actin-binding protein localized in intestinal and kidney brush borders. In vitro, villin has been demonstrated to bundle and sever F-actin in a Ca2+-dependent manner. We generated knockout mice to study the role of villin in vivo. In villin-null mice, no noticeable changes were observed in the ultrastructure of the microvilli or in the localization and expression of the actin-binding and membrane proteins of the intestine. Interestingly, the response to elevated intracellular Ca2+ differed significantly between mutant and normal mice. In wild-type animals, isolated brush borders were disrupted by the addition of Ca2+, whereas Ca2+ had no effect in villin-null isolates. Moreover, increase in intracellular Ca2+ by serosal carbachol or mucosal Ca2+ ionophore A23187 application abolished the F-actin labeling only in the brush border of wild-type animals. This F-actin disruption was also observed in physiological fasting/refeeding experiments. Oral administration of dextran sulfate sodium, an agent that causes colonic epithelial injury, induced large mucosal lesions resulting in a higher death probability in mice lacking villin, 36 ± 9.6%, compared with wild-type mice, 70 ± 8.8%, at day 13. These results suggest that in vivo, villin is not necessary for the bundling of F-actin microfilaments, whereas it is necessary for the reorganization elicited by various signals. We postulate that this property might be involved in cellular plasticity related to cell injury. PMID:10459016

F-actin clustering and cell dysmotility induced by the pathological W148R missense mutation of filamin B at the actin-binding domain.

PubMed

Zhao, Yongtong; Shapiro, Sandor S; Eto, Masumi

2016-01-01

Filamin B (FLNB) is a dimeric actin-binding protein that orchestrates the reorganization of the actin cytoskeleton. Congenital mutations of FLNB at the actin-binding domain (ABD) are known to cause abnormalities of skeletal development, such as atelosteogenesis types I and III and Larsen's syndrome, although the underlying mechanisms are poorly understood. Here, using fluorescence microscopy, we characterized the reorganization of the actin cytoskeleton in cells expressing each of six pathological FLNB mutants that have been linked to skeletal abnormalities. The subfractionation assay showed a greater accumulation of the FLNB ABD mutants W148R and E227K than the wild-type protein to the cytoskeleton. Ectopic expression of FLNB-W148R and, to a lesser extent, FLNB-E227K induced prominent F-actin accumulations and the consequent rearrangement of focal adhesions, myosin II, and septin filaments and results in a delayed directional migration of the cells. The W148R protein-induced cytoskeletal rearrangement was partially attenuated by the inhibition of myosin II, p21-activated protein kinase, or Rho-associated protein kinase. The expression of a single-head ABD fragment with the mutations partially mimicked the rearrangement induced by the dimer. The F-actin clustering through the interaction with the mutant FLNB ABD may limit the cytoskeletal reorganization, preventing normal skeletal development. Copyright © 2016 the American Physiological Society.

Damage effects of protoporphyrin IX - sonodynamic therapy on the cytoskeletal F-actin of Ehrlich ascites carcinoma cells.

PubMed

Zhao, Xia; Liu, Quanhong; Tang, Wei; Wang, Xiaobing; Wang, Pan; Gong, Liyan; Wang, Yuan

2009-01-01

In this study, we report evidence of the damage effects of sonodynamic therapy (SDT) on a novel intracellular target, cytoskeletal F-actin, that has great importance for cancer treatment. Ehrlich ascites carcinoma (EAC) cells suspended in PBS were exposed to ultrasound at 1.34 MHz for up to 60s in the presence and absence of protoporphyrin IX (PPIX). To evaluate the polymeric state and distribution of actin filaments (AF) we employed FITC-Phalloidin staining. The percentage of cells with intact AF was decreased with 10-80 microM PPIX after ultrasonic exposure, while only few cells with disturbed F-actin were observed with 80 microM PPIX alone. The fluorescence intensity of FITC-Phalloidin labeled cells was detected by flow cytometry. The morphological changes of EAC cells were observed by scanning electron microscope (SEM). The nuclei were stained with Hoechst 33258 to determine apoptosis. Cytoskeletal F-actin and cell morphological changes were dependent on the time after SDT. Some cells suffered deformations of plasma membrane as blebs that reacted positively to FITC-Phalloidin at 2h after SDT treatment. Many of the cells showed the typically apoptotic chromatin fragmentation. The alterations were more significant 4h later. Our results showed that cytoskeletal F-actin might represent an important target for the SDT treatment and the observed effect on F-actin and the subsequent bleb formation mainly due to apoptosis formation due to the treatment.

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.

The MARVEL domain protein Nce102 regulates actin organization and invasive growth of Candida albicans.

PubMed

Douglas, Lois M; Wang, Hong X; Konopka, James B

2013-11-26

Invasive growth of the fungal pathogen Candida albicans into tissues promotes disseminated infections in humans. The plasma membrane is essential for pathogenesis because this important barrier mediates morphogenesis and invasive growth, as well as secretion of virulence factors, cell wall synthesis, nutrient import, and other processes. Previous studies showed that the Sur7 tetraspan protein that localizes to MCC (membrane compartment occupied by Can1)/eisosome subdomains of the plasma membrane regulates a broad range of key functions, including cell wall synthesis, morphogenesis, and resistance to copper. Therefore, a distinct tetraspan protein found in MCC/eisosomes, Nce102, was investigated. Nce102 belongs to the MARVEL domain protein family, which is implicated in regulating membrane structure and function. Deletion of NCE102 did not cause the broad defects seen in sur7Δ cells. Instead, the nce102Δ mutant displayed a unique phenotype in that it was defective in forming hyphae and invading low concentrations of agar but could invade well in higher agar concentrations. This phenotype was likely due to a defect in actin organization that was observed by phalloidin staining. In support of this, the invasive growth defect of a bni1Δ mutant that mislocalizes actin due to lack of the Bni1 formin was also reversed at high agar concentrations. This suggests that a denser matrix provides a signal that compensates for the actin defects. The nce102Δ mutant displayed decreased virulence and formed abnormal hyphae in mice. These studies identify novel ways that Nce102 and the physical environment surrounding C. albicans regulate morphogenesis and pathogenesis. The plasma membrane promotes virulence of the human fungal pathogen Candida albicans by acting as a protective barrier around the cell and mediating dynamic activities, such as morphogenesis, cell wall synthesis, secretion of virulence factors, and nutrient uptake. To better understand how the plasma membrane

Spire, an actin nucleation factor, regulates cell division during Drosophila heart development.

PubMed

Xu, Peng; Johnson, Tamara L; Stoller-Conrad, Jessica R; Schulz, Robert A

2012-01-01

The Drosophila dorsal vessel is a beneficial model system for studying the regulation of early heart development. Spire (Spir), an actin-nucleation factor, regulates actin dynamics in many developmental processes, such as cell shape determination, intracellular transport, and locomotion. Through protein expression pattern analysis, we demonstrate that the absence of spir function affects cell division in Myocyte enhancer factor 2-, Tinman (Tin)-, Even-skipped- and Seven up (Svp)-positive heart cells. In addition, genetic interaction analysis shows that spir functionally interacts with Dorsocross, tin, and pannier to properly specify the cardiac fate. Furthermore, through visualization of double heterozygous embryos, we determines that spir cooperates with CycA for heart cell specification and division. Finally, when comparing the spir mutant phenotype with that of a CycA mutant, the results suggest that most Svp-positive progenitors in spir mutant embryos cannot undergo full cell division at cell cycle 15, and that Tin-positive progenitors are arrested at cell cycle 16 as double-nucleated cells. We conclude that Spir plays a crucial role in controlling dorsal vessel formation and has a function in cell division during heart tube morphogenesis.

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.

Daam1 regulates fascin for actin assembly in mouse oocyte meiosis.

PubMed

Lu, Yujie; Zhang, Yu; Pan, Meng-Hao; Kim, Nam-Hyung; Sun, Shao-Chen; Cui, Xiang-Shun

2017-07-18

As a formin protein, Daam1 (Dishevelled-associated activator of morphogenesis 1) is reported to regulate series of cell processes like endocytosis, cell morphology and migration via its effects on actin assembly in mitosis. However, whether Daam1 plays roles in female meiosis remains uncertain. In this study, we investigated the expression and functions of Daam1 during mouse oocyte meiosis. Our results indicated that Daam1 localized at the cortex of oocytes, which was similar with actin filaments. After Daam1 morpholino (MO) microinjection, the expression of Daam1 significantly decreased, which resulted in the failure of oocyte polar body extrusion. These results might be due to the defects of actin assembly, since the decreased fluorescence intensity of actin filaments in oocyte cortex and cytoplasm were observed. However, Daam1 knockdown seemed not to affect the meiotic spindle movement. In addition, we found that fascin might be the down effector of Daam1, since the protein expression of fascin decreased after Daam1 knockdown. Thus, our data suggested that Daam1 affected actin assembly during oocyte meiotic division via the regulation of fascin expression.

Knockdown of microtubule actin crosslinking factor 1 inhibits cell proliferation in MC3T3-E1 osteoblastic cells

PubMed Central

Hu, Lifang; Su, Peihong; Li, Runzhi; Yan, Kun; Chen, Zhihao; Shang, Peng; Qian, Airong

2015-01-01

Microtubule actin crosslinking factor 1 (MACF1), a widely expressed cytoskeletal linker, plays important roles in various cells by regulating cytoskeleton dynamics. However, its role in osteoblastic cells is not well understood. Based on our previous findings that the association of MACF1 with F-actin and microtubules in osteoblast-like cells was altered under magnetic force conditions, here, by adopting a stable MACF1-knockdown MC3T3-E1 osteoblastic cell line, we found that MACF1 knockdown induced large cells with a binuclear/multinuclear structure. Further, immunofluorescence staining showed disorganization of F-actin and microtubules in MACF1-knockdown cells. Cell counting revealed significant decrease of cell proliferation and cell cycle analysis showed an S phase cell cycle arrest in MACF1-knockdown cells. Moreover and interestingly, MACF1 knockdown showed a potential effect on cellular MTT reduction activity and mitochondrial content, suggesting an impact on cellular metabolic activity. These results together indicate an important role of MACF1 in regulating osteoblastic cell morphology and function. [BMB Reports 2015; 48(10): 583-588] PMID:26277981

Knockdown of microtubule actin crosslinking factor 1 inhibits cell proliferation in MC3T3-E1 osteoblastic cells.

PubMed

Hu, Lifang; Su, Peihong; Li, Runzhi; Yan, Kun; Chen, Zhihao; Shang, Peng; Qian, Airong

2015-10-01

Microtubule actin crosslinking factor 1 (MACF1), a widely expressed cytoskeletal linker, plays important roles in various cells by regulating cytoskeleton dynamics. However, its role in osteoblastic cells is not well understood. Based on our previous findings that the association of MACF1 with F-actin and microtubules in osteoblast-like cells was altered under magnetic force conditions, here, by adopting a stable MACF1-knockdown MC3T3-E1 osteoblastic cell line, we found that MACF1 knockdown induced large cells with a binuclear/multinuclear structure. Further, immunofluorescence staining showed disorganization of F-actin and microtubules in MACF1-knockdown cells. Cell counting revealed significant decrease of cell proliferation and cell cycle analysis showed an S phase cell cycle arrest in MACF1-knockdown cells. Moreover and interestingly, MACF1 knockdown showed a potential effect on cellular MTT reduction activity and mitochondrial content, suggesting an impact on cellular metabolic activity. These results together indicate an important role of MACF1 in regulating osteoblastic cell morphology and function.

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

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).

Distinct structural changes detected by X-ray fiber diffraction in stabilization of F-actin by lowering pH and increasing ionic strength.

PubMed

Oda, T; Makino, K; Yamashita, I; Namba, K; Maéda, Y

2001-02-01

Lowering pH or raising salt concentration stabilizes the F-actin structure by increasing the free energy change associated with its polymerization. To understand the F-actin stabilization mechanism, we studied the effect of pH, salt concentration, and cation species on the F-actin structure. X-ray fiber diffraction patterns recorded from highly ordered F-actin sols at high density enabled us to detect minute changes of diffraction intensities and to precisely determine the helical parameters. F-actin in a solution containing 30 mM NaCl at pH 8 was taken as the control. F-actin at pH 8, 30 to 90 mM NaCl or 30 mM KCl showed a helical symmetry of 2.161 subunits per turn of the 1-start helix (12.968 subunits/6 turns). Lowering pH from 8 to 6 or replacing NaCl by LiCl altered the helical symmetry to 2.159 subunits per turn (12.952/6). The diffraction intensity associated with the 27-A meridional layer-line increased as the pH decreased but decreased as the NaCl concentration increased. None of the solvent conditions tested gave rise to significant changes in the pitch of the left-handed 1-start helix (approximately 59.8 A). The present results indicate that the two factors that stabilize F-actin, relatively low pH and high salt concentration, have distinct effects on the F-actin structure. Possible mechanisms will be discussed to understand how F-actin is stabilized under these conditions.

STEF/TIAM2-mediated Rac1 activity at the nuclear envelope regulates the perinuclear actin cap.

PubMed

Woroniuk, Anna; Porter, Andrew; White, Gavin; Newman, Daniel T; Diamantopoulou, Zoi; Waring, Thomas; Rooney, Claire; Strathdee, Douglas; Marston, Daniel J; Hahn, Klaus M; Sansom, Owen J; Zech, Tobias; Malliri, Angeliki

2018-05-29

The perinuclear actin cap is an important cytoskeletal structure that regulates nuclear morphology and re-orientation during front-rear polarisation. The mechanisms regulating the actin cap are currently poorly understood. Here, we demonstrate that STEF/TIAM2, a Rac1 selective guanine nucleotide exchange factor, localises at the nuclear envelope, co-localising with the key perinuclear proteins Nesprin-2G and Non-muscle myosin IIB (NMMIIB), where it regulates perinuclear Rac1 activity. We show that STEF depletion reduces apical perinuclear actin cables (a phenotype rescued by targeting active Rac1 to the nuclear envelope), increases nuclear height and impairs nuclear re-orientation. STEF down-regulation also reduces perinuclear pMLC and decreases myosin-generated tension at the nuclear envelope, suggesting that STEF-mediated Rac1 activity regulates NMMIIB activity to promote stabilisation of the perinuclear actin cap. Finally, STEF depletion decreases nuclear stiffness and reduces expression of TAZ-regulated genes, indicating an alteration in mechanosensing pathways as a consequence of disruption of the actin cap.

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

PHD3-mediated prolyl hydroxylation of nonmuscle actin impairs polymerization and cell motility

PubMed Central

Luo, Weibo; Lin, Benjamin; Wang, Yingfei; Zhong, Jun; O'Meally, Robert; Cole, Robert N.; Pandey, Akhilesh; Levchenko, Andre; Semenza, Gregg L.

2014-01-01

Actin filaments play an essential role in cell movement, and many posttranslational modifications regulate actin filament assembly. Here we report that prolyl hydroxylase 3 (PHD3) interacts with nonmuscle actin in human cells and catalyzes hydroxylation of actin at proline residues 307 and 322. Blocking PHD3 expression or catalytic activity by short hairpin RNA knockdown or pharmacological inhibition, respectively, decreased actin prolyl hydroxylation. PHD3 knockdown increased filamentous F-actin assembly, which was reversed by PHD3 overexpression. PHD3 knockdown increased cell velocity and migration distance. Inhibition of PHD3 prolyl hydroxylase activity by dimethyloxalylglycine also increased actin polymerization and cell migration. These data reveal a novel role for PHD3 as a negative regulator of cell motility through posttranslational modification of nonmuscle actins. PMID:25079693

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.

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

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.

Importance of a Lys113–Glu195 Intermonomer Ionic Bond in F-actin Stabilization and Regulation by Yeast Formins Bni1p and Bnr1p*

PubMed Central

Wen, Kuo-Kuang; McKane, Melissa; Rubenstein, Peter A.

2013-01-01

Proper actin cytoskeletal function requires actin's ability to generate a stable filament and requires that this reaction be regulated by actin-binding proteins via allosteric effects on the actin. A proposed ionic interaction in the actin filament interior between Lys113 of one monomer and Glu195 of a monomer in the apposing strand potentially fosters cross-strand stabilization and allosteric communication between the filament interior and exterior. We interrupted the potential interaction by creating either K113E or E195K actin. By combining the two, we also reversed the interaction with a K113E/E195K (E/K) mutant. In all cases, we isolated viable cells expressing only the mutant actin. Either single mutant cell displays significantly decreased growth in YPD medium. This deficit is rescued in the double mutant. All three mutants display abnormal phalloidin cytoskeletal staining. K113E actin exhibits a critical concentration of polymerization 4 times higher than WT actin, nucleates more poorly, and forms shorter filaments. Restoration of the ionic bond, E/K, eliminates most of these problems. E195K actin behaves much more like WT actin, indicating accommodation of the neighboring lysines. Both Bni1 and Bnr1 formin FH1-FH2 fragment accelerate polymerization of WT, E/K, and to a lesser extent E195K actin. Bni1p FH1-FH2 dramatically inhibits K113E actin polymerization, consistent with barbed end capping. However, Bnr1p FH1-FH2 restores K113E actin polymerization, forming single filaments. In summary, the proposed ionic interaction plays an important role in filament stabilization and in the propagation of allosteric changes affecting formin regulation in an isoform-specific fashion. PMID:23653364

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.

A nuclear F-actin scaffold stabilizes ribonucleoprotein droplets against gravity in large cells.

PubMed

Feric, Marina; Brangwynne, Clifford P

2013-10-01

The size of a typical eukaryotic cell is of the order of ∼10 μm. However, some cell types grow to very large sizes, including oocytes (immature eggs) of organisms from humans to starfish. For example, oocytes of the frog Xenopus laevis grow to a diameter ≥1 mm. They have a correspondingly large nucleus (germinal vesicle) of ∼450 μm in diameter, which is similar to smaller somatic nuclei, but contains a significantly higher concentration of actin. The form and structure of this nuclear actin remain controversial, and its potential mechanical role within these large nuclei is unknown. Here, we use a microrheology and quantitative imaging approach to show that germinal vesicles contain an elastic F-actin scaffold that mechanically stabilizes these large nuclei against gravitational forces, which are usually considered negligible within cells. We find that on actin disruption, ribonucleoprotein droplets, including nucleoli and histone locus bodies, undergo gravitational sedimentation and fusion. We develop a model that reveals how gravity becomes an increasingly potent force as cells and their nuclei grow larger than ∼10 μm, explaining the requirement for a stabilizing nuclear F-actin scaffold in large Xenopus oocytes. All life forms are subject to gravity, and our results may have broad implications for cell growth and size control.

A nuclear F-actin scaffold stabilizes RNP droplets against gravity in large cells

PubMed Central

Feric, Marina; Brangwynne, Clifford P.

2013-01-01

The size of a typical eukaryotic cell is on the order of ≈10 μm. However, some cell types grow to very large sizes, including oocytes (immature eggs) of organisms from humans to starfish. For example, oocytes of the frog X. laevis grow to a diameter ≥1 mm. They contain a correspondingly large nucleus (germinal vesicle, GV) of ≈450 μm in diameter, which is similar to smaller somatic nuclei, but contains a significantly higher concentration of actin. The form and structure of this nuclear actin remain controversial, and its potential mechanical role within these large nuclei is unknown. Here, we use a microrheology and quantitative imaging approach to show that GVs contain an elastic F-actin scaffold that mechanically stabilizes these large nuclei against gravitational forces, which are usually considered negligible within cells. We find that upon actin disruption, RNA/protein droplets, including nucleoli and histone locus bodies (HLBs), undergo gravitational sedimentation and fusion. We develop a model that reveals how gravity becomes an increasingly potent force as cells and their nuclei grow larger than ≈10 μm, explaining the requirement for a stabilizing nuclear F-actin scaffold in large X. laevis ooctyes. All life forms are subject to gravity, and our results may have broad implications for cell growth and size control. PMID:23995731

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 capping protein CAPZB regulates cell morphology, differentiation, and neural crest migration in craniofacial morphogenesis†

PubMed Central

Mukherjee, Kusumika; Ishii, Kana; Pillalamarri, Vamsee; Kammin, Tammy; Atkin, Joan F.; Hickey, Scott E.; Xi, Qiongchao J.; Zepeda, Cinthya J.; Gusella, James F.; Talkowski, Michael E.; Morton, Cynthia C.; Maas, Richard L.; Liao, Eric C.

2016-01-01

CAPZB is an actin-capping protein that caps the growing end of F-actin and modulates the cytoskeleton and tethers actin filaments to the Z-line of the sarcomere in muscles. Whole-genome sequencing was performed on a subject with micrognathia, cleft palate and hypotonia that harbored a de novo, balanced chromosomal translocation that disrupts the CAPZB gene. The function of capzb was analyzed in the zebrafish model. capzb−/− mutants exhibit both craniofacial and muscle defects that recapitulate the phenotypes observed in the human subject. Loss of capzb affects cell morphology, differentiation and neural crest migration. Differentiation of both myogenic stem cells and neural crest cells requires capzb. During palate morphogenesis, defective cranial neural crest cell migration in capzb−/− mutants results in loss of the median cell population, creating a cleft phenotype. capzb is also required for trunk neural crest migration, as evident from melanophores disorganization in capzb−/− mutants. In addition, capzb over-expression results in embryonic lethality. Therefore, proper capzb dosage is important during embryogenesis, and regulates both cell behavior and tissue morphogenesis. PMID:26758871

Actin Isoform-specific Conformational Differences Observed with Hydrogen/Deuterium Exchange and Mass Spectrometry*

PubMed Central

Stokasimov, Ema; Rubenstein, Peter A.

2009-01-01

Actin can exist in multiple conformations necessary for normal function. Actin isoforms, although highly conserved in sequence, exhibit different biochemical properties and cellular roles. We used amide proton hydrogen/deuterium (HD) exchange detected by mass spectrometry to analyze conformational differences between Saccharomyces cerevisiae and muscle actins in the G and F forms to gain insight into these differences. We also utilized HD exchange to study interdomain and allosteric communication in yeast-muscle hybrid actins to better understand the conformational dynamics of actin. Areas showing differences in HD exchange between G- and F-actins are areas of intermonomer contacts, consistent with the current filament models. Our results showed greater exchange for yeast G-actin compared with muscle actin in the barbed end pivot region and areas in subdomains 1 and 2 and for F-actin in monomer-monomer contact areas. These results suggest greater flexibility of the yeast actin monomer and filament compared with muscle actin. For hybrid G-actins, the muscle-like and yeastlike parts of the molecule generally showed exchange characteristics resembling their parent actins. A few exceptions were a peptide on top of subdomain 2 and the pivot region between subdomains 1 and 3 with muscle actin-like exchange characteristics although the areas were yeastlike. These results demonstrate that there is cross-talk between subdomains 1 and 2 and the large and small domains. Hybrid F-actin data showing greater exchange compared with both yeast and muscle actins are consistent with mismatched yeast-muscle interfaces resulting in decreased stability of the hybrid filament contacts. PMID:19605362

Actin dynamics involved in gravity perception in Arabidopsis inflorescense stem

NASA Astrophysics Data System (ADS)

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

The amyloplasts sedimentation in the endodermal cells is important for gravity perception in Arabidopsis shoot. Our previous study suggests that SGR5(SHOOT GRAVITROPISM 5) and SGR9 are synergistically involved in regulation of amyloplast movement in these cells, and shows that sgr5 sgr9 double mutant completely loses gravitropic response. SGR5 encodes putative transcription factor and SGR9 encodes a ring finger containing protein, which surrounds amyloplasts. It has been reported that amyloplasts are surrounded by actin microfilaments (MFs), and that treatment with actin polymerization inhibitor enhances gravitropic organ curvature. However, not only the molecular link between amyolplasts and MFs, but also regulatory role of MFs in gravitropic response is still unclear. Here, we found that treatment with actin polymerization inhibitor restored gravitropic response of sgr5 sgr9 double mutant stems. The result suggests that abnormal amyloplasts movement in the double mutant could result from inhibition of MFs depolymerization, leading to abnormal gravitropism. We are investigating whether SGR5 and SGR9 are involved in amyloplasts movement by regulating actin remodeling in gravity perceptive cells.

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.

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

The F-BAR domains from srGAP1, srGAP2 and srGAP3 regulate membrane deformation differently

PubMed Central

Coutinho-Budd, Jaeda; Ghukasyan, Vladimir; Zylka, Mark J.; Polleux, Franck

2012-01-01

Summary Coordination of membrane deformation and cytoskeletal dynamics lies at the heart of many biological processes critical for cell polarity, motility and morphogenesis. We have recently shown that Slit-Robo GTPase-activating protein 2 (srGAP2) regulates neuronal morphogenesis through the ability of its F-BAR domain to regulate membrane deformation and induce filopodia formation. Here, we demonstrate that the F-BAR domains of two closely related family members, srGAP1 and srGAP3 [designated F-BAR(1) and F-BAR(3), respectively] display significantly different membrane deformation properties in non-neuronal COS7 cells and in cortical neurons. F-BAR(3) induces filopodia in both cell types, though less potently than F-BAR(2), whereas F-BAR(1) prevents filopodia formation in cortical neurons and reduces plasma membrane dynamics. These three F-BAR domains can heterodimerize, and they act synergistically towards filopodia induction in COS7 cells. As measured by fluorescence recovery after photobleaching, F-BAR(2) displays faster molecular dynamics than F-BAR(3) and F-BAR(1) at the plasma membrane, which correlates well with its increased potency to induce filopodia. We also show that the molecular dynamic properties of F-BAR(2) at the membrane are partially dependent on F-Actin. Interestingly, acute phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] depletion in cells does not interfere with plasma membrane localization of F-BAR(2), which is compatible with our result showing that F-BAR(2) binds to a broad range of negatively-charged phospholipids present at the plasma membrane, including phosphatidylserine (PtdSer). Overall, our results provide novel insights into the functional diversity of the membrane deformation properties of this subclass of F-BAR-domains required for cell morphogenesis. PMID:22467852

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

PKCθ links proximal T cell and Notch signaling through localized regulation of the actin cytoskeleton

PubMed Central

Britton, Graham J; Ambler, Rachel; Clark, Danielle J; Hill, Elaine V; Tunbridge, Helen M; McNally, Kerrie E; Burton, Bronwen R; Butterweck, Philomena; Sabatos-Peyton, Catherine; Hampton-O’Neil, Lea A; Verkade, Paul; Wülfing, Christoph; Wraith, David Cameron

2017-01-01

Notch is a critical regulator of T cell differentiation and is activated through proteolytic cleavage in response to ligand engagement. Using murine myelin-reactive CD4 T cells, we demonstrate that proximal T cell signaling modulates Notch activation by a spatiotemporally constrained mechanism. The protein kinase PKCθ is a critical mediator of signaling by the T cell antigen receptor and the principal costimulatory receptor CD28. PKCθ selectively inactivates the negative regulator of F-actin generation, Coronin 1A, at the center of the T cell interface with the antigen presenting cell (APC). This allows for effective generation of the large actin-based lamellum required for recruitment of the Notch-processing membrane metalloproteinase ADAM10. Such enhancement of Notch activation is critical for efficient T cell proliferation and Th17 differentiation. We reveal a novel mechanism that, through modulation of the cytoskeleton, controls Notch activation at the T cell:APC interface thereby linking T cell receptor and Notch signaling pathways. DOI: http://dx.doi.org/10.7554/eLife.20003.001 PMID:28112644

SDF1 Reduces Interneuron Leading Process Branching through Dual Regulation of Actin and Microtubules

PubMed Central

Lysko, Daniel E.; Putt, Mary

2014-01-01

Normal cerebral cortical function requires a highly ordered balance between projection neurons and interneurons. During development these two neuronal populations migrate from distinct progenitor zones to form the cerebral cortex, with interneurons originating in the more distant ganglionic eminences. Moreover, deficits in interneurons have been linked to a variety of neurodevelopmental disorders underscoring the importance of understanding interneuron development and function. We, and others, have identified SDF1 signaling as one important modulator of interneuron migration speed and leading process branching behavior in mice, although how SDF1 signaling impacts these behaviors remains unknown. We previously found SDF1 inhibited leading process branching while increasing the rate of migration. We have now mechanistically linked SDF1 modulation of leading process branching behavior to a dual regulation of both actin and microtubule organization. We find SDF1 consolidates actin at the leading process tip by de-repressing calpain protease and increasing proteolysis of branched-actin-supporting cortactin. Additionally, SDF1 stabilizes the microtubule array in the leading process through activation of the microtubule-associated protein doublecortin (DCX). DCX stabilizes the microtubule array by bundling microtubules within the leading process, reducing branching. These data provide mechanistic insight into the regulation of interneuron leading process dynamics during neuronal migration in mice and provides insight into how cortactin and DCX, a known human neuronal migration disorder gene, participate in this process. PMID:24695713

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

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

Myofibril growth during cardiac hypertrophy is regulated through dual phosphorylation and acetylation of the actin capping protein CapZ

PubMed Central

Lin, Ying-Hsi; Warren, Chad M.; Li, Jieli; McKinsey, Timothy A.; Russell, Brenda

2016-01-01

The mechanotransduction signaling pathways initiated in heart muscle by increased mechanical loading are known to lead to long-term transcriptional changes and hypertrophy, but the rapid events for adaptation at the sarcomeric level are not fully understood. The goal of this study was to test the hypothesis that actin filament assembly during cardiomyocyte growth is regulated by post-translational modifications (PTMs) of CapZβ1. In rapidly hypertrophying neonatal rat ventricular myocytes (NRVMs) stimulated by phenylephrine (PE), two-dimensional gel electrophoresis (2DGE) of CapZβ1 revealed a shift toward more negative charge. Consistent with this, mass spectrometry identified CapZβ1 phosphorylation on serine-204 and acetylation on lysine-199, two residues which are near the actin binding surface of CapZβ1. Ectopic expression of dominant negative PKCε (dnPKCε) in NRVMs blunted the PE-induced increase in CapZ dynamics, as evidenced by the kinetic constant (Kfrap) of fluorescence recovery after photobleaching (FRAP), and concomitantly reduced phosphorylation and acetylation of CapZβ1. Furthermore, inhibition of class I histone deacetylases (HDACs) increased lysine-199 acetylation on CapZβ1, which increased Kfrap of CapZ and stimulated actin dynamics. Finally, we show that PE treatment of NRVMs results in decreased binding of HDAC3 to myofibrils, suggesting a signal-dependent mechanism for the regulation of sarcomere-associated CapZβ1 acetylation. Taken together, this dual regulation through phosphorylation and acetylation of CapZβ1 provides a novel model for the regulation of myofibril growth during cardiac hypertrophy. PMID:27185186

F-BAR family proteins, emerging regulators for cell membrane dynamic changes-from structure to human diseases.

PubMed

Liu, Suxuan; Xiong, Xinyu; Zhao, Xianxian; Yang, Xiaofeng; Wang, Hong

2015-05-09

Eukaryotic cell membrane dynamics change in curvature during physiological and pathological processes. In the past ten years, a novel protein family, Fes/CIP4 homology-Bin/Amphiphysin/Rvs (F-BAR) domain proteins, has been identified to be the most important coordinators in membrane curvature regulation. The F-BAR domain family is a member of the Bin/Amphiphysin/Rvs (BAR) domain superfamily that is associated with dynamic changes in cell membrane. However, the molecular basis in membrane structure regulation and the biological functions of F-BAR protein are unclear. The pathophysiological role of F-BAR protein is unknown. This review summarizes the current understanding of structure and function in the BAR domain superfamily, classifies F-BAR family proteins into nine subfamilies based on domain structure, and characterizes F-BAR protein structure, domain interaction, and functional relevance. In general, F-BAR protein binds to cell membrane via F-BAR domain association with membrane phospholipids and initiates membrane curvature and scission via Src homology-3 (SH3) domain interaction with its partner proteins. This process causes membrane dynamic changes and leads to seven important cellular biological functions, which include endocytosis, phagocytosis, filopodium, lamellipodium, cytokinesis, adhesion, and podosome formation, via distinct signaling pathways determined by specific domain-binding partners. These cellular functions play important roles in many physiological and pathophysiological processes. We further summarize F-BAR protein expression and mutation changes observed in various diseases and developmental disorders. Considering the structure feature and functional implication of F-BAR proteins, we anticipate that F-BAR proteins modulate physiological and pathophysiological processes via transferring extracellular materials, regulating cell trafficking and mobility, presenting antigens, mediating extracellular matrix degradation, and transmitting

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

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

Interactions of histatin-3 and histatin-5 with actin.

PubMed

Blotnick, Edna; Sol, Asaf; Bachrach, Gilad; Muhlrad, Andras

2017-03-06

Histatins are histidine rich polypeptides produced in the parotid and submandibular gland and secreted into the saliva. Histatin-3 and -5 are the most important polycationic histatins. They possess antimicrobial activity against fungi such as Candida albicans. Histatin-5 has a higher antifungal activity than histatin-3 while histatin-3 is mostly involved in wound healing in the oral cavity. We found that these histatins, like other polycationic peptides and proteins, such as LL-37, lysozyme and histones, interact with extracellular actin. Histatin-3 and -5 polymerize globular actin (G-actin) to filamentous actin (F-actin) and bundle F-actin filaments. Both actin polymerization and bundling by histatins is pH sensitive due to the high histidine content of histatins. In spite of the equal number of net positive charges and histidine residues in histatin-3 and -5, less histatin-3 is needed than histatin-5 for polymerization and bundling of actin. The efficiency of actin polymerization and bundling by histatins greatly increases with decreasing pH. Histatin-3 and -5 induced actin bundles are dissociated by 100 and 50 mM NaCl, respectively. The relatively low NaCl concentration required to dissociate histatin-induced bundles implies that the actin-histatin filaments bind to each other mainly by electrostatic forces. The binding of histatin-3 to F-actin is stronger than that of histatin-5 showing that hydrophobic forces have also some role in histatin-3- actin interaction. Histatins affect the fluorescence of probes attached to the D-loop of G-actin indicating histatin induced changes in actin structure. Transglutaminase cross-links histatins to actin. Competition and limited proteolysis experiments indicate that the main histatin cross-linking site on actin is glutamine-49 on the D-loop of actin. Both histatin-3 and -5 interacts with actin, however, histatin 3 binds stronger to actin and affects actin structure at lower concentration than histatin-5 due to the extra 8

Identification of interfaces involved in weak interactions with application to F-actin-aldolase rafts.

PubMed

Hu, Guiqing; Taylor, Dianne W; Liu, Jun; Taylor, Kenneth A

2018-03-01

Macromolecular interactions occur with widely varying affinities. Strong interactions form well defined interfaces but weak interactions are more dynamic and variable. Weak interactions can collectively lead to large structures such as microvilli via cooperativity and are often the precursors of much stronger interactions, e.g. the initial actin-myosin interaction during muscle contraction. Electron tomography combined with subvolume alignment and classification is an ideal method for the study of weak interactions because a 3-D image is obtained for the individual interactions, which subsequently are characterized collectively. Here we describe a method to characterize heterogeneous F-actin-aldolase interactions in 2-D rafts using electron tomography. By forming separate averages of the two constituents and fitting an atomic structure to each average, together with the alignment information which relates the raw motif to the average, an atomic model of each crosslink is determined and a frequency map of contact residues is computed. The approach should be applicable to any large structure composed of constituents that interact weakly and heterogeneously. Copyright © 2017 Elsevier Inc. All rights reserved.

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).

Anti-inflammatory Mechanism of Geniposide: Inhibiting the Hyperpermeability of Fibroblast-Like Synoviocytes via the RhoA/p38MAPK/NF-κB/F-Actin Signal Pathway

PubMed Central

Deng, Ran; Li, Feng; Wu, Hong; Wang, Wen-yu; Dai, Li; Zhang, Zheng-rong; Fu, Jun

2018-01-01

its anti-inflammatory and immunoregulatory effects via regulating the relative equilibrium of pro-inflammatory cytokines and anti-inflammatory cytokines. GE attenuated the hyperpermeability of FLSs. The down-regulation of the conduction of RhoA/p38MAPK/NF-κB/F-actin signal may play a critical role in the mechanisms of GE on RA. GE could be an effective therapeutic agent for the treatment of RA. PMID:29497378

A novel function of twins, B subunit of protein phosphatase 2A, in regulating actin polymerization.

PubMed

Yeh, Po-An; Chang, Ching-Jin

2017-01-01

Actin is an important component of the cytoskeleton and its polymerization is delicately regulated by several kinases and phosphatases. Heterotrimeric protein phosphatase 2A (PP2A) is a potent phosphatase that is crucial for cell proliferation, apoptosis, tumorigenesis, signal transduction, cytoskeleton arrangement, and neurodegeneration. To facilitate these varied functions, different regulators determine the different targets of PP2A. Among these regulators of PP2A, the B subunits in particular may be involved in cytoskeleton arrangement. However, little is known about the role of PP2A in actin polymerization in vivo. Using sophisticated fly genetics, we demonstrated a novel function for the fly B subunit, twins, to promote actin polymerization in varied tissue types, suggesting a broad and conserved effect. Furthermore, our genetic data suggest that twins may act upstream of the actin-polymerized-proteins, Moesin and Myosin-light-chain, and downstream of Rho to promote actin polymerization. This work opens a new avenue for exploring the biological functions of a PP2A regulator, twins, in cytoskeleton regulation.

A novel function of twins, B subunit of protein phosphatase 2A, in regulating actin polymerization

PubMed Central

Chang, Ching-Jin

2017-01-01

Actin is an important component of the cytoskeleton and its polymerization is delicately regulated by several kinases and phosphatases. Heterotrimeric protein phosphatase 2A (PP2A) is a potent phosphatase that is crucial for cell proliferation, apoptosis, tumorigenesis, signal transduction, cytoskeleton arrangement, and neurodegeneration. To facilitate these varied functions, different regulators determine the different targets of PP2A. Among these regulators of PP2A, the B subunits in particular may be involved in cytoskeleton arrangement. However, little is known about the role of PP2A in actin polymerization in vivo. Using sophisticated fly genetics, we demonstrated a novel function for the fly B subunit, twins, to promote actin polymerization in varied tissue types, suggesting a broad and conserved effect. Furthermore, our genetic data suggest that twins may act upstream of the actin-polymerized-proteins, Moesin and Myosin-light-chain, and downstream of Rho to promote actin polymerization. This work opens a new avenue for exploring the biological functions of a PP2A regulator, twins, in cytoskeleton regulation. PMID:28977036

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

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

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

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.

Actin grips: circular actin-rich cytoskeletal structures that mediate the wrapping of polymeric microfibers by endothelial cells.

PubMed

Jones, Desiree; Park, DoYoung; Anghelina, Mirela; Pécot, Thierry; Machiraju, Raghu; Xue, Ruipeng; Lannutti, John J; Thomas, Jessica; Cole, Sara L; Moldovan, Leni; Moldovan, Nicanor I

2015-06-01

Interaction of endothelial-lineage cells with three-dimensional substrates was much less studied than that with flat culture surfaces. We investigated the in vitro attachment of both mature endothelial cells (ECs) and of less differentiated EC colony-forming cells to poly-ε-capro-lactone (PCL) fibers with diameters in 5-20 μm range ('scaffold microfibers', SMFs). We found that notwithstanding the poor intrinsic adhesiveness to PCL, both cell types completely wrapped the SMFs after long-term cultivation, thus attaining a cylindrical morphology. In this system, both EC types grew vigorously for more than a week and became increasingly more differentiated, as shown by multiplexed gene expression. Three-dimensional reconstructions from multiphoton confocal microscopy images using custom software showed that the filamentous (F) actin bundles took a conspicuous ring-like organization around the SMFs. Unlike the classical F-actin-containing stress fibers, these rings were not associated with either focal adhesions or intermediate filaments. We also demonstrated that plasma membrane boundaries adjacent to these circular cytoskeletal structures were tightly yet dynamically apposed to the SMFs, for which reason we suggest to call them 'actin grips'. In conclusion, we describe a particular form of F-actin assembly with relevance for cytoskeletal organization in response to biomaterials, for endothelial-specific cell behavior in vitro and in vivo, and for tissue engineering. Copyright © 2015 Elsevier Ltd. All rights reserved.

Effects of dexamethasone on angiotensin II-induced changes of monolayer permeability and F-actin distribution in glomerular endothelial cells.

PubMed

Fang, Junyan; Wang, Miao; Zhang, Wei; Wang, Yingdeng

2013-11-01

The aim of this study was to investigate the changes in monolayer permeability and F-actin distribution caused by angiotensin II (Ang II)-induced injury in glomerular endothelial cells (GENCs) and the effects of dexamethasone on these changes. GENCs isolated and cultured from Wistar rats were used to examine the changes in monolayer permeability and F-actin distribution induced by Ang II. GENC permeability was evaluated by measuring the diffusion of biotin-conjugated bovine serum albumin (biotin-BSA) across a cell monolayer. The expression levels and distribution of F-actin were assessed by flow cytometry. The biotin-BSA concentrations were measured by capture enzyme-linked immunosorbent assay. Ang II at a concentration of 10 mg/l increased the permeability of the GENC monolayer at 6 h and 12 h (P<0.05 and P<0.01, respectively) and caused F-actin depolymerisation at 6 h and 12 h (P<0.01). The two effects attributed to Ang II were significantly inhibited by dexamethasone treatment (P<0.01). The increased permeability of the GENC monolayer induced by Ang II was significantly correlated with the depolymerisation of F-actin. Dexamethasone abrogated the Ang II-mediated damage to GENCs indicating that it may play an important role in protecting GENCs from injury.

Flexibility of myosin attachment to surfaces influences F-actin motion.

PubMed Central

Winkelmann, D A; Bourdieu, L; Ott, A; Kinose, F; Libchaber, A

1995-01-01

We have analyzed the dependence of actin filament sliding movement on the mode of myosin attachment to surfaces. Monoclonal antibodies (mAbs) that bind to three distinct sites were used to tether myosin to nitrocellulose-coated glass. One antibody reacts with an epitope on the regulatory light chain (LC2) located at the head-rod junction. The other two react with sites in the rod domain, one in the S2 region near the S2-LMM hinge, and the other at the C terminus of the myosin rod. This method of attachment provides a means of controlling the flexibility and density of myosin on the surface. Fast skeletal muscle myosin monomers were bound to the surfaces through the specific interaction with these mAbs, and the sliding movement of fluorescently labeled actin filaments was analyzed by video microscopy. Each of these antibodies produced stable myosin-coated surfaces that supported uniform motion of actin over the course of several hours. Attachment of myosin through the anti-S2 and anti-LMM mAbs yielded significantly higher velocities (10 microns/s at 30 degrees C) than attachment through anti-LC2 (4-5 microns/s at 30 degrees C). For each antibody, we observed a characteristic value of the myosin density for the onset of F-actin motion and a second critical density for velocity saturation. The specific mode of attachment influences the velocity of actin filaments and the characteristic surface density needed to support movement. Images FIGURE 1 FIGURE 4 FIGURE 8 PMID:7544167

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

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

Perfluorooctanesulfonate (PFOS) Perturbs Male Rat Sertoli Cell Blood-Testis Barrier Function by Affecting F-Actin Organization via p-FAK-Tyr407: An in Vitro Study

PubMed Central

Wan, Hin-Ting; Mruk, Dolores D.; Wong, Chris K. C.

2014-01-01

Environmental toxicants such as perfluorooctanesulfonate (PFOS) have been implicated in male reproductive dysfunction, including reduced sperm count and semen quality, in humans. However, the underlying mechanism(s) remains unknown. Herein PFOS at 10–20 μM (∼5–10 μg/mL) was found to be more potent than bisphenol A (100 μM) in perturbing the blood-testis barrier (BTB) function by disrupting the Sertoli cell tight junction-permeability barrier without detectable cytotoxicity. We also delineated the underlying molecular mechanism by which PFOS perturbed Sertoli cell BTB function using an in vitro model that mimics the BTB in vivo. First, PFOS perturbed F-actin organization in Sertoli cells, causing truncation of actin filaments at the BTB. Thus, the actin-based cytoskeleton was no longer capable of supporting the distribution and/or localization of actin-regulatory and adhesion proteins at the cell-cell interface necessary to maintain BTB integrity. Second, PFOS was found to perturb inter-Sertoli cell gap junction (GJ) communication based on a dye-transfer assay by down-regulating the expression of connexin-43, a GJ integral membrane protein. Third, phosphorylated focal adhesion kinase (FAK)-Tyr407 was found to protect the BTB from the destructive effects of PFOS as shown in a study via an overexpression of an FAK Y407E phosphomimetic mutant. Also, transfection of Sertoli cells with an FAK-specific microRNA, miR-135b, to knock down the expression of phosphorylated FAK-Tyr407 was found to worsen PFOS-mediated Sertoli cell tight junction disruption. In summary, PFOS-induced BTB disruption is mediated by down-regulating phosphorylated FAK-Tyr407 and connexin-43, which in turn perturbed F-actin organization and GJ-based intercellular communication, leading to mislocalization of actin-regulatory and adhesion proteins at the BTB. PMID:24169556

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.

OPC-compounds prevent oxidant-induced carbonylation and depolymerization of the F-actin cytoskeleton and intestinal barrier hyperpermeability.

PubMed

Banan, A; Fitzpatrick, L; Zhang, Y; Keshavarzian, A

2001-02-01

Rebamipide (OPC-12759), a quinolone derivative, and OPC-6535, a thiazol-carboxylic acid derivative, are compounds with ability to protect gastrointestinal (GI) mucosal integrity against reactive oxygen metabolites (ROM). The underlying mechanism of OPC-mediated protection remains poorly understood. It is now established that ROM can injure the mucosa by disruption of the cytoskeletal network, a key component of mucosal barrier integrity. We, therefore, investigated whether OPC compounds prevent the oxidation, disassembly, and instability of the cytoskeletal protein actin and, in turn, protect intestinal barrier function against ROM. Human intestinal (Caco-2) cell monolayers were pretreated with OPC (-12759 or -6535) prior to incubation with ROM (H2O2) or HOCl). Effects on cell integrity (ethidium homodimer-1), epithelial barrier function (fluorescein sulfonic acid clearance), and actin cytoskeletal integrity (high-resolution laser confocal) were then determined. Cells were also processed for quantitative immunoblotting of G- and F-actin to measure oxidation (carbonylation) and disassembly of actin. In monolayers exposed to ROM, preincubation with OPC compounds prevented actin oxidation, decreased depolymerized G-actin, and enhanced the stable F-actin. Concomitantly, OPC agents abolished both actin cytoskeletal disruption and monolayer barrier dysfunction. Data suggest for the first time that OPC drugs prevent oxidation of actin and lead to the protection of actin cytoskeleton and intestinal barrier integrity against oxidant insult. Accordingly, these compounds may be used as novel therapeutic agents for the treatment of a variety of oxidative inflammatory intestinal disorders with an abnormal mucosal barrier such as inflammatory bowel disease.

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

PubMed Central

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

2016-01-01