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Sample records for actin filament binding

  1. Side-binding proteins modulate actin filament dynamics.

    PubMed

    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. PMID:25706231

  2. Direct Observation of Tropomyosin Binding to Actin Filaments

    PubMed Central

    Schmidt, William M.; Lehman, William; Moore, Jeffrey R.

    2015-01-01

    Tropomyosin is an elongated α-helical coiled-coil that binds to seven consecutive actin subunits along the long-pitch helix of actin filaments. Once bound, tropomyosin polymerizes end-to-end and both stabilizes F-actin and regulates access of various actin binding proteins including myosin in muscle and non-muscle cells. Single tropomyosin molecules bind weakly to F-actin with millimolar Kd, whereas the end-to-end linked tropomyosin associates with about a one thousand-fold greater affinity. Despite years of study, the assembly mechanism of tropomyosin onto actin filaments remains unclear. In the current study, we used total internal reflection fluorescence (TIRF) microscopy to directly monitor the cooperative binding of fluorescently labeled tropomyosin molecules to phalloidin-stabilized actin filaments. We find that tropomyosin molecules assemble from multiple growth sites following random low affinity binding of single molecules to actin. As the length of the tropomyosin chain increases, the probability of detachment decreases, which leads to further chain growth. Tropomyosin chain extension is linearly dependent on tropomyosin concentration, occurring at approximately 100 monomers/(μM*s). The random tropomyosin binding to F-actin leads to discontinuous end-to-end association where gaps in the chain continuity smaller than the required seven sequential actin monomers are available. Direct observation of tropomyosin detachment revealed the number of gaps in actin-bound tropomyosin, the time course of gap annealing, and the eventual filament saturation process. PMID:26033920

  3. Filament Assembly by Spire: Key Residues and Concerted Actin Binding

    PubMed Central

    Rasson, Amy S.; Bois, Justin S.; Pham, Duy Stephen L.; Yoo, Haneul; Quinlan, Margot E.

    2014-01-01

    The most recently identified class of actin nucleators, WASp Homology domain 2 (WH2) – nucleators, use tandem repeats of monomeric actin-binding WH2 domains to facilitate actin nucleation. WH2 domains are involved in a wide variety of actin regulatory activities. Structurally, they are expected to clash with interprotomer contacts within the actin filament. Thus, the discovery of their role in nucleation was surprising. Here we use Drosophila Spire (Spir) as a model system to investigate both how tandem WH2 domains can nucleate actin and what differentiates nucleating WH2-containing proteins from their non-nucleating counterparts. We found that the third WH2 domain in Spir (Spir-C or Sc), plays a unique role. In the context of a short nucleation construct (containing only two WH2 domains), placement of Sc in the N-terminal position was required for the most potent nucleation. We found that the native organization of the WH2 domains with respect to each other is necessary for binding to actin with positive cooperativity. We identified two residues within Sc that are critical for its activity. Using this information we were able to convert a weak synthetic nucleator into one with activity equal to a native Spir construct. Lastly, we found evidence that Sc binds actin filaments, in addition to monomers. PMID:25234086

  4. Filament assembly by Spire: key residues and concerted actin binding.

    PubMed

    Rasson, Amy S; Bois, Justin S; Pham, Duy Stephen L; Yoo, Haneul; Quinlan, Margot E

    2015-02-27

    The most recently identified class of actin nucleators, WASp homology domain 2 (WH2) nucleators, use tandem repeats of monomeric actin-binding WH2 domains to facilitate actin nucleation. WH2 domains are involved in a wide variety of actin regulatory activities. Structurally, they are expected to clash with interprotomer contacts within the actin filament. Thus, the discovery of their role in nucleation was surprising. Here we use Drosophila Spire (Spir) as a model system to investigate both how tandem WH2 domains can nucleate actin and what differentiates nucleating WH2-containing proteins from their non-nucleating counterparts. We found that the third WH2 domain in Spir (Spir-C or SC) plays a unique role. In the context of a short nucleation construct (containing only two WH2 domains), placement of SC in the N-terminal position was required for the most potent nucleation. We found that the native organization of the WH2 domains with respect to each other is necessary for binding to actin with positive cooperativity. We identified two residues within SC that are critical for its activity. Using this information, we were able to convert a weak synthetic nucleator into one with activity equal to a native Spir construct. Lastly, we found evidence that SC binds actin filaments, in addition to monomers.

  5. Vinculin Is a Dually Regulated Actin Filament Barbed End-capping and Side-binding Protein

    PubMed Central

    Le Clainche, Christophe; Dwivedi, Satya Prakash; Didry, Dominique; Carlier, Marie-France

    2010-01-01

    The focal adhesion protein vinculin is an actin-binding protein involved in the mechanical coupling between the actin cytoskeleton and the extracellular matrix. An autoinhibitory interaction between the N-terminal head (Vh) and the C-terminal tail (Vt) of vinculin masks an actin filament side-binding domain in Vt. The binding of several proteins to Vh disrupts this intramolecular interaction and exposes the actin filament side-binding domain. Here, by combining kinetic assays and microscopy observations, we show that Vt inhibits actin polymerization by blocking the barbed ends of actin filaments. In low salt conditions, Vt nucleates actin filaments capped at their barbed ends. We determined that the interaction between vinculin and the barbed end is characterized by slow association and dissociation rate constants. This barbed end capping activity requires C-terminal amino acids of Vt that are dispensable for actin filament side binding. Like the side-binding domain, the capping domain of vinculin is masked by an autoinhibitory interaction between Vh and Vt. In contrast to the side-binding domain, the capping domain is not unmasked by the binding of a talin domain to Vh and requires the dissociation of an additional autoinhibitory interaction. Finally, we show that vinculin and the formin mDia1, which is involved in the processive elongation of actin filaments in focal adhesions, compete for actin filament barbed ends. PMID:20484056

  6. The architecture of actin filaments and the ultrastructural location of actin-binding protein in the periphery of lung macrophages.

    PubMed

    Hartwig, J H; Shevlin, P

    1986-09-01

    A highly branched filament network is the principal structure in the periphery of detergent-extracted cytoskeletons of macrophages that have been spread on a surface and either freeze or critical point dried, and then rotary shadowed with platinum-carbon. This array of filaments completely fills lamellae extended from the cell and bifurcates to form 0.2-0.5 micron thick layers on the top and bottom of the cell body. Reaction of the macrophage cytoskeletons with anti-actin IgG and with anti-IgG bound to colloidal gold produces dense staining of these filaments, and incubation with myosin subfragment 1 uniformly decorates these filaments, identifying them as actin. 45% of the total cellular actin and approximately 70% of actin-binding protein remains in the detergent-insoluble cell residue. The soluble actin is not filamentous as determined by sedimentation analysis, the DNAase I inhibition assay, and electron microscopy, indicating that the cytoskeleton is not fragmented by detergent extraction. The spacing between the ramifications of the actin network is 94 +/- 47 nm and 118 +/- 72 nm in cytoskeletons prepared for electron microscopy by freeze drying and critical point drying, respectively. Free filament ends are rare, except for a few which project upward from the body of the network or which extend down to the substrate. Filaments of the network intersect predominantly at right angles to form either T-shaped and X-shaped overlaps having striking perpendicularity or else Y-shaped intersections composed of filaments intersecting at 120-130 degrees angles. The actin filament concentration in the lamellae is high, with an average value of 12.5 mg/ml. The concentration was much more uniform in freeze-dried preparations than in critical point-dried specimens, indicating that there is less collapse associated with the freezing technique. The orthogonal actin network of the macrophage cortical cytoplasm resembles actin gels made with actin-binding protein. Reaction of

  7. A Mechanism for Actin Filament Severing by Malaria Parasite Actin Depolymerizing Factor 1 via a Low Affinity Binding Interface*

    PubMed Central

    Wong, Wilson; Webb, Andrew I.; Olshina, Maya A.; Infusini, Giuseppe; Tan, Yan Hong; Hanssen, Eric; Catimel, Bruno; Suarez, Cristian; Condron, Melanie; Angrisano, Fiona; NebI, Thomas; Kovar, David R.; Baum, Jake

    2014-01-01

    Actin depolymerizing factor (ADF)/cofilins are essential regulators of actin turnover in eukaryotic cells. These multifunctional proteins facilitate both stabilization and severing of filamentous (F)-actin in a concentration-dependent manner. At high concentrations ADF/cofilins bind stably to F-actin longitudinally between two adjacent actin protomers forming what is called a decorative interaction. Low densities of ADF/cofilins, in contrast, result in the optimal severing of the filament. To date, how these two contrasting modalities are achieved by the same protein remains uncertain. Here, we define the proximate amino acids between the actin filament and the malaria parasite ADF/cofilin, PfADF1 from Plasmodium falciparum. PfADF1 is unique among ADF/cofilins in being able to sever F-actin but do so without stable filament binding. Using chemical cross-linking and mass spectrometry (XL-MS) combined with structure reconstruction we describe a previously overlooked binding interface on the actin filament targeted by PfADF1. This site is distinct from the known binding site that defines decoration. Furthermore, total internal reflection fluorescence (TIRF) microscopy imaging of single actin filaments confirms that this novel low affinity site is required for F-actin severing. Exploring beyond malaria parasites, selective blocking of the decoration site with human cofilin (HsCOF1) using cytochalasin D increases its severing rate. HsCOF1 may therefore also use a decoration-independent site for filament severing. Thus our data suggest that a second, low affinity actin-binding site may be universally used by ADF/cofilins for actin filament severing. PMID:24371134

  8. Probing the flexibility of tropomyosin and its binding to filamentous actin using molecular dynamics simulations.

    PubMed

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

    2013-10-15

    Tropomyosin (Tm) is a coiled-coil protein that binds to filamentous actin (F-actin) and regulates its interactions with actin-binding proteins like myosin by moving between three positions on F-actin (the blocked, closed, and open positions). To elucidate the molecular details of Tm flexibility in relation to its binding to F-actin, we conducted extensive molecular dynamics simulations for both Tm alone and Tm-F-actin complex in the presence of explicit solvent (total simulation time >400 ns). Based on the simulations, we systematically analyzed the local flexibility of the Tm coiled coil using multiple parameters. We found a good correlation between the regions with high local flexibility and a number of destabilizing regions in Tm, including six clusters of core alanines. Despite the stabilization by F-actin binding, the distribution of local flexibility in Tm is largely unchanged in the absence and presence of F-actin. Our simulations showed variable fluctuations of individual Tm periods from the closed position toward the open position. In addition, we performed Tm-F-actin binding calculations based on the simulation trajectories, which support the importance of Tm flexibility to Tm-F-actin binding. We identified key residues of Tm involved in its dynamic interactions with F-actin, many of which have been found in recent mutational studies to be functionally important, and the rest of which will make promising targets for future mutational experiments.

  9. Gestalt-binding of tropomyosin on actin during thin filament activation.

    PubMed

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

    2013-08-01

    Our thesis is that thin filament function can only be fully understood and muscle regulation then elucidated if atomic structures of the thin filament are available to reveal the positions of tropomyosin on actin in all physiological states. After all, it is tropomyosin influenced by troponin that regulates myosin-crossbridge cycling on actin and therefore controls contraction in all muscles. In addition, we maintain that a complete appreciation of thin filament activation also requires that the mechanical properties of tropomyosin itself are recognized and then related to the effect of myosin-association on actin. Taking the Gestalt-binding of tropomyosin into account, coupled with our electron microscopy structures and computational chemistry, we propose a comprehensive mechanism for tropomyosin regulatory movement over the actin filament surface that explains the cooperative muscle activation process. In fact, well-known point mutations of critical amino acids on the actin-tropomyosin binding interface disrupt Gestalt-binding and are associated with a number of inherited myopathies. Moreover, dysregulation of tropomyosin may also be a factor that interferes with the gatekeeping operation of non-muscle tropomyosin in the controlling interactions of a wide variety of cellular actin-binding proteins. The clinical relevance of Gestalt-binding is discussed in articles by the Marston and the Gunning groups in this special journal issue devoted to the impact of tropomyosin on biological systems.

  10. Coordination of the Filament Stabilizing Versus Destabilizing Activities of Cofilin Through its Secondary Binding Site on Actin

    PubMed Central

    Aggeli, Dimitra; Kish-Trier, Erik; Lin, Meng Chi; Haarer, Brian; Cingolani, Gino; Cooper, John A.; Wilkens, Stephan; Amberg, David C.

    2014-01-01

    Cofilin is a ubiquitous modulator of actin cytoskeleton dynamics that can both stabilize and destabilize actin filaments depending on its concentration and/or the presence of regulatory co-factors. Three charge-reversal mutants of yeast cofilin, located in cofilin’s filament-specific secondary binding site, were characterized in order to understand why disruption of this site leads to enhanced filament disassembly. Crystal structures of the mutants showed that the mutations specifically affect the secondary actin-binding interface, leaving the primary binding site unaltered. The mutant cofilins show enhanced activity compared to wild-type cofilin in severing and disassembling actin filaments. Electron microscopy and image analysis revealed long actin filaments in the presence of wild-type cofilin, while the mutants induced many short filaments, consistent with enhanced severing. Real-time fluorescence microscopy of labeled actin filaments confirmed that the mutants, unlike wild-type cofilin, were functioning as constitutively active severing proteins. In cells, the mutant cofilins delayed endocytosis, which depends on rapid actin turnover. We conclude that mutating cofilin’s secondary actin-binding site increases cofilin’s ability to sever and depolymerize actin filaments. We hypothesize that activators of cofilin severing, like Aip1p, may act by disrupting the interface between cofilin’s secondary actin-binding site and the actin filament. PMID:24943913

  11. Allosteric regulation by cooperative conformational changes of actin filaments drives mutually exclusive binding with cofilin and myosin

    PubMed Central

    Ngo, Kien Xuan; Umeki, Nobuhisa; Kijima, Saku T.; Kodera, Noriyuki; Ueno, Hiroaki; Furutani-Umezu, Nozomi; Nakajima, Jun; Noguchi, Taro Q. P.; Nagasaki, Akira; Tokuraku, Kiyotaka; Uyeda, Taro Q. P.

    2016-01-01

    Heavy meromyosin (HMM) of myosin II and cofilin each binds to actin filaments cooperatively and forms clusters along the filaments, but it is unknown whether the two cooperative bindings are correlated and what physiological roles they have. Fluorescence microscopy demonstrated that HMM-GFP and cofilin-mCherry each bound cooperatively to different parts of actin filaments when they were added simultaneously in 0.2 μM ATP, indicating that the two cooperative bindings are mutually exclusive. In 0.1 mM ATP, the motor domain of myosin (S1) strongly inhibited the formation of cofilin clusters along actin filaments. Under this condition, most actin protomers were unoccupied by S1 at any given moment, suggesting that transiently bound S1 alters the structure of actin filaments cooperatively and/or persistently to inhibit cofilin binding. Consistently, cosedimentation experiments using copolymers of actin and actin-S1 fusion protein demonstrated that the fusion protein affects the neighboring actin protomers, reducing their affinity for cofilin. In reciprocal experiments, cofilin-actin fusion protein reduced the affinity of neighboring actin protomers for S1. Thus, allosteric regulation by cooperative conformational changes of actin filaments contributes to mutually exclusive cooperative binding of myosin II and cofilin to actin filaments, and presumably to the differential localization of both proteins in cells. PMID:27762277

  12. The Actin Filament-Binding Protein Coronin Regulates Motility in Plasmodium Sporozoites

    PubMed Central

    Bane, Kartik S.; Singer, Mirko; Reinig, Miriam; Klug, Dennis; Heiss, Kirsten; Baum, Jake; Mueller, Ann-Kristin; Frischknecht, Friedrich

    2016-01-01

    Parasites causing malaria need to migrate in order to penetrate tissue barriers and enter host cells. Here we show that the actin filament-binding protein coronin regulates gliding motility in Plasmodium berghei sporozoites, the highly motile forms of a rodent malaria-causing parasite transmitted by mosquitoes. Parasites lacking coronin show motility defects that impair colonization of the mosquito salivary glands but not migration in the skin, yet result in decreased transmission efficiency. In non-motile sporozoites low calcium concentrations mediate actin-independent coronin localization to the periphery. Engagement of extracellular ligands triggers an intracellular calcium release followed by the actin-dependent relocalization of coronin to the rear and initiation of motility. Mutational analysis and imaging suggest that coronin organizes actin filaments for productive motility. Using coronin-mCherry as a marker for the presence of actin filaments we found that protein kinase A contributes to actin filament disassembly. We finally speculate that calcium and cAMP-mediated signaling regulate a switch from rapid parasite motility to host cell invasion by differentially influencing actin dynamics. PMID:27409081

  13. Actin filament organization and myosin head labelling patterns in vertebrate skeletal muscles in the rigor and weak binding states.

    PubMed

    Squire, J M; Harford, J J

    1988-08-01

    The structures of vertebrate skeletal muscles (particularly from frog and fish) in the rigor state are analysed in terms of the concept of target areas on actin filaments. Assuming that 100% of the heads are to be attached to actin in rigor, then satisfactory qualitative low-resolution modelling of observed X-ray diffraction data is obtained if the outer ends of these myosin heads can move axially (total range about 200A) and azimuthally (total range less than 60 degrees) from their original lattice sites on the myosin filament surface to attach in defined target areas on the actin filaments. On this basis, each actin target area comprises about four actin monomers along one of the two long-pitched helical strands of the actin filament (about 200 A) or an azimuthal range of actin binding sites of about 100 degrees around the thin filament axis. If myosin heads simply label in a non-specific way the nearest actin monomers to them, as could occur with non-specific transient attachment in a 'weak binding' state, then the predicted X-ray diffraction pattern would comprise layer lines at the same axial spacings (orders of 429 A) as those seen in patterns from resting muscle. It is shown that actin target areas in vertebrate skeletal muscles are probably arranged on an approximate 62 (right-handed) helix of pitch (P) of about 720 A, subunit translation P/6 and near repeat P/2. Troponin position need not be considered in defining the labelling pattern of cross-bridges on this 62 helix of target areas; the target areas appear to be defined solely by the azimuthal position of the actin binding sites. The distribution of actin filament labelling patterns could be regular in fish muscle which has a 'crystalline' A-band, but will be irregular in higher vertebrate muscles such as frog sartorius muscle.

  14. A Peek into Tropomyosin Binding and Unfolding on the Actin Filament

    PubMed Central

    Singh, Abhishek; Hitchcock-DeGregori, Sarah E.

    2009-01-01

    Background Tropomyosin is a prototypical coiled coil along its length with subtle variations in structure that allow interactions with actin and other proteins. Actin binding globally stabilizes tropomyosin. Tropomyosin-actin interaction occurs periodically along the length of tropomyosin. However, it is not well understood how tropomyosin binds actin. Principal Findings Tropomyosin's periodic binding sites make differential contributions to two components of actin binding, cooperativity and affinity, and can be classified as primary or secondary sites. We show through mutagenesis and analysis of recombinant striated muscle α-tropomyosins that primary actin binding sites have a destabilizing coiled-coil interface, typically alanine-rich, embedded within a non-interface recognition sequence. Introduction of an Ala cluster in place of the native, more stable interface in period 2 and/or period 3 sites (of seven) increased the affinity or cooperativity of actin binding, analysed by cosedimentation and differential scanning calorimetry. Replacement of period 3 with period 5 sequence, an unstable region of known importance for cooperative actin binding, increased the cooperativity of binding. Introduction of the fluorescent probe, pyrene, near the mutation sites in periods 2 and 3 reported local instability, stabilization by actin binding, and local unfolding before or coincident with dissociation from actin (measured using light scattering), and chain dissociation (analyzed using circular dichroism). Conclusions This, and previous work, suggests that regions of tropomyosin involved in binding actin have non-interface residues specific for interaction with actin and an unstable interface that is locally stabilized upon binding. The destabilized interface allows residues on the coiled-coil surface to obtain an optimal conformation for interaction with actin by increasing the number of local substates that the side chains can sample. We suggest that local disorder is a

  15. Effect of phosphorylation of phosphatidylinositol on myelin basic protein-mediated binding of actin filaments to lipid bilayers in vitro.

    PubMed

    Boggs, Joan M; Rangaraj, Godha; Dicko, Awa

    2012-09-01

    Myelin basic protein (MBP) binds to negatively charged lipids on the cytosolic surface of oligodendrocytes and is believed to be responsible for adhesion of these surfaces in the multilayered myelin sheath. It can also assemble actin filaments and tether them to lipid bilayers through electrostatic interactions. Here we investigate the effect of increased negative charge of the lipid bilayer due to phosphorylation of phosphatidylinositol (PI) on MBP-mediated binding of actin to the lipid bilayer, by substituting phosphatidylinositol 4-phosphate or phosphatidylinositol 4,5-bisphosphate for PI in phosphatidylcholine/phosphatidylglycerol lipid vesicles. Phosphorylation of PI caused dissociation of the MBP/actin complex from the lipid vesicles due to repulsion of the negatively charged complex from the negatively charged membrane surface. An effect of phosphorylation could be detected even if the inositol lipid was only 2mol% of the total lipid. Calcium-calmodulin dissociated actin from the MBP-lipid vesicles and phosphorylation of PI increased the amount dissociated. These results show that changes to the lipid composition of myelin, which could occur during signaling or other physiological events, could regulate the ability of MBP to act as a scaffolding protein and bind actin filaments to the lipid bilayer.

  16. Filamentous actin and its associated binding proteins are the stimulatory site for 6-phosphofructo-1-kinase association within the membrane of human erythrocytes.

    PubMed

    Real-Hohn, Antonio; Zancan, Patricia; Da Silva, Daniel; Martins, Eliane R; Salgado, Leonardo T; Mermelstein, Claudia S; Gomes, Andre M O; Sola-Penna, Mauro

    2010-05-01

    Glycolytic enzymes reversibly associate with the human erythrocyte membrane (EM) as part of their regulatory mechanism. The site for this association has been described as the amino terminus of band 3, a transmembrane anion transporter. Binding of glycolytic enzymes to this site is recognized to inhibit glycolysis, since binding inhibits the catalytic activity of these enzymes, including the rate-limiting enzyme 6-phosphofructo-1-kinase (PFK). However, the existence of a putative stimulatory site for glycolytic enzymes within the EM has been proposed. PFK has been described as able to reversibly associate with other proteins, such as microtubules, which inhibit the enzyme, and filamentous actin, which activates the enzyme. Here, it is demonstrated that PFK also binds to actin filaments and its associated binding proteins in the protein meshwork that forms the erythrocyte cytoskeleton. Through fluorescence resonance energy transfer experiments using either confocal microscopy or fluorescence spectroscopy, we show that, within the EM, PFK and actin filaments containing its associated binding proteins are located close enough to propose binding between them. Moreover, specifically blocking PFK binding to band 3 results in an association of the enzyme with the EM that increases the enzyme's catalytic activity. Conversely, disruption of the association between PFK and actin filaments containing its associated binding proteins potentiates the inhibitory action of the EM on the enzyme. Furthermore, it is shown that insulin signaling increases the association of PFK to actin filaments and its associated binding proteins, revealing that this event may play a role on the stimulatory effects of insulin on erythrocyte glycolysis. In summary, the present work presents evidence that filamentous actin and its associated binding proteins are the stimulatory site for PFK within the EM.

  17. Loss of cargo binding in the human myosin VI deafness mutant (R1166X) leads to increased actin filament binding

    PubMed Central

    Arden, Susan D.; Tumbarello, David A.; Butt, Tariq; Kendrick-Jones, John; Buss, Folma

    2016-01-01

    Mutations in myosin VI have been associated with autosomal-recessive (DFNB37) and autosomal-dominant (DFNA22) deafness in humans. Here, we characterise an myosin VI nonsense mutation (R1166X) that was identified in a family with hereditary hearing loss in Pakistan. This mutation leads to the deletion of the C-terminal 120 amino acids of the myosin VI cargo-binding domain, which includes the WWY-binding motif for the adaptor proteins LMTK2, Tom1 as well as Dab2. Interestingly, compromising myosin VI vesicle-binding ability by expressing myosin VI with the R1166X mutation or with single point mutations in the adaptor-binding sites leads to increased F-actin binding of this myosin in vitro and in vivo. As our results highlight the importance of cargo attachment for regulating actin binding to the motor domain, we perform a detailed characterisation of adaptor protein binding and identify single amino acids within myosin VI required for binding to cargo adaptors. We not only show that the adaptor proteins can directly interact with the cargo-binding tail of myosin VI, but our in vitro studies also suggest that multiple adaptor proteins can bind simultaneously to non-overlapping sites in the myosin VI tail. In conclusion, our characterisation of the human myosin VI deafness mutant (R1166X) suggests that defects in cargo binding may leave myosin VI in a primed/activated state with an increased actin-binding ability. PMID:27474411

  18. Loss of cargo binding in the human myosin VI deafness mutant (R1166X) leads to increased actin filament binding.

    PubMed

    Arden, Susan D; Tumbarello, David A; Butt, Tariq; Kendrick-Jones, John; Buss, Folma

    2016-10-01

    Mutations in myosin VI have been associated with autosomal-recessive (DFNB37) and autosomal-dominant (DFNA22) deafness in humans. Here, we characterise an myosin VI nonsense mutation (R1166X) that was identified in a family with hereditary hearing loss in Pakistan. This mutation leads to the deletion of the C-terminal 120 amino acids of the myosin VI cargo-binding domain, which includes the WWY-binding motif for the adaptor proteins LMTK2, Tom1 as well as Dab2. Interestingly, compromising myosin VI vesicle-binding ability by expressing myosin VI with the R1166X mutation or with single point mutations in the adaptor-binding sites leads to increased F-actin binding of this myosin in vitro and in vivo As our results highlight the importance of cargo attachment for regulating actin binding to the motor domain, we perform a detailed characterisation of adaptor protein binding and identify single amino acids within myosin VI required for binding to cargo adaptors. We not only show that the adaptor proteins can directly interact with the cargo-binding tail of myosin VI, but our in vitro studies also suggest that multiple adaptor proteins can bind simultaneously to non-overlapping sites in the myosin VI tail. In conclusion, our characterisation of the human myosin VI deafness mutant (R1166X) suggests that defects in cargo binding may leave myosin VI in a primed/activated state with an increased actin-binding ability. PMID:27474411

  19. Boolean gates on actin filaments

    NASA Astrophysics Data System (ADS)

    Siccardi, Stefano; Tuszynski, Jack A.; Adamatzky, Andrew

    2016-01-01

    Actin is a globular protein which forms long polar filaments in the eukaryotic cytoskeleton. Actin networks play a key role in cell mechanics and cell motility. They have also been implicated in information transmission and processing, memory and learning in neuronal cells. The actin filaments have been shown to support propagation of voltage pulses. Here we apply a coupled nonlinear transmission line model of actin filaments to study interactions between voltage pulses. To represent digital information we assign a logical TRUTH value to the presence of a voltage pulse in a given location of the actin filament, and FALSE to the pulse's absence, so that information flows along the filament with pulse transmission. When two pulses, representing Boolean values of input variables, interact, then they can facilitate or inhibit further propagation of each other. We explore this phenomenon to construct Boolean logical gates and a one-bit half-adder with interacting voltage pulses. We discuss implications of these findings on cellular process and technological applications.

  20. Effect of Tropomyosin on Formin-Bound Actin Filaments

    PubMed Central

    Ujfalusi, Zoltán; Vig, Andrea; Hild, Gábor; Nyitrai, Miklós

    2009-01-01

    Abstract Formins are conservative proteins with important roles in the regulation of the microfilament system in eukaryotic cells. Previous studies showed that the binding of formins to actin made the structure of actin filaments more flexible. Here, the effects of tropomyosin on formin-induced changes in actin filaments were investigated using fluorescence spectroscopic methods. The temperature dependence of the Förster-type resonance energy transfer showed that the formin-induced increase of flexibility of actin filaments was diminished by the binding of tropomyosin to actin. Fluorescence anisotropy decay measurements also revealed that the structure of flexible formin-bound actin filaments was stabilized by the binding of tropomyosin. The stabilizing effect reached its maximum when all binding sites on actin were occupied by tropomyosin. The effect of tropomyosin on actin filaments was independent of ionic strength, but became stronger as the magnesium concentration increased. Based on these observations, we propose that in cells there is a molecular mechanism in which tropomyosin binding to actin plays an important role in forming mechanically stable actin filaments, even in the case of formin-induced rapid filament assembly. PMID:18931257

  1. Mechanism of Actin Filament Bundling by Fascin

    SciTech Connect

    Jansen, Silvia; Collins, Agnieszka; Yang, Changsong; Rebowski, Grzegorz; Svitkina, Tatyana; Dominguez, Roberto

    2013-03-07

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

  2. Tropomyosin - master regulator of actin filament function in the cytoskeleton.

    PubMed

    Gunning, Peter W; Hardeman, Edna C; Lappalainen, Pekka; Mulvihill, Daniel P

    2015-08-15

    Tropomyosin (Tpm) isoforms are the master regulators of the functions of individual actin filaments in fungi and metazoans. Tpms are coiled-coil parallel dimers that form a head-to-tail polymer along the length of actin filaments. Yeast only has two Tpm isoforms, whereas mammals have over 40. Each cytoskeletal actin filament contains a homopolymer of Tpm homodimers, resulting in a filament of uniform Tpm composition along its length. Evidence for this 'master regulator' role is based on four core sets of observation. First, spatially and functionally distinct actin filaments contain different Tpm isoforms, and recent data suggest that members of the formin family of actin filament nucleators can specify which Tpm isoform is added to the growing actin filament. Second, Tpms regulate whole-organism physiology in terms of morphogenesis, cell proliferation, vesicle trafficking, biomechanics, glucose metabolism and organ size in an isoform-specific manner. Third, Tpms achieve these functional outputs by regulating the interaction of actin filaments with myosin motors and actin-binding proteins in an isoform-specific manner. Last, the assembly of complex structures, such as stress fibers and podosomes involves the collaboration of multiple types of actin filament specified by their Tpm composition. This allows the cell to specify actin filament function in time and space by simply specifying their Tpm isoform composition.

  3. Tropomyosin - master regulator of actin filament function in the cytoskeleton.

    PubMed

    Gunning, Peter W; Hardeman, Edna C; Lappalainen, Pekka; Mulvihill, Daniel P

    2015-08-15

    Tropomyosin (Tpm) isoforms are the master regulators of the functions of individual actin filaments in fungi and metazoans. Tpms are coiled-coil parallel dimers that form a head-to-tail polymer along the length of actin filaments. Yeast only has two Tpm isoforms, whereas mammals have over 40. Each cytoskeletal actin filament contains a homopolymer of Tpm homodimers, resulting in a filament of uniform Tpm composition along its length. Evidence for this 'master regulator' role is based on four core sets of observation. First, spatially and functionally distinct actin filaments contain different Tpm isoforms, and recent data suggest that members of the formin family of actin filament nucleators can specify which Tpm isoform is added to the growing actin filament. Second, Tpms regulate whole-organism physiology in terms of morphogenesis, cell proliferation, vesicle trafficking, biomechanics, glucose metabolism and organ size in an isoform-specific manner. Third, Tpms achieve these functional outputs by regulating the interaction of actin filaments with myosin motors and actin-binding proteins in an isoform-specific manner. Last, the assembly of complex structures, such as stress fibers and podosomes involves the collaboration of multiple types of actin filament specified by their Tpm composition. This allows the cell to specify actin filament function in time and space by simply specifying their Tpm isoform composition. PMID:26240174

  4. Phosphatidylinositol 3-Kinase-Associated Protein (PI3KAP)/XB130 Crosslinks Actin Filaments through Its Actin Binding and Multimerization Properties In Vitro and Enhances Endocytosis in HEK293 Cells.

    PubMed

    Yamanaka, Daisuke; Akama, Takeshi; Chida, Kazuhiro; Minami, Shiro; Ito, Koichi; Hakuno, Fumihiko; Takahashi, Shin-Ichiro

    2016-01-01

    Actin-crosslinking proteins control actin filament networks and bundles and contribute to various cellular functions including regulation of cell migration, cell morphology, and endocytosis. Phosphatidylinositol 3-kinase-associated protein (PI3KAP)/XB130 has been reported to be localized to actin filaments (F-actin) and required for cell migration in thyroid carcinoma cells. Here, we show a role for PI3KAP/XB130 as an actin-crosslinking protein. First, we found that the carboxyl terminal region of PI3KAP/XB130 containing amino acid residues 830-840 was required and sufficient for localization to F-actin in NIH3T3 cells, and this region is directly bound to F-actin in vitro. Moreover, actin-crosslinking assay revealed that recombinant PI3KAP/XB130 crosslinked F-actin. In general, actin-crosslinking proteins often multimerize to assemble multiple actin-binding sites. We then investigated whether PI3KAP/XB130 could form a multimer. Blue native-PAGE analysis showed that recombinant PI3KAP/XB130 was detected at 250-1200 kDa although the molecular mass was approximately 125 kDa, suggesting that PI3KAP/XB130 formed multimers. Furthermore, we found that the amino terminal 40 amino acids were required for this multimerization by co-immunoprecipitation assay in HEK293T cells. Deletion mutants of PI3KAP/XB130 lacking the actin-binding region or the multimerizing region did not crosslink actin filaments, indicating that actin binding and multimerization of PI3KAP/XB130 were necessary to crosslink F-actin. Finally, we examined roles of PI3KAP/XB130 on endocytosis, an actin-related biological process. Overexpression of PI3KAP/XB130 enhanced dextran uptake in HEK 293 cells. However, most of the cells transfected with the deletion mutant lacking the actin-binding region incorporated dextran to a similar extent as control cells. Taken together, these results demonstrate that PI3KAP/XB130 crosslinks F-actin through both its actin-binding region and multimerizing region and plays

  5. Bundling of actin filaments by elongation factor 1 alpha inhibits polymerization at filament ends

    PubMed Central

    1996-01-01

    Elongation factor 1 alpha (EF1 alpha) is an abundant protein that binds aminoacyl-tRNA and ribosomes in a GTP-dependent manner. EF1 alpha also interacts with the cytoskeleton by binding and bundling actin filaments and microtubules. In this report, the effect of purified EF1 alpha on actin polymerization and depolymerization is examined. At molar ratios present in the cytosol, EF1 alpha significantly blocks both polymerization and depolymerization of actin filaments and increases the final extent of actin polymer, while at high molar ratios to actin, EF1 alpha nucleates actin polymerization. Although EF1 alpha binds actin monomer, this monomer-binding activity does not explain the effects of EF1 alpha on actin polymerization at physiological molar ratios. The mechanism for the inhibition of polymerization is related to the actin-bundling activity of EF1 alpha. Both ends of the actin filament are inhibited for polymerization and both bundling and the inhibition of actin polymerization are affected by pH within the same physiological range; at high pH both bundling and the inhibition of actin polymerization are reduced. Additionally, it is seen that the binding of aminoacyl-tRNA to EF1 alpha releases EF1 alpha's inhibiting effect on actin polymerization. These data demonstrate that EF1 alpha can alter the assembly of F-actin, a filamentous scaffold on which non- membrane-associated protein translation may be occurring in vivo. PMID:8947553

  6. αT-Catenin Is a Constitutive Actin-binding α-Catenin That Directly Couples the Cadherin·Catenin Complex to Actin Filaments*

    PubMed Central

    Wickline, Emily D.; Dale, Ian W.; Merkel, Chelsea D.; Heier, Jonathon A.; Stolz, Donna B.

    2016-01-01

    α-Catenin is the primary link between the cadherin·catenin complex and the actin cytoskeleton. Mammalian αE-catenin is allosterically regulated: the monomer binds the β-catenin·cadherin complex, whereas the homodimer does not bind β-catenin but interacts with F-actin. As part of the cadherin·catenin complex, αE-catenin requires force to bind F-actin strongly. It is not known whether these properties are conserved across the mammalian α-catenin family. Here we show that αT (testes)-catenin, a protein unique to amniotes that is expressed predominantly in the heart, is a constitutive actin-binding α-catenin. We demonstrate that αT-catenin is primarily a monomer in solution and that αT-catenin monomer binds F-actin in cosedimentation assays as strongly as αE-catenin homodimer. The β-catenin·αT-catenin heterocomplex also binds F-actin with high affinity unlike the β-catenin·αE-catenin complex, indicating that αT-catenin can directly link the cadherin·catenin complex to the actin cytoskeleton. Finally, we show that a mutation in αT-catenin linked to arrhythmogenic right ventricular cardiomyopathy, V94D, promotes homodimerization, blocks β-catenin binding, and in cardiomyocytes disrupts localization at cell-cell contacts. Together, our data demonstrate that αT-catenin is a constitutively active actin-binding protein that can physically couple the cadherin·catenin complex to F-actin in the absence of tension. We speculate that these properties are optimized to meet the demands of cardiomyocyte adhesion. PMID:27231342

  7. Interaction of profilin with the barbed end of actin filaments.

    PubMed

    Courtemanche, Naomi; Pollard, Thomas D

    2013-09-17

    Profilin binds not only to actin monomers but also to the barbed end of the actin filament, where it inhibits association of subunits. To address open questions about the interactions of profilin with barbed ends, we measured the effects of a wide range of concentrations of Homo sapiens profilin 1 on the rate of elongation of individual skeletal muscle actin filaments by total internal reflection fluorescence microscopy. Much higher concentrations of profilin were required to stop elongation by AMP-PNP-actin monomers than ADP-actin monomers. High concentrations of profilin depolymerized barbed ends at a rate much faster than the spontaneous dissociation rates of Mg-ATP-, Mg-AMP-PNP-, Mg-ADP-Pi-, and Mg-ADP-actin subunits. Fitting a thermodynamic model to these data allowed us to determine the affinities of profilin and profilin-actin for barbed ends and the influence of the nucleotide bound to actin on these interactions. Profilin has a much higher affinity for ADP-actin filament barbed ends (Kd = 1 μM) than AMP-PNP-actin filament barbed ends (Kd = 226 μM). ADP-actin monomers associated with profilin bind to ADP-actin filament barbed ends 10% as fast as free ADP-actin monomers, but bound profilin does not affect the rate of association of AMP-PNP-actin monomers with barbed ends. The differences in the affinities of AMP-PNP- and ADP-bound barbed ends for profilin and profilin-actin suggest that conformations of barbed end subunits differ from those of monomers and change upon nucleotide hydrolysis and phosphate release. A structural model revealed minor steric clashes between profilin and actin subunits at the barbed end that explain the biochemical results.

  8. Actin filament organization of foot processes in vertebrate glomerular podocytes.

    PubMed

    Ichimura, Koichiro; Kurihara, Hidetake; Sakai, Tatsuo

    2007-09-01

    We investigated the actin filament organization and immunolocalization of actin-binding proteins (alpha-actinin and cortactin) in the podocyte foot processes of eight vertebrate species (lamprey, carp, newt, frog, gecko, turtle, quail, and rat). Three types of actin cytoskeleton were found in these foot processes. (1) A cortical actin network with cortactin filling the space between the plasma membrane and the other actin cytoskeletons described below was found in all of the species examined here. The data indicated that the cortical actin network was the minimal essential actin cytoskeleton for the formation and maintenance of the foot processes in vertebrate podocytes. (2) An actin bundle with alpha-actinin existing along the longitudinal axis of foot process above the level of slit diaphragms was only observed in quail and rat. (3) An actin fascicle consisting of much fewer numbers of actin filaments than that of the actin bundle was observed in the species other than quail and rat, but at various frequencies. These findings suggest that the actin bundle is an additional actin cytoskeleton reflecting a functional state peculiar to quail and rat glomeruli. Considering the higher intraglomerular pressure and the extremely thin filtration barrier in birds and mammals, the foot processes probably mainly protect the thinner filtration barrier from the higher internal pressure occurring in quail and rat glomeruli. Therefore, we consider that the actin bundle plays a crucial role in the mechanical protection of the filtration barrier. Moreover, the actin fascicle may be a potential precursor of the actin bundle.

  9. Caenorhabditis elegans Kettin, a Large Immunoglobulin-like Repeat Protein, Binds to Filamentous Actin and Provides Mechanical Stability to the Contractile Apparatuses in Body Wall Muscle

    PubMed Central

    Ono, Kanako; Yu, Robinson; Mohri, Kurato

    2006-01-01

    Kettin is a large actin-binding protein with immunoglobulin-like (Ig) repeats, which is associated with the thin filaments in arthropod muscles. Here, we report identification and functional characterization of kettin in the nematode Caenorhabditis elegans. We found that one of the monoclonal antibodies that were raised against C. elegans muscle proteins specifically reacts with kettin (Ce-kettin). We determined the entire cDNA sequence of Ce-kettin that encodes a protein of 472 kDa with 31 Ig repeats. Arthropod kettins are splice variants of much larger connectin/titin-related proteins. However, the gene for Ce-kettin is independent of other connectin/titin-related genes. Ce-kettin localizes to the thin filaments near the dense bodies in both striated and nonstriated muscles. The C-terminal four Ig repeats and the adjacent non-Ig region synergistically bind to actin filaments in vitro. RNA interference of Ce-kettin caused weak disorganization of the actin filaments in body wall muscle. This phenotype was suppressed by inhibiting muscle contraction by a myosin mutation, but it was enhanced by tetramisole-induced hypercontraction. Furthermore, Ce-kettin was involved in organizing the cytoplasmic portion of the dense bodies in cooperation with α-actinin. These results suggest that kettin is an important regulator of myofibrillar organization and provides mechanical stability to the myofibrils during contraction. PMID:16597697

  10. Control of actin-based motility through localized actin binding.

    PubMed

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

    2013-12-01

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

  11. Actin filament nucleation and elongation factors--structure-function relationships.

    PubMed

    Dominguez, Roberto

    2009-01-01

    The spontaneous and unregulated polymerization of actin filaments is inhibited in cells by actin monomer-binding proteins such as profilin and Tbeta4. Eukaryotic cells and certain pathogens use filament nucleators to stabilize actin polymerization nuclei, whose formation is rate-limiting. Known filament nucleators include the Arp2/3 complex and its large family of nucleation promoting factors (NPFs), formins, Spire, Cobl, VopL/VopF, TARP and Lmod. These molecules control the time and location for polymerization, and additionally influence the structures of the actin networks that they generate. Filament nucleators are generally unrelated, but with the exception of formins they all use the WASP-Homology 2 domain (WH2 or W), a small and versatile actin-binding motif, for interaction with actin. A common architecture, found in Spire, Cobl and VopL/VopF, consists of tandem W domains that bind three to four actin subunits to form a nucleus. Structural considerations suggest that NPFs-Arp2/3 complex can also be viewed as a specialized form of tandem W-based nucleator. Formins are unique in that they use the formin-homology 2 (FH2) domain for interaction with actin and promote not only nucleation, but also processive barbed end elongation. In contrast, the elongation function among W-based nucleators has been "outsourced" to a dedicated family of proteins, Eva/VASP, which are related to WASP-family NPFs.

  12. Lamellipodin promotes actin assembly by clustering Ena/VASP proteins and tethering them to actin filaments.

    PubMed

    Hansen, Scott D; Mullins, R Dyche

    2015-01-01

    Enabled/Vasodilator (Ena/VASP) proteins promote actin filament assembly at multiple locations, including: leading edge membranes, focal adhesions, and the surface of intracellular pathogens. One important Ena/VASP regulator is the mig-10/Lamellipodin/RIAM family of adaptors that promote lamellipod formation in fibroblasts and drive neurite outgrowth and axon guidance in neurons. To better understand how MRL proteins promote actin network formation we studied the interactions between Lamellipodin (Lpd), actin, and VASP, both in vivo and in vitro. We find that Lpd binds directly to actin filaments and that this interaction regulates its subcellular localization and enhances its effect on VASP polymerase activity. We propose that Lpd delivers Ena/VASP proteins to growing barbed ends and increases their polymerase activity by tethering them to filaments. This interaction represents one more pathway by which growing actin filaments produce positive feedback to control localization and activity of proteins that regulate their assembly.

  13. Single Filaments to Reveal the Multiple Flavors of Actin.

    PubMed

    Jégou, Antoine; Romet-Lemonne, Guillaume

    2016-05-24

    A number of key cell processes rely on specific assemblies of actin filaments, which are all constructed from nearly identical building blocks: the abundant and extremely conserved actin protein. A central question in the field is to understand how different filament networks can coexist and be regulated. Discoveries in science are often related to technical advances. Here, we focus on the ongoing single filament revolution and discuss how these techniques have greatly contributed to our understanding of actin assembly. In particular, we highlight how they have refined our understanding of the many protein-based regulatory mechanisms that modulate actin assembly. It is now becoming apparent that other factors give filaments a specific identity that determines which proteins will bind to them. We argue that single filament techniques will play an essential role in the coming years as we try to understand the many ways actin filaments can take different flavors and unveil how these flavors modulate the action of regulatory proteins. We discuss different factors known to make actin filaments distinguishable by regulatory proteins and speculate on their possible consequences.

  14. Clamped-filament elongation model for actin-based motors.

    PubMed Central

    Dickinson, Richard B; Purich, Daniel L

    2002-01-01

    Although actin-based motility drives cell crawling and intracellular locomotion of organelles and certain pathogens, the underlying mechanism of force generation remains a mystery. Recent experiments demonstrated that Listeria exhibit episodes of 5.4-nm stepwise motion corresponding to the periodicity of the actin filament subunits, and extremely small positional fluctuations during the intermittent pauses [S. C. Kuo and J. L. McGrath. 2000. Nature. 407:1026-1029]. These findings suggest that motile bacteria remain firmly bound to actin filament ends as they elongate, a behavior that appears to rule out previous models for actin-based motility. We propose and analyze a new mechanochemical model (called the "Lock, Load & Fire" mechanism) for force generation by means of affinity-modulated, clamped-filament elongation. During the locking step, the filament's terminal ATP-containing subunit binds tightly to a clamp situated on the surface of a motile object; in the loading step, actin.ATP monomer(s) bind to the filament end, an event that triggers the firing step, wherein ATP hydrolysis on the clamped subunit attenuates the filament's affinity for the clamp. This last step initiates translocation of the new ATP-containing terminus to the clamp, whereupon another cycle begins anew. This model explains how surface-tethered filaments can grow while exerting flexural or tensile force on the motile surface. Moreover, stochastic simulations of the model reproduce the signature motions of Listeria. This elongation motor, which we term actoclampin, exploits actin's intrinsic ATPase activity to provide a simple, high-fidelity enzymatic reaction cycle for force production that does not require elongating filaments to dissociate from the motile surface. This mechanism may operate whenever actin polymerization is called upon to generate the forces that drive cell crawling or intracellular organelle motility. PMID:11806905

  15. 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.

  16. How capping protein enhances actin filament growth and nucleation on biomimetic beads

    NASA Astrophysics Data System (ADS)

    Wang, Ruizhe; Carlsson, Anders E.

    2015-12-01

    Capping protein (CP), which caps the growing ends of actin filaments, accelerates actin-based motility. Recent experiments on biomimetic beads have shown that CP also enhances the rate of actin filament nucleation. Proposed explanations for these phenomena include (i) the actin funneling hypothesis (AFH), in which the presence of CP increases the free-actin concentration, and (ii) the monomer gating model, in which CP binding to actin filament barbed ends makes more monomers available for filament nucleation. To establish how CP increases the rates of filament elongation and nucleation on biomimetic beads, we perform a quantitative modeling analysis of actin polymerization, using rate equations that include actin filament nucleation, polymerization and capping, as modified by monomer depletion near the surface of the bead. With one adjustable parameter, our simulation results match previously measured time courses of polymerized actin and filament number. The results support a version of the AFH where CP increases the local actin monomer concentration at the bead surface, but leaves the global free-actin concentration nearly constant. Because the rate of filament nucleation increases with the monomer concentration, the increased local monomer concentration enhances actin filament nucleation. We derive a closed-form formula for the characteristic CP concentration where the local free-actin concentration reaches half the bulk value, and find it to be comparable to the global Arp2/3 complex concentration. We also propose an experimental protocol for distinguishing branching nucleation of filaments from spontaneous nucleation.

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

    PubMed

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

    2011-07-29

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

  18. Evolutionarily Divergent, Unstable Filamentous Actin Is Essential for Gliding Motility in Apicomplexan Parasites

    PubMed Central

    Skillman, Kristen M.; Diraviyam, Karthikeyan; Khan, Asis; Tang, Keliang; Sept, David; Sibley, L. David

    2011-01-01

    Apicomplexan parasites rely on a novel form of actin-based motility called gliding, which depends on parasite actin polymerization, to migrate through their hosts and invade cells. However, parasite actins are divergent both in sequence and function and only form short, unstable filaments in contrast to the stability of conventional actin filaments. The molecular basis for parasite actin filament instability and its relationship to gliding motility remain unresolved. We demonstrate that recombinant Toxoplasma (TgACTI) and Plasmodium (PfACTI and PfACTII) actins polymerized into very short filaments in vitro but were induced to form long, stable filaments by addition of equimolar levels of phalloidin. Parasite actins contain a conserved phalloidin-binding site as determined by molecular modeling and computational docking, yet vary in several residues that are predicted to impact filament stability. In particular, two residues were identified that form intermolecular contacts between different protomers in conventional actin filaments and these residues showed non-conservative differences in apicomplexan parasites. Substitution of divergent residues found in TgACTI with those from mammalian actin resulted in formation of longer, more stable filaments in vitro. Expression of these stabilized actins in T. gondii increased sensitivity to the actin-stabilizing compound jasplakinolide and disrupted normal gliding motility in the absence of treatment. These results identify the molecular basis for short, dynamic filaments in apicomplexan parasites and demonstrate that inherent instability of parasite actin filaments is a critical adaptation for gliding motility. PMID:21998582

  19. Actin Filament Segmentation Using Dynamic Programming

    PubMed Central

    Li, Hongsheng; Shen, Tian; Huang, Xiaolei

    2011-01-01

    We introduce a novel algorithm for actin filament segmentation in 2D TIRFM image sequences. This problem is difficult because actin filaments dynamically change shapes during their growth, and the TIRFM images are usually noisy. We ask a user to specify the two tips of a filament of interest in the first frame. We then model the segmentation problem in an image sequence as a temporal chain, where its states are tip locations; given candidate tip locations, actin filaments' body points are inferred by a dynamic programming method, which adaptively generates candidate solutions. Combining candidate tip locations and their inferred body points, the temporal chain model is efficiently optimized using another dynamic programming method. Evaluation on noisy TIRFM image sequences demonstrates the accuracy and robustness of this approach. PMID:21761674

  20. Auxin Stimulates Its Own Transport by Shaping Actin Filaments1

    PubMed Central

    Nick, Peter; Han, Min-Jung; An, Gyeunhung

    2009-01-01

    The directional transport of the plant hormone auxin has been identified as central element of axis formation and patterning in plants. This directionality of transport depends on gradients, across the cell, of auxin-efflux carriers that continuously cycle between plasma membrane and intracellular compartments. This cycling has been proposed to depend on actin filaments. However, the role of actin for the polarity of auxin transport has been disputed. The organization of actin, in turn, has been shown to be under control of auxin. By overexpression of the actin-binding protein talin, we have generated transgenic rice (Oryza sativa) lines, where actin filaments are bundled to variable extent and, in consequence, display a reduced dynamics. We show that this bundling of actin filaments correlates with impaired gravitropism and reduced longitudinal transport of auxin. We can restore a normal actin configuration by addition of exogenous auxins and restore gravitropism as well as polar auxin transport. This rescue is mediated by indole-3-acetic acid and 1-naphthyl acetic acid but not by 2,4-dichlorophenoxyacetic acid. We interpret these findings in the context of a self-referring regulatory circuit between polar auxin transport and actin organization. This circuit might contribute to the self-amplification of auxin transport that is a central element in current models of auxin-dependent patterning. PMID:19633235

  1. Human Muscle LIM Protein Dimerizes along the Actin Cytoskeleton and Cross-Links Actin Filaments

    PubMed Central

    Hoffmann, Céline; Moreau, Flora; Moes, Michèle; Luthold, Carole; Dieterle, Monika; Goretti, Emeline; Neumann, Katrin; Steinmetz, André

    2014-01-01

    The muscle LIM protein (MLP) is a nucleocytoplasmic shuttling protein playing important roles in the regulation of myocyte remodeling and adaptation to hypertrophic stimuli. Missense mutations in human MLP or its ablation in transgenic mice promotes cardiomyopathy and heart failure. The exact function(s) of MLP in the cytoplasmic compartment and the underlying molecular mechanisms remain largely unknown. Here, we provide evidence that MLP autonomously binds to, stabilizes, and bundles actin filaments (AFs) independently of calcium and pH. Using total internal reflection fluorescence microscopy, we have shown how MLP cross-links actin filaments into both unipolar and mixed-polarity bundles. Quantitative analysis of the actin cytoskeleton configuration confirmed that MLP substantially promotes actin bundling in live myoblasts. In addition, bimolecular fluorescence complementation (BiFC) assays revealed MLP self-association. Remarkably, BiFC complexes mostly localize along actin filament-rich structures, such as stress fibers and sarcomeres, supporting a functional link between MLP self-association and actin cross-linking. Finally, we have demonstrated that MLP self-associates through its N-terminal LIM domain, whereas it binds to AFs through its C-terminal LIM domain. Together our data support that MLP contributes to the maintenance of cardiomyocyte cytoarchitecture by a mechanism involving its self-association and actin filament cross-linking. PMID:24934443

  2. Correlative nanoscale imaging of actin filaments and their complexes

    PubMed Central

    Zhu, Huanqi; Grintsevich, Elena E.; Reisler, Emil

    2014-01-01

    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. PMID:23727693

  3. Correlative nanoscale imaging of actin filaments and their complexes.

    PubMed

    Sharma, Shivani; Zhu, Huanqi; Grintsevich, Elena E; Reisler, Emil; Gimzewski, James K

    2013-07-01

    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.

  4. The EGF receptor is an actin-binding protein

    PubMed Central

    1992-01-01

    In a number of recent studies it has been shown that in vivo part of the EGF receptor (EGFR) population is associated to the actin filament system. In this paper we demonstrate that the purified EGFR can be cosedimented with purified filamentous actin (F-actin) indicating a direct association between EGFR and actin. A truncated EGFR, previously shown not to be associated to the cytoskeleton, was used as a control and this receptor did not cosediment with actin filaments. Determination of the actin-binding domain of the EGFR was done by measuring competition of either a polyclonal antibody or synthetic peptides on EGFR cosedimentation with F-actin. A synthetic peptide was made homologous to amino acid residues 984-996 (HL-33) of the EGFR which shows high homology with the actin-binding domain of Acanthamoeba profilin. A polyclonal antibody raised against HL-33 was found to prevent cosedimentation of EGFR with F-actin. This peptide HL-33 was shown to bind directly to actin in contrast with a synthetic peptide homologous to residues 1001-1013 (HL-34). During cosedimentation, HL-33 competed for actin binding of the EGFR and HL-34 did not, indicating that the EGFR contains one actin-binding site. These results demonstrate that the EGFR is an actin-binding protein which binds to actin via a domain containing amino acids residues 984-996. PMID:1383230

  5. Calcium Regulation Of Actin Filament Speed In Vitro

    NASA Astrophysics Data System (ADS)

    Lamadrid, M. A.; Gordon, A. M.; Chase, P. B.; Chen, Y.; Luo, Z.

    1998-03-01

    Using an in-vitro motility assay, we have studied the Ca regulation of the gliding speed of actin filaments with regulatory proteins troponin and tropomyosin. In skeletal muscle, Ca binding to the troponin/tropomyosin system serves as the switch which enables a myosin head to bind to actin and create a power stroke. Fluorescently labeled filaments were observed using video fluorescence microscopy and speeds measured for different calcium concentrations and ionic strengths. In contrast to F-actin (for which speed was unaffected by [Ca]), the speed increased with increasing [Ca] in a non-linear manner. By comparing the behavior of short and long filaments, we also found that there was no length dependence to the observed non-zero speeds, but filaments shorter than 3 um had a higher tendency to undergo stop-go motion. Analysis of the data in the context of protein friction suggests that Ca affects not only the number of binding motors but also the lifetime of the strong binding state between actin and myosin.

  6. Persistent nuclear actin filaments inhibit transcription by RNA polymerase II.

    PubMed

    Serebryannyy, Leonid A; Parilla, Megan; Annibale, Paolo; Cruz, Christina M; Laster, Kyle; Gratton, Enrico; Kudryashov, Dmitri; Kosak, Steven T; Gottardi, Cara J; de Lanerolle, Primal

    2016-09-15

    Actin is abundant in the nucleus and it is clear that nuclear actin has important functions. However, mystery surrounds the absence of classical actin filaments in the nucleus. To address this question, we investigated how polymerizing nuclear actin into persistent nuclear actin filaments affected transcription by RNA polymerase II. Nuclear filaments impaired nuclear actin dynamics by polymerizing and sequestering nuclear actin. Polymerizing actin into stable nuclear filaments disrupted the interaction of actin with RNA polymerase II and correlated with impaired RNA polymerase II localization, dynamics, gene recruitment, and reduced global transcription and cell proliferation. Polymerizing and crosslinking nuclear actin in vitro similarly disrupted the actin-RNA-polymerase-II interaction and inhibited transcription. These data rationalize the general absence of stable actin filaments in mammalian somatic nuclei. They also suggest a dynamic pool of nuclear actin is required for the proper localization and activity of RNA polymerase II.

  7. Fatal congenital myopathy with actin filament deposits.

    PubMed

    Bornemann, A; Petersen, M B; Schmalbruch, H

    1996-07-01

    We present the clinical and morphological findings in a case of progressive congenital myopathy. The symptoms present at birth included severe general muscular hypotonia, diffuse muscular atrophy, arthrogryposis, absence of spontaneous movements, and left ventricular hypertrophy. A biopsy specimen taken from the gastrocnemius muscle when the patient was 2 weeks old revealed deposits which consisted of actin filaments as shown by electron microscopy. The infant was occasionally respirator dependent but was mostly able to breathe unassisted. At the age of 5 months he died of respiratory failure. The actin filament deposits may explain the clinical findings.

  8. Arabidopsis ACTIN-DEPOLYMERIZING FACTOR7 Severs Actin Filaments and Regulates Actin Cable Turnover to Promote Normal Pollen Tube Growth[W

    PubMed Central

    Zheng, Yiyan; Xie, Yurong; Jiang, Yuxiang; Qu, Xiaolu; Huang, Shanjin

    2013-01-01

    Actin filaments are often arranged into higher-order structures, such as the longitudinal actin cables that generate the reverse fountain cytoplasmic streaming pattern present in pollen tubes. While several actin binding proteins have been implicated in the generation of these cables, the mechanisms that regulate their dynamic turnover remain largely unknown. Here, we show that Arabidopsis thaliana ACTIN-DEPOLYMERIZING FACTOR7 (ADF7) is required for turnover of longitudinal actin cables. In vitro biochemical analyses revealed that ADF7 is a typical ADF that prefers ADP-G-actin over ATP-G-actin. ADF7 inhibits nucleotide exchange on actin and severs filaments, but its filament severing and depolymerizing activities are less potent than those of the vegetative ADF1. ADF7 primarily decorates longitudinal actin cables in the shanks of pollen tubes. Consistent with this localization pattern, the severing frequency and depolymerization rate of filaments significantly decreased, while their maximum lifetime significantly increased, in adf7 pollen tube shanks. Furthermore, an ADF7–enhanced green fluorescent protein fusion with defective severing activity but normal G-actin binding activity could not complement adf7, providing compelling evidence that the severing activity of ADF7 is vital for its in vivo functions. These observations suggest that ADF7 evolved to promote turnover of longitudinal actin cables by severing actin filaments in pollen tubes. PMID:24058157

  9. Formin DAAM1 Organizes Actin Filaments in the Cytoplasmic Nodal Actin Network

    PubMed Central

    Luo, Weiwei; Lieu, Zi Zhao; Manser, Ed; Bershadsky, Alexander D.; Sheetz, Michael P.

    2016-01-01

    A nodal cytoplasmic actin network underlies actin cytoplasm cohesion in the absence of stress fibers. We previously described such a network that forms upon Latrunculin A (LatA) treatment, in which formin DAAM1 was localized at these nodes. Knock down of DAAM1 reduced the mobility of actin nodes but the nodes remained. Here we have investigated DAAM1 containing nodes after LatA washout. DAAM1 was found to be distributed between the cytoplasm and the plasma membrane. The membrane binding likely occurs through an interaction with lipid rafts, but is not required for F-actin assembly. Interesting the forced interaction of DAAM1 with plasma membrane through a rapamycin-dependent linkage, enhanced F-actin assembly at the cell membrane (compared to the cytoplasm) after the LatA washout. However, immediately after addition of both rapamycin and LatA, the cytoplasmic actin nodes formed transiently, before DAAM1 moved to the membrane. This was consistent with the idea that DAAM1 was initially anchored to cytoplasmic actin nodes. Further, photoactivatable tracking of DAAM1 showed DAAM1 was immobilized at these actin nodes. Thus, we suggest that DAAM1 organizes actin filaments into a nodal complex, and such nodal complexes seed actin network recovery after actin depolymerization. PMID:27760153

  10. A Robust Actin Filaments Image Analysis Framework

    PubMed Central

    Alioscha-Perez, Mitchel; Benadiba, Carine; Goossens, Katty; Kasas, Sandor; Dietler, Giovanni; Willaert, Ronnie; Sahli, Hichem

    2016-01-01

    The cytoskeleton is a highly dynamical protein network that plays a central role in numerous cellular physiological processes, and is traditionally divided into three components according to its chemical composition, i.e. actin, tubulin and intermediate filament cytoskeletons. Understanding the cytoskeleton dynamics is of prime importance to unveil mechanisms involved in cell adaptation to any stress type. Fluorescence imaging of cytoskeleton structures allows analyzing the impact of mechanical stimulation in the cytoskeleton, but it also imposes additional challenges in the image processing stage, such as the presence of imaging-related artifacts and heavy blurring introduced by (high-throughput) automated scans. However, although there exists a considerable number of image-based analytical tools to address the image processing and analysis, most of them are unfit to cope with the aforementioned challenges. Filamentous structures in images can be considered as a piecewise composition of quasi-straight segments (at least in some finer or coarser scale). Based on this observation, we propose a three-steps actin filaments extraction methodology: (i) first the input image is decomposed into a ‘cartoon’ part corresponding to the filament structures in the image, and a noise/texture part, (ii) on the ‘cartoon’ image, we apply a multi-scale line detector coupled with a (iii) quasi-straight filaments merging algorithm for fiber extraction. The proposed robust actin filaments image analysis framework allows extracting individual filaments in the presence of noise, artifacts and heavy blurring. Moreover, it provides numerous parameters such as filaments orientation, position and length, useful for further analysis. Cell image decomposition is relatively under-exploited in biological images processing, and our study shows the benefits it provides when addressing such tasks. Experimental validation was conducted using publicly available datasets, and in osteoblasts

  11. A Robust Actin Filaments Image Analysis Framework.

    PubMed

    Alioscha-Perez, Mitchel; Benadiba, Carine; Goossens, Katty; Kasas, Sandor; Dietler, Giovanni; Willaert, Ronnie; Sahli, Hichem

    2016-08-01

    The cytoskeleton is a highly dynamical protein network that plays a central role in numerous cellular physiological processes, and is traditionally divided into three components according to its chemical composition, i.e. actin, tubulin and intermediate filament cytoskeletons. Understanding the cytoskeleton dynamics is of prime importance to unveil mechanisms involved in cell adaptation to any stress type. Fluorescence imaging of cytoskeleton structures allows analyzing the impact of mechanical stimulation in the cytoskeleton, but it also imposes additional challenges in the image processing stage, such as the presence of imaging-related artifacts and heavy blurring introduced by (high-throughput) automated scans. However, although there exists a considerable number of image-based analytical tools to address the image processing and analysis, most of them are unfit to cope with the aforementioned challenges. Filamentous structures in images can be considered as a piecewise composition of quasi-straight segments (at least in some finer or coarser scale). Based on this observation, we propose a three-steps actin filaments extraction methodology: (i) first the input image is decomposed into a 'cartoon' part corresponding to the filament structures in the image, and a noise/texture part, (ii) on the 'cartoon' image, we apply a multi-scale line detector coupled with a (iii) quasi-straight filaments merging algorithm for fiber extraction. The proposed robust actin filaments image analysis framework allows extracting individual filaments in the presence of noise, artifacts and heavy blurring. Moreover, it provides numerous parameters such as filaments orientation, position and length, useful for further analysis. Cell image decomposition is relatively under-exploited in biological images processing, and our study shows the benefits it provides when addressing such tasks. Experimental validation was conducted using publicly available datasets, and in osteoblasts grown in

  12. Unconventional actin conformations localize on intermediate filaments in mitosis

    SciTech Connect

    Hubert, Thomas; Vandekerckhove, Joel; Gettemans, Jan

    2011-03-04

    Research highlights: {yields} Unconventional actin conformations colocalize with vimentin on a cage-like structure in metaphase HEK 293T cells. {yields} These conformations are detected with the anti-actin antibodies 1C7 ('lower dimer') and 2G2 ('nuclear actin'), but not C4 (monomeric actin). {yields} Mitotic unconventional actin cables are independent of filamentous actin or microtubules. {yields} Unconventional actin colocalizes with vimentin on a nocodazole-induced perinuclear dense mass of cables. -- Abstract: Different structural conformations of actin have been identified in cells and shown to reside in distinct subcellular locations of cells. In this report, we describe the localization of actin on a cage-like structure in metaphase HEK 293T cells. Actin was detected with the anti-actin antibodies 1C7 and 2G2, but not with the anti-actin antibody C4. Actin contained in this structure is independent of microtubules and actin filaments, and colocalizes with vimentin. Taking advantage of intermediate filament collapse into a perinuclear dense mass of cables when microtubules are depolymerized, we were able to relocalize actin to such structures. We hypothesize that phosphorylation of intermediate filaments at mitosis entry triggers the recruitment of different actin conformations to mitotic intermediate filaments. Storage and partition of the nuclear actin and antiparallel 'lower dimer' actin conformations between daughter cells possibly contribute to gene transcription and transient actin filament dynamics at G1 entry.

  13. Stretching Actin Filaments within Cells Enhances their Affinity for the Myosin II Motor Domain

    PubMed Central

    Uyeda, Taro Q. P.; Iwadate, Yoshiaki; Umeki, Nobuhisa; Nagasaki, Akira; Yumura, Shigehiko

    2011-01-01

    To test the hypothesis that the myosin II motor domain (S1) preferentially binds to specific subsets of actin filaments in vivo, we expressed GFP-fused S1 with mutations that enhanced its affinity for actin in Dictyostelium cells. Consistent with the hypothesis, the GFP-S1 mutants were localized along specific portions of the cell cortex. Comparison with rhodamine-phalloidin staining in fixed cells demonstrated that the GFP-S1 probes preferentially bound to actin filaments in the rear cortex and cleavage furrows, where actin filaments are stretched by interaction with endogenous myosin II filaments. The GFP-S1 probes were similarly enriched in the cortex stretched passively by traction forces in the absence of myosin II or by external forces using a microcapillary. The preferential binding of GFP-S1 mutants to stretched actin filaments did not depend on cortexillin I or PTEN, two proteins previously implicated in the recruitment of myosin II filaments to stretched cortex. These results suggested that it is the stretching of the actin filaments itself that increases their affinity for the myosin II motor domain. In contrast, the GFP-fused myosin I motor domain did not localize to stretched actin filaments, which suggests different preferences of the motor domains for different structures of actin filaments play a role in distinct intracellular localizations of myosin I and II. We propose a scheme in which the stretching of actin filaments, the preferential binding of myosin II filaments to stretched actin filaments, and myosin II-dependent contraction form a positive feedback loop that contributes to the stabilization of cell polarity and to the responsiveness of the cells to external mechanical stimuli. PMID:22022566

  14. CASEIN KINASE1-LIKE PROTEIN2 Regulates Actin Filament Stability and Stomatal Closure via Phosphorylation of Actin Depolymerizing Factor.

    PubMed

    Zhao, Shuangshuang; Jiang, Yuxiang; Zhao, Yang; Huang, Shanjin; Yuan, Ming; Zhao, Yanxiu; Guo, Yan

    2016-06-01

    The opening and closing of stomata are crucial for plant photosynthesis and transpiration. Actin filaments undergo dynamic reorganization during stomatal closure, but the underlying mechanism for this cytoskeletal reorganization remains largely unclear. In this study, we identified and characterized Arabidopsis thaliana casein kinase 1-like protein 2 (CKL2), which responds to abscisic acid (ABA) treatment and participates in ABA- and drought-induced stomatal closure. Although CKL2 does not bind to actin filaments directly and has no effect on actin assembly in vitro, it colocalizes with and stabilizes actin filaments in guard cells. Further investigation revealed that CKL2 physically interacts with and phosphorylates actin depolymerizing factor 4 (ADF4) and inhibits its activity in actin filament disassembly. During ABA-induced stomatal closure, deletion of CKL2 in Arabidopsis alters actin reorganization in stomata and renders stomatal closure less sensitive to ABA, whereas deletion of ADF4 impairs the disassembly of actin filaments and causes stomatal closure to be more sensitive to ABA Deletion of ADF4 in the ckl2 mutant partially recues its ABA-insensitive stomatal closure phenotype. Moreover, Arabidopsis ADFs from subclass I are targets of CKL2 in vitro. Thus, our results suggest that CKL2 regulates actin filament reorganization and stomatal closure mainly through phosphorylation of ADF. PMID:27268429

  15. Myosin and Tropomyosin Stabilize the Conformation of Formin-nucleated Actin Filaments*

    PubMed Central

    Ujfalusi, Zoltán; Kovács, Mihály; Nagy, Nikolett T.; Barkó, Szilvia; Hild, Gábor; Lukács, András; Nyitrai, Miklós; Bugyi, Beáta

    2012-01-01

    The conformational elasticity of the actin cytoskeleton is essential for its versatile biological functions. Increasing evidence supports that the interplay between the structural and functional properties of actin filaments is finely regulated by actin-binding proteins; however, the underlying mechanisms and biological consequences are not completely understood. Previous studies showed that the binding of formins to the barbed end induces conformational transitions in actin filaments by making them more flexible through long range allosteric interactions. These conformational changes are accompanied by altered functional properties of the filaments. To get insight into the conformational regulation of formin-nucleated actin structures, in the present work we investigated in detail how binding partners of formin-generated actin structures, myosin and tropomyosin, affect the conformation of the formin-nucleated actin filaments using fluorescence spectroscopic approaches. Time-dependent fluorescence anisotropy and temperature-dependent Förster-type resonance energy transfer measurements revealed that heavy meromyosin, similarly to tropomyosin, restores the formin-induced effects and stabilizes the conformation of actin filaments. The stabilizing effect of heavy meromyosin is cooperative. The kinetic analysis revealed that despite the qualitatively similar effects of heavy meromyosin and tropomyosin on the conformational dynamics of actin filaments the mechanisms of the conformational transition are different for the two proteins. Heavy meromyosin stabilizes the formin-nucleated actin filaments in an apparently single step reaction upon binding, whereas the stabilization by tropomyosin occurs after complex formation. These observations support the idea that actin-binding proteins are key elements of the molecular mechanisms that regulate the conformational and functional diversity of actin filaments in living cells. PMID:22753415

  16. Lamellipodin promotes actin assembly by clustering Ena/VASP proteins and tethering them to actin filaments

    PubMed Central

    Hansen, Scott D; Mullins, R Dyche

    2015-01-01

    Enabled/Vasodilator (Ena/VASP) proteins promote actin filament assembly at multiple locations, including: leading edge membranes, focal adhesions, and the surface of intracellular pathogens. One important Ena/VASP regulator is the mig-10/Lamellipodin/RIAM family of adaptors that promote lamellipod formation in fibroblasts and drive neurite outgrowth and axon guidance in neurons. To better understand how MRL proteins promote actin network formation we studied the interactions between Lamellipodin (Lpd), actin, and VASP, both in vivo and in vitro. We find that Lpd binds directly to actin filaments and that this interaction regulates its subcellular localization and enhances its effect on VASP polymerase activity. We propose that Lpd delivers Ena/VASP proteins to growing barbed ends and increases their polymerase activity by tethering them to filaments. This interaction represents one more pathway by which growing actin filaments produce positive feedback to control localization and activity of proteins that regulate their assembly. DOI: http://dx.doi.org/10.7554/eLife.06585.001 PMID:26295568

  17. Actin-binding proteins: the long road to understanding the dynamic landscape of cellular actin networks.

    PubMed

    Lappalainen, Pekka

    2016-08-15

    The actin cytoskeleton supports a vast number of cellular processes in nonmuscle cells. It is well established that the organization and dynamics of the actin cytoskeleton are controlled by a large array of actin-binding proteins. However, it was only 40 years ago that the first nonmuscle actin-binding protein, filamin, was identified and characterized. Filamin was shown to bind and cross-link actin filaments into higher-order structures and contribute to phagocytosis in macrophages. Subsequently many other nonmuscle actin-binding proteins were identified and characterized. These proteins regulate almost all steps of the actin filament assembly and disassembly cycles, as well as the arrangement of actin filaments into diverse three-dimensional structures. Although the individual biochemical activities of most actin-regulatory proteins are relatively well understood, knowledge of how these proteins function together in a common cytoplasm to control actin dynamics and architecture is only beginning to emerge. Furthermore, understanding how signaling pathways and mechanical cues control the activities of various actin-binding proteins in different cellular, developmental, and pathological processes will keep researchers busy for decades. PMID:27528696

  18. Cleavage furrow: timing of emergence of contractile ring actin filaments and establishment of the contractile ring by filament bundling in sea urchin eggs.

    PubMed

    Mabuchi, I

    1994-07-01

    Cleavage furrow formation at the first cell division of sea urchin and sand dollar eggs was investigated in detail by fluorescence staining of actin filaments with rhodamine-phalloidin of either whole eggs or isolated egg cortices. Cortical actin filaments were clustered at anaphase and then the clusters became fibrillar at the end of anaphase. The timing when the contractile ring actin filaments appear was precisely determined in the course of mitosis: accumulation of the contractile ring actin filaments at the equatorial cell cortex is first noticed at the beginning of telophase (shortly before furrow formation), when the chromosomal vesicles are fusing with each other. The accumulated actin filaments were not well organized at the early stage but were organized into parallel bundles as the furrowing progressed. The bundles were finally fused into a tightly packed filament belt. Wheat germ agglutinin (WGA)-binding sites were distributed on the surface of the egg in a manner similar to the actin filaments after anaphase. The WGA-binding sites became accumulated in the contractile ring together with the contractile ring actin filaments, indicating an intimate relationship between these sites and actin filament-anchoring sites on the plasma membrane. Myosin also appeared in the contractile ring together with the actin filaments. The 'cleavage stimulus', a signal hypothesized by Rappaport (reviewed by R. Rappaport (1986) Int. Rev. Cytol. 105, 245-281) was suggested to induce aggregation or bundling of the actin filaments in the cortical layer.

  19. Site-specific cation release drives actin filament severing by vertebrate cofilin

    PubMed Central

    Kang, Hyeran; Bradley, Michael J.; Cao, Wenxiang; Zhou, Kaifeng; Grintsevich, Elena E.; Michelot, Alphée; Sindelar, Charles V.; Hochstrasser, Mark; De La Cruz, Enrique M.

    2014-01-01

    Actin polymerization powers the directed motility of eukaryotic cells. Sustained motility requires rapid filament turnover and subunit recycling. The essential regulatory protein cofilin accelerates network remodeling by severing actin filaments and increasing the concentration of ends available for elongation and subunit exchange. Although cofilin effects on actin filament assembly dynamics have been extensively studied, the molecular mechanism of cofilin-induced filament severing is not understood. Here we demonstrate that actin filament severing by vertebrate cofilin is driven by the linked dissociation of a single cation that controls filament structure and mechanical properties. Vertebrate cofilin only weakly severs Saccharomyces cerevisiae actin filaments lacking this “stiffness cation” unless a stiffness cation-binding site is engineered into the actin molecule. Moreover, vertebrate cofilin rescues the viability of a S. cerevisiae cofilin deletion mutant only when the stiffness cation site is simultaneously introduced into actin, demonstrating that filament severing is the essential function of cofilin in cells. This work reveals that site-specific interactions with cations serve a key regulatory function in actin filament fragmentation and dynamics. PMID:25468977

  20. Single turnovers of fluorescent ATP bound to bipolar myosin filament during actin filaments sliding.

    PubMed

    Maruta, Takahiro; Kobatake, Takahiro; Okubo, Hiroyuki; Chaen, Shigeru

    2013-01-01

    The nucleotide turnover rates of bipolar myosin thick filament along which actin filament slides were measured by the displacement of prebound fluorescent ATP analog 2'(3')-O-[N-[2-[(Cy3)]amindo]ethyl] carbamoyl]-adenosine 5' triphosphate (Cy3-EDA-ATP) upon flash photolysis of caged ATP. The fluorescence of the thick filament where actin filament slides decayed with two exponential processes. The slower rate constant was the same as that without actin filament. Along bipolar myosin thick filament, actin filaments slide at a fast speed towards the central bare zone (forward), but more slowly away from the bare zone (backward). The displacement rate constant of fluorescent ATP from the myosin filament where actin filament moved forward was 5.0 s(-1), whereas the rate constant where the actin filament slid backward was 1.7 s(-1). These findings suggest that the slow ADP release rate is responsible for the slow backward sliding movement.

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

    PubMed Central

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

    2016-01-01

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

  2. Profilin Interaction with Actin Filament Barbed End Controls Dynamic Instability, Capping, Branching, and Motility

    PubMed Central

    Pernier, Julien; Shekhar, Shashank; Jegou, Antoine; Guichard, Bérengère; Carlier, Marie-France

    2016-01-01

    Summary Cell motility and actin homeostasis depend on the control of polarized growth of actin filaments. Profilin, an abundant regulator of actin dynamics, supports filament assembly at barbed ends by binding G-actin. Here, we demonstrate how, by binding and destabilizing filament barbed ends at physiological concentrations, profilin also controls motility, cell migration, and actin homeostasis. Profilin enhances filament length fluctuations. Profilin competes with Capping Protein at barbed ends, which generates a lower amount of profilin-actin than expected if barbed ends were tightly capped. Profilin competes with barbed end polymerases, such as formins and VopF, and inhibits filament branching by WASP-Arp2/3 complex by competition for filament barbed ends, accounting for its as-yet-unknown effects on motility and metastatic cell migration observed in this concentration range. In conclusion, profilin is a major coordinator of polarized growth of actin filaments, controlled by competition between barbed end cappers, trackers, destabilizers, and filament branching machineries. PMID:26812019

  3. Structural characterization of a capping protein interaction motif defines a family of actin filament regulators

    PubMed Central

    Hernandez-Valladares, Maria; Kim, Taekyung; Kannan, Balakrishnan; Tung, Alvin; Aguda, Adeleke H; Larsson, Mårten; Cooper, John A; Robinson, Robert C

    2011-01-01

    Capping protein (CP) regulates actin dynamics by binding the barbed ends of actin filaments. Removal of CP may be one means to harness actin polymerization for processes such as cell movement and endocytosis. Here we structurally and biochemically investigated a CP interaction (CPI) motif present in the otherwise unrelated proteins CARMIL and CD2AP. The CPI motif wraps around the stalk of the mushroom-shaped CP at a site distant from the actin-binding interface, which lies on the top of the mushroom cap. We propose that the CPI motif may act as an allosteric modulator, restricting CP to a low-affinity, filament-binding conformation. Structure-based sequence alignments extend the CPI motif–containing family to include CIN85, CKIP-1, CapZIP and a relatively uncharacterized protein, WASHCAP (FAM21). Peptides comprising these CPI motifs are able to inhibit CP and to uncap CP-bound actin filaments. PMID:20357771

  4. Multiple actin binding domains of Ena/VASP proteins determine actin network stiffening.

    PubMed

    Gentry, Brian S; van der Meulen, Stef; Noguera, Philippe; Alonso-Latorre, Baldomero; Plastino, Julie; Koenderink, Gijsje H

    2012-11-01

    Vasodilator-stimulated phosphoprotein (Ena/VASP) is an actin binding protein, important for actin dynamics in motile cells and developing organisms. Though VASP's main activity is the promotion of barbed end growth, it has an F-actin binding site and can form tetramers, and so could additionally play a role in actin crosslinking and bundling in the cell. To test this activity, we performed rheology of reconstituted actin networks in the presence of wild-type VASP or mutants lacking the ability to tetramerize or to bind G-actin and/or F-actin. We show that increasing amounts of wild-type VASP increase network stiffness up to a certain point, beyond which stiffness actually decreases with increasing VASP concentration. The maximum stiffness is 10-fold higher than for pure actin networks. Confocal microscopy shows that VASP forms clustered actin filament bundles, explaining the reduction in network elasticity at high VASP concentration. Removal of the tetramerization site results in significantly reduced bundling and bundle clustering, indicating that VASP's flexible tetrameric structure causes clustering. Removing either the F-actin or the G-actin binding site diminishes VASP's effect on elasticity, but does not eliminate it. Mutating the F-actin and G-actin binding site together, or mutating the F-actin binding site and saturating the G-actin binding site with monomeric actin, eliminates VASP's ability to increase network stiffness. We propose that, in the cell, VASP crosslinking confers only moderate increases in linear network elasticity, and unlike other crosslinkers, VASP's network stiffening activity may be tuned by the local concentration of monomeric actin.

  5. Proteolytic cleavage of actin within the DNase-I-binding loop changes the conformation of F-actin and its sensitivity to myosin binding.

    PubMed

    Borovikov, Y S; Moraczewska, J; Khoroshev, M I; Strzelecka-Gołaszewska, H

    2000-03-16

    Effects of subtilisin cleavage of actin between residues 47 and 48 on the conformation of F-actin and on its changes occurring upon binding of myosin subfragment-1 (S1) were investigated by measuring polarized fluorescence from rhodamine-phalloidin- or 1, 5-IAEDANS-labeled actin filaments reconstructed from intact or subtilisin-cleaved actin in myosin-free muscle fibers (ghost fibers). In separate experiments, polarized fluorescence from 1, 5-IAEDANS-labeled S1 bound to non-labeled actin filaments in ghost fibers was measured. The measurements revealed differences between the filaments of cleaved and intact actin in the orientation of rhodamine probe on the rhodamine-phalloidin-labeled filaments, orientation and mobility of the C-terminus of actin, filament flexibility, and orientation and mobility of the myosin heads bound to F-actin. The changes in the filament flexibility and orientation of the actin-bound fluorophores produced by S1 binding to actin in the absence of ATP were substantially diminished by subtilisin cleavage of actin. The results suggest that loop 38-52 plays an important role, not only in maintaining the F-actin structure, but also in the conformational transitions in actin accompanying the strong binding of the myosin heads that may be essential for the generation of force and movement during actin-myosin interaction.

  6. Dynamic Regulation of Sarcomeric Actin Filaments in Striated Muscle

    PubMed Central

    Ono, Shoichiro

    2010-01-01

    In striated muscle, the actin cytoskeleton is differentiated into myofibrils. Actin and myosin filaments are organized in sarcomeres and specialized for producing contractile forces. Regular arrangement of actin filaments with uniform length and polarity is critical for the contractile function. However, the mechanisms of assembly and maintenance of sarcomeric actin filaments in striated muscle are not completely understood. Live imaging of actin in striated muscle has revealed that actin subunits within sarcomeric actin filaments are dynamically exchanged without altering overall sarcomeric structures. A number of regulators for actin dynamics have been identified, and malfunction of these regulators often result in disorganization of myofibril structures or muscle diseases. Therefore, proper regulation of actin dynamics in striated muscle is critical for assembly and maintenance of functional myofibrils. Recent studies have suggested that both enhancers of actin dynamics and stabilizers of actin filaments are important for sarcomeric actin organization. Further investigation of the regulatory mechanism of actin dynamics in striated muscle should be a key to understanding how myofibrils develop and operate. © 2010 Wiley-Liss, Inc. PMID:20737540

  7. Structural basis of thymosin-β4/profilin exchange leading to actin filament polymerization

    PubMed Central

    Xue, Bo; Leyrat, Cedric; Grimes, Jonathan M.; Robinson, Robert C.

    2014-01-01

    Thymosin-β4 (Tβ4) and profilin are the two major sequestering proteins that maintain the pool of monomeric actin (G-actin) within cells of higher eukaryotes. Tβ4 prevents G-actin from joining a filament, whereas profilin:actin only supports barbed-end elongation. Here, we report two Tβ4:actin structures. The first structure shows that Tβ4 has two helices that bind at the barbed and pointed faces of G-actin, preventing the incorporation of the bound G-actin into a filament. The second structure displays a more open nucleotide binding cleft on G-actin, which is typical of profilin:actin structures, with a concomitant disruption of the Tβ4 C-terminal helix interaction. These structures, combined with biochemical assays and molecular dynamics simulations, show that the exchange of bound actin between Tβ4 and profilin involves both steric and allosteric components. The sensitivity of profilin to the conformational state of actin indicates a similar allosteric mechanism for the dissociation of profilin during filament elongation. PMID:25313062

  8. 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.

  9. Fullerenol Nanoparticles with Structural Activity Induce Variable Intracellular Actin Filament Morphologies.

    PubMed

    Jin, Junjiang; Dong, Ying; Wang, Ying; Xia, Lin; Gu, Weihong; Bai, Xue; Chang, Yanan; Zhang, Mingyi; Chen, Kui; Li, Juan; Zhao, Lina; Xing, Gengmei

    2016-06-01

    Fullerenol nanoparticles are promising for various biological applications; many studies have shown that they induce variable and diverse biological effects including side effects. Separation and purification of two fractions of fullerenols has demonstrated that they have varied chemical structures on the surfaces of their carbon cages. Actin is an important structural protein that is able to transform functional structures under varied physiological conditions. We assessed the abilities of the two fractions of fullerenols to attach to actin and induce variable morphological features in actin filament structures. Specifically the fullerenol fraction with a surface electric charge of -1.913 ± 0.008q (x10(-6) C) has percentages of C-OH and C=O on the carbon cage of 16.14 ± 0.60 and 17.55 ± 0.69. These features allow it to form intermolecular hydrogen bonds with actin at a stoichiometric ratio of four fullerenols per actin subunit. Molecular simulations revealed these specific binding sites and binding modes in atomic details in the interaction between the active fullerenol and actin filament. Conversely, these interactions were not possible for the other fraction of fullerenol with that percentages of C-OH and C=O on the carbon cage were 15.59 ± 0.01 and 1.94 ± 0.11. Neither sample induced appreciable cytotoxicity or acute cell death. After entering cells, active fullerenol binding to actin induces variable morphological features and may transform ATP-actin to ADP-actin. These changes facilitate the binding of ADF/cofilin, allowing cofilin to sever actin filaments to form cofilin/actin/fullerenol rods. Our findings suggest that fullerenol with structural activity binding disturbs actin filament structure, which may inhibit locomotion of cell or induce chronic side effects in to cells. PMID:27319217

  10. Bending Flexibility of Actin Filaments during Motor-Induced Sliding

    PubMed Central

    Vikhorev, Petr G.; Vikhoreva, Natalia N.; Månsson, Alf

    2008-01-01

    Muscle contraction and other forms of cell motility occur as a result of cyclic interactions between myosin molecules and actin filaments. Force generation is generally attributed to ATP-driven structural changes in myosin, whereas a passive role is ascribed to actin. However, some results challenge this view, predicting structural changes in actin during motor activity, e.g., when the actin filaments slide on a myosin-coated surface in vitro. Here, we analyzed statistical properties of the sliding filament paths, allowing us to detect changes of this type. It is interesting to note that evidence for substantial structural changes that led to increased bending flexibility of the filaments was found in phalloidin-stabilized, but not in phalloidin-free, actin filaments. The results are in accordance with the idea that a high-flexibility structural state of actin is a prerequisite for force production, but not the idea that a low-to-high flexibility transition of the actin filament should be an important component of the force-generating step per se. Finally, our data challenge the general view that phalloidin-stabilized filaments behave as native actin filaments in their interaction with myosin. This has important implications, since phalloidin stabilization is a routine procedure in most studies of actomyosin function. PMID:18835897

  11. The effects of formins on the conformation of subdomain 1 in actin filaments.

    PubMed

    Ujfalusi, Zoltán; Barkó, Szilvia; Hild, Gábor; Nyitrai, Miklós

    2010-01-21

    In this study we investigated the effects of formins on the conformation of actin filaments by using the method of fluorescence quenching. Actin was labelled with IAEDANS at Cys(374) and the quencher was acrylamide. The results showed that formin binding induced structural changes in the subdomain 1 of actin protomers which were reflected by greater quenching constants (K(SV)). Simultaneously the fraction of the fluorophore population accessible for the quencher (alpha) decreased. These observations suggest that the conformational distribution characteristic for the actin protomers became broader after the binding of formins, for which the structural framework was provided by a more flexible protein matrix in the microenvironment of the label. The effects of formins depended on the formin:actin molar ratio, and also on the ionic strength of the medium. These observations are in agreement with previous results and underline the importance of the intramolecular conformational changes induced by formins in the structure of actin filaments.

  12. Identification of sucrose synthase as an actin-binding protein

    NASA Technical Reports Server (NTRS)

    Winter, H.; Huber, J. L.; Huber, S. C.; Davies, E. (Principal Investigator)

    1998-01-01

    Several lines of evidence indicate that sucrose synthase (SuSy) binds both G- and F-actin: (i) presence of SuSy in the Triton X-100-insoluble fraction of microsomal membranes (i.e. crude cytoskeleton fraction); (ii) co-immunoprecipitation of actin with anti-SuSy monoclonal antibodies; (iii) association of SuSy with in situ phalloidin-stabilized F-actin filaments; and (iv) direct binding to F-actin, polymerized in vitro. Aldolase, well known to interact with F-actin, interfered with binding of SuSy, suggesting that a common or overlapping binding site may be involved. We postulate that some of the soluble SuSy in the cytosol may be associated with the actin cytoskeleton in vivo.

  13. Arabidopsis AtADF1 is functionally affected by mutations on actin binding sites.

    PubMed

    Dong, Chun-Hai; Tang, Wei-Ping; Liu, Jia-Yao

    2013-03-01

    The plant actin depolymerizing factor (ADF) binds to both monomeric and filamentous actin, and is directly involved in the depolymerization of actin filaments. To better understand the actin binding sites of the Arabidopsis thaliana L. AtADF1, we generated mutants of AtADF1 and investigated their functions in vitro and in vivo. Analysis of mutants harboring amino acid substitutions revealed that charged residues (Arg98 and Lys100) located at the α-helix 3 and forming an actin binding site together with the N-terminus are essential for both G- and F-actin binding. The basic residues on the β-strand 5 (K82/A) and the α-helix 4 (R135/A, R137/A) form another actin binding site that is important for F-actin binding. Using transient expression of CFP-tagged AtADF1 mutant proteins in onion (Allium cepa) peel epidermal cells and transgenic Arabidopsis thaliana L. plants overexpressing these mutants, we analyzed how these mutant proteins regulate actin organization and affect seedling growth. Our results show that the ADF mutants with a lower affinity for actin filament binding can still be functional, unless the affinity for actin monomers is also affected. The G-actin binding activity of the ADF plays an essential role in actin binding, depolymerization of actin polymers, and therefore in the control of actin organization. PMID:23190411

  14. Positioning and stretching of actin filaments by electric fields

    NASA Astrophysics Data System (ADS)

    Wigge, Christoph; Hinssen, Horst; Reiss, Günter; Herth, Simone

    2010-06-01

    The alignment of biological filaments on surfaces offers a high potential for controllable geometries in lab-on-a-chip-structures and micrototal analysis systems. Actin is a polar filamentous protein with a diameter of 7-8 nm that can be manipulated with strong electric fields. It is demonstrated that with the use of microelectrodes or nanoelectrodes and electric fields of 20 kV/m single actin filaments can be manipulated, stretched, and positioned between gold electrodes.

  15. Force Generation, Polymerization Dynamics and Nucleation of Actin Filaments

    NASA Astrophysics Data System (ADS)

    Wang, Ruizhe

    We study force generation and actin filament dynamics using stochastic and deterministic methods. First, we treat force generation of bundled actin filaments by polymerization via molecular-level stochastic simulations. In the widely-used Brownian Ratchet model, actin filaments grow freely whenever the tip-obstacle gap created by thermal fluctuation exceeds the monomer size. We name this model the Perfect Brownian Ratchet (PBR) model. In the PBR model, actin monomer diffusion is treated implicitly. We perform a series of simulations based on the PBR, in which obstacle motion is treated explicitly; in most previous studies, obstacle motion has been treated implicitly. We find that the cooperativity of filaments is generally weak in the PBR model, meaning that more filaments would grow more slowly given the same force per filament. Closed-form formulas are also developed, which match the simulation results. These portable and accurate formulas provide guidance for experiments and upper and lower bounds for theoretical analyses. We also studied a variation of the PBR, called the Diffusing Brownian Ratchet (DBR) model, in which both actin monomer and obstacle diffusion are treated explicitly. We find that the growth rate of multiple filaments is even lower, compared with that in PBR. This finding challenges the widely-accepted PBR assumption and suggests that pushing the study of actin dynamics down to the sub-nanometer level yields new insights. We subsequently used a rate equation approach to model the effect of local depletion of actin monomers on the nucleation of actin filaments on biomimetic beads, and how the effect is regulated by capping protein (CP). We find that near the bead surface, a higher CP concentration increases local actin concentration, which leads to an enhanced activities of actin filaments' nucleation. Our model analysis matches the experimental results and lends support to an important but undervalued hypothesis proposed by Carlier and

  16. Bundling actin filaments from membranes: some novel players

    PubMed Central

    Thomas, Clément

    2012-01-01

    Progress in live-cell imaging of the cytoskeleton has significantly extended our knowledge about the organization and dynamics of actin filaments near the plasma membrane of plant cells. Noticeably, two populations of filamentous structures can be distinguished. On the one hand, fine actin filaments which exhibit an extremely dynamic behavior basically characterized by fast polymerization and prolific severing events, a process referred to as actin stochastic dynamics. On the other hand, thick actin bundles which are composed of several filaments and which are comparatively more stable although they constantly remodel as well. There is evidence that the actin cytoskeleton plays critical roles in trafficking and signaling at both the cell cortex and organelle periphery but the exact contribution of actin bundles remains unclear. A common view is that actin bundles provide the long-distance tracks used by myosin motors to deliver their cargo to growing regions and accordingly play a particularly important role in cell polarization. However, several studies support that actin bundles are more than simple passive highways and display multiple and dynamic roles in the regulation of many processes, such as cell elongation, polar auxin transport, stomatal and chloroplast movement, and defense against pathogens. The list of identified plant actin-bundling proteins is ever expanding, supporting that plant cells shape structurally and functionally different actin bundles. Here I review the most recently characterized actin-bundling proteins, with a particular focus on those potentially relevant to membrane trafficking and/or signaling. PMID:22936939

  17. Bacterial actin MreB forms antiparallel double filaments

    PubMed Central

    van den Ent, Fusinita; Izoré, Thierry; Bharat, Tanmay AM; Johnson, Christopher M; Löwe, Jan

    2014-01-01

    Filaments of all actin-like proteins known to date are assembled from pairs of protofilaments that are arranged in a parallel fashion, generating polarity. In this study, we show that the prokaryotic actin homologue MreB forms pairs of protofilaments that adopt an antiparallel arrangement in vitro and in vivo. We provide an atomic view of antiparallel protofilaments of Caulobacter MreB as apparent from crystal structures. We show that a protofilament doublet is essential for MreB's function in cell shape maintenance and demonstrate by in vivo site-specific cross-linking the antiparallel orientation of MreB protofilaments in E. coli. 3D cryo-EM shows that pairs of protofilaments of Caulobacter MreB tightly bind to membranes. Crystal structures of different nucleotide and polymerisation states of Caulobacter MreB reveal conserved conformational changes accompanying antiparallel filament formation. Finally, the antimicrobial agents A22/MP265 are shown to bind close to the bound nucleotide of MreB, presumably preventing nucleotide hydrolysis and destabilising double protofilaments. DOI: http://dx.doi.org/10.7554/eLife.02634.001 PMID:24843005

  18. Actin-crosslinking protein regulation of filament movement in motility assays: a theoretical model.

    PubMed Central

    Janson, L W; Taylor, D L

    1994-01-01

    The interaction of single actin filaments on a myosin-coated coverslip has been modeled by several authors. One model adds a component of "frictional drag" by myosin heads that oppose movement of the actin filaments. We have extended this concept by including the resistive drag from actin crosslinking proteins to understand better the relationship among crosslinking number, actin-myosin force generation, and motility. The validity of this model is supported by agreement with the experimental results from a previous study in which crosslinking proteins were added with myosin molecules under otherwise standard motility assay conditions. The theoretical relationship provides a means to determine many physical parameters that characterize the interaction between a single actin filament and a single actin-crosslinking molecule (various types). In particular, the force constant of a single filamin molecule is calculated as 1.105 pN, approximately 3 times less than a driving myosin head (3.4 pN). Knowledge of this parameter and others derived from this model allows a better understanding of the interaction between myosin and the actin/actin-binding protein cytoskeleton and the role of actin-binding proteins in the regulation and modulation of motility. PMID:7811954

  19. Actin filament organization in the fish keratocyte lamellipodium

    PubMed Central

    1995-01-01

    From recent studies of locomoting fish keratocytes it was proposed that the dynamic turnover of actin filaments takes place by a nucleation- release mechanism, which predicts the existence of short (less than 0.5 microns) filaments throughout the lamellipodium (Theriot, J. A., and T. J. Mitchison. 1991. Nature (Lond.). 352:126-131). We have tested this model by investigating the structure of whole mount keratocyte cytoskeletons in the electron microscope and phalloidin-labeled cells, after various fixations, in the light microscope. Micrographs of negatively stained keratocyte cytoskeletons produced by Triton extraction showed that the actin filaments of the lamellipodium are organized to a first approximation in a two-dimensional orthogonal network with the filaments subtending an angle of around 45 degrees to the cell front. Actin filament fringes grown onto the front edge of keratocyte cytoskeletons by the addition of exogenous actin showed a uniform polarity when decorated with myosin subfragment-1, consistent with the fast growing ends of the actin filaments abutting the anterior edge. A steady drop in filament density was observed from the mid- region of the lamellipodium to the perinuclear zone and in images of the more posterior regions of lower filament density many of the actin filaments could be seen to be at least several microns in length. Quantitative analysis of the intensity distribution of fluorescent phalloidin staining across the lamellipodium revealed that the gradient of filament density as well as the absolute content of F-actin was dependent on the fixation method. In cells first fixed and then extracted with Triton, a steep gradient of phalloidin staining was observed from the front to the rear of the lamellipodium. With the protocol required to obtain the electron microscope images, namely Triton extraction followed by fixation, phalloidin staining was, significantly and preferentially reduced in the anterior part of the lamellipodium. This

  20. 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.

  1. Molecular mechanism of Ena/VASP-mediated actin-filament elongation.

    PubMed

    Breitsprecher, Dennis; Kiesewetter, Antje K; Linkner, Joern; Vinzenz, Marlene; Stradal, Theresia E B; Small, John Victor; Curth, Ute; Dickinson, Richard B; Faix, Jan

    2011-02-01

    Ena/VASP proteins are implicated in a variety of fundamental cellular processes including axon guidance and cell migration. In vitro, they enhance elongation of actin filaments, but at rates differing in nearly an order of magnitude according to species, raising questions about the molecular determinants of rate control. Chimeras from fast and slow elongating VASP proteins were generated and their ability to promote actin polymerization and to bind G-actin was assessed. By in vitro TIRF microscopy as well as thermodynamic and kinetic analyses, we show that the velocity of VASP-mediated filament elongation depends on G-actin recruitment by the WASP homology 2 motif. Comparison of the experimentally observed elongation rates with a quantitative mathematical model moreover revealed that Ena/VASP-mediated filament elongation displays a saturation dependence on the actin monomer concentration, implying that Ena/VASP proteins, independent of species, are fully saturated with actin in vivo and generally act as potent filament elongators. Moreover, our data showed that spontaneous addition of monomers does not occur during processive VASP-mediated filament elongation on surfaces, suggesting that most filament formation in cells is actively controlled.

  2. Single turnovers of fluorescent ATP bound to bipolar myosin filament during actin filaments sliding

    PubMed Central

    Maruta, Takahiro; Kobatake, Takahiro; Okubo, Hiroyuki; Chaen, Shigeru

    2013-01-01

    The nucleotide turnover rates of bipolar myosin thick filament along which actin filament slides were measured by the displacement of prebound fluorescent ATP analog 2′(3′)-O-[N-[2-[(Cy3)]amindo]ethyl] carbamoyl]-adenosine 5′ triphosphate (Cy3-EDA-ATP) upon flash photolysis of caged ATP. The fluorescence of the thick filament where actin filament slides decayed with two exponential processes. The slower rate constant was the same as that without actin filament. Along bipolar myosin thick filament, actin filaments slide at a fast speed towards the central bare zone (forward), but more slowly away from the bare zone (backward). The displacement rate constant of fluorescent ATP from the myosin filament where actin filament moved forward was 5.0 s−1, whereas the rate constant where the actin filament slid backward was 1.7 s−1. These findings suggest that the slow ADP release rate is responsible for the slow backward sliding movement. PMID:27493536

  3. Mechanical Heterogeneity Favors Fragmentation of Strained Actin Filaments

    PubMed Central

    De La Cruz, Enrique M.; Martiel, Jean-Louis; Blanchoin, Laurent

    2015-01-01

    We present a general model of actin filament deformation and fragmentation in response to compressive forces. The elastic free energy density along filaments is determined by their shape and mechanical properties, which were modeled in terms of bending, twisting, and twist-bend coupling elasticities. The elastic energy stored in filament deformation (i.e., strain) tilts the fragmentation-annealing reaction free-energy profile to favor fragmentation. The energy gradient introduces a local shear force that accelerates filament intersubunit bond rupture. The severing protein, cofilin, renders filaments more compliant in bending and twisting. As a result, filaments that are partially decorated with cofilin are mechanically heterogeneous (i.e., nonuniform) and display asymmetric shape deformations and energy profiles distinct from mechanically homogenous (i.e., uniform), bare actin, or saturated cofilactin filaments. The local buckling strain depends on the relative size of the compliant segment as well as the bending and twisting rigidities of flanking regions. Filaments with a single bare/cofilin-decorated boundary localize energy and force adjacent to the boundary, within the compliant cofilactin segment. Filaments with small cofilin clusters were predicted to fragment within the compliant cofilactin rather than at boundaries. Neglecting contributions from twist-bend coupling elasticity underestimates the energy density and gradients along filaments, and thus the net effects of filament strain to fragmentation. Spatial confinement causes compliant cofilactin segments and filaments to adopt higher deformation modes and store more elastic energy, thereby promoting fragmentation. The theory and simulations presented here establish a quantitative relationship between actin filament fragmentation thermodynamics and elasticity, and reveal how local discontinuities in filament mechanical properties introduced by regulatory proteins can modulate both the severing efficiency

  4. Golgi-derived vesicles from developing epithelial cells bind actin filaments and possess myosin-I as a cytoplasmically oriented peripheral membrane protein

    PubMed Central

    1993-01-01

    In the intestinal brush border, the mechanoenzyme myosin-I links the microvillus core actin filaments with the plasma membrane. Previous immunolocalization shows that myosin-I is associated with vesicles in mature enterocytes (Drenckhahn, D., and R. Dermietzel. 1988. J. Cell Biol. 107:1037-1048) suggesting a potential role mediating vesicle motility. We now report that myosin-I is associated with Golgi-derived vesicles isolated from cells that are rapidly assembling brush borders in intestinal crypts. Crypt cells were isolated in hyperosmotic buffer, homogenized, and fractionated using differential- and equilibrium- density centrifugation. Fractions containing 50-100-nm vesicles, a similar size to those observed in situ, were identified by EM and were shown to contain myosin-I as demonstrated by immunoblotting and immunolabel negative staining. Galactosyltransferase, a marker enzyme for trans-Golgi membranes was present in these fractions, as was alkaline phosphatase, which is an apical membrane targeted enzyme. Galactosyltransferase was also present in vesicles immuno-purified with antibodies to myosin-I. Villin, a marker for potential contamination from fragmented microvilli, was absent. Myosin-I was found to reside on the vesicle "outer" or cytoplasmic surface for it was accessible to exogenous proteases and intact vesicles could be immunolabeled with myosin-I antibodies in solution. The bound myosin-I could be extracted from the vesicles using NaCl, KI and Na2CO3, suggesting that it is a vesicle peripheral membrane protein. These vesicles were shown to bundle actin filaments in an ATP-dependent manner. These results are consistent with a role for myosin-I as an apically targeted motor for vesicle translocation in epithelial cells. PMID:8416982

  5. Synthetic chondramide A analogues stabilize filamentous actin and block invasion by Toxoplasma gondii.

    PubMed

    Ma, Christopher I; Diraviyam, Karthikeyan; Maier, Martin E; Sept, David; Sibley, L David

    2013-09-27

    Apicomplexan parasites such as Toxoplasma gondii rely on actin-based motility to cross biological barriers and invade host cells. Key structural and biochemical differences in host and parasite actins make this an attractive target for small-molecule inhibitors. Here we took advantage of recent advances in the synthesis of cyclic depsipeptide compounds that stabilize filamentous actin to test the ability of chondramides to disrupt growth of T. gondii in vitro. Structural modeling of chondramide A (2) binding to an actin filament model revealed variations in the binding site between host and parasite actins. A series of 10 previously synthesized analogues (2b-k) with substitutions in the β-tyrosine moiety blocked parasite growth on host cell monolayers with EC₅₀ values that ranged from 0.3 to 1.3 μM. In vitro polymerization assays using highly purified recombinant actin from T. gondii verified that synthetic and natural product chondramides target the actin cytoskeleton. Consistent with this, chondramide treatment blocked parasite invasion into host cells and was more rapidly effective than pyrimethamine, a standard therapeutic agent. Although the current compounds lack specificity for parasite vs host actin, these studies provide a platform for the future design and synthesis of synthetic cyclic peptide inhibitors that selectively disrupt actin dynamics in parasites. PMID:24020843

  6. Synthetic Chondramide A Analogues Stabilize Filamentous Actin and Block Invasion by Toxoplasma gondii

    PubMed Central

    2013-01-01

    Apicomplexan parasites such as Toxoplasma gondii rely on actin-based motility to cross biological barriers and invade host cells. Key structural and biochemical differences in host and parasite actins make this an attractive target for small-molecule inhibitors. Here we took advantage of recent advances in the synthesis of cyclic depsipeptide compounds that stabilize filamentous actin to test the ability of chondramides to disrupt growth of T. gondii in vitro. Structural modeling of chondramide A (2) binding to an actin filament model revealed variations in the binding site between host and parasite actins. A series of 10 previously synthesized analogues (2b–k) with substitutions in the β-tyrosine moiety blocked parasite growth on host cell monolayers with EC50 values that ranged from 0.3 to 1.3 μM. In vitro polymerization assays using highly purified recombinant actin from T. gondii verified that synthetic and natural product chondramides target the actin cytoskeleton. Consistent with this, chondramide treatment blocked parasite invasion into host cells and was more rapidly effective than pyrimethamine, a standard therapeutic agent. Although the current compounds lack specificity for parasite vs host actin, these studies provide a platform for the future design and synthesis of synthetic cyclic peptide inhibitors that selectively disrupt actin dynamics in parasites. PMID:24020843

  7. Demonstration of prominent actin filaments in the root columella.

    PubMed

    Collings, D A; Zsuppan, G; Allen, N S; Blancaflor, E B

    2001-02-01

    The distribution of actin filaments within the gravity-sensing columella cells of plant roots remains poorly understood, with studies over numerous years providing inconsistent descriptions of actin organization in these cells. This uncertainty in actin organization, and thus in actin's role in graviperception and gravisignaling, has led us to investigate actin arrangements in the columella cells of Zea mays L., Medicago truncatula Gaertn., Linum usitatissiilium L. and Nicotianla benthamiana Domin. Actin organization was examined using a combination of optimized immunofluorescence techniques, and an improved fluorochrome-conjugated phalloidin labeling method reliant on 3-maleimidobenzoyl-N-hydroxy-succinimide ester (MBS) cross-linking combined with glycerol permeabilization. Confocal microscopy of root sections labeled with anti-actin antibodies revealed patterns suggestive of actin throughout the columella region. These patterns included short and fragmented actin bundles, fluorescent rings around amyloplasts and intense fluorescence originating from the nucleus. Additionally, confocal microscopy of MBS-stabilized and Alexa Fluor-phalloidin-labeled root sections revealed a previously undetected state of actin organization in the columella. Discrete actin structures surrounded the amyloplasts and prominent actin cables radiated from the nuclear surface toward the cell periphery. Furthermore, the cortex of the columella cells contained fine actin bundles (or single filaments) that had a predominant transverse orientation. We also used confocal microscopy of plant roots expressing endoplasmic reticulum (ER)-targeted green fluorescent protein to demonstrate rapid ER movements within the columella cells, suggesting that the imaged actin network is functional. The successful identification of discrete actin structures in the root columella cells forms the perception and signaling. PMID:11289604

  8. Demonstration of prominent actin filaments in the root columella

    NASA Technical Reports Server (NTRS)

    Collings, D. A.; Zsuppan, G.; Allen, N. S.; Blancaflor, E. B.; Brown, C. S. (Principal Investigator)

    2001-01-01

    The distribution of actin filaments within the gravity-sensing columella cells of plant roots remains poorly understood, with studies over numerous years providing inconsistent descriptions of actin organization in these cells. This uncertainty in actin organization, and thus in actin's role in graviperception and gravisignaling, has led us to investigate actin arrangements in the columella cells of Zea mays L., Medicago truncatula Gaertn., Linum usitatissiilium L. and Nicotianla benthamiana Domin. Actin organization was examined using a combination of optimized immunofluorescence techniques, and an improved fluorochrome-conjugated phalloidin labeling method reliant on 3-maleimidobenzoyl-N-hydroxy-succinimide ester (MBS) cross-linking combined with glycerol permeabilization. Confocal microscopy of root sections labeled with anti-actin antibodies revealed patterns suggestive of actin throughout the columella region. These patterns included short and fragmented actin bundles, fluorescent rings around amyloplasts and intense fluorescence originating from the nucleus. Additionally, confocal microscopy of MBS-stabilized and Alexa Fluor-phalloidin-labeled root sections revealed a previously undetected state of actin organization in the columella. Discrete actin structures surrounded the amyloplasts and prominent actin cables radiated from the nuclear surface toward the cell periphery. Furthermore, the cortex of the columella cells contained fine actin bundles (or single filaments) that had a predominant transverse orientation. We also used confocal microscopy of plant roots expressing endoplasmic reticulum (ER)-targeted green fluorescent protein to demonstrate rapid ER movements within the columella cells, suggesting that the imaged actin network is functional. The successful identification of discrete actin structures in the root columella cells forms the perception and signaling.

  9. A small molecule inhibitor of tropomyosin dissociates actin binding from tropomyosin-directed regulation of actin dynamics

    PubMed Central

    Bonello, Teresa T.; Janco, Miro; Hook, Jeff; Byun, Alex; Appaduray, Mark; Dedova, Irina; Hitchcock-DeGregori, Sarah; Hardeman, Edna C.; Stehn, Justine R.; Böcking, Till; Gunning, Peter W.

    2016-01-01

    The tropomyosin family of proteins form end-to-end polymers along the actin filament. Tumour cells rely on specific tropomyosin-containing actin filament populations for growth and survival. To dissect out the role of tropomyosin in actin filament regulation we use the small molecule TR100 directed against the C terminus of the tropomyosin isoform Tpm3.1. TR100 nullifies the effect of Tpm3.1 on actin depolymerisation but surprisingly Tpm3.1 retains the capacity to bind F-actin in a cooperative manner. In vivo analysis also confirms that, in the presence of TR100, fluorescently tagged Tpm3.1 recovers normally into stress fibers. Assembling end-to-end along the actin filament is thereby not sufficient for tropomyosin to fulfil its function. Rather, regulation of F-actin stability by tropomyosin requires fidelity of information communicated at the barbed end of the actin filament. This distinction has significant implications for perturbing tropomyosin-dependent actin filament function in the context of anti-cancer drug development. PMID:26804624

  10. Septins promote F-actin ring formation by crosslinking actin filaments into curved bundles.

    PubMed

    Mavrakis, Manos; Azou-Gros, Yannick; Tsai, Feng-Ching; Alvarado, José; Bertin, Aurélie; Iv, Francois; Kress, Alla; Brasselet, Sophie; Koenderink, Gijsje H; Lecuit, Thomas

    2014-04-01

    Animal cell cytokinesis requires a contractile ring of crosslinked actin filaments and myosin motors. How contractile rings form and are stabilized in dividing cells remains unclear. We address this problem by focusing on septins, highly conserved proteins in eukaryotes whose precise contribution to cytokinesis remains elusive. We use the cleavage of the Drosophila melanogaster embryo as a model system, where contractile actin rings drive constriction of invaginating membranes to produce an epithelium in a manner akin to cell division. In vivo functional studies show that septins are required for generating curved and tightly packed actin filament networks. In vitro reconstitution assays show that septins alone bundle actin filaments into rings, accounting for the defects in actin ring formation in septin mutants. The bundling and bending activities are conserved for human septins, and highlight unique functions of septins in the organization of contractile actomyosin rings.

  11. Actin filaments elongate from their membrane-associated ends

    PubMed Central

    Tilney, LG; Bonder, EM; DeRosier, DJ

    1981-01-01

    In limulus sperm an actin filament bundle 55 mum in length extends from the acrosomal vacuole membrane through a canal in the nucleus and then coils in a regular fashion around the base of the nucleus. The bundle expands systematically from 15 filaments near the acrosomal vacuole to 85 filaments at the basal end. Thin sections of sperm fixed during stages in spermatid maturation reveal that the filament bundle begins to assemble on dense material attached to the acrosomal vacuole membrane. In micrographs fo these early stages in maturation, short bundles are seen extending posteriorly from the dense material. The significance is that these short, developing bundles have about 85 filaments, suggesting that the 85-filament end of the bundle is assembled first. By using filament bundles isolated and incubated in vitro with G actin from muscle, we can determine the end “preferred” for addition of actin monomers during polymerization. The end that would be associated with the acrosomal vacuole membrane, a membrane destined to be continuous with the plasma membrane, is preferred about 10 times over the other, thicker end. Decoration of the newly polymerized portions of the filament bundle with subfragment 1 of myosin reveals that the arrowheads point away from the acrosomal vacuole membrane, as is true of other actin filament bundles attached to membranes. From these observations we conclude that the bundle is nucleated from the dense material associated with the acrosomal vacuole and that monomers are added to the membrane-associated end. As monomers are added at the dense material, the thick first-made end of the filament bundle is pushed down through the nucleus where, upon reaching the base of the nucleus, it coils up. Tapering is brought about by the capping of the peripheral filaments in the bundle. PMID:7197276

  12. The impact of tropomyosins on actin filament assembly is isoform specific.

    PubMed

    Janco, Miro; Bonello, Teresa T; Byun, Alex; Coster, Adelle C F; Lebhar, Helene; Dedova, Irina; Gunning, Peter W; Böcking, Till

    2016-07-01

    Tropomyosin (Tpm) is an α helical coiled-coil dimer that forms a co-polymer along the actin filament. Tpm is involved in the regulation of actin's interaction with binding proteins as well as stabilization of the actin filament and its assembly kinetics. Recent studies show that multiple Tpm isoforms also define the functional properties of distinct actin filament populations within a cell. Subtle structural variations within well conserved Tpm isoforms are the key to their functional specificity. Therefore, we purified and characterized a comprehensive set of 8 Tpm isoforms (Tpm1.1, Tpm1.12, Tpm1.6, Tpm1.7, Tpm1.8, Tpm2.1, Tpm3.1, and Tpm4.2), using well-established actin co-sedimentation and pyrene fluorescence polymerization assays. We observed that the apparent affinity (Kd(app)) to filamentous actin varied in all Tpm isoforms between ∼0.1-5 μM with similar values for both, skeletal and cytoskeletal actin filaments. The data did not indicate any correlation between affinity and size of Tpm molecules, however high molecular weight (HMW) isoforms Tpm1.1, Tpm1.6, Tpm1.7 and Tpm2.1, showed ∼3-fold higher cooperativity compared to low molecular weight (LMW) isoforms Tpm1.12, Tpm1.8, Tpm3.1, and Tpm4.2. The rate of actin filament elongation in the presence of Tpm2.1 increased, while all other isoforms decreased the elongation rate by 27-85 %. Our study shows that the biochemical properties of Tpm isoforms are finely tuned and depend on sequence variations in alternatively spliced regions of Tpm molecules.

  13. The impact of tropomyosins on actin filament assembly is isoform specific.

    PubMed

    Janco, Miro; Bonello, Teresa T; Byun, Alex; Coster, Adelle C F; Lebhar, Helene; Dedova, Irina; Gunning, Peter W; Böcking, Till

    2016-07-01

    Tropomyosin (Tpm) is an α helical coiled-coil dimer that forms a co-polymer along the actin filament. Tpm is involved in the regulation of actin's interaction with binding proteins as well as stabilization of the actin filament and its assembly kinetics. Recent studies show that multiple Tpm isoforms also define the functional properties of distinct actin filament populations within a cell. Subtle structural variations within well conserved Tpm isoforms are the key to their functional specificity. Therefore, we purified and characterized a comprehensive set of 8 Tpm isoforms (Tpm1.1, Tpm1.12, Tpm1.6, Tpm1.7, Tpm1.8, Tpm2.1, Tpm3.1, and Tpm4.2), using well-established actin co-sedimentation and pyrene fluorescence polymerization assays. We observed that the apparent affinity (Kd(app)) to filamentous actin varied in all Tpm isoforms between ∼0.1-5 μM with similar values for both, skeletal and cytoskeletal actin filaments. The data did not indicate any correlation between affinity and size of Tpm molecules, however high molecular weight (HMW) isoforms Tpm1.1, Tpm1.6, Tpm1.7 and Tpm2.1, showed ∼3-fold higher cooperativity compared to low molecular weight (LMW) isoforms Tpm1.12, Tpm1.8, Tpm3.1, and Tpm4.2. The rate of actin filament elongation in the presence of Tpm2.1 increased, while all other isoforms decreased the elongation rate by 27-85 %. Our study shows that the biochemical properties of Tpm isoforms are finely tuned and depend on sequence variations in alternatively spliced regions of Tpm molecules. PMID:27420374

  14. Interactions of actin, myosin, and an actin-binding protein of chronic myelogenous leukemia leukocytes.

    PubMed Central

    Boxer, L A; Stossel, T P

    1976-01-01

    Actin, myosin, and a high molecular weight actin-binding protein were purified from chronic myelogenous leukemia (CML) leukocytes. CML leukocyte actin resembled skeletal muscle and other cytoplasmic actins by its subunit molecular weight, by its ability to polymerize in the presence of salts, and to activate the Mg2+-ATPase activity of rabbit skeletal muscle myosin. CML leukocyte myosin was similar to other vertebrate cytoplasmic myosins in having heavy chains and two light subunits. However, its apparent heavy-chain molecular weight and Stokes radius suggested that it was variably degraded during purification. Purified CML leukocyte myosin had average specific EDTA- AND Ca2+-activated ATPase activities of 125 and 151 nmol Pi released/mg protein per min, respectively and low specific Mg2+-ATPase activity. The Mg2+-ATPase activity of CML myosin was increased 200-fold by rabbit skeletal muscle F-actin, but the specific activity relative to that of actin-activated rabbit skeletal muscle myosin was low. CML leukocyte myosin, like other vertebrate cytoplasmic myosins, formed filaments in 0.1 M KCl solutions. Reduced and denatured CML leukocyte-actin-binding protein had a single high molecular weight subunit like a recently described actin-binding protein of rabbit pulmonary macrophages which promotes the polymerization and gelation of actin. Cytoplasmic extracts of CML leukocytes prepared with ice-cold 0.34-M sucrose solutions containing Mg2+-ATP, dithiothreitol, and EDTA at pH 7.0 underwent rapid gelation when warmed to 25 degrees C. Initially, the gel could be liquified by cooling to ice-bath temperature. With time, warmed cytoplasmic extract gels shrunk ("contracted") into aggregates. The following findings indicated that CML leukocyte actin-binding protein promoted the temperature-dependent gelation of actin in the cytoplasmic extracts and that CML leukocyte myosin was involved in the contraction of the actin gels: (a) Cytoplasmic extract gels initially contained

  15. The role of formin tails in actin nucleation, processive elongation, and filament bundling.

    PubMed

    Vizcarra, Christina L; Bor, Batbileg; Quinlan, Margot E

    2014-10-31

    Formins are multidomain proteins that assemble actin in a wide variety of biological processes. They both nucleate and remain processively associated with growing filaments, in some cases accelerating filament growth. The well conserved formin homology 1 and 2 domains were originally thought to be solely responsible for these activities. Recently a role in nucleation was identified for the Diaphanous autoinhibitory domain (DAD), which is C-terminal to the formin homology 2 domain. The C-terminal tail of the Drosophila formin Cappuccino (Capu) is conserved among FMN formins but distinct from other formins. It does not have a DAD domain. Nevertheless, we find that Capu-tail plays a role in filament nucleation similar to that described for mDia1 and other formins. Building on this, replacement of Capu-tail with DADs from other formins tunes nucleation activity. Capu-tail has low-affinity interactions with both actin monomers and filaments. Removal of the tail reduces actin filament binding and bundling. Furthermore, when the tail is removed, we find that processivity is compromised. Despite decreased processivity, the elongation rate of filaments is unchanged. Again, replacement of Capu-tail with DADs from other formins tunes the processive association with the barbed end, indicating that this is a general role for formin tails. Our data show a role for the Capu-tail domain in assembling the actin cytoskeleton, largely mediated by electrostatic interactions. Because of its multifunctionality, the formin tail is a candidate for regulation by other proteins during cytoskeletal rearrangements.

  16. Involvement of actin filaments in rhizoid morphogenesis of Spirogyra.

    PubMed

    Yoshida, Katsuhisa; Shimmen, Teruo

    2009-01-01

    The role of actin filaments in rhizoid morphogenesis was studied in Spirogyra. When the algal filaments were severed, new terminal cells started tip growth and finally formed rhizoids. Actin inhibitors, latrunculin B and cytochalasin D, reversibly inhibited the process. A mesh-like structure of actin filaments (AFs) was formed at the tip region. Gd(3+) inhibited tip growth and decreased AFs in the tip region. Either a decrease in turgor pressure or lowering of the external Ca(2+) concentration also induced similar results. It was suggested that the mesh-like AF structure is indispensable for the elongation of rhizoids. A possible organization mechanism of the mesh-like AF structure was discussed.

  17. Septin 9 Exhibits Polymorphic Binding to F-Actin and Inhibits Myosin and Cofilin Activity.

    PubMed

    Smith, Clayton; Dolat, Lee; Angelis, Dimitrios; Forgacs, Eva; Spiliotis, Elias T; Galkin, Vitold E

    2015-10-01

    Septins are a highly conserved family of proteins in eukaryotes that is recognized as a novel component of the cytoskeleton. Septin 9 (SEPT9) interacts directly with actin filaments and functions as an actin stress fiber cross-linking protein that promotes the maturation of nascent focal adhesions and cell migration. However, the molecular details of how SEPT9 interacts with F-actin remain unknown. Here, we use electron microscopy and image analysis to show that SEPT9 binds to F-actin in a highly polymorphic fashion. We demonstrate that the basic domain (B-domain) of the N-terminal tail of SEPT9 is responsible for actin cross-linking, while the GTP-binding domain (G-domain) does not bundle F-actin. We show that the B-domain of SEPT9 binds to three sites on F-actin, and the two of these sites overlap with the binding regions of myosin and cofilin. SEPT9 inhibits actin-dependent ATPase activity of myosin and competes with the weakly bound state of myosin for binding to F-actin. At the same time, SEPT9 significantly reduces the extent of F-actin depolymerization by cofilin. Taken together, these data suggest that SEPT9 protects actin filaments from depolymerization by cofilin and myosin and indicate a mechanism by which SEPT9 could maintain the integrity of growing and contracting actin filaments.

  18. The Switch-associated Protein 70 (SWAP-70) Bundles Actin Filaments and Contributes to the Regulation of F-actin Dynamics*

    PubMed Central

    Chacón-Martínez, Carlos Andrés; Kiessling, Nadine; Winterhoff, Moritz; Faix, Jan; Müller-Reichert, Thomas; Jessberger, Rolf

    2013-01-01

    Coordinated assembly and disassembly of actin into filaments and higher order structures such as stress fibers and lamellipodia are fundamental for cell migration and adhesion. However, the precise spatiotemporal regulation of F-actin structures is not completely understood. SWAP-70, a phosphatidylinositol 3,4,5-trisphosphate-interacting, F-actin-binding protein, participates in actin rearrangements through yet unknown mechanisms. Here, we show that SWAP-70 is an F-actin-bundling protein that oligomerizes through a Gln/Glu-rich stretch within a coiled-coil region. SWAP-70 bundles filaments in parallel and anti-parallel fashion through its C-terminal F-actin binding domain and delays dilution-induced F-actin depolymerization. We further demonstrate that SWAP-70 co-localizes and directly interacts with cofilin, an F-actin severing and depolymerization factor, and contributes to the regulation of cofilin activity in vivo. In line with these activities, upon stem cell factor stimulation, murine bone marrow-derived mast cells lacking SWAP-70 display aberrant regulation of F-actin and actin free barbed ends dynamics. Moreover, proper stem cell factor-dependent cofilin activation via dephosphorylation and subcellular redistribution into a detergent-resistant cytoskeletal compartment also require SWAP-70. Together, these findings reveal an important role of SWAP-70 in the dynamic spatiotemporal regulation of F-actin networks. PMID:23921380

  19. How actin binds and assembles onto plasma membranes from Dictyostelium discoideum

    PubMed Central

    1988-01-01

    We have shown previously (Schwartz, M. A., and E. J. Luna. 1986. J. Cell Biol. 102: 2067-2075) that actin binds with positive cooperativity to plasma membranes from Dictyostelium discoideum. Actin is polymerized at the membrane surface even at concentrations well below the critical concentration for polymerization in solution. Low salt buffer that blocks actin polymerization in solution also prevents actin binding to membranes. To further explore the relationship between actin polymerization and binding to membranes, we prepared four chemically modified actins that appear to be incapable of polymerizing in solution. Three of these derivatives also lost their ability to bind to membranes. The fourth derivative (EF actin), in which histidine-40 is labeled with ethoxyformic anhydride, binds to membranes with reduced affinity. Binding curves exhibit positive cooperativity, and cross- linking experiments show that membrane-bound actin is multimeric. Thus, binding and polymerization are tightly coupled, and the ability of these membranes to polymerize actin is dramatically demonstrated. EF actin coassembles weakly with untreated actin in solution, but coassembles well on membranes. Binding by untreated actin and EF actin are mutually competitive, indicating that they bind to the same membrane sites. Hill plots indicate that an actin trimer is the minimum assembly state required for tight binding to membranes. The best explanation for our data is a model in which actin oligomers assemble by binding to clustered membrane sites with successive monomers on one side of the actin filament bound to the membrane. Individual binding affinities are expected to be low, but the overall actin-membrane avidity is high, due to multivalency. Our results imply that extracellular factors that cluster membrane proteins may create sites for the formation of actin nuclei and thus trigger actin polymerization in the cell. PMID:3392099

  20. Structural Modeling and Molecular Dynamics Simulation of the Actin Filament

    SciTech Connect

    Splettstoesser, Thomas; Holmes, Kenneth; Noe, Frank; Smith, Jeremy C

    2011-01-01

    Actin is a major structural protein of the eukaryotic cytoskeleton and enables cell motility. Here, we present a model of the actin filament (F-actin) that not only incorporates the global structure of the recently published model by Oda et al. but also conserves internal stereochemistry. A comparison is made using molecular dynamics simulation of the model with other recent F-actin models. A number of structural determents such as the protomer propeller angle, the number of hydrogen bonds, and the structural variation among the protomers are analyzed. The MD comparison is found to reflect the evolution in quality of actin models over the last 6 years. In addition, simulations of the model are carried out in states with both ADP or ATP bound and local hydrogen-bonding differences characterized.

  1. Echinococcus granulosus: Cloning and Functional in Vitro Characterization of an Actin Filament Fragmenting Protein.

    PubMed

    Cortez-Herrera, E; Yamamoto, R R; Rodrigues, J J; Farias, S E; Ferreira, H B; Zaha, A

    2001-04-01

    We report the isolation and characterization of an Echinococcus granulosus gene that codes for a protein with actin filament fragmenting and nucleating activities (EgAFFP). The genomic region corresponding to the EgAFFP gene presents a coding sequence of 1110 bp that is interrupted by eight introns. The EgAFFP deduced amino acid sequence is about 40% homologous to those of several members of the gelsolin family, such as Physarum polycephalum fragmin, Dictyostelium discoideum severin, and Lumbricus terrestris actin modulator. As do other proteins of the same family, EgAFFP presents three repeated domains, each one characterized by internal conserved amino acid motifs. Assays with fluorescence-labeled actin showed that the full-length recombinant EgAFFP effectively binds actin monomers in both a calcium-dependent and calcium-independent manner and also presents actin nucleating and severing activities.

  2. A second Las17 monomeric actin-binding motif functions in Arp2/3-dependent actin polymerization during endocytosis.

    PubMed

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

    2015-04-01

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

  3. Concentration profiles of actin-binding molecules in lamellipodia

    NASA Astrophysics Data System (ADS)

    Falcke, Martin

    2016-04-01

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

  4. The "Le Chatelier's principle"-governed response of actin filaments to osmotic stress.

    PubMed

    Ito, Tadanao; Yamazaki, Masahito

    2006-07-13

    Actin filaments inhibit osmotic stress-driven water flow across a semipermeable membrane in proportion to the filament concentration (Ito, T.; Zaner, K. S.; Stossel, T. P. Biophys. J. 1987, 51, 745). When the filaments are cross-linked by F-actin binding protein, filamin A, this flow is stopped completely (Ito, T.; Suzuki, A.; Stossel, T. P. Biophys. J. 1992, 61, 1301). No conventional theory accurately accounts for these results. Here, this response is analyzed by formulating the entropy of the system under osmotic stress. Results demonstrate that the response of the actin filaments to osmotic stress is governed by the Le Chatelier's principle, which states that an external interaction that disturbs the equilibrium brings about processes in the body that tend to reduce the effects of this interaction. In the present case, disrupting equilibrium by osmotic stress brings about a reaction that decreases the chemical potential of water in the F-actin solution, reducing the effect of the applied osmotic disturbance. This decrease in the chemical potential of the water in the F-actin solution is caused by an increase in the chemical potential of F-actin, which is induced by isothermal absorption of heat by F-actin aided by work done by osmotic stress. As a result, F-actin has an inhibitory effect on the osmotic stress-driven water flow, and can even completely stop the flow when it is cross-linked. This is the first report demonstrating that the Le Chatelier's principle applies to the reaction of biopolymers against equilibrium disturbances such as osmotic stress. PMID:16821884

  5. Actin Filaments Regulate Exocytosis at the Hair Cell Ribbon Synapse.

    PubMed

    Guillet, Marie; Sendin, Gaston; Bourien, Jérôme; Puel, Jean-Luc; Nouvian, Régis

    2016-01-20

    Exocytosis at the inner hair cell ribbon synapse is achieved through the functional coupling between calcium channels and glutamate-filled synaptic vesicles. Using membrane capacitance measurements, we investigated whether the actin network regulates the exocytosis of synaptic vesicles at the mouse auditory hair cell. Our results suggest that actin network disruption increases exocytosis and that actin filaments may spatially organize a subfraction of synaptic vesicles with respect to the calcium channels. Significance statement: Inner hair cells (IHCs), the auditory sensory cells of the cochlea, release glutamate onto the afferent auditory nerve fibers to encode sound stimulation. To achieve this task, the IHC relies on the recruitment of glutamate-filled vesicles that can be located in close vicinity to the calcium channels or more remotely from them. The molecular determinants responsible for organizing these vesicle pools are not fully identified. Using pharmacological tools in combination with membrane capacitance measurements, we show that actin filament disruption increases exocytosis in IHCs and that actin filaments most likely position a fraction of vesicles away from the calcium channels. PMID:26791198

  6. Utilization of paramagnetic relaxation enhancements for structural analysis of actin-binding proteins in complex with actin

    PubMed Central

    Huang, Shuxian; Umemoto, Ryo; Tamura, Yuki; Kofuku, Yutaka; Uyeda, Taro Q. P.; Nishida, Noritaka; Shimada, Ichio

    2016-01-01

    Actin cytoskeleton dynamics are controlled by various actin binding proteins (ABPs) that modulate the polymerization of the monomeric G-actin and the depolymerization of filamentous F-actin. Although revealing the structures of the actin/ABP complexes is crucial to understand how the ABPs regulate actin dynamics, the X-ray crystallography and cryoEM methods are inadequate to apply for the ABPs that interact with G- or F-actin with lower affinity or multiple binding modes. In this study, we aimed to establish the alternative method to build a structural model of G-actin/ABP complexes, utilizing the paramagnetic relaxation enhancement (PRE) experiments. Thymosin β4 (Tβ4) was used as a test case for validation, since its structure in complex with G-actin was reported recently. Recombinantly expressed G-actin, containing a cysteine mutation, was conjugated with a nitroxyl spin label at the specific site. Based on the intensity ratio of the 1H-15N HSQC spectra of Tβ4 in the complex with G-actin in the paramagnetic and diamagnetic states, the distances between the amide groups of Tβ4 and the spin label of G-actin were estimated. Using the PRE-derived distance constraints, we were able to compute a well-converged docking structure of the G-actin/Tβ4 complex that shows great accordance with the reference structure. PMID:27654858

  7. 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

  8. 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. PMID:27414648

  9. Actin filaments target the oligomeric maturation of the dynamin GTPase Drp1 to mitochondrial fission sites.

    PubMed

    Ji, Wei-ke; Hatch, Anna L; Merrill, Ronald A; Strack, Stefan; Higgs, Henry N

    2015-11-26

    While the dynamin GTPase Drp1 plays a critical role during mitochondrial fission, mechanisms controlling its recruitment to fission sites are unclear. A current assumption is that cytosolic Drp1 is recruited directly to fission sites immediately prior to fission. Using live-cell microscopy, we find evidence for a different model, progressive maturation of Drp1 oligomers on mitochondria through incorporation of smaller mitochondrially-bound Drp1 units. Maturation of a stable Drp1 oligomer does not forcibly lead to fission. Drp1 oligomers also translocate directionally along mitochondria. Ionomycin, a calcium ionophore, causes rapid mitochondrial accumulation of actin filaments followed by Drp1 accumulation at the fission site, and increases fission rate. Inhibiting actin polymerization, myosin IIA, or the formin INF2 reduces both un-stimulated and ionomycin-induced Drp1 accumulation and mitochondrial fission. Actin filaments bind purified Drp1 and increase GTPase activity in a manner that is synergistic with the mitochondrial protein Mff, suggesting a role for direct Drp1/actin interaction. We propose that Drp1 is in dynamic equilibrium on mitochondria in a fission-independent manner, and that fission factors such as actin filaments target productive oligomerization to fission sites.

  10. Actin filaments target the oligomeric maturation of the dynamin GTPase Drp1 to mitochondrial fission sites

    PubMed Central

    Ji, Wei-ke; Hatch, Anna L; Merrill, Ronald A; Strack, Stefan; Higgs, Henry N

    2015-01-01

    While the dynamin GTPase Drp1 plays a critical role during mitochondrial fission, mechanisms controlling its recruitment to fission sites are unclear. A current assumption is that cytosolic Drp1 is recruited directly to fission sites immediately prior to fission. Using live-cell microscopy, we find evidence for a different model, progressive maturation of Drp1 oligomers on mitochondria through incorporation of smaller mitochondrially-bound Drp1 units. Maturation of a stable Drp1 oligomer does not forcibly lead to fission. Drp1 oligomers also translocate directionally along mitochondria. Ionomycin, a calcium ionophore, causes rapid mitochondrial accumulation of actin filaments followed by Drp1 accumulation at the fission site, and increases fission rate. Inhibiting actin polymerization, myosin IIA, or the formin INF2 reduces both un-stimulated and ionomycin-induced Drp1 accumulation and mitochondrial fission. Actin filaments bind purified Drp1 and increase GTPase activity in a manner that is synergistic with the mitochondrial protein Mff, suggesting a role for direct Drp1/actin interaction. We propose that Drp1 is in dynamic equilibrium on mitochondria in a fission-independent manner, and that fission factors such as actin filaments target productive oligomerization to fission sites. DOI: http://dx.doi.org/10.7554/eLife.11553.001 PMID:26609810

  11. Multiscale impact of nucleotides and cations on the conformational equilibrium, elasticity and rheology of actin filaments and crosslinked networks.

    PubMed

    Bidone, Tamara Carla; Kim, Taeyoon; Deriu, Marco A; Morbiducci, Umberto; Kamm, Roger D

    2015-10-01

    Cells are able to respond to mechanical forces and deformations. The actin cytoskeleton, a highly dynamic scaffolding structure, plays an important role in cell mechano-sensing. Thus, understanding rheological behaviors of the actin cytoskeleton is critical for delineating mechanical behaviors of cells. The actin cytoskeleton consists of interconnected actin filaments (F-actin) that form via self-assembly of actin monomers. It has been shown that molecular changes of the monomer subunits impact the rigidity of F-actin. However, it remains inconclusive whether or not the molecular changes can propagate to the network level and thus alter the rheological properties of actin networks. Here, we focus on how cation binding and nucleotide state tune the molecular conformation and rigidity of F-actin and a representative rheological behavior of actin networks, strain-stiffening. We employ a multiscale approach by combining established computational techniques: molecular dynamics, normal mode analysis and Brownian dynamics. Our findings indicate that different combinations of nucleotide (ATP, ADP or ADP-Pi) and cation [Formula: see text] or [Formula: see text] at one or multiple sites) binding change the molecular conformation of F-actin by varying inter- and intra-strand interactions which bridge adjacent subunits between and within F-actin helical strands. This is reflected in the rigidity of actin filaments against bending and stretching. We found that differences in extension and bending rigidity of F-actin induced by cation binding to the low-, intermediate- and high-affinity sites vary the strain-stiffening response of actin networks crosslinked by rigid crosslinkers, such as scruin, whereas they minimally impact the strain-stiffening response when compliant crosslinkers, such as filamin A or [Formula: see text]-actinin, are used.

  12. Multiscale impact of nucleotides and cations on the conformational equilibrium, elasticity and rheology of actin filaments and crosslinked networks.

    PubMed

    Bidone, Tamara Carla; Kim, Taeyoon; Deriu, Marco A; Morbiducci, Umberto; Kamm, Roger D

    2015-10-01

    Cells are able to respond to mechanical forces and deformations. The actin cytoskeleton, a highly dynamic scaffolding structure, plays an important role in cell mechano-sensing. Thus, understanding rheological behaviors of the actin cytoskeleton is critical for delineating mechanical behaviors of cells. The actin cytoskeleton consists of interconnected actin filaments (F-actin) that form via self-assembly of actin monomers. It has been shown that molecular changes of the monomer subunits impact the rigidity of F-actin. However, it remains inconclusive whether or not the molecular changes can propagate to the network level and thus alter the rheological properties of actin networks. Here, we focus on how cation binding and nucleotide state tune the molecular conformation and rigidity of F-actin and a representative rheological behavior of actin networks, strain-stiffening. We employ a multiscale approach by combining established computational techniques: molecular dynamics, normal mode analysis and Brownian dynamics. Our findings indicate that different combinations of nucleotide (ATP, ADP or ADP-Pi) and cation [Formula: see text] or [Formula: see text] at one or multiple sites) binding change the molecular conformation of F-actin by varying inter- and intra-strand interactions which bridge adjacent subunits between and within F-actin helical strands. This is reflected in the rigidity of actin filaments against bending and stretching. We found that differences in extension and bending rigidity of F-actin induced by cation binding to the low-, intermediate- and high-affinity sites vary the strain-stiffening response of actin networks crosslinked by rigid crosslinkers, such as scruin, whereas they minimally impact the strain-stiffening response when compliant crosslinkers, such as filamin A or [Formula: see text]-actinin, are used. PMID:25708806

  13. Moesin, ezrin, and p205 are actin-binding proteins associated with neutrophil plasma membranes.

    PubMed Central

    Pestonjamasp, K; Amieva, M R; Strassel, C P; Nauseef, W M; Furthmayr, H; Luna, E J

    1995-01-01

    Actin-binding proteins in bovine neutrophil plasma membranes were identified using blot overlays with 125I-labeled F-actin. Along with surface-biotinylated proteins, membranes were enriched in major actin-binding polypeptides of 78, 81, and 205 kDa. Binding was specific for F-actin because G-actin did not bind. Further, unlabeled F-actin blocked the binding of 125I-labeled F-actin whereas other acidic biopolymers were relatively ineffective. Binding also was specifically inhibited by myosin subfragment 1, but not by CapZ or plasma gelsolin, suggesting that the membrane proteins, like myosin, bind along the sides of the actin filaments. The 78- and 81-kDa polypeptides were identified as moesin and ezrin, respectively, by co-migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoprecipitation with antibodies specific for moesin and ezrin. Although not present in detectable amounts in bovine neutrophils, radixin (a third and closely related member of this gene family) also bound 125I-labeled F-actin on blot overlays. Experiments with full-length and truncated bacterial fusion proteins localized the actin-binding site in moesin to the extreme carboxy terminus, a highly conserved sequence. Immunofluorescence micrographs of permeabilized cells and cell "footprints" showed moesin co-localization with actin at the cytoplasmic surface of the plasma membrane, consistent with a role as a membrane-actin-linking protein. Images PMID:7612961

  14. Actin filaments on myosin beds: The velocity distribution

    NASA Astrophysics Data System (ADS)

    Bourdieu, L.; Magnasco, M. O.; Winkelmann, D. A.; Libchaber, A.

    1995-12-01

    In vitro studies of actin filaments sliding on a myosin-coated surface are analyzed, filament by filament, at a sampling rate of 30 per second. For each filament, the mean arc length coordinate is computed and histograms of instantaneous velocities, along the arc length, are established. Two types of motion are observed, depending on the experimental conditions. The first one is characterized by a homogeneous flow, with well defined velocities. In this regime, specific defects are a constitutive part of the flow. It is observed at high temperature, at high myosin coverage, and with a particular mode of attachment of myosin to the surface. The second regime shows no clear velocity selection, but a broadband distribution. It is characterized by high friction and is observed at low temperature or low myosin density. (c) 1995 The American Physical Society

  15. Equilibrium muscle cross-bridge behavior. Theoretical considerations. II. Model describing the behavior of strongly-binding cross-bridges when both heads of myosin bind to the actin filament.

    PubMed Central

    Schoenberg, M

    1991-01-01

    A model has been developed for characterizing the interaction between strongly-binding myosin cross-bridges and actin in muscle fibers under equilibrium conditions where both heads of the myosin cross-bridge bind to actin. The model, that of Anderson and Schoenberg (1987. Biophys. J. 52:1077-1082) is quite similar to that of Schoenberg (1985. Biophys. J. 48:467-475), except that explicit account is taken of the fact that each crossbridge has two heads which can bind to actin. The key assumption that allows this model to explain a large body of data unexplained by the Schoenberg (1985) model is that the two crossbridge heads are not totally independent of one another after attachment. After the first head attaches, the second head is then free to attach only to an actin site distal to the first head. This means that when the more distally attached head subsequently detaches and reattaches (as the heads continually do), it will not reattach in a position of lesser strain and reduce the force it supports, but instead will remain attached in its strained position until the proximally attached head also detaches. This model gives an explanation for two important and otherwise unexplained observations made previously: it explains why at ionic strengths in the range of 50-120 mM, (a) the rate constant of force decay after a small stretch is a sigmoidal function of nucleotide analogue concentration, and (b) why in the presence of analogues or in rigor the rate constant of force decay after a small stretch is significantly slower than the rate constant for myosin subfragment-1 detachment from actin in solution. PMID:1932554

  16. Myosin-induced volume increase of the hyper-mobile water surrounding actin filaments.

    PubMed

    Suzuki, Makoto; Kabir, Syed Rashel; Siddique, Md Shahjahan Parvez; Nazia, Umme Salma; Miyazaki, Takashi; Kodama, Takao

    2004-09-10

    Microwave dielectric spectroscopy can measure the rotational mobility of water molecules that hydrate proteins and the hydration-shell volume. Using this technique, we have recently shown that apart from typical hydrating water molecules with lowered mobility there are other water molecules around the actin filaments (F-actin) which have a much higher mobility than that of bulk water [Biophys. J. 85 (2003) 3154]. We report here that the volume of this water component (hyper-mobile water) markedly increases without significant change of the volume of the ordinary hydration shell when the myosin motor-domain (S1, myosin subfragment-1) binds to F-actin. No hyper-mobile component was found in the hydration shell of S1 itself. The present results strongly suggest that the solvent space around S1 bound to F-actin is diffusionally asymmetric, which supports our model of force generation by actomyosin proposed previously [op. cit.]. PMID:15313212

  17. Real-Time Measurements of Actin Filament Polymerization by Total Internal Reflection Fluorescence Microscopy

    PubMed Central

    Kuhn, Jeffrey R.; Pollard, Thomas D.

    2005-01-01

    Understanding the mechanism of actin polymerization and its regulation by associated proteins requires an assay to monitor polymerization dynamics and filament topology simultaneously. The only assay meeting these criteria is total internal reflection fluorescence microscopy (Amann and Pollard, 2001; Fujiwara et al., 2002). The fluorescence signal is fourfold stronger with actin labeled on Cys-374 with Oregon green rather than rhodamine. To distinguish growth at barbed and pointed ends we used image drift correction and maximum intensity projections to reveal points where single N-ethylmaleimide inactivated myosins attach filaments to the glass coverslip. We estimated association rates at high actin concentrations and dissociation rates near and below the critical actin concentration. At the barbed end, the association rate constant for Mg-ATP-actin is 7.4 μM−1 s−1 and the dissociation rate constant is 0.89 s−1. At the pointed end the association and dissociation rate constants are 0.56 μM−1 s−1 and 0.19 s−1. When vitamin D binding protein sequesters all free monomers, ADP-actin dissociates from barbed ends at 1.4 s−1 and from pointed ends at 0.16 s−1 regardless of buffer nucleotide. PMID:15556992

  18. Drebrin-like protein DBN-1 is a sarcomere component that stabilizes actin filaments during muscle contraction.

    PubMed

    Butkevich, Eugenia; Bodensiek, Kai; Fakhri, Nikta; von Roden, Kerstin; Schaap, Iwan A T; Majoul, Irina; Schmidt, Christoph F; Klopfenstein, Dieter R

    2015-07-06

    Actin filament organization and stability in the sarcomeres of muscle cells are critical for force generation. Here we identify and functionally characterize a Caenorhabditis elegans drebrin-like protein DBN-1 as a novel constituent of the muscle contraction machinery. In vitro, DBN-1 exhibits actin filament binding and bundling activity. In vivo, DBN-1 is expressed in body wall muscles of C. elegans. During the muscle contraction cycle, DBN-1 alternates location between myosin- and actin-rich regions of the sarcomere. In contracted muscle, DBN-1 is accumulated at I-bands where it likely regulates proper spacing of α-actinin and tropomyosin and protects actin filaments from the interaction with ADF/cofilin. DBN-1 loss of function results in the partial depolymerization of F-actin during muscle contraction. Taken together, our data show that DBN-1 organizes the muscle contractile apparatus maintaining the spatial relationship between actin-binding proteins such as α-actinin, tropomyosin and ADF/cofilin and possibly strengthening actin filaments by bundling.

  19. Actin filament dynamics impacts keratinocyte stem cell maintenance

    PubMed Central

    Nanba, Daisuke; Toki, Fujio; Matsushita, Natsuki; Matsushita, Sachi; Higashiyama, Shigeki; Barrandon, Yann

    2013-01-01

    Cultured human epidermal keratinocyte stem cells (holoclones) are crucial for regenerative medicine for burns and genetic disorders. In serial culture, holoclones progressively lose their proliferative capacity to become transient amplifying cells with limited growth (paraclones), a phenomenon termed clonal conversion. Although it negatively impacts the culture lifespan and the success of cell transplantation, little is known on the molecular mechanism underlying clonal conversion. Here, we show that holoclones and paraclones differ in their actin filament organization, with actin bundles distributed radially in holoclones and circumferentially in paraclones. Moreover, actin organization sets the stage for a differing response to epidermal growth factor (EGF), since EGF signalling induces a rapid expansion of colony size in holoclones and a significant reduction in paraclones. Furthermore, inhibition of PI3K or Rac1 in holoclones results in the reorganization of actin filaments in a pattern that is similar to that of paraclones. Importantly, continuous Rac1 inhibition in holoclones results in clonal conversion and reduction of growth potential. Together, our data connect loss of stem cells to EGF-induced colony dynamics governed by Rac1. PMID:23554171

  20. Gamma Interferon-Induced Guanylate Binding Protein 1 Is a Novel Actin Cytoskeleton Remodeling Factor

    PubMed Central

    Ostler, Nicole; Britzen-Laurent, Nathalie; Liebl, Andrea; Naschberger, Elisabeth; Lochnit, Günter; Ostler, Markus; Forster, Florian; Kunzelmann, Peter; Ince, Semra; Supper, Verena; Praefcke, Gerrit J. K.; Schubert, Dirk W.; Stockinger, Hannes; Herrmann, Christian

    2014-01-01

    Gamma interferon (IFN-γ) regulates immune defenses against viruses, intracellular pathogens, and tumors by modulating cell proliferation, migration, invasion, and vesicle trafficking processes. The large GTPase guanylate binding protein 1 (GBP-1) is among the cellular proteins that is the most abundantly induced by IFN-γ and mediates its cell biologic effects. As yet, the molecular mechanisms of action of GBP-1 remain unknown. Applying an interaction proteomics approach, we identified actin as a strong and specific binding partner of GBP-1. Furthermore, GBP-1 colocalized with actin at the subcellular level and was both necessary and sufficient for the extensive remodeling of the fibrous actin structure observed in IFN-γ-exposed cells. These effects were dependent on the oligomerization and the GTPase activity of GBP-1. Purified GBP-1 and actin bound to each other, and this interaction was sufficient to impair the formation of actin filaments in vitro, as demonstrated by atomic force microscopy, dynamic light scattering, and fluorescence-monitored polymerization. Cosedimentation and band shift analyses demonstrated that GBP-1 binds robustly to globular actin and slightly to filamentous actin. This indicated that GBP-1 may induce actin remodeling via globular actin sequestering and/or filament capping. These results establish GBP-1 as a novel member within the family of actin-remodeling proteins specifically mediating IFN-γ-dependent defense strategies. PMID:24190970

  1. Binding of actin to lens alpha crystallins

    NASA Technical Reports Server (NTRS)

    Gopalakrishnan, S.; Takemoto, L.; Spooner, B. S. (Principal Investigator)

    1992-01-01

    Actin has been coupled to a cyanogen bromide-activated Sepharose 4B column, then tested for binding to alpha, beta, and gamma crystallin preparations from the bovine lens. Alpha, but not beta or gamma, crystallins bound to the actin affinity column in a time dependent and saturable manner. Subfractionation of the alpha crystallin preparation into the alpha-A and alpha-B species, followed by incubation with the affinity column, demonstrated that both species bound approximately the same. Together, these studies demonstrate a specific and saturable binding of lens alpha-A and alpha-B with actin.

  2. Actin filaments growing against a barrier with fluctuating shape

    NASA Astrophysics Data System (ADS)

    Sadhu, Raj Kumar; Chatterjee, Sakuntala

    2016-06-01

    We study force generation by a set of parallel actin filaments growing against a nonrigid obstacle, in the presence of an external load. The filaments polymerize by either moving the whole obstacle, with a large energy cost, or by causing local distortion in its shape which costs much less energy. The nonrigid obstacle also has local thermal fluctuations due to which its shape can change with time and we describe this using fluctuations in the height profile of a one-dimensional interface with Kardar-Parisi-Zhang dynamics. We find the shape fluctuations of the barrier strongly affect the force generation mechanism. The qualitative nature of the force-velocity curve is crucially determined by the relative time scale of filament and barrier dynamics. The height profile of the barrier also shows interesting variation with the external load. Our analytical calculations within mean-field theory show reasonable agreement with our simulation results.

  3. Arabidopsis FIM5 decorates apical actin filaments and regulates their organization in the pollen tube

    PubMed Central

    Zhang, Meng; Zhang, Ruihui; Qu, Xiaolu; Huang, Shanjin

    2016-01-01

    The actin cytoskeleton is increasingly recognized as a major regulator of pollen tube growth. Actin filaments have distinct distribution patterns and dynamic properties within different regions of the pollen tube. Apical actin filaments are highly dynamic and crucial for pollen tube growth. However, how apical actin filaments are generated and properly constructed remains an open question. Here we showed that Arabidopsis fimbrin5 (FIM5) decorates filamentous structures throughout the entire tube but is apically concentrated. Apical actin structures are disorganized to different degrees in the pollen tubes of fim5 loss-of-function mutants. Further observations suggest that apical actin structures are not constructed properly because apical actin filaments cannot be maintained at the cortex of fim5 pollen tubes. Actin filaments appeared to be more curved in fim5 pollen tubes and this was confirmed by measurements showing that the convolutedness and the rate of change of convolutedness of actin filaments was significantly increased in fim5 pollen tubes. This suggests that the rigidity of the actin filaments may be compromised in fim5 pollen tubes. Further, the apical cell wall composition is altered, implying that tip-directed vesicle trafficking events are impaired in fim5 pollen tubes. Thus, we found that FIM5 decorates apical actin filaments and regulates their organization in order to drive polarized pollen tube growth. PMID:27117336

  4. 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

    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.

  5. Mechanisms of leiomodin 2-mediated regulation of actin filament in muscle cells.

    PubMed

    Chen, Xiaorui; Ni, Fengyun; Kondrashkina, Elena; Ma, Jianpeng; Wang, Qinghua

    2015-10-13

    Leiomodin (Lmod) is a class of potent tandem-G-actin-binding nucleators in muscle cells. Lmod mutations, deletion, or instability are linked to lethal nemaline myopathy. However, the lack of high-resolution structures of Lmod nucleators in action severely hampered our understanding of their essential cellular functions. Here we report the crystal structure of the actin-Lmod2162-495 nucleus. The structure contains two actin subunits connected by one Lmod2162-495 molecule in a non-filament-like conformation. Complementary functional studies suggest that the binding of Lmod2 stimulates ATP hydrolysis and accelerates actin nucleation and polymerization. The high level of conservation among Lmod proteins in sequence and functions suggests that the mechanistic insights of human Lmod2 uncovered here may aid in a molecular understanding of other Lmod proteins. Furthermore, our structural and mechanistic studies unraveled a previously unrecognized level of regulation in mammalian signal transduction mediated by certain tandem-G-actin-binding nucleators. PMID:26417072

  6. Srv2/cyclase-associated protein forms hexameric shurikens that directly catalyze actin filament severing by cofilin

    PubMed Central

    Chaudhry, Faisal; Breitsprecher, Dennis; Little, Kristin; Sharov, Grigory; Sokolova, Olga; Goode, Bruce L.

    2013-01-01

    Actin filament severing is critical for the dynamic turnover of cellular actin networks. Cofilin severs filaments, but additional factors may be required to increase severing efficiency in vivo. Srv2/cyclase-associated protein (CAP) is a widely expressed protein with a role in binding and recycling actin monomers ascribed to domains in its C-terminus (C-Srv2). In this paper, we report a new biochemical and cellular function for Srv2/CAP in directly catalyzing cofilin-mediated severing of filaments. This function is mediated by its N-terminal half (N-Srv2), and is physically and genetically separable from C-Srv2 activities. Using dual-color total internal reflection fluorescence microscopy, we determined that N-Srv2 stimulates filament disassembly by increasing the frequency of cofilin-mediated severing without affecting cofilin binding to filaments. Structural analysis shows that N-Srv2 forms novel hexameric star-shaped structures, and disrupting oligomerization impairs N-Srv2 activities and in vivo function. Further, genetic analysis shows that the combined activities of N-Srv2 and Aip1 are essential in vivo. These observations define a novel mechanism by which the combined activities of cofilin and Srv2/CAP lead to enhanced filament severing and support an emerging view that actin disassembly is controlled not by cofilin alone, but by a more complex set of factors working in concert. PMID:23135996

  7. Reverse actin sliding triggers strong myosin binding that moves tropomyosin

    SciTech Connect

    Bekyarova, T.I.; Reedy, M.C.; Baumann, B.A.J.; Tregear, R.T.; Ward, A.; Krzic, U.; Prince, K.M.; Perz-Edwards, R.J.; Reconditi, M.; Gore, D.; Irving, T.C.; Reedy, M.K.

    2008-09-03

    Actin/myosin interactions in vertebrate striated muscles are believed to be regulated by the 'steric blocking' mechanism whereby the binding of calcium to the troponin complex allows tropomyosin (TM) to change position on actin, acting as a molecular switch that blocks or allows myosin heads to interact with actin. Movement of TM during activation is initiated by interaction of Ca{sup 2+} with troponin, then completed by further displacement by strong binding cross-bridges. We report x-ray evidence that TM in insect flight muscle (IFM) moves in a manner consistent with the steric blocking mechanism. We find that both isometric contraction, at high [Ca{sup 2+}], and stretch activation, at lower [Ca{sup 2+}], develop similarly high x-ray intensities on the IFM fourth actin layer line because of TM movement, coinciding with x-ray signals of strong-binding cross-bridge attachment to helically favored 'actin target zones.' Vanadate (Vi), a phosphate analog that inhibits active cross-bridge cycling, abolishes all active force in IFM, allowing high [Ca{sup 2+}] to elicit initial TM movement without cross-bridge attachment or other changes from relaxed structure. However, when stretched in high [Ca{sup 2+}], Vi-'paralyzed' fibers produce force substantially above passive response at pCa {approx} 9, concurrent with full conversion from resting to active x-ray pattern, including x-ray signals of cross-bridge strong-binding and TM movement. This argues that myosin heads can be recruited as strong-binding 'brakes' by backward-sliding, calcium-activated thin filaments, and are as effective in moving TM as actively force-producing cross-bridges. Such recruitment of myosin as brakes may be the major mechanism resisting extension during lengthening contractions.

  8. Ha-VP39 binding to actin and the influence of F-actin on assembly of progeny virions.

    PubMed

    Lu, S; Ge, G; Qi, Y

    2004-11-01

    We present evidence that actin is necessary for the successful assembly of HaNPV virions. Purified nucleocapsid protein Ha-VP39 of Heliothis armigera nuclear polyhedrosis virus (HaNPV) was found to be able to bind to actin in vitro without assistance, as demonstrated by Western blot and isothermal titration calorimeter. DeltaH and binding constants (K) detected by isothermal titration calorimeter strongly suggested that Ha-VP39 first binds actin to seed the formation of hexamer complex of actin, and the hexamers then link to each other to form filaments, and the filaments finally twist into cable structures. The proliferation of HaNPV was completely inhibited in Hz-AM1 cells cultivated in the medium containing 0.5 microg/ml cytochalasin D (CD) to prevent polymerization of actin, while its yield was reduced to 10(-4) in the presence of 0.1 microg/ml CD. Actin concentration and the viral DNA synthesis were not significantly affected by CD even though the progeny virions assembled in the CD treated cells were morphologically different from normal ones and resulted in fewer plaques in plaque assay.

  9. Modeling of the motion of the actin filament on the myosin motility assays

    NASA Astrophysics Data System (ADS)

    Young, Yuan; Shelley, Mike

    2007-11-01

    In motility assays, cytoskeletal actin filaments (actin filaments) glide over a surface coated with motor proteins, and the different modes of motion provide a simple measure of the force exerted by the motor proteins (Bourdieu, 1995). Motivated by these experiments, we consider the actin filament as a slender, elastic filament immersed in Stokesian flow, driven by a tangential forcing that mimics the force by the motor proteins. We find qualitative agreement on several points between our analysis and simulations and experimental observations. Furthermore, we study the correlation between filament transport and the characteristics of motion with the spatial pattern of motor protein density.

  10. Myosin-I moves actin filaments on a phospholipid substrate: implications for membrane targeting

    PubMed Central

    1992-01-01

    Acanthamoeba myosin-I bound to substrates of nitrocellulose or planar lipid membranes on glass moved actin filaments at an average velocity of 0.2 micron/s. This movement required ATP and phosphorylation of the myosin-I heavy chain. We prepared planar lipid membranes on a glass support by passive fusion of lipid vesicles (Brian, A. A., and H. M. McConnell. 1984. Proc. Natl. Acad. Sci. USA. 81:6159-6163) composed of phosphatidylcholine and containing 0-40% phosphatidylserine. The mass of lipid that bound to the glass was the same for membranes of 2 and 20% phosphatidylserine in phosphatidylcholine and was sufficient to form a single bilayer. Myosin-I moved actin filaments on planar membranes of 5-40% but not 0-2% phosphatidylserine. At the low concentrations of phosphatidylserine, actin filaments tended to detach suggesting that less myosin-I was bound. We used the cooperative activation of Acanthamoeba myosin-I ATPase by low concentrations of actin to assess the association of phospholipids with myosin-I. Under conditions where activity depends on the binding of actin to the tail of myosin-I (Albanesi, J. P., H. Fujisaki, and E. D. Korn. 1985. J. Biol. Chem. 260:11174-11179), phospholipid vesicles with 5-40% phosphatidylserine inhibited ATPase activity. The motility and ATPase results demonstrate a specific interaction of the tail of myosin-I with physiological concentrations of phosphatidylserine. This interaction is sufficient to support motility and may provide a mechanism to target myosin-I to biological membranes. PMID:1530945

  11. Assembly and Turnover of Short Actin Filaments by the Formin INF2 and Profilin*

    PubMed Central

    Gurel, Pinar S.; A, Mu; Guo, Bingqian; Shu, Rui; Mierke, Dale F.; Higgs, Henry N.

    2015-01-01

    INF2 (inverted formin 2) is a formin protein with unique biochemical effects on actin. In addition to the common formin ability to accelerate actin nucleation and elongation, INF2 can also sever filaments and accelerate their depolymerization. Although we understand key attributes of INF2-mediated severing, we do not understand the mechanism by which INF2 accelerates depolymerization subsequent to severing. Here, we show that INF2 can create short filaments (<60 nm) that continuously turn over actin subunits through a combination of barbed end elongation, severing, and WH2 motif-mediated depolymerization. This pseudo-steady state condition occurs whether starting from actin filaments or monomers. The rate-limiting step of the cycle is nucleotide exchange of ADP for ATP on actin monomers after release from the INF2/actin complex. Profilin addition has two effects: 1) to accelerate filament turnover 6-fold by accelerating nucleotide exchange and 2) to shift the equilibrium toward polymerization, resulting in longer filaments. In sum, our findings show that the combination of multiple interactions of INF2 with actin can work in concert to increase the ATP turnover rate of actin. Depending on the ratio of INF2:actin, this increased flux can result in rapid filament depolymerization or maintenance of short filaments. We also show that high concentrations of cytochalasin D accelerate ATP turnover by actin but through a different mechanism from that of INF2. PMID:26124273

  12. Assembly and turnover of short actin filaments by the formin INF2 and profilin.

    PubMed

    Gurel, Pinar S; A, Mu; Guo, Bingqian; Shu, Rui; Mierke, Dale F; Higgs, Henry N

    2015-09-11

    INF2 (inverted formin 2) is a formin protein with unique biochemical effects on actin. In addition to the common formin ability to accelerate actin nucleation and elongation, INF2 can also sever filaments and accelerate their depolymerization. Although we understand key attributes of INF2-mediated severing, we do not understand the mechanism by which INF2 accelerates depolymerization subsequent to severing. Here, we show that INF2 can create short filaments (<60 nm) that continuously turn over actin subunits through a combination of barbed end elongation, severing, and WH2 motif-mediated depolymerization. This pseudo-steady state condition occurs whether starting from actin filaments or monomers. The rate-limiting step of the cycle is nucleotide exchange of ADP for ATP on actin monomers after release from the INF2/actin complex. Profilin addition has two effects: 1) to accelerate filament turnover 6-fold by accelerating nucleotide exchange and 2) to shift the equilibrium toward polymerization, resulting in longer filaments. In sum, our findings show that the combination of multiple interactions of INF2 with actin can work in concert to increase the ATP turnover rate of actin. Depending on the ratio of INF2:actin, this increased flux can result in rapid filament depolymerization or maintenance of short filaments. We also show that high concentrations of cytochalasin D accelerate ATP turnover by actin but through a different mechanism from that of INF2.

  13. Formin and capping protein together embrace the actin filament in a ménage à trois

    PubMed Central

    Shekhar, Shashank; Kerleau, Mikael; Kühn, Sonja; Pernier, Julien; Romet-Lemonne, Guillaume; Jégou, Antoine; Carlier, Marie-France

    2015-01-01

    Proteins targeting actin filament barbed ends play a pivotal role in motile processes. While formins enhance filament assembly, capping protein (CP) blocks polymerization. On their own, they both bind barbed ends with high affinity and very slow dissociation. Their barbed-end binding is thought to be mutually exclusive. CP has recently been shown to be present in filopodia and controls their morphology and dynamics. Here we explore how CP and formins may functionally coregulate filament barbed-end assembly. We show, using kinetic analysis of individual filaments by microfluidics-assisted fluorescence microscopy, that CP and mDia1 formin are able to simultaneously bind barbed ends. This is further confirmed using single-molecule imaging. Their mutually weakened binding enables rapid displacement of one by the other. We show that formin FMNL2 behaves similarly, thus suggesting that this is a general property of formins. Implications in filopodia regulation and barbed-end structural regulation are discussed. PMID:26564775

  14. Fluorescence resonance energy transfer between points on tropomyosin and actin in skeletal muscle thin filaments: does tropomyosin move?

    PubMed

    Miki, M; Miura, T; Sano, K; Kimura, H; Kondo, H; Ishida, H; Maéda, Y

    1998-06-01

    Fluorescence resonance energy transfer (FRET) spectroscopy has been used to determine spatial relationships between residues on tropomyosin and actin in reconstituted muscle thin filament, and to detect a positional change of tropomyosin relative to actin on the thin filament in the presence and absence of Ca2+ ions. In addition to Cys-190 which is a single cysteine residue in rabbit skeletal muscle alpha-tropomyosin, a new site, Cys-87 which is a unique cysteine residue in a mutant alpha-tropomyosin, was labeled with a resonance energy donor molecule, 5-(2-iodoacetylaminoethyl)aminonaphthalene 1-sulfonic acid (IAEDANS). On the other hand, Gln-41, Lys-61, Cys-374, and the ATP-binding site of actin were selectively labeled with acceptor probes: fluorescein cadaverine, fluorescein 5-isothiocyanate, 4-dimethyl-aminophenylazophenyl 4'-maleimide, and TNP-ATP (or TNP-ADP), respectively. The distances between probes attached to position 87 of the mutant tropomyosin and Gln-41, Lys-61, Cys-374, or the nucleotide-binding site of actin on the reconstituted thin filament in the presence of Ca2+ ion were measured to be 43.2, 49.7, 45.4, and 35.2 A, respectively, and the distance between probes attached to position 190 of tropomyosin and Gln-41 or the nucleotide-binding site of actin were 51.6 and 43.1 A, respectively. The transfer efficiencies between these donor and acceptor molecules were large, so that the efficiency should be very sensitive to changes in distance between probes attached to tropomyosin and actin. However, the transfer efficiency did not change appreciably upon removal of Ca2+ ions, suggesting that tropomyosin does not change its position on the reconstituted thin filament in response to a change in Ca2+ ion concentration. The present results do not support the notion of tropomyosin movement on skeletal muscle thin filaments as proposed in the steric blocking theory.

  15. Importance of internal regions and the overall length of tropomyosin for actin binding and regulatory function.

    PubMed

    Hitchcock-DeGregori, S E; Song, Y; Moraczewska, J

    2001-02-20

    Tropomyosin (Tm) binds along actin filaments, one molecule spanning four to seven actin monomers, depending on the isoform. Periodic repeats in the sequence have been proposed to correspond to actin binding sites. To learn the functional importance of length and the internal periods we made a series of progressively shorter Tms, deleting from two up to six of the internal periods from rat striated alpha-TM (dAc2--3, dAc2--4, dAc3--5, dAc2--5, dAc2--6, dAc1.5--6.5). Recombinant Tms (unacetylated) were expressed in Escherichia coli. Tropomyosins that are four or more periods long (dAc2--3, dAc2--4, and dAc3--5) bound well to F-actin with troponin (Tn). dAc2--5 bound weakly (with EGTA) and binding of shorter mutants was undetectable in any condition. Myosin S1-induced binding of Tm to actin in the tight Tm-binding "open" state did not correlate with actin binding. dAc3--5 and dAc2--5 did not bind to actin even when the filament was saturated with S1. In contrast, dAc2--3 and dAc2--4 did, like wild-type-Tm, requiring about 3 mol of S1/mol of Tm for half-maximal binding. The results show the critical importance of period 5 (residues 166--207) for myosin S1-induced binding. The Tms that bound to actin (dAc2--3, dAc2--4, and dAc3--5) all fully inhibited the actomyosin ATPase (+Tn) in EGTA. In the presence of Ca(2+), relief of inhibition by these Tms was incomplete. We conclude (1) four or more actin periods are required for Tm to bind to actin with reasonable affinity and (2) that the structural requirements of Tm for the transition of the regulated filament from the blocked-to-closed/open (relief of inhibition by Ca(2+)) and the closed-to-open states (strong Tm binding to actin-S1) are different. PMID:11329279

  16. The Interaction of Arp2/3 Complex with Actin: Nucleation, High Affinity Pointed End Capping, and Formation of Branching Networks of Filaments

    NASA Astrophysics Data System (ADS)

    Dyche Mullins, R.; Heuser, John A.; Pollard, Thomas D.

    1998-05-01

    The Arp2/3 complex is a stable assembly of seven protein subunits including two actin-related proteins (Arp2 and Arp3) and five novel proteins. Previous work showed that this complex binds to the sides of actin filaments and is concentrated at the leading edges of motile cells. Here, we show that Arp2/3 complex purified from Acanthamoeba caps the pointed ends of actin filaments with high affinity. Arp2/3 complex inhibits both monomer addition and dissociation at the pointed ends of actin filaments with apparent nanomolar affinity and increases the critical concentration for polymerization at the pointed end from 0.6 to 1.0 μ M. The high affinity of Arp2/3 complex for pointed ends and its abundance in amoebae suggest that in vivo all actin filament pointed ends are capped by Arp2/3 complex. Arp2/3 complex also nucleates formation of actin filaments that elongate only from their barbed ends. From kinetic analysis, the nucleation mechanism appears to involve stabilization of polymerization intermediates (probably actin dimers). In electron micrographs of quick-frozen, deep-etched samples, we see Arp2/3 bound to sides and pointed ends of actin filaments and examples of Arp2/3 complex attaching pointed ends of filaments to sides of other filaments. In these cases, the angle of attachment is a remarkably constant 70 ± 7 degrees. From these in vitro biochemical properties, we propose a model for how Arp2/3 complex controls the assembly of a branching network of actin filaments at the leading edge of motile cells.

  17. Identification of Actin-Binding Proteins from Maize Pollen

    SciTech Connect

    Staiger, C.J.

    2004-01-13

    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 ABPs (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.

  18. A three-dimensional FRET analysis to construct an atomic model of the actin-tropomyosin complex on a reconstituted thin filament.

    PubMed

    Miki, Masao; Makimura, Satoshi; Saitoh, Takahiro; Bunya, Masashi; Sugahara, Yasuyuki; Ueno, Yutaka; Kimura-Sakiyama, Chieko; Tobita, Hidetaka

    2011-12-16

    Fluorescence resonance energy transfer (FRET) was used to construct an atomic model of the actin-tropomyosin (Tm) complex on a reconstituted thin filament. We generated five single-cysteine mutants in the 146-174 region of rabbit skeletal muscle α-Tm. An energy donor probe was attached to a single-cysteine Tm residue, while an energy acceptor probe was located in actin Gln41, actin Cys374, or the actin nucleotide binding site. From these donor-acceptor pairs, FRET efficiencies were determined with and without Ca(2+). Using the atomic coordinates for F-actin and Tm, we searched all possible arrangements for Tm segment 146-174 on F-actin to calculate the FRET efficiency for each donor-acceptor pair in each arrangement. By minimizing the squared sum of deviations for the calculated FRET efficiencies from the observed FRET efficiencies, we determined the location of the Tm segment on the F-actin filament. Furthermore, we generated a set of five single-cysteine mutants in each of the four Tm regions 41-69, 83-111, 216-244, and 252-279. Using the same procedures, we determined each segment's location on the F-actin filament. In the best-fit model, Tm runs along actin residues 217-236, which were reported to compose the Tm binding site. Electrostatic, hydrogen-bonding, and hydrophobic interactions are involved in actin and Tm binding. The C-terminal region of Tm was observed to contact actin more closely than did the N-terminal region. Tm contacts more residues on actin without Ca(2+) than with it. Ca(2+)-induced changes on the actin-Tm contact surface strongly affect the F-actin structure, which is important for muscle regulation.

  19. Cytoplasmic intermediate filaments mediate actin-driven positioning of the nucleus.

    PubMed

    Dupin, Isabelle; Sakamoto, Yasuhisa; Etienne-Manneville, Sandrine

    2011-03-15

    The localization of the nucleus is precisely regulated, and defects in nuclear positioning are observed in diseases such as lissencephaly, cerebellar ataxia and dysplasia. We show here that cytoplasmic intermediate filaments are essential players in actin-dependent positioning of the nucleus. The actin retrograde flow is relayed by a flow of intermediate filaments that accumulate asymmetrically around the nuclear envelope. Perturbations of the intermediate filament network alter positioning of the nucleus in both migrating and immobile astrocytes. This function of intermediate filaments might be crucial for regulating cell motility, in particular in tumor cells expressing high levels of cytoplasmic intermediate filaments.

  20. Inhibition of tobacco mosaic virus movement by expression of an actin-binding protein.

    PubMed

    Hofmann, Christina; Niehl, Annette; Sambade, Adrian; Steinmetz, André; Heinlein, Manfred

    2009-04-01

    The tobacco mosaic virus (TMV) movement protein (MP) required for the cell-to-cell spread of viral RNA interacts with the endoplasmic reticulum (ER) as well as with the cytoskeleton during infection. Whereas associations of MP with ER and microtubules have been intensely investigated, research on the role of actin has been rather scarce. We demonstrate that Nicotiana benthamiana plants transgenic for the actin-binding domain 2 of Arabidopsis (Arabidopsis thaliana) fimbrin (AtFIM1) fused to green fluorescent protein (ABD2:GFP) exhibit a dynamic ABD2:GFP-labeled actin cytoskeleton and myosin-dependent Golgi trafficking. These plants also support the movement of TMV. In contrast, both myosin-dependent Golgi trafficking and TMV movement are dominantly inhibited when ABD2:GFP is expressed transiently. Inhibition is mediated through binding of ABD2:GFP to actin filaments, since TMV movement is restored upon disruption of the ABD2:GFP-labeled actin network with latrunculin B. Latrunculin B shows no significant effect on the spread of TMV infection in either wild-type plants or ABD2:GFP transgenic plants under our treatment conditions. We did not observe any binding of MP along the length of actin filaments. Collectively, these observations demonstrate that TMV movement does not require an intact actomyosin system. Nevertheless, actin-binding proteins appear to have the potential to exert control over TMV movement through the inhibition of myosin-associated protein trafficking along the ER membrane.

  1. Unidirectional movement of an actin filament taking advantage of temperature gradients.

    PubMed

    Kawaguchi, Tomoaki; Honda, Hajime

    2007-01-01

    An actin filament with heat acceptors attached to its Cys374 residue in each actin monomer could move unidirectionally even under heat pulsation alone, while in the total absence of both ATP and myosin. The prime driver for the movement was temperature gradients operating between locally heated portions on an actin filament and its cooler surroundings. In this report, we investigated how the mitigation of the temperature gradients induces a unidirectional movement of an actin filament. We then observed the transversal fluctuations of the filament in response to heat pulsation and their transition into longitudinally unidirectional movement. The transition was significantly accelerated when Cys374 and Lys336 were simultaneously excited within an actin monomer. These results suggest that the mitigation of the temperature gradients within each actin monomer first went through the energy transformation to transversal fluctuations of the filament, and then followed by the transformation further down to longitudinal movements of the filament. The faster mitigation of temperature gradients within actin monomer helps build up the transition from the transversal to longitudinal movements of the filament by coordinating the interaction between the neighboring monomers. PMID:17030086

  2. Myosin binding protein-C activates thin filaments and inhibits thick filaments in heart muscle cells.

    PubMed

    Kampourakis, Thomas; Yan, Ziqian; Gautel, Mathias; Sun, Yin-Biao; Irving, Malcolm

    2014-12-30

    Myosin binding protein-C (MyBP-C) is a key regulatory protein in heart muscle, and mutations in the MYBPC3 gene are frequently associated with cardiomyopathy. However, the mechanism of action of MyBP-C remains poorly understood, and both activating and inhibitory effects of MyBP-C on contractility have been reported. To clarify the function of the regulatory N-terminal domains of MyBP-C, we determined their effects on the structure of thick (myosin-containing) and thin (actin-containing) filaments in intact sarcomeres of heart muscle. We used fluorescent probes on troponin C in the thin filaments and on myosin regulatory light chain in the thick filaments to monitor structural changes associated with activation of demembranated trabeculae from rat ventricle by the C1mC2 region of rat MyBP-C. C1mC2 induced larger structural changes in thin filaments than calcium activation, and these were still present when active force was blocked with blebbistatin, showing that C1mC2 directly activates the thin filaments. In contrast, structural changes in thick filaments induced by C1mC2 were smaller than those associated with calcium activation and were abolished or reversed by blebbistatin. Low concentrations of C1mC2 did not affect resting force but increased calcium sensitivity and reduced cooperativity of force and structural changes in both thin and thick filaments. These results show that the N-terminal region of MyBP-C stabilizes the ON state of thin filaments and the OFF state of thick filaments and lead to a novel hypothesis for the physiological role of MyBP-C in the regulation of cardiac contractility.

  3. Probing the dynamic responses of individual actin filaments under fluidic mechanical stimulation via microfluidics

    NASA Astrophysics Data System (ADS)

    Cheng, Chao-Min; Yang, Chung-Yao; Kim, YongTae; LeDuc, Philip R.

    2013-05-01

    Herein, we demonstrate an easy-to-handle approach that employs a combination of microcurvilinear flow and fluorescence microscopy for probing the dynamic responses of individual synthesized actin filaments. We observed morphological changes of single actin filaments with different spatiotemporal responses when they were elongated with rotation or underwent significant bending during fluidic shear stress, and found that they may initially increase their curvature but then start releasing the external force immediately thereafter. Our approach allowed us to visibly examine the dynamic responses of individual actin filaments under simultaneous forces of rotation and elongation, as well as bending resulting from fluidic shear stress.

  4. Fluorescence energy transfer between Cys-10 residues in F-actin filaments.

    PubMed

    Miki, M; Barden, J A; Hambly, B D; dos Remedios, C G

    1986-05-01

    Fluorescence energy transfer was measured between Cys-10 residues in an F-actin filament using 5-[2-((iodoacetyl)amino)-ethyl]aminonaphthalene-1-sulphonic acid (1,5-IAEDANS) as a fluorescence energy donor and 4-dimethylaminophenylazophenyl-4'-maleimide (DABMI) as the acceptor. Both labels were covalently attached to Cys-10 residues in an F-actin filament. Taking the helical structure of the F-actin filament into consideration, the radial coordinate of Cys-10 was calculated to be 23 A. This corresponds to a distance between adjacent sites along the long pitch helix of 56.1 A and along the genetic helix of 53.3 A.

  5. WAVE binds Ena/VASP for enhanced Arp2/3 complex-based actin assembly.

    PubMed

    Havrylenko, Svitlana; Noguera, Philippe; Abou-Ghali, Majdouline; Manzi, John; Faqir, Fahima; Lamora, Audrey; Guérin, Christophe; Blanchoin, Laurent; Plastino, Julie

    2015-01-01

    The WAVE complex is the main activator of the Arp2/3 complex for actin filament nucleation and assembly in the lamellipodia of moving cells. Other important players in lamellipodial protrusion are Ena/VASP proteins, which enhance actin filament elongation. Here we examine the molecular coordination between the nucleating activity of the Arp2/3 complex and the elongating activity of Ena/VASP proteins for the formation of actin networks. Using an in vitro bead motility assay, we show that WAVE directly binds VASP, resulting in an increase in Arp2/3 complex-based actin assembly. We show that this interaction is important in vivo as well, for the formation of lamellipodia during the ventral enclosure event of Caenorhabditis elegans embryogenesis. Ena/VASP's ability to bind F-actin and profilin-complexed G-actin are important for its effect, whereas Ena/VASP tetramerization is not necessary. Our data are consistent with the idea that binding of Ena/VASP to WAVE potentiates Arp2/3 complex activity and lamellipodial actin assembly.

  6. WAVE binds Ena/VASP for enhanced Arp2/3 complex–based actin assembly

    PubMed Central

    Havrylenko, Svitlana; Noguera, Philippe; Abou-Ghali, Majdouline; Manzi, John; Faqir, Fahima; Lamora, Audrey; Guérin, Christophe; Blanchoin, Laurent; Plastino, Julie

    2015-01-01

    The WAVE complex is the main activator of the Arp2/3 complex for actin filament nucleation and assembly in the lamellipodia of moving cells. Other important players in lamellipodial protrusion are Ena/VASP proteins, which enhance actin filament elongation. Here we examine the molecular coordination between the nucleating activity of the Arp2/3 complex and the elongating activity of Ena/VASP proteins for the formation of actin networks. Using an in vitro bead motility assay, we show that WAVE directly binds VASP, resulting in an increase in Arp2/3 complex–based actin assembly. We show that this interaction is important in vivo as well, for the formation of lamellipodia during the ventral enclosure event of Caenorhabditis elegans embryogenesis. Ena/VASP's ability to bind F-actin and profilin-complexed G-actin are important for its effect, whereas Ena/VASP tetramerization is not necessary. Our data are consistent with the idea that binding of Ena/VASP to WAVE potentiates Arp2/3 complex activity and lamellipodial actin assembly. PMID:25355952

  7. Banding and polarity of actin filaments in interphase and cleaving cells

    PubMed Central

    1980-01-01

    Heavy meromyosin (HMM) decoration of actin filaments was used to detect the polarity of microfilaments in interphase and cleaving rat kangaroo (PtK2) cells. Ethanol at -20 degrees C was used to make the cells permeable to HMM followed by tannic acid-glutaraldehyde fixation for electron microscopy. Uniform polarity of actin filaments was observed at cell junctions and central attachment plaques with the HMM arrowheads always pointing away from the junction or plaque. Stress fibers were banded in appearance with their component microfilaments exhibiting both parallel and antiparallel orientation with respect to one another. Identical banding of microfilament bundles was also seen in cleavage furrows with the same variation in filament polarity as found in stress fibers. Similarly banded fibers were not seen outside the cleavage furrow in mitotic cells. By the time that a mid-body was present, the actin filaments in the cleavage furrow were no longer in banded fibers. The alternating dark and light bands of both the stress fibers and cleavage furrow fibers are approximately equal in length, each measuring approximately 0.16 micrometer. Actin filaments were present in both bands, and individual decorated filaments could sometimes be traced through four band lengths. Undecorated filaments, 10 nm in diameter, could often be seen within the light bands. A model is proposed to explain the arrangement of filaments in stress fibers and cleavage furrows based on the striations observed with tannic acid and the polarity of the actin filaments. PMID:6995468

  8. Isolation of an actin-binding protein from membranes of Dictyostelium discoideum

    PubMed Central

    1985-01-01

    We prepared a probe of radiolabeled, glutaraldehyde cross-linked filamentous actin (F-actin) to study binding of actin to membranes of Dictyostelium discoideum. The probe bound to membranes or detergent extracts of membranes with a high affinity and in a saturable manner. The binding could be reduced by boiling of either the actin probe or the membranes, or by addition of excess native F-actin, but not by addition of an equivalent amount of bovine serum albumin, to the assay. The probe labeled several proteins when used to overlay sodium dodecyl sulfate gels of Dictyostelium membranes. One of these labeled proteins was a 24,000-mol-wt protein (p24), which was soluble only in the presence of a high concentration of sodium deoxycholate (5%, wt/vol) at room temperature or above. The p24 was purified by selective detergent extraction and column chromatography. When tested in a novel two-phase binding assay, p24 bound both native monomeric actin (G-actin) and F- actin in a specific manner. In this assay, G-actin bound p24 with a submicromolar affinity. PMID:3972891

  9. [Conformational changes of actin induced by strong or weak myosin subfragment-1 binding].

    PubMed

    Dedova, I V; Avrova, S V; Vikhoreva, N N; Vikhorev, R G; Hazlett, T L; Van der Meer, W; Dos Remedios, C G; Borovikov, Iu S

    2004-01-01

    Movements of different areas of polypeptide chains within F-actin monomers induced by S1 or pPDM-S1 binding were studied by polarized fluorimetry. Thin filaments of ghost muscle were reconstructed by adding G-actin labeled with fluorescent probes attached alternatively to different sites of actin molecule. These sites were: Cys-374 labeled with 1,5-IAEDANS, TMRIA or 5-IAF; Lys-373 labeled with NBD-Cl; Lys-113 labeled with Alexa-488; Lys-61 labeled with FITC; Gln-41 labeled with DED and Cys-10 labeled with 1,5-IAEDANS, 5-IAF or fluorescein-maleimid. In addition, we used TRITC-, FITC-falloidin and e-ADP that were located, respectively, in filament groove and interdomain cleft. The data were analysed by model-dependent and model-independent methods (see appendixes). The orientation and mobility of fluorescent probes were significantly changed when actin and myosin interacted, depending on fluorophore location and binding site of actomyosin. Strong binding of S with actin leads to 1) a decrease in the orientation of oscillators of derivatives of falloidin (TRITC-falloidin, FITC-falloidin) and actin-bound nucleotide (e-ADP); 2) an increase in the orientation of dye oscillators located in the "front' surface of the small domain (where actin is viewed in the standard orientation with subdomains 1/2 and 3/4 oriented to the right and to the left, respectively); 3) a decrease in the angles of dye oscillators located on the "back" surface of subdomain-1. In contrast, a weak binding of S1 to actin induces the opposite effects in orientation of these probes. These data suggest that during the ATP hydrolysis cycle myosin heads induce a change in actin monomer (a tilt and twisting of its small domain). Presumably, these alterations in F-actin conformation play an important role in muscle contraction.

  10. Arabidopsis Actin Depolymerizing Factor4 Modulates the Stochastic Dynamic Behavior of Actin Filaments in the Cortical Array of Epidermal Cells[C][W

    PubMed Central

    Henty, Jessica L.; Bledsoe, Samuel W.; Khurana, Parul; Meagher, Richard B.; Day, Brad; Blanchoin, Laurent; Staiger, Christopher J.

    2011-01-01

    Actin filament arrays are constantly remodeled as the needs of cells change as well as during responses to biotic and abiotic stimuli. Previous studies demonstrate that many single actin filaments in the cortical array of living Arabidopsis thaliana epidermal cells undergo stochastic dynamics, a combination of rapid growth balanced by disassembly from prolific severing activity. Filament turnover and dynamics are well understood from in vitro biochemical analyses and simple reconstituted systems. However, the identification in living cells of the molecular players involved in controlling actin dynamics awaits the use of model systems, especially ones where the power of genetics can be combined with imaging of individual actin filaments at high spatial and temporal resolution. Here, we test the hypothesis that actin depolymerizing factor (ADF)/cofilin contributes to stochastic filament severing and facilitates actin turnover. A knockout mutant for Arabidopsis ADF4 has longer hypocotyls and epidermal cells when compared with wild-type seedlings. This correlates with a change in actin filament architecture; cytoskeletal arrays in adf4 cells are significantly more bundled and less dense than in wild-type cells. Several parameters of single actin filament turnover are also altered. Notably, adf4 mutant cells have a 2.5-fold reduced severing frequency as well as significantly increased actin filament lengths and lifetimes. Thus, we provide evidence that ADF4 contributes to the stochastic dynamic turnover of actin filaments in plant cells. PMID:22010035

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

    SciTech Connect

    Gomibuchi, Yuki; Uyeda, Taro Q.P.; Wakabayashi, Takeyuki

    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-143 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 (ASA

  12. Moving and stationary actin filaments are involved in spreading of postmitotic PtK2 cells

    PubMed Central

    1993-01-01

    We have investigated spreading of postmitotic PtK2 cells and the behavior of actin filaments in this system by time-lapse microscopy and photoactivation of fluorescence. During mitosis PtK2 cells round up and at cytokinesis the daughter cells spread back to regain their interphase morphology. Normal spreading edges are quite homogenous and are not comprised of two distinct areas (lamellae and lamellipodia) as found in moving edges of interphase motile cells. Spreading edges are connected to a network of long, thin, actin-rich fibers called retraction fibers. A role for retraction fibers in spreading was tested by mechanical disruption of fibers ahead of a spreading edge. Spreading is inhibited over the region of disruption, but not over neighboring intact fibers. Using photoactivation of fluorescence to mark actin filaments, we have determined that the majority of actin filaments move forward in spreading edges at the same rate as the edge. As far as we are aware, this is the first time that forward movement of a cell edge has been correlated with forward movement of actin filaments. In contrast, actin filaments in retraction fibers remain stationary with respect to the substrate. Thus there are at least two dynamic populations of actin polymer in spreading postmitotic cells. This is supported by the observation that actin filaments in some spreading edges not only move forward, but also separate into two fractions or broaden with time. A small fraction of postmitotic cells have a spreading edge with a distinct lamellipodium. In these edges, marked actin polymer fluxes backward with respect to substrate. We suggest that forward movement of actin filaments may participate in generating force for spreading in postmitotic cells and perhaps more generally for cell locomotion. PMID:8349733

  13. Stabilization of actin filaments prevents germinal vesicle breakdown and affects microtubule organization in Xenopus oocytes.

    PubMed

    Okada, Iyo; Fujiki, Saburo; Iwase, Shohei; Abe, Hiroshi

    2012-05-01

    In Xenopus oocytes, extremely giant nuclei, termed germinal vesicles, contain a large amount of actin filaments most likely for mechanical integrity. Here, we show that microinjection of phalloidin, an F-actin-stabilizing drug, prevents the germinal vesicle breakdown (GVBD) in oocytes treated with progesterone. These nuclei remained for more 12 h after control oocytes underwent GVBD. Immunostaining showed significant elevation of actin in the remaining nuclei and many actin filament bundles in the cytoplasm. Furthermore, microtubules formed unusual structures in both nuclei and cytoplasm of phalloidin-injected oocytes stimulated by progesterone. Cytoplasmic microtubule arrays and intranuclear microtubules initially formed in phalloidin-injected oocytes as control oocytes exhibited white maturation spots; these structures gradually disappeared and finally converged upon intranuclear short bundles when control oocytes completed maturation. In contrast, treatment of oocytes with jasplakinolide, a cell membrane-permeable actin filament-stabilizing drug, did not affect GVBD. This drug preferentially induced accumulation of actin filaments at the cortex without any increase in cytoplasmic actin staining. Based on these results, intranuclear and cytoplasmic actin filament dynamics appear to be required for the completion of GVBD and critically involved in the regulation of microtubule assembly during oocyte maturation in Xenopus laevis. PMID:22422719

  14. Axon initial segment cytoskeleton comprises a multiprotein submembranous coat containing sparse actin filaments

    PubMed Central

    Jones, Steven L.; Korobova, Farida

    2014-01-01

    The axon initial segment (AIS) of differentiated neurons regulates action potential initiation and axon–dendritic polarity. The latter function depends on actin dynamics, but actin structure and functions at the AIS remain unclear. Using platinum replica electron microscopy (PREM), we have characterized the architecture of the AIS cytoskeleton in mature and developing hippocampal neurons. The AIS cytoskeleton assembly begins with bundling of microtubules and culminates in formation of a dense, fibrillar–globular coat over microtubule bundles. Immunogold PREM revealed that the coat contains a network of known AIS proteins, including ankyrin G, spectrin βIV, neurofascin, neuronal cell adhesion molecule, voltage-gated sodium channels, and actin filaments. Contrary to existing models, we find neither polarized actin arrays, nor dense actin meshworks in the AIS. Instead, the AIS contains two populations of sparse actin filaments: short, stable filaments and slightly longer dynamic filaments. We propose that stable actin filaments play a structural role for formation of the AIS diffusion barrier, whereas dynamic actin may promote AIS coat remodeling. PMID:24711503

  15. ER transport on actin filaments in squid giant axon: implications for signal transduction at synapse.

    PubMed

    Langford, G M

    1999-12-01

    The smooth endoplasmic reticulum (S-ER) is transported on actin filaments in the giant axon of the squid. The identity of the myosin motors that transport S-ER in the squid giant axon has been determined. Our recent studies have shown that the motor for movement of S-ER vesicles on actin filaments is Myosin-V (1). These findings grew out of a series of studies that began with the initial observation that vesicles in the giant axon of the squid move on both microtubules and actin filaments (2). These initial studies documented the ability of individual vesicles to move from microtubules to actin filaments and led to the development of the dual filament model of vesicle transport (3, 4). The model proposes that long-range movement of vesicles occurs on microtubules and short-range movement on actin filaments. S-ER vesicles were identified as the major population of vesicles in the axon that use myosin-V for movement on actin filaments. The S-ER is the primary site of calcium storage, and it regulates the local cytosolic calcium concentration. Calcium release from the S-ER in neurons couples electrical excitation to signal transduction cascades. The signaling cascades triggered by the release of calcium from S-ER in dendritic spines are postulated to initiate the cellular mechanisms that lead to learning and memory.

  16. Possible association of actin filaments with chloroplasts of spinach mesophyll cells in vivo and in vitro.

    PubMed

    Kumatani, T; Sakurai-Ozato, N; Miyawaki, N; Yokota, E; Shimmen, T; Terashima, I; Takagi, S

    2006-11-01

    In palisade mesophyll cells of spinach (Spinacia oleracea L.) kept under low-intensity white light, chloroplasts were apparently immobile and seemed to be surrounded by fine bundles of actin filaments. High-intensity blue light induced actin-dependent chloroplast movement concomitant with the appearance of a couple of long, straight bundles of actin filaments in each cell, whereas high-intensity red light was essentially ineffective in inducing these responses. The actin organization observed under low-intensity white light has been postulated to function in anchoring chloroplasts at proper intracellular positions through direct interaction with the chloroplasts. Intact chloroplasts, which retained their outer envelopes, were isolated after homogenization of leaves and Percoll centrifugation. No endogenous actin was detected by immunoblotting in the final intact-chloroplast fraction prepared from the leaves kept under low-intensity white light or in darkness. In cosedimentation assays with exogenously added skeletal muscle filamentous actin, however, actin was detected in the intact-chloroplast fraction precipitated after low-speed centrifugation. The association of actin with chloroplasts was apparently dependent on incubation time and chloroplast density. After partial disruption of the outer envelope of isolated chloroplasts by treatment with trypsin, actin was no longer coprecipitated. The results suggest that chloroplasts in spinach leaves can directly interact with actin, and that this interaction may be involved in the regulation of intracellular positioning of chloroplasts.

  17. Tissue Expression and Actin Binding of a Novel N-Terminal Utrophin Isoform

    PubMed Central

    Zuellig, Richard A.; Bornhauser, Beat C.; Amstutz, Ralf; Constantin, Bruno; Schaub, Marcus C.

    2011-01-01

    Utrophin and dystrophin present two large proteins that link the intracellular actin cytoskeleton to the extracellular matrix via the C-terminal-associated protein complex. Here we describe a novel short N-terminal isoform of utrophin and its protein product in various rat tissues (N-utro, 62 kDa, amino acids 1–539, comprising the actin-binding domain plus the first two spectrin repeats). Using different N-terminal recombinant utrophin fragments, we show that actin binding exhibits pronounced negative cooperativity (affinity constants K1 = ∼5 × 106 and K2 = ∼1 × 105 M−1) and is Ca2+-insensitive. Expression of the different fragments in COS7 cells and in myotubes indicates that the actin-binding domain alone binds exlusively to actin filaments. The recombinant N-utro analogue binds in vitro to actin and in the cells associates to the membranes. The results indicate that N-utro may be responsible for the anchoring of the cortical actin cytoskeleton to the membranes in muscle and other tissues. PMID:22228988

  18. Analysis of the human cofilin 1 structure reveals conformational changes required for actin binding

    PubMed Central

    Klejnot, Marta; Gabrielsen, Mads; Cameron, Jenifer; Mleczak, Andrzej; Talapatra, Sandeep K.; Kozielski, Frank; Pannifer, Andrew; Olson, Michael F.

    2013-01-01

    The actin cytoskeleton is the chassis that gives a cell its shape and structure, and supplies the power for numerous dynamic processes including motility, endocytosis, intracellular transport and division. To perform these activities, the cytoskeleton undergoes constant remodelling and reorganization. One of the major actin-remodelling families are the cofilin proteins, made up of cofilin 1, cofilin 2 and actin-depolymerizing factor (ADF), which sever aged ADP-associated actin filaments to reduce filament length and provide new potential nucleation sites. Despite the significant interest in cofilin as a central node in actin-cytoskeleton dynamics, to date the only forms of cofilin for which crystal structures have been solved are from the yeast, Chromalveolata and plant kingdoms; none have previously been reported for an animal cofilin protein. Two distinct regions in animal cofilin are significantly larger than in the forms previously crystallized, suggesting that they would be uniquely organized. Therefore, it was sought to determine the structure of human cofilin 1 by X-ray crystallography to elucidate how it could interact with and regulate dynamic actin-cytoskeletal structures. Although wild-type human cofilin 1 proved to be recalcitrant, a C147A point mutant yielded crystals that diffracted to 2.8 Å resolution. These studies revealed how the actin-binding helix undergoes a conformational change that increases the number of potential hydrogen bonds available for substrate binding. PMID:23999301

  19. F actin bundles in Drosophila bristles. I. Two filament cross-links are involved in bundling

    PubMed Central

    1995-01-01

    Transverse sections though Drosophila bristles reveal 7-11 nearly round, plasma membrane-associated bundles of actin filaments. These filaments are hexagonally packed and in a longitudinal section they show a 12-nm periodicity in both the 1.1 and 1.0 views. From earlier studies this periodicity is attributable to cross-links and indicates that the filaments are maximally cross-linked, singed mutants also have 7-11 bundles, but the bundles are smaller, flattened, and the filaments within the bundles are randomly packed (not hexagonal); no periodicity can be detected in longitudinal sections. Another mutant, forked (f36a), also has 7-11 bundles but even though the bundles are very small, the filaments within them are hexagonally packed and display a 12-nm periodicity in longitudinal section. The singed-forked double mutant lacks filament bundles. Thus there are at least two species of cross-links between adjacent actin filaments. Hints of why two species of cross-links are necessary can be gleaned by studying bristle formation. Bristles sprout with only microtubules within them. A little later in development actin filaments appear. At early stages the filaments in the bundles are randomly packed. Later the filaments in the bundles become hexagonally packed and maximally cross-linked. We consider that the forked proteins may be necessary early in development to tie the filaments together in a bundle so that they can be subsequently zippered together by fascin (the singed gene product). PMID:7622563

  20. Direct dynamin–actin interactions regulate the actin cytoskeleton

    PubMed Central

    Gu, Changkyu; Yaddanapudi, Suma; Weins, Astrid; Osborn, Teresia; Reiser, Jochen; Pollak, Martin; Hartwig, John; Sever, Sanja

    2010-01-01

    The large GTPase dynamin assembles into higher order structures that are thought to promote endocytosis. Dynamin also regulates the actin cytoskeleton through an unknown, GTPase-dependent mechanism. Here, we identify a highly conserved site in dynamin that binds directly to actin filaments and aligns them into bundles. Point mutations in the actin-binding domain cause aberrant membrane ruffling and defective actin stress fibre formation in cells. Short actin filaments promote dynamin assembly into higher order structures, which in turn efficiently release the actin-capping protein (CP) gelsolin from barbed actin ends in vitro, allowing for elongation of actin filaments. Together, our results support a model in which assembled dynamin, generated through interactions with short actin filaments, promotes actin polymerization via displacement of actin-CPs. PMID:20935625

  1. In vivo dynamics of the F-actin-binding protein neurabin-II.

    PubMed Central

    Stephens, D J; Banting, G

    2000-01-01

    Neurabin-II (spinophilin) is a ubiquitously expressed F-actin-binding protein containing an N-terminal actin-binding domain, a PDZ (PSD95/discs large/ZO-1) domain and a C-terminal domain predicted to form a coiled-coil structure. We have stably expressed a green fluorescent protein (GFP)-tagged version of neurabin-II in PC12 cells, and characterized the in vivo dynamics of this actin-binding protein using confocal fluorescence microscopy. We show that GFP-neurabin-II localizes to actin filaments, especially at cortical sites and areas underlying sites of active membrane remodelling. GFP-neurabin-II labels only a subset of F-actin within these cells, as indicated by rhodamine-phalloidin staining. Both actin filaments and small, highly motile structures within the cell body are seen. Photobleaching experiments show that GFP-neurabin-II also exhibits highly dynamic behaviour when bound to actin filaments. Latrunculin B treatment results in rapid relocalization of GFP-neurabin-II to the cytosol, whereas cytochalasin D treatment causes the collapse of GFP-neurabin-II fluorescence to intensely fluorescent foci of F-actin within the cell body. This collapse is reversed on cytochalasin D removal, recovery from which is greatly accelerated by stimulation of cells with epidermal growth factor (EGF). Furthermore, we show that this EGF-induced relocalization of GFP-neurabin-II is dependent on the activity of the small GTPase Rac1 but not the activity of ADP-ribosylation factor 6. PMID:10620493

  2. Single-molecule visualization of a formin-capping protein ‘decision complex' at the actin filament barbed end

    PubMed Central

    Bombardier, Jeffrey P.; Eskin, Julian A.; Jaiswal, Richa; Corrêa, Ivan R.; Xu, Ming-Qun; Goode, Bruce L.; Gelles, Jeff

    2015-01-01

    Precise control of actin filament length is essential to many cellular processes. Formins processively elongate filaments, whereas capping protein (CP) binds to barbed ends and arrests polymerization. While genetic and biochemical evidence has indicated that these two proteins function antagonistically, the mechanism underlying the antagonism has remained unresolved. Here we use multi-wavelength single-molecule fluorescence microscopy to observe the fully reversible formation of a long-lived ‘decision complex' in which a CP dimer and a dimer of the formin mDia1 simultaneously bind the barbed end. Further, mDia1 displaced from the barbed end by CP can randomly slide along the filament and later return to the barbed end to re-form the complex. Quantitative kinetic analysis reveals that the CP-mDia1 antagonism that we observe in vitro occurs through the decision complex. Our observations suggest new molecular mechanisms for the control of actin filament length and for the capture of filament barbed ends in cells. PMID:26566078

  3. High Speed Depolymerization at Actin Filament Ends Jointly Catalyzed by Twinfilin and Srv2/CAP

    PubMed Central

    Johnston, Adam B.; Collins, Agnieszka; Goode, Bruce L.

    2015-01-01

    Purified actin filaments depolymerize slowly, and cytosolic conditions strongly favor actin assembly over disassembly, which has left our understanding of how actin filaments are rapidly turned over in vivo incomplete 1,2. One mechanism for driving filament disassembly is severing by factors such as Cofilin. However, even after severing, pointed end depolymerization remains slow and unable to fully account for observed rates of actin filament turnover in vivo. Here we describe a mechanism by which Twinfilin and Cyclase-associated protein work in concert to accelerate depolymerization of actin filaments by 3-fold and 17-fold at their barbed and pointed ends, respectively. This mechanism occurs even under assembly conditions, allowing reconstitution and direct visualization of individual filaments undergoing tunable, accelerated treadmilling. Further, we use specific mutations to demonstrate that this activity is critical for Twinfilin function in vivo. These findings fill a major gap in our knowledge of mechanisms, and suggest that depolymerization and severing may be deployed separately or together to control the dynamics and architecture of distinct actin networks. PMID:26458246

  4. Mechanisms of leiomodin 2-mediated regulation of actin filament in muscle cells

    PubMed Central

    Chen, Xiaorui; Ni, Fengyun; Kondrashkina, Elena; Ma, Jianpeng; Wang, Qinghua

    2015-01-01

    Leiomodin (Lmod) is a class of potent tandem-G-actin–binding nucleators in muscle cells. Lmod mutations, deletion, or instability are linked to lethal nemaline myopathy. However, the lack of high-resolution structures of Lmod nucleators in action severely hampered our understanding of their essential cellular functions. Here we report the crystal structure of the actin–Lmod2162–495 nucleus. The structure contains two actin subunits connected by one Lmod2162–495 molecule in a non–filament-like conformation. Complementary functional studies suggest that the binding of Lmod2 stimulates ATP hydrolysis and accelerates actin nucleation and polymerization. The high level of conservation among Lmod proteins in sequence and functions suggests that the mechanistic insights of human Lmod2 uncovered here may aid in a molecular understanding of other Lmod proteins. Furthermore, our structural and mechanistic studies unraveled a previously unrecognized level of regulation in mammalian signal transduction mediated by certain tandem-G-actin–binding nucleators. PMID:26417072

  5. Human endothelial actin-binding protein (ABP-280, nonmuscle filamin): a molecular leaf spring

    PubMed Central

    1990-01-01

    Actin-binding protein (ABP-280, nonmuscle filamin) is a ubiquitous dimeric actin cross-linking phosphoprotein of peripheral cytoplasm, where it promotes orthogonal branching of actin filaments and links actin filaments to membrane glycoproteins. The complete nucleotide sequence of human endothelial cell ABP cDNA predicts a polypeptide subunit chain of 2,647 amino acids, corresponding to 280 kD, also the mass derived from physical measurements of the native protein. The actin-binding domain is near the amino-terminus of the subunit where the amino acid sequence is similar to other actin filament binding proteins, including alpha-actinin, beta-spectrin, dystrophin, and Dictyostelium abp-120. The remaining 90% of the sequence comprises 24 repeats, each approximately 96 residues long, predicted to have stretches of beta-sheet secondary structure interspersed with turns. The first 15 repeats may have substantial intrachain hydrophobic interactions and overlap in a staggered fashion to yield a backbone with mechanical resilience. Sequence insertions immediately before repeats 16 and 24 predict two hinges in the molecule near points where rotary-shadowed molecules appear to swivel in electron micrographs. Both putative hinge regions are susceptible to cleavage by proteases and the second also contains the site that binds the platelet glycoprotein Ib/IX complex. Phosphorylation consensus sequences are also located in the hinges or near them. Degeneracy within every even- numbered repeat between 16 and 24 and the insertion before repeat 24 may convert interactions within chains to interactions between chains to account for dimer formation within a domain of 7 kD at the carboxy- terminus. The structure of ABP dimers resembles a leaf spring. Interchain interactions hold the leaves firmly together at one end, whereas intrachain hydrophobic bonds reinforce the arms of the spring where the leaves diverge, making it sufficiently stiff to promote high- angle branching of actin

  6. 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

  7. The molybdenum cofactor biosynthesis complex interacts with actin filaments via molybdenum insertase Cnx1 as anchor protein in Arabidopsis thaliana.

    PubMed

    Kaufholdt, David; Baillie, Christin-Kirsty; Bikker, Rolf; Burkart, Valentin; Dudek, Christian-Alexander; von Pein, Linn; Rothkegel, Martin; Mendel, Ralf R; Hänsch, Robert

    2016-03-01

    The pterin based molybdenum cofactor (Moco) plays an essential role in almost all organisms. Its biosynthesis is catalysed by six enzymes in a conserved four step reaction pathway. The last three steps are located in the cytoplasm, where a multimeric protein complex is formed to protect the intermediates from degradation. Bimolecular fluorescence complementation was used to test for cytoskeleton association of the Moco biosynthesis enzymes with actin filaments and microtubules using known cytoskeleton associated proteins, thus permitting non-invasive in vivo studies. Coding sequences of binding proteins were cloned via the GATEWAY system. No Moco biosynthesis enzyme showed any interaction with microtubules. However, alone the two domain protein Cnx1 exhibited interaction with actin filaments mediated by both domains with the Cnx1G domain displaying a stronger interaction. Cnx6 showed actin association only if unlabelled Cnx1 was co-expressed in comparable amounts. So Cnx1 is likely to be the anchor protein for the whole biosynthesis complex on actin filaments. A stabilization of the whole Moco biosynthesis complex on the cytoskeleton might be crucial. In addition a micro-compartmentation might either allow a localisation near the mitochondrial ATM3 exporter providing the first Moco intermediate or near one of the three molybdate transporters enabling efficient molybdate incorporation.

  8. Identification of an ATP-controlled allosteric switch that controls actin filament nucleation by Arp2/3 complex

    PubMed Central

    Rodnick-Smith, Max; Liu, Su-Ling; Balzer, Connor J.; Luan, Qing; Nolen, Brad J.

    2016-01-01

    Nucleation of branched actin filaments by Arp2/3 complex is tightly regulated to control actin assembly in cells. Arp2/3 complex activation involves conformational changes brought about by ATP, Nucleation Promoting Factor (NPF) proteins, actin filaments and NPF-recruited actin monomers. To understand how these factors promote activation, we must first understand how the complex is held inactive in their absence. Here we demonstrate that the Arp3 C-terminal tail is a structural switch that prevents Arp2/3 complex from adopting an active conformation. The interaction between the tail and a hydrophobic groove in Arp3 blocks movement of Arp2 and Arp3 into an activated filament-like (short pitch) conformation. Our data indicate ATP binding destabilizes this interaction via an allosteric link between the Arp3 nucleotide cleft and the hydrophobic groove, thereby promoting the short-pitch conformation. Our results help explain how Arp2/3 complex is locked in an inactive state without activators and how autoinhibition is relieved. PMID:27417392

  9. Identification of an ATP-controlled allosteric switch that controls actin filament nucleation by Arp2/3 complex.

    PubMed

    Rodnick-Smith, Max; Liu, Su-Ling; Balzer, Connor J; Luan, Qing; Nolen, Brad J

    2016-01-01

    Nucleation of branched actin filaments by Arp2/3 complex is tightly regulated to control actin assembly in cells. Arp2/3 complex activation involves conformational changes brought about by ATP, Nucleation Promoting Factor (NPF) proteins, actin filaments and NPF-recruited actin monomers. To understand how these factors promote activation, we must first understand how the complex is held inactive in their absence. Here we demonstrate that the Arp3 C-terminal tail is a structural switch that prevents Arp2/3 complex from adopting an active conformation. The interaction between the tail and a hydrophobic groove in Arp3 blocks movement of Arp2 and Arp3 into an activated filament-like (short pitch) conformation. Our data indicate ATP binding destabilizes this interaction via an allosteric link between the Arp3 nucleotide cleft and the hydrophobic groove, thereby promoting the short-pitch conformation. Our results help explain how Arp2/3 complex is locked in an inactive state without activators and how autoinhibition is relieved. PMID:27417392

  10. Identification of a new actin binding surface on vinculin that mediates mechanical cell and focal adhesion properties

    PubMed Central

    Thompson, Peter M.; Tolbert, Caitlin E.; Shen, Kai; Kota, Pradeep; Palmer, Sean M.; Plevock, Karen M.; Orlova, Albina; Galkin, Vitold E.; Burridge, Keith; Egelman, Edward H.; Dokholyan, Nikolay V.; Superfine, Richard; Campbell, Sharon L.

    2014-01-01

    SUMMARY Vinculin, a cytoskeletal scaffold protein essential for embryogenesis and cardiovascular function, localizes to focal adhesions and adherens junctions, connecting cell surface receptors to the actin cytoskeleton. While vinculin interacts with many adhesion proteins, its interaction with filamentous actin regulates cell morphology, motility, and mechanotransduction. Disruption of this interaction lowers cell traction forces and enhances actin flow rates. Although a model for the vinculin:actin complex exists, we recently identified actin-binding deficient mutants of vinculin outside sites predicted to bind actin, and developed an alternative model to better define this novel actin-binding surface, using negative-stain EM, discrete molecular dynamics, and mutagenesis. Actin-binding deficient vinculin variants expressed in vinculin knockout fibroblasts fail to rescue cell-spreading defects and reduce cellular response to external force. These findings highlight the importance of this new actin-binding surface and provide the molecular basis for elucidating additional roles of this interaction, including actin-induced conformational changes which promote actin bundling. PMID:24685146

  11. Actin-binding protein (ABP-280) filamin gene (FLN) maps telomeric to the color vision locus (R/GCP) and centromeric to G6PD in Xq28

    SciTech Connect

    Gorlin, J.B. Dana-Farber Cancer Institute, Boston, MA ); Henske, E.; Hartwig, J.H.; Kwiatkowski, D.J. ); Warren, S.T.; Kunst, C.B. ); D'Urso, M.; Palmieri, G. ); Bruns, G. )

    1993-08-01

    Actin-binding protein-280 (ABP-280) is a dimeric actin filament-crosslinking protein that promotes orthogonal branching of actin filaments and links actin filaments to membrane glycoproteins. The authors have mapped the ABP-280 filamin gene (FLN) to Xq28 by Southern blot analysis of somatic cell hybrid lines, by fluorescence in situ hybridization, and through identification of portions of the FLN gene within cosmids and YACs mapped to Xq28. The FLN gene is found within a 200-kb region centromeric to the G6PD locus and telomeric to DSX52 and the color vision locus. 23 refs., 2 figs.

  12. Enhancing the staggered fluctuations of an actin filament sliding on Chara myosin.

    PubMed

    Hatori, Kuniyuki; Okeno, Yusuke; Honda, Hajime; Shimada, Katsuhiko; Matsuno, Koichiro

    2004-06-01

    We examined both longitudinal and transversal fluctuations of displacements of an actin filament sliding upon Chara myosin molecules. Although the magnitude of transversal fluctuations remained rather independent of ATP concentration, the longitudinal ones were found to increase their magnitude as the concentration increased. In addition, the longitudinal fluctuations gradually increased as the sliding velocity of the filament increased.

  13. Intermonomer flexibility of Ca- and Mg-actin filaments at different pH values.

    PubMed

    Hild, Gábor; Nyitrai, Miklós; Somogyi, Béla

    2002-02-01

    The fluorescence resonance energy transfer parameter, f, is defined as the efficiency of the energy transfer normalized by the quantum yield of the donor in the presence of acceptor. It is possible to characterize the flexibility of the protein matrix between the appropriate fluorescent probes by monitoring the temperature dependence of f. The intermonomer flexibility of the Ca-actin and Mg-actin filaments was characterized by using this method at pH values of 6.5 and 7.4. The protomers were labeled on Cys374 with donor [N-(((iodoacetyl)amino)ethyl)-5-naphthylamine-1-sulfonate; IAEDANS] or acceptor [5-(iodoacetamido)fluorescein; IAF] molecules. The temperature profile of f suggested that the intermonomer flexibility of actin filaments was larger at pH 7.4 than pH 6.5 in the case of Mg-F-actin while this difference was absent in the case of Ca-F-actin. More rigid intermonomer connection was identified at both pH values between the protomers of Mg-F-actin compared to the Ca-F-actin. The results were further supported by time dependent fluorescence measurements made on IAEDANS and IAF labeled Mg- and Ca-actin filaments at pH 6.5 and 7.4. Our spectroscopic results may suggest that the altered function of muscle following the change of pH within the muscle cells under physiological or pathological conditions might be affected by the modified dynamic properties of the magnesium saturated actin filaments. The change of the intracellular pH does not have an effect on the intermonomer flexibility of the Ca-actin filaments.

  14. Structural and Functional Dissection of the Abp1 ADFH Actin-binding Domain Reveals Versatile In Vivo Adapter Functions

    SciTech Connect

    Quintero-Monzon,O.; Rodal, A.; Strokopytov, B.; Almo, S.; Goode, B.

    2005-01-01

    Abp1 is a multidomain protein that regulates the Arp2/3 complex and links proteins involved in endocytosis to the actin cytoskeleton. All of the proposed cellular functions of Abp1 involve actin filament binding, yet the actin binding site(s) on Abp1 have not been identified, nor has the importance of actin binding for Abp1 localization and function in vivo been tested. Here, we report the crystal structure of the Saccharomyces cerevisiae Abp1 actin-binding actin depolymerizing factor homology (ADFH) domain and dissect its activities by mutagenesis. Abp1-ADFH domain and ADF/cofilin structures are similar, and they use conserved surfaces to bind actin; however, there are also key differences that help explain their differential effects on actin dynamics. Using point mutations, we demonstrate that actin binding is required for localization of Abp1 in vivo, the lethality caused by Abp1 overexpression, and the ability of Abp1 to activate Arp2/3 complex. Furthermore, we genetically uncouple ABP1 functions that overlap with SAC6, SLA1, and SLA2, showing they require distinct combinations of activities and interactions. Together, our data provide the first structural and functional view of the Abp1-actin interaction and show that Abp1 has distinct cellular roles as an adapter, linking different sets of ligands for each function.

  15. In vitro and in vivo evidence for actin association of the naphthylphthalamic acid-binding protein from zucchini hypocotyls

    NASA Technical Reports Server (NTRS)

    Butler, J. H.; Hu, S.; Brady, S. R.; Dixon, M. W.; Muday, G. K.

    1998-01-01

    The N-1-naphthylphthalamic acid (NPA)-binding protein is part of the auxin efflux carrier, the protein complex that controls polar auxin transport in plant tissues. This study tested the hypothesis that the NPA-binding protein (NBP) is associated with the actin cytoskeleton in vitro and that an intact actin cytoskeleton is required for polar auxin transport in vivo. Cytoskeletal polymerization was altered in extracts of zucchini hypocotyls with reagents that stabilized either the polymeric or monomeric forms of actin or tubulin. Phalloidin treatment altered actin polymerization, as demonstrated by immunoblot analyses following native and denaturing electrophoresis. Phalloidin increased both filamentous actin (F-actin) and NPA-binding activity, while cytochalasin D and Tris decreased both F-actin and NPA-binding activity in cytoskeletal pellets. The microtubule stabilizing drug taxol increased pelletable tubulin, but did not alter either the amount of pelletable actin or NPA-binding activity. Treatment of etiolated zucchini hypocotyls with cytochalasin D decreased the amount of auxin transport and its regulation by NPA. These experimental results are consistent with an in vitro actin cytoskeletal association of the NPA-binding protein and with the requirement of an intact actin cytoskeleton for maximal polar auxin transport in vivo.

  16. Phosphatase and actin regulator 4 is associated with intermediate filaments in adult neural stem cells and their progenitor astrocytes.

    PubMed

    Cho, Hyo Min; Kim, Joo Yeon; Kim, Hyun; Sun, Woong

    2014-10-01

    Phosphatase and actin regulator 4 (Phactr4) is a newly discovered protein that inhibits protein phosphatase 1 and shows actin-binding activity. We previously found that Phactr4 is expressed in the neurogenic niche in adult mice, although its precise subcellular localization and possible function in neural stem cells (NSCs) is not yet understood. Here, we show that Phactr4 formed punctiform clusters in the cytosol of subventricular zone-derived adult NSCs and their progeny in vitro. These Phactr4 signals were not associated with F-actin fibers but were closely associated with intermediate filaments such as nestin and glial fibrillary acidic protein (GFAP) fibers. Direct binding of Phactr4 with nestin and GFAP filaments was demonstrated using Duolink protein interaction analyses and immunoprecipitation assays. Interestingly, when nestin fibers were de-polymerized during the mitosis or by the phosphatase inhibitor, Phactr4 appeared to be dissociated from nestin, suggesting that their protein interaction is regulated by the protein phosphorylation. These results suggest that Phactr4 forms functional associations with intermediate filament networks in adult NSCs.

  17. Three-dimensional architecture of actin filaments in Listeria monocytogenes comet tails

    PubMed Central

    Jasnin, Marion; Asano, Shoh; Gouin, Edith; Hegerl, Reiner; Plitzko, Jürgen M.; Villa, Elizabeth; Cossart, Pascale; Baumeister, Wolfgang

    2013-01-01

    The intracellular bacterial pathogen Listeria monocytogenes is capable of remodelling the actin cytoskeleton of its host cells such that “comet tails” are assembled powering its movement within cells and enabling cell-to-cell spread. We used cryo-electron tomography to visualize the 3D structure of the comet tails in situ at the level of individual filaments. We have performed a quantitative analysis of their supramolecular architecture revealing the existence of bundles of nearly parallel hexagonally packed filaments with spacings of 12–13 nm. Similar configurations were observed in stress fibers and filopodia, suggesting that nanoscopic bundles are a generic feature of actin filament assemblies involved in motility; presumably, they provide the necessary stiffness. We propose a mechanism for the initiation of comet tail assembly and two scenarios that occur either independently or in concert for the ensuing actin-based motility, both emphasizing the role of filament bundling. PMID:24306931

  18. Molecular dynamics simulation of a myosin subfragment-1 docking with an actin filament.

    PubMed

    Masuda, Tadashi

    2013-09-01

    Myosins are typical molecular motor proteins, which convert the chemical energy of ATP into mechanical work. The fundamental mechanism of this energy conversion is still unknown. To explain the experimental results observed in molecular motors, Masuda has proposed a theory called the "Driven by Detachment (DbD)" mechanism for the working principle of myosins. Based on this theory, the energy used during the power stroke of the myosins originates from the attractive force between a detached myosin head and an actin filament, and does not directly arise from the energy of ATP. According to this theory, every step in the myosin working process may be reproduced by molecular dynamics (MD) simulations, except for the ATP hydrolysis step. Therefore, MD simulations were conducted to reproduce the docking process of a myosin subfragment-1 (S1) against an actin filament. A myosin S1 directed toward the barbed end of an actin filament was placed at three different positions by shifting it away from the filament axis. After 30 ns of MD simulations, in three cases out of ten trials on average, the myosin made a close contact with two actin monomers by changing the positions and the orientation of both the myosin and the actin as predicted in previous studies. Once the docking was achieved, the distance between the myosin and the actin showed smaller fluctuations, indicating that the docking is stable over time. If the docking was not achieved, the myosin moved randomly around the initial position or moved away from the actin filament. MD simulations thus successfully reproduced the docking of a myosin S1 with an actin filament. By extending the similar MD simulations to the other steps of the myosin working process, the validity of the DbD theory may be computationally demonstrated.

  19. Myosin binding surface on actin probed by hydroxyl radical footprinting and site-directed labels.

    PubMed

    Oztug Durer, Zeynep A; Kamal, J K Amisha; Benchaar, Sabrina; Chance, Mark R; Reisler, Emil

    2011-11-25

    Actin and myosin are the two main proteins required for cell motility and muscle contraction. The structure of their strongly bound complex-rigor state-is a key for delineating the functional mechanism of actomyosin motor. Current knowledge of that complex is based on models obtained from the docking of known atomic structures of actin and myosin subfragment 1 (S1; the head and neck region of myosin) into low-resolution electron microscopy electron density maps, which precludes atomic- or side-chain-level information. Here, we use radiolytic protein footprinting for global mapping of sites across the actin molecules that are impacted directly or allosterically by myosin binding to actin filaments. Fluorescence and electron paramagnetic resonance spectroscopies and cysteine actin mutants are used for independent, residue-specific probing of S1 effects on two structural elements of actin. We identify actin residue candidates involved in S1 binding and provide experimental evidence to discriminate between the regions of hydrophobic and electrostatic interactions. Focusing on the role of the DNase I binding loop (D-loop) and the W-loop residues of actin in their interactions with S1, we found that the emission properties of acrylodan and the mobility of electron paramagnetic resonance spin labels attached to cysteine mutants of these residues change strongly and in a residue-specific manner upon S1 binding, consistent with the recently proposed direct contacts of these loops with S1. As documented in this study, the direct and indirect changes on actin induced by myosin are more extensive than known until now and attest to the importance of actin dynamics to actomyosin function. PMID:21986200

  20. Actin binding proteins, spermatid transport and spermiation.

    PubMed

    Qian, Xiaojing; Mruk, Dolores D; Cheng, Yan-Ho; Tang, Elizabeth I; Han, Daishu; Lee, Will M; Wong, Elissa W P; Cheng, C Yan

    2014-06-01

    The transport of germ cells across the seminiferous epithelium is composed of a series of cellular events during the epithelial cycle essential to the completion of spermatogenesis. Without the timely transport of spermatids during spermiogenesis, spermatozoa that are transformed from step 19 spermatids in the rat testis fail to reach the luminal edge of the apical compartment and enter the tubule lumen at spermiation, thereby arriving the epididymis for further maturation. Step 19 spermatids and/or sperms that remain in the epithelium beyond stage VIII of the epithelial cycle will be removed by the Sertoli cell via phagocytosis to form phagosomes and be degraded by lysosomes, leading to subfertility and/or infertility. However, the biology of spermatid transport, in particular the final events that lead to spermiation remain elusive. Based on recent data in the field, we critically evaluate the biology of spermiation herein by focusing on the actin binding proteins (ABPs) that regulate the organization of actin microfilaments at the Sertoli-spermatid interface, which is crucial for spermatid transport during this event. The hypothesis we put forth herein also highlights some specific areas of research that can be pursued by investigators in the years to come.

  1. Actin filament turnover drives leading edge growth during myelin sheath formation in the central nervous system.

    PubMed

    Nawaz, Schanila; Sánchez, Paula; Schmitt, Sebastian; Snaidero, Nicolas; Mitkovski, Mišo; Velte, Caroline; Brückner, Bastian R; Alexopoulos, Ioannis; Czopka, Tim; Jung, Sang Y; Rhee, Jeong S; Janshoff, Andreas; Witke, Walter; Schaap, Iwan A T; Lyons, David A; Simons, Mikael

    2015-07-27

    During CNS development, oligodendrocytes wrap their plasma membrane around axons to generate multilamellar myelin sheaths. To drive growth at the leading edge of myelin at the interface with the axon, mechanical forces are necessary, but the underlying mechanisms are not known. Using an interdisciplinary approach that combines morphological, genetic, and biophysical analyses, we identified a key role for actin filament network turnover in myelin growth. At the onset of myelin biogenesis, F-actin is redistributed to the leading edge, where its polymerization-based forces push out non-adhesive and motile protrusions. F-actin disassembly converts protrusions into sheets by reducing surface tension and in turn inducing membrane spreading and adhesion. We identified the actin depolymerizing factor ADF/cofilin1, which mediates high F-actin turnover rates, as an essential factor in this process. We propose that F-actin turnover is the driving force in myelin wrapping by regulating repetitive cycles of leading edge protrusion and spreading.

  2. Yeast actin filaments display ATP-dependent sliding movement over surfaces coated with rabbit muscle myosin.

    PubMed Central

    Kron, S J; Drubin, D G; Botstein, D; Spudich, J A

    1992-01-01

    The yeast Saccharomyces cerevisiae has been used to study the function of components of the actin cytoskeleton in vivo, mainly because it is easy to derive and characterize mutations affecting these proteins. In contrast, biochemical studies have generally used proteins derived from higher eukaryotes. We have devised a simple procedure to prepare, in high yield, homogeneous native actin from wild-type and act1 mutant yeast. Using intensified video fluorescence microscopy, we found that actin filaments polymerized from these preparations exhibit ATP-dependent sliding movement over surfaces coated with rabbit skeletal muscle myosin. The rates of sliding movement of the wild-type and mutant yeast actins were each about half that of rabbit skeletal muscle actin under similar conditions. We conclude that over the large evolutionary distance between yeast and mammals there has been significant conservation of actin function, specifically the ability to be moved by interaction with myosin. Images PMID:1533933

  3. The N-terminal tropomyosin- and actin-binding sites are important for leiomodin 2’s function

    PubMed Central

    Ly, Thu; Moroz, Natalia; Pappas, Christopher T.; Novak, Stefanie M.; Tolkatchev, Dmitri; Wooldridge, Dayton; Mayfield, Rachel M.; Helms, Gregory; Gregorio, Carol C.; Kostyukova, Alla S.

    2016-01-01

    Leiomodin is a potent actin nucleator related to tropomodulin, a capping protein localized at the pointed end of the thin filaments. Mutations in leiomodin-3 are associated with lethal nemaline myopathy in humans, and leiomodin-2–knockout mice present with dilated cardiomyopathy. The arrangement of the N-terminal actin- and tropomyosin-binding sites in leiomodin is contradictory and functionally not well understood. Using one-dimensional nuclear magnetic resonance and the pointed-end actin polymerization assay, we find that leiomodin-2, a major cardiac isoform, has an N-terminal actin-binding site located within residues 43–90. Moreover, for the first time, we obtain evidence that there are additional interactions with actin within residues 124–201. Here we establish that leiomodin interacts with only one tropomyosin molecule, and this is the only site of interaction between leiomodin and tropomyosin. Introduction of mutations in both actin- and tropomyosin-binding sites of leiomodin affected its localization at the pointed ends of the thin filaments in cardiomyocytes. On the basis of our new findings, we propose a model in which leiomodin regulates actin poly­merization dynamics in myocytes by acting as a leaky cap at thin filament pointed ends. PMID:27307584

  4. The N-terminal tropomyosin- and actin-binding sites are important for leiomodin 2's function.

    PubMed

    Ly, Thu; Moroz, Natalia; Pappas, Christopher T; Novak, Stefanie M; Tolkatchev, Dmitri; Wooldridge, Dayton; Mayfield, Rachel M; Helms, Gregory; Gregorio, Carol C; Kostyukova, Alla S

    2016-08-15

    Leiomodin is a potent actin nucleator related to tropomodulin, a capping protein localized at the pointed end of the thin filaments. Mutations in leiomodin-3 are associated with lethal nemaline myopathy in humans, and leiomodin-2-knockout mice present with dilated cardiomyopathy. The arrangement of the N-terminal actin- and tropomyosin-binding sites in leiomodin is contradictory and functionally not well understood. Using one-dimensional nuclear magnetic resonance and the pointed-end actin polymerization assay, we find that leiomodin-2, a major cardiac isoform, has an N-terminal actin-binding site located within residues 43-90. Moreover, for the first time, we obtain evidence that there are additional interactions with actin within residues 124-201. Here we establish that leiomodin interacts with only one tropomyosin molecule, and this is the only site of interaction between leiomodin and tropomyosin. Introduction of mutations in both actin- and tropomyosin-binding sites of leiomodin affected its localization at the pointed ends of the thin filaments in cardiomyocytes. On the basis of our new findings, we propose a model in which leiomodin regulates actin poly-merization dynamics in myocytes by acting as a leaky cap at thin filament pointed ends.

  5. The N-terminal tropomyosin- and actin-binding sites are important for leiomodin 2's function.

    PubMed

    Ly, Thu; Moroz, Natalia; Pappas, Christopher T; Novak, Stefanie M; Tolkatchev, Dmitri; Wooldridge, Dayton; Mayfield, Rachel M; Helms, Gregory; Gregorio, Carol C; Kostyukova, Alla S

    2016-08-15

    Leiomodin is a potent actin nucleator related to tropomodulin, a capping protein localized at the pointed end of the thin filaments. Mutations in leiomodin-3 are associated with lethal nemaline myopathy in humans, and leiomodin-2-knockout mice present with dilated cardiomyopathy. The arrangement of the N-terminal actin- and tropomyosin-binding sites in leiomodin is contradictory and functionally not well understood. Using one-dimensional nuclear magnetic resonance and the pointed-end actin polymerization assay, we find that leiomodin-2, a major cardiac isoform, has an N-terminal actin-binding site located within residues 43-90. Moreover, for the first time, we obtain evidence that there are additional interactions with actin within residues 124-201. Here we establish that leiomodin interacts with only one tropomyosin molecule, and this is the only site of interaction between leiomodin and tropomyosin. Introduction of mutations in both actin- and tropomyosin-binding sites of leiomodin affected its localization at the pointed ends of the thin filaments in cardiomyocytes. On the basis of our new findings, we propose a model in which leiomodin regulates actin poly-merization dynamics in myocytes by acting as a leaky cap at thin filament pointed ends. PMID:27307584

  6. Emerging roles of sumoylation in the regulation of actin, microtubules, intermediate filaments, and septins.

    PubMed

    Alonso, Annabel; Greenlee, Matt; Matts, Jessica; Kline, Jake; Davis, Kayla J; Miller, Rita K

    2015-07-01

    Sumoylation is a powerful regulatory system that controls many of the critical processes in the cell, including DNA repair, transcriptional regulation, nuclear transport, and DNA replication. Recently, new functions for SUMO have begun to emerge. SUMO is covalently attached to components of each of the four major cytoskeletal networks, including microtubule-associated proteins, septins, and intermediate filaments, in addition to nuclear actin and actin-regulatory proteins. However, knowledge of the mechanisms by which this signal transduction system controls the cytoskeleton is still in its infancy. One story that is beginning to unfold is that SUMO may regulate the microtubule motor protein dynein by modification of its adaptor Lis1. In other instances, cytoskeletal elements can both bind to SUMO non-covalently and also be conjugated by it. The molecular mechanisms for many of these new functions are not yet clear, but are under active investigation. One emerging model links the function of MAP sumoylation to protein degradation through SUMO-targeted ubiquitin ligases, also known as STUbL enzymes. Other possible functions for cytoskeletal sumoylation are also discussed.

  7. Transport of ER vesicles on actin filaments in neurons by myosin V.

    PubMed

    Tabb, J S; Molyneaux, B J; Cohen, D L; Kuznetsov, S A; Langford, G M

    1998-11-01

    Axoplasmic organelles in the giant axon of the squid have been shown to move on both actin filaments and microtubules and to switch between actin filaments and microtubules during fast axonal transport. The objectives of this investigation were to identify the specific classes of axoplasmic organelles that move on actin filaments and the myosin motors involved. We developed a procedure to isolate endoplasmic reticulum (ER) from extruded axoplasm and to reconstitute its movement in vitro. The isolated ER vesicles moved on exogenous actin filaments adsorbed to coverslips in an ATP-dependent manner without the addition of soluble factors. Therefore myosin was tightly bound and not extracted during isolation. These vesicles were identified as smooth ER by use of an antibody to an ER-resident protein, ERcalcistorin/protein disulfide isomerase (EcaSt/PDI). Furthermore, an antibody to squid myosin V was used in immunogold EM studies to show that myosin V localized to these vesicles. The antibody was generated to a squid brain myosin (p196) that was classified as myosin V based on comparisons of amino acid sequences of tryptic peptides of this myosin with those of other known members of the myosin V family. Dual labeling with the squid myosin V antibody and a kinesin heavy chain antibody showed that the two motors colocalized on the same vesicles. Finally, antibody inhibition experiments were performed with two myosin V-specific antibodies to show that myosin V motor activity is required for transport of vesicles on actin filaments in axoplasm. One antibody was made to a peptide in the globular tail domain and the other to the globular head fragment of myosin V. Both antibodies inhibited vesicle transport on actin filaments by greater than 90% compared to controls. These studies provide the first direct evidence that ER vesicles are transported on actin filaments by myosin V. These data confirm the role of actin filaments in fast axonal transport and provide support for

  8. A mechanical model for the motility of actin filaments on myosin

    NASA Astrophysics Data System (ADS)

    Nicolau, Dan V., Jr.; Fulga, Florin; Nicolau, Dan V.

    2004-03-01

    The interaction of actin filaments with myosin is crucial to cell motility, muscular contraction, cell division and other processes. The in vitro motility assay involves the motion of actin filaments on a substrate coated with myosin, and is used extensively to investigate the dynamics of the actomyosin system. Following on from previous work, we propose a new mechanical model of actin motility on myosin, wherein a filament is modeled as a chain of beads connected by harmonic springs. This imposes a limitation on the "stretching" of the filament. The rotation of one bead with respect to its neighbours is also constrained in similar way. We implemented this model and used Monte Carlo simulations to determine whether it can predict the directionality of filament motion. The principal advantages of this model over our previous one are that we have removed the empirically correct but artificial assumption that the filament moves like a "worm" i.e. the head determines the direction of movement and the rest of the filament "follows" the head as well as the inclusion of dependencies on experimental rate constants (and so also on e.g. ATP concentration) via the cross-bridge cycle.

  9. Single-filament kinetic studies provide novel insights into regulation of actin-based motility

    PubMed Central

    Shekhar, Shashank; Carlier, Marie-France

    2016-01-01

    Polarized assembly of actin filaments forms the basis of actin-based motility and is regulated both spatially and temporally. Cells use a variety of mechanisms by which intrinsically slower processes are accelerated, and faster ones decelerated, to match rates observed in vivo. Here we discuss how kinetic studies of individual reactions and cycles that drive actin remodeling have provided a mechanistic and quantitative understanding of such processes. We specifically consider key barbed-end regulators such as capping protein and formins as illustrative examples. We compare and contrast different kinetic approaches, such as the traditional pyrene-polymerization bulk assays, as well as more recently developed single-filament and single-molecule imaging approaches. Recent development of novel biophysical methods for sensing and applying forces will in future allow us to address the very important relationship between mechanical stimulus and kinetics of actin-based motility. PMID:26715420

  10. Red light, Phot1 and JAC1 modulate Phot2-dependent reorganization of chloroplast actin filaments and chloroplast avoidance movement.

    PubMed

    Ichikawa, Satoshi; Yamada, Noboru; Suetsugu, Noriyuki; Wada, Masamitsu; Kadota, Akeo

    2011-08-01

    The phototropin (phot)-dependent intracellular relocation of chloroplasts is a ubiquitous phenomenon in plants. We have previously revealed the involvement of a short cp-actin (chloroplast actin) filament-based mechanism in this movement. Here, the reorganization of cp-actin filaments during the avoidance movement of chloroplasts was analyzed in higher time resolution under blue GFP (green fluorescent protein) excitation light in an actin filament-visualized line of Arabidopsis thaliana. Under standard background red light of 89 μmol m(-2) s(-1), cp-actin filaments transiently disappeared at approximately 30 s and reappeared in a biased configuration on chloroplasts approximately 70 s after blue excitation light irradiation. The timing of biased cp-actin reappearance was delayed under the background of strong red light or in the absence of red light. Consistently, chloroplast movement was delayed under these conditions. In phot1 mutants, acceleration of both the disappearance and reappearance of cp-actin filaments occurred, indicating an inhibitory action of phot1 on reorganization of cp-actin filaments. Avoidance movements began sooner in phot1 than in wild-type plants. No reorganization of cp-actin filaments was seen in phot2 or phot1phot2 mutants lacking phot2, which is responsible for avoidance movements. Surprisingly, jac1 (j-domain protein required for chloroplast accumulation response 1) mutants, lacking the accumulation response, showed no avoidance movements under the whole-cell irradiation condition for GFP observation. Cp-actin filaments in jac1 did not show a biased distribution, with a small or almost no transient decrease in the number. These results indicate a close association between the biased distribution of cp-actin filaments and chloroplast movement. Further, JAC1 is suggested to function in the biased cp-actin filament distribution by regulating their appearance and disappearance.

  11. Phosphorylation of platelet actin-binding protein during platelet activation

    SciTech Connect

    Carroll, R.C.; Gerrard, J.M.

    1982-03-01

    In this study we have followed the 32P-labeling of actin-binding protein as a function of platelet activation. Utilizing polyacrylamide-sodium dodecyl sulfate gel electrophoresis to resolve total platelet protein samples, we found 2 to 3-fold labeling increases in actin-binding protein 30 to 60 sec after thrombin stimulation. Somewhat larger increases were observed for 40,000 and 20,000 apparent molecular weight peptides. The actin-binding protein was identified on the gels by coelectrophoresis with purified actin-binding protein, its presence in cytoskeletal cores prepared by detergent extraction of activated 32P-labeled platelets, and by direct immunoprecipitation with antibodies against guinea pig vas deferens filamin (actin-binding protein). In addition, these cytoskeletal cores indicated that the 32P-labeled actin-binding protein was closely associated with the activated platelet's cytoskeleton. Following the 32P-labeling of actin-binding protein over an 8-min time course revealed that in aggregating platelet samples rapid dephosphorylation to almost initial levels occurred between 3 and 5 min. A similar curve was obtained for the 20,000 apparent molecular weight peptide. However, rapid dephosphorylation was not observed if platelet aggregation was prevented by chelating external calcium or by using thrombasthenic platelets lacking the aggregation response. Thus, cell-cell contact would seem to be crucial in initiating the rapid dephosphorylation response.

  12. [Congenital myopathies - skeletal muscle diseases related to disorder of actin filament structure and functions].

    PubMed

    Robaszkiewicz, Katarzyna; Moraczewska, Joanna

    2011-01-01

    Congenital myopathies are clinically and genetically heterogeneous disorders characterized by muscle structural abnormalities, muscle weakness and deformities. The clinical spectrum of the disease ranges from severe cases with early death to adult-onset cases with slow progression. In the skeletal muscle fibers, the specific structural changes are rod-shaped structures present in the sarcoplasm (nemaline myopathy – NM) or nuclei (intranuclear rod myopathy – IRM), cap-like structures peripherally located within muscle fibers (cap disease – CD), accumulations of actin filaments (actin myopathy – AM), changes in the fiber type proportion and size (congenital fiber type disproportion – CFTD), irregularity of Z-lines and abnormal localization of myofiber nuclei. Mutations in several genes encoding muscle proteins have been linked to congenital myopathy. These genes include a-skeletal actin (ACTA1), tropomyosin (TPM2 and TPM3), troponin (TNNT1) and nebulin (NEB). In vitro and in vivo studies show that mutations identified within these genes have varying impacts on thin filament protein structure, which affect polymerization and stabilization of actin filament, actin cellular localization and regulation of actin-myosin activity. Many lines of evidence suggest that mutated proteins have "toxic" effects. Unfortunately, there is no existing simple correlation between the degree of protein disruption, muscle pathologies and disease severity. PMID:21677359

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

    PubMed

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

    2015-06-16

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

  14. Novel actin filaments from Bacillus thuringiensis form nanotubules for plasmid DNA segregation

    PubMed Central

    Jiang, Shimin; Narita, Akihiro; Popp, David; Ghoshdastider, Umesh; Lee, Lin Jie; Srinivasan, Ramanujam; Balasubramanian, Mohan K.; Oda, Toshiro; Koh, Fujiet; Larsson, Mårten; Robinson, Robert C.

    2016-01-01

    Here we report the discovery of a bacterial DNA-segregating actin-like protein (BtParM) from Bacillus thuringiensis, which forms novel antiparallel, two-stranded, supercoiled, nonpolar helical filaments, as determined by electron microscopy. The BtParM filament features of supercoiling and forming antiparallel double-strands are unique within the actin fold superfamily, and entirely different to the straight, double-stranded, polar helical filaments of all other known ParMs and of eukaryotic F-actin. The BtParM polymers show dynamic assembly and subsequent disassembly in the presence of ATP. BtParR, the DNA-BtParM linking protein, stimulated ATP hydrolysis/phosphate release by BtParM and paired two supercoiled BtParM filaments to form a cylinder, comprised of four strands with inner and outer diameters of 57 Å and 145 Å, respectively. Thus, in this prokaryote, the actin fold has evolved to produce a filament system with comparable features to the eukaryotic chromosome-segregating microtubule. PMID:26873105

  15. Spatial Localisation of Actin Filaments across Developmental Stages of the Malaria Parasite

    PubMed Central

    Angrisano, Fiona; Delves, Michael J.; Zuccala, Elizabeth S.; Turnbull, Lynne; Dekiwadia, Chaitali; Olshina, Maya A.; Marapana, Danushka S.; Wong, Wilson; Mollard, Vanessa; Bradin, Clare H.; Tonkin, Christopher J.; Gunning, Peter W.; Ralph, Stuart A.; Whitchurch, Cynthia B.; Sinden, Robert E.; Cowman, Alan F.; McFadden, Geoffrey I.; Baum, Jake

    2012-01-01

    Actin dynamics have been implicated in a variety of developmental processes during the malaria parasite lifecycle. Parasite motility, in particular, is thought to critically depend on an actomyosin motor located in the outer pellicle of the parasite cell. Efforts to understand the diverse roles actin plays have, however, been hampered by an inability to detect microfilaments under native conditions. To visualise the spatial dynamics of actin we generated a parasite-specific actin antibody that shows preferential recognition of filamentous actin and applied this tool to different lifecycle stages (merozoites, sporozoites and ookinetes) of the human and mouse malaria parasite species Plasmodium falciparum and P. berghei along with tachyzoites from the related apicomplexan parasite Toxoplasma gondii. Actin filament distribution was found associated with three core compartments: the nuclear periphery, pellicular membranes of motile or invasive parasite forms and in a ring-like distribution at the tight junction during merozoite invasion of erythrocytes in both human and mouse malaria parasites. Localisation at the nuclear periphery is consistent with an emerging role of actin in facilitating parasite gene regulation. During invasion, we show that the actin ring at the parasite-host cell tight junction is dependent on dynamic filament turnover. Super-resolution imaging places this ring posterior to, and not concentric with, the junction marker rhoptry neck protein 4. This implies motor force relies on the engagement of dynamic microfilaments at zones of traction, though not necessarily directly through receptor-ligand interactions at sites of adhesion during invasion. Combined, these observations extend current understanding of the diverse roles actin plays in malaria parasite development and apicomplexan cell motility, in particular refining understanding on the linkage of the internal parasite gliding motor with the extra-cellular milieu. PMID:22389687

  16. Flexural rigidity of microtubules and actin filaments measured from thermal fluctuations in shape

    PubMed Central

    1993-01-01

    Microtubules are long, proteinaceous filaments that perform structural functions in eukaryotic cells by defining cellular shape and serving as tracks for intracellular motor proteins. We report the first accurate measurements of the flexural rigidity of microtubules. By analyzing the thermally driven fluctuations in their shape, we estimated the mean flexural rigidity of taxol-stabilized microtubules to be 2.2 x 10(-23) Nm2 (with 6.4% uncertainty) for seven unlabeled microtubules and 2.1 x 10(-23) Nm2 (with 4.7% uncertainty) for eight rhodamine-labeled microtubules. These values are similar to earlier, less precise estimates of microtubule bending stiffness obtained by modeling flagellar motion. A similar analysis on seven rhodamine-phalloidin- labeled actin filaments gave a flexural rigidity of 7.3 x 10(-26) Nm2 (with 6% uncertainty), consistent with previously reported results. The flexural rigidity of these microtubules corresponds to a persistence length of 5,200 microns showing that a microtubule is rigid over cellular dimensions. By contrast, the persistence length of an actin filament is only approximately 17.7 microns, perhaps explaining why actin filaments within cells are usually cross-linked into bundles. The greater flexural rigidity of a microtubule compared to an actin filament mainly derives from the former's larger cross-section. If tubulin were homogeneous and isotropic, then the microtubule's Young's modulus would be approximately 1.2 GPa, similar to Plexiglas and rigid plastics. Microtubules are expected to be almost inextensible: the compliance of cells is due primarily to filament bending or sliding between filaments rather than the stretching of the filaments themselves. PMID:8432732

  17. The Disruption of the Cytoskeleton during Semaphorin 3A induced Growth Cone Collapse Correlates with Differences in Actin Organization and Associated Binding Proteins

    PubMed Central

    Brown, Jacquelyn A; Bridgman, Paul C

    2010-01-01

    Repulsive guidance cues induce growth cone collapse or collapse and retraction. Collapse results from disruption and loss of the actin cytoskeleton. Actin rich regions of growth cones contain binding proteins that influence filament organization, such as Arp2/3, cortactin, and fascin, but little is known about the role that these proteins play in collapse. Here we show that Semaphorin 3A (Sema 3A), which is repulsive to mouse dorsal root ganglion neurons, has unequal effects on actin binding proteins and their associated filaments. The immunofluorescence staining intensity of Arp-2 and cortactin decreases relative to total protein, while in unextracted growth cones fascin increases. Fascin and myosin IIB staining redistribute and show increased overlap. The degree of actin filament loss during collapse correlates with filament superstructures detected by rotary shadow electron microscopy. Collapse results in the loss of branched f-actin meshworks, while actin bundles are partially retained to varying degrees. Taken together with the known affects of Sema 3A on actin, this suggests a model for collapse that follows a sequence; depolymerization of actin meshworks followed by partial depolymerization of fascin associated actin bundles and their movement to the neurite to complete collapse. The relocated fascin associated actin bundles may provide the substrate for actomyosin contractions that produce retraction. PMID:19513995

  18. Actin Filaments at the Leading Edge of Cancer Cells Are Characterized by a High Mobile Fraction and Turnover Regulation by Profilin I

    PubMed Central

    Lorente, Gisela; Syriani, Emilio; Morales, Miguel

    2014-01-01

    Cellular motility is the basis for cancer cell invasion and metastasis. In the case of breast cancer, the most common type of cancer among women, metastasis represents the most devastating stage of the disease. The central role of cellular motility in cancer development emphasizes the importance of understanding the specific mechanisms involved in this process. In this context, tumor development and metastasis would be the consequence of a loss or defect of the mechanisms that control cytoskeletal remodeling. Profilin I belongs to a family of small actin binding proteins that are thought to assist in actin filament elongation at the leading edge of migrating cells. Traditionally, Profilin I has been considered to be an essential control element for actin polymerization and cell migration. Expression of Profilin I is down-regulated in breast and various other cancer cells. In MDA-MB-231 cells, a breast cancer cell line, further inhibition of Profilin I expression promotes hypermotility and metastatic spread, a finding that contrasts with the proposed role of Profilin in enhancing polymerization. In this report, we have taken advantage of the fluorescence recovery after photobleaching (FRAP) of GFP-actin to quantify and compare actin dynamics at the leading edge level in both cancer and non-cancer cell models. Our results suggest that (i) a high level of actin dynamics (i.e., a large mobile fraction of actin filaments and a fast turnover) is a common characteristic of some cancer cells; (ii) actin polymerization shows a high degree of independence from the presence of extracellular growth factors; and (iii) our results also corroborate the role of Profilin I in regulating actin polymerization, as raising the intracellular levels of Profilin I decreased the mobile fraction ratio of actin filaments and slowed their polymerization rate; furthermore, increased Profilin levels also led to reduced individual cell velocity and directionality. PMID:24465723

  19. The structural basis of actin filament branching by the Arp2/3 complex

    PubMed Central

    Rouiller, Isabelle; Xu, Xiao-Ping; Amann, Kurt J.; Egile, Coumaran; Nickell, Stephan; Nicastro, Daniela; Li, Rong; Pollard, Thomas D.; Volkmann, Niels; Hanein, Dorit

    2008-01-01

    The actin-related protein 2/3 (Arp2/3) complex mediates the formation of branched actin filaments at the leading edge of motile cells and in the comet tails moving certain intracellular pathogens. Crystal structures of the Arp2/3 complex are available, but the architecture of the junction formed by the Arp2/3 complex at the base of the branch was not known. In this study, we use electron tomography to reconstruct the branch junction with sufficient resolution to show how the Arp2/3 complex interacts with the mother filament. Our analysis reveals conformational changes in both the mother filament and Arp2/3 complex upon branch formation. The Arp2 and Arp3 subunits reorganize into a dimer, providing a short-pitch template for elongation of the daughter filament. Two subunits of the mother filament undergo conformational changes that increase stability of the branch. These data provide a rationale for why branch formation requires cooperative interactions among the Arp2/3 complex, nucleation-promoting factors, an actin monomer, and the mother filament. PMID:18316411

  20. The structural basis of actin filament branching by the Arp2/3 complex.

    PubMed

    Rouiller, Isabelle; Xu, Xiao-Ping; Amann, Kurt J; Egile, Coumaran; Nickell, Stephan; Nicastro, Daniela; Li, Rong; Pollard, Thomas D; Volkmann, Niels; Hanein, Dorit

    2008-03-10

    The actin-related protein 2/3 (Arp2/3) complex mediates the formation of branched actin filaments at the leading edge of motile cells and in the comet tails moving certain intracellular pathogens. Crystal structures of the Arp2/3 complex are available, but the architecture of the junction formed by the Arp2/3 complex at the base of the branch was not known. In this study, we use electron tomography to reconstruct the branch junction with sufficient resolution to show how the Arp2/3 complex interacts with the mother filament. Our analysis reveals conformational changes in both the mother filament and Arp2/3 complex upon branch formation. The Arp2 and Arp3 subunits reorganize into a dimer, providing a short-pitch template for elongation of the daughter filament. Two subunits of the mother filament undergo conformational changes that increase stability of the branch. These data provide a rationale for why branch formation requires cooperative interactions among the Arp2/3 complex, nucleation-promoting factors, an actin monomer, and the mother filament.

  1. Phosphorylation of tropomodulin1 contributes to the regulation of actin filament architecture in cardiac muscle

    PubMed Central

    Bliss, Katherine T.; Tsukada, Takehiro; Novak, Stefanie Mares; Dorovkov, Maxim V.; Shah, Samar P.; Nworu, Chinedu; Kostyukova, Alla S.; Gregorio, Carol C.

    2014-01-01

    Tropomodulin1 (Tmod1) is an actin-capping protein that plays an important role in actin filament pointed-end dynamics and length in striated muscle. No mechanisms have been identified to explain how Tmod1's functional properties are regulated. The purpose of this investigation was to explore the functional significance of the phosphorylation of Tmod1 at previously identified Thr54. Rat cardiomyocytes were assessed for phosphorylation of Tmod1 using Pro-Q Diamond staining and 32P labeling. Green fluorescent protein-tagged phosphorylation-mimic (T54E) and phosphorylation-deficient (T54A) versions of Tmod1 were expressed in cultured cardiomyocytes, and the ability of these mutants to assemble and restrict actin lengths was observed. We report for the first time that Tmod1 is phosphorylated endogenously in cardiomyocytes, and phosphorylation at Thr54 causes a significant reduction in the ability of Tmod1 to assemble to the pointed end compared with that of the wild type (WT; 48 vs. 78%, respectively). In addition, overexpression of Tmod1-T54E restricts actin filament lengths by only ∼3%, whereas Tmod1-WT restricts the lengths significantly by ∼8%. Finally, Tmod1-T54E altered the actin filament-capping activity in polymerization assays. Taken together, our data suggest that pointed-end assembly and Tmod1's thin filament length regulatory function are regulated by its phosphorylation state.—Bliss, K. T., Tsukada, T., Novak, S. M., Dorovkov, M. V., Shah, S. P., Nworu, C., Kostyukova, A. S., Gregorio, C. C. Phosphorylation of tropomodulin1 contributes to the regulation of actin filament architecture in cardiac muscle. PMID:24891520

  2. Drosophila myosin-XX functions as an actin-binding protein to facilitate the interaction between Zyx102 and actin.

    PubMed

    Cao, Yang; White, Howard D; Li, Xiang-Dong

    2014-01-21

    The class XX myosin is a member of the diverse myosin superfamily and exists in insects and several lower invertebrates. DmMyo20, the class XX myosin in Drosophila, is encoded by dachs, which functions as a crucial downstream component of the Fat signaling pathway, influencing growth, affinity, and gene expression during development. Sequence analysis shows that DmMyo20 contains a unique N-terminal extension, the motor domain, followed by one IQ motif, and a C-terminal tail. To investigate the biochemical properties of DmMyo20, we expressed several DmMyo20 truncated constructs containing the motor domain in the baculovirus/Sf9 system. We found that the motor domain of DmMyo20 had neither ATPase activity nor the ability to bind to ATP, suggesting that DmMyo20 does not function as a molecular motor. We found that the motor domain of DmMyo20 could specifically bind to actin filaments in an ATP-independent manner and enhance the interaction between actin filaments and Zyx102, a downstream component of DmMyo20 in the Fat signaling pathway. These results suggest that DmMyo20 functions as a scaffold protein, but not as a molecular motor, in a signaling pathway controlling cell differentiation.

  3. Actin filament tracking based on particle filters and stretching open active contour models.

    PubMed

    Li, Hongsheng; Shen, Tian; Vavylonis, Dimitrios; Huang, Xiaolei

    2009-01-01

    We introduce a novel algorithm for actin filament tracking and elongation measurement. Particle Filters (PF) and Stretching Open Active Contours (SOAC) work cooperatively to simplify the modeling of PF in a one-dimensional state space while naturally integrating filament body constraints to tip estimation. Our algorithm reduces the PF state spaces to one-dimensional spaces by tracking filament bodies using SOAC and probabilistically estimating tip locations along the curve length of SOACs. Experimental evaluation on TIRFM image sequences with very low SNRs demonstrates the accuracy and robustness of this approach. PMID:20426170

  4. Actin filaments and microtubules play different roles during bristle elongation in Drosophila.

    PubMed

    Tilney, L G; Connelly, P S; Vranich, K A; Shaw, M K; Guild, G M

    2000-04-01

    Developing bristles in Drosophila pupae contain 7-11 bundles of crosslinked actin filaments and a large population of microtubules. During bristle growth the rate of cell elongation increases with bristle length. Thin section EM shows that bundle size is correlated with the amount of cytoplasm at all points along the bristle. Thus, as the bristle elongates and tapers, fewer actin filaments are used. To ensure penetration of inhibitors we isolated thoraces and cultured them in vitro; bristles elongate at rates identical to bristles growing in situ. Interestingly, inhibitors of actin filament assembly (cytochalasin D and latrunculin A) dramatically curtailed bristle elongation while a filament stabilizer (jasplakinolide) accelerated elongation. In contrast, inhibitors of microtubule dynamics (nocodazole, vinblastine, colchicine and taxol) did not affect bristle elongation. Surprisingly, the bristle microtubules are stable and do not turn over. Furthermore, the density of microtubules decreases as the bristle elongates. These two facts coupled with calculations and kinetics of elongation and the fact that the microtubules are short indicate that the microtubules are assembled early in development and then transported distally as the bristle grows. We conclude that actin assembly is crucial for bristle cell elongation and that microtubules must furnish other functions such as to provide bulk to the bristle cytoplasm as well as playing a role in vesicle transport.

  5. Nanoscopy of filamentous actin in cortical dendrites of a living mouse.

    PubMed

    Willig, Katrin I; Steffens, Heinz; Gregor, Carola; Herholt, Alexander; Rossner, Moritz J; Hell, Stefan W

    2014-01-01

    We demonstrate superresolution fluorescence microscopy (nanoscopy) of protein distributions in a mammalian brain in vivo. Stimulated emission depletion microscopy reveals the morphology of the filamentous actin in dendritic spines down to 40 μm in the molecular layer of the visual cortex of an anesthetized mouse. Consecutive recordings at 43-70 nm resolution reveal dynamical changes in spine morphology.

  6. Shortening actin filaments cause force generation in actomyosin network to change from contractile to extensile

    NASA Astrophysics Data System (ADS)

    Kumar, Nitin; Gardel, Margaret

    Motor proteins in conjunction with filamentous proteins convert biochemical energy into mechanical energy which serves a number of cellular processes including cell motility, force generation and intracellular cargo transport. In-vitro experiments suggest that the forces generated by kinesin motors on microtubule bundles are extensile in nature whereas myosin motors on actin filaments are contractile. It is not clear how qualitatively similar systems can show completely different behaviors in terms of the nature of force generation. In order to answer this question, we carry out in vitro experiments where we form quasi 2D filamentous actomyosin networks and vary the length of actin filaments by adding capping protein. We show that when filaments are much shorter than their typical persistence length (approximately 10 microns), the forces generated are extensile and we see active nematic defect propagation, as seen in the microtubule-kinesin system. Based on this observation, we claim that the rigidity of rods plays an important role in dictating the nature of force generation in such systems. In order to understand this transition, we selectively label individual filaments and find that longer filaments show considerable bending and buckling, making them difficult to slide and extend along their length.

  7. Extracellular Inhibitors, Repellents, and Semaphorin/Plexin/MICAL-mediated Actin Filament Disassembly

    PubMed Central

    Hung, Ruei-Jiun; Terman, Jonathan R.

    2011-01-01

    Multiple extracellular signals have been identified that regulate actin dynamics within motile cells, but how these instructive cues present on the cell surface exert their precise effects on the internal actin cytoskeleton is still poorly understood. One particularly interesting class of these cues is a group of extracellular proteins that negatively alter the movement of cells and their processes. Over the years, these types of events have been described using a variety of terms and herein we provide an overview of inhibitory/repulsive cellular phenomena and highlight the largest known protein family of repulsive extracellular cues, the Semaphorins. Specifically, the Semaphorins (Semas) utilize Plexin cell-surface receptors to dramatically collapse the actin cytoskeleton and we summarize what is known of the direct molecular and biochemical mechanisms of Sema-triggered actin filament (F-actin) disassembly. We also discuss new observations from our lab that reveal that the multi-domain oxidoreductase (Redox) enzyme MICAL, an important mediator of Sema/Plexin repulsion, is a novel F-actin disassembly factor. Our results indicate that MICAL triggers Sema/Plexin-mediated reorganization of the F-actin cytoskeleton and suggest a role for specific Redox signaling events in regulating actin dynamics. PMID:21800438

  8. AFAP-1L1-mediated actin filaments crosslinks hinder Trypanosoma cruzi cell invasion and intracellular multiplication.

    PubMed

    de Araújo, Karine Canuto Loureiro; Teixeira, Thaise Lara; Machado, Fabrício Castro; da Silva, Aline Alves; Quintal, Amanda Pifano Neto; da Silva, Claudio Vieira

    2016-10-01

    Host actin cytoskeleton polymerization has been shown to play an important role during Trypanosoma cruzi internalization into mammalian cell. The structure and dynamics of the actin cytoskeleton in cells are regulated by a vast number of actin-binding proteins. Here we aimed to verify the impact of AFAP-1L1, during invasion and multiplication of T. cruzi. Knocking-down AFAP-1L1 increased parasite cell invasion and intracellular multiplication. Thus, we have shown that the integrity of the machinery formed by AFAP-1L1 in actin cytoskeleton polymerization is important to hinder parasite infection.

  9. Electron tomography and simulation of baculovirus actin comet tails support a tethered filament model of pathogen propulsion.

    PubMed

    Mueller, Jan; Pfanzelter, Julia; Winkler, Christoph; Narita, Akihiro; Le Clainche, Christophe; Nemethova, Maria; Carlier, Marie-France; Maeda, Yuichiro; Welch, Matthew D; Ohkawa, Taro; Schmeiser, Christian; Resch, Guenter P; Small, J Victor

    2014-01-01

    Several pathogens induce propulsive actin comet tails in cells they invade to disseminate their infection. They achieve this by recruiting factors for actin nucleation, the Arp2/3 complex, and polymerization regulators from the host cytoplasm. Owing to limited information on the structural organization of actin comets and in particular the spatial arrangement of filaments engaged in propulsion, the underlying mechanism of pathogen movement is currently speculative and controversial. Using electron tomography we have resolved the three-dimensional architecture of actin comet tails propelling baculovirus, the smallest pathogen yet known to hijack the actin motile machinery. Comet tail geometry was also mimicked in mixtures of virus capsids with purified actin and a minimal inventory of actin regulators. We demonstrate that propulsion is based on the assembly of a fishbone-like array of actin filaments organized in subsets linked by branch junctions, with an average of four filaments pushing the virus at any one time. Using an energy-minimizing function we have simulated the structure of actin comet tails as well as the tracks adopted by baculovirus in infected cells in vivo. The results from the simulations rule out gel squeezing models of propulsion and support those in which actin filaments are continuously tethered during branch nucleation and polymerization. Since Listeria monocytogenes, Shigella flexneri, and Vaccinia virus among other pathogens use the same common toolbox of components as baculovirus to move, we suggest they share the same principles of actin organization and mode of propulsion. PMID:24453943

  10. Interactions between actin and myosin filaments in skeletal muscle visualized in frozen-hydrated thin sections.

    PubMed

    Trus, B L; Steven, A C; McDowall, A W; Unser, M; Dubochet, J; Podolsky, R J

    1989-04-01

    For the purpose of determining net interactions between actin and myosin filaments in muscle cells, perhaps the single most informative view of the myofilament lattice is its averaged axial projection. We have studied frozen-hydrated transverse thin sections with the goal of obtaining axial projections that are not subject to the limitations of conventional thin sectioning (suspect preservation of native structure) or of equatorial x-ray diffraction analysis (lack of experimental phases). In principle, good preservation of native structure may be achieved with fast freezing, followed by low-dose electron imaging of unstained vitrified cryosections. In practice, however, cryosections undergo large-scale distortions, including irreversible compression; furthermore, phase contrast imaging results in a nonlinear relationship between the projected density of the specimen and the optical density of the micrograph. To overcome these limitations, we have devised methods of image restoration and generalized correlation averaging, and applied them to cryosections of rabbit psoas fibers in both the relaxed and rigor states. Thus visualized, myosin filaments appear thicker than actin filaments by a much smaller margin than in conventional thin sections, and particularly so for rigor muscle. This may result from a significant fraction of the myosin S1-cross-bridges averaging out in projection and thus contributing only to the baseline of projected density. Entering rigor incurs a loss of density from an annulus around the myosin filament, with a compensating accumulation of density around the actin filament. This redistribution of mass represents attachment of the fraction of cross-bridges that are visible above background. Myosin filaments in the "nonoverlap" zone appear to broaden on entering rigor, suggesting that on deprivation of ATP, cross-bridges in situ move outwards even without actin in their immediate proximity.

  11. Unconventional actins and actin-binding proteins in human protozoan parasites.

    PubMed

    Gupta, C M; Thiyagarajan, S; Sahasrabuddhe, A A

    2015-06-01

    Actin and its regulatory proteins play a key role in several essential cellular processes such as cell movement, intracellular trafficking and cytokinesis in most eukaryotes. While these proteins are highly conserved in higher eukaryotes, a number of unicellular eukaryotic organisms contain divergent forms of these proteins which have highly unusual biochemical and structural properties. Here, we review the biochemical and structural properties of these unconventional actins and their core binding proteins which are present in commonly occurring human protozoan parasites.

  12. A function for filamentous alpha-smooth muscle actin: retardation of motility in fibroblasts.

    PubMed

    Rønnov-Jessen, L; Petersen, O W

    1996-07-01

    Actins are known to comprise six mammalian isoforms of which beta- and gamma-nonmuscle actins are present in all cells, whereas alpha-smooth muscle (alpha-sm) actin is normally restricted to cells of the smooth muscle lineages. alpha-Sm actin has been found also to be expressed transiently in certain nonmuscle cells, in particular fibroblasts, which are referred to as myofibroblasts. The functional significance of alpha-sm actin in fibroblasts is unknown. However, myofibroblasts appear to play a prominent role in stromal reaction in breast cancer, at the site of wound repair, and in fibrotic reactions. Here, we show that the presence of alpha-sm actin is a signal for retardation of migratory behavior in fibroblasts. Comparison in a migration assay of fibroblast cell strains with and without alpha-sm actin revealed migratory restraint in alpha-sm actin-positive fibroblasts. Electroporation of monoclonal antibody (mAb) 1A4, which recognizes specifically the NH2-terminal Ac-EEED sequence of alpha-sm actin, significantly increased the frequency of migrating cells over that obtained with an unrelated antibody or a mAb against beta-actin. Time-lapse video microscopy revealed migratory rates of 4.8 and 3.0 microns/h, respectively. To knock out the alpha-sm actin protein, several antisense phosphorothioate oligodeoxynucleotide (ODNs) were tested. One of these, 3'UTI, which is complementary to a highly evolutionary conserved 3' untranslated (3'UT) sequence of alpha-sm actin mRNA, was found to block alpha-sm actin synthesis completely without affecting the synthesis of any other proteins as analyzed by two-dimensional gel electrophoresis. Targeting by antisense 3'UTI significantly increased motility compared with the corresponding sense ODN. alpha-Sm actin inhibition also led to the formation of less prominent focal adhesions as revealed by immunofluorescence staining against vinculin, talin, and beta1-integrin. We propose that an important function of filamentous alpha

  13. Mechanism of actin filament nucleation by the bacterial effector VopL

    SciTech Connect

    Yu, Bingke; Cheng, Hui-Chun; Brautigam, Chad A.; Tomchick, Diana R.; Rosen, Michael K.

    2012-05-02

    Vibrio parahaemolyticus protein L (VopL) is an actin nucleation factor that induces stress fibers when injected into eukaryotic host cells. VopL contains three N-terminal Wiskott-Aldrich homology 2 (WH2) motifs and a unique VopL C-terminal domain (VCD). We describe crystallographic and biochemical analyses of filament nucleation by VopL. The WH2 element of VopL does not nucleate on its own and requires the VCD for activity. The VCD forms a U-shaped dimer in the crystal, stabilized by a terminal coiled coil. Dimerization of the WH2 motifs contributes strongly to nucleation activity, as do contacts of the VCD to actin. Our data lead to a model in which VopL stabilizes primarily lateral (short-pitch) contacts between actin monomers to create the base of a two-stranded filament. Stabilization of lateral contacts may be a common feature of actin filament nucleation by WH2-based factors.

  14. Dense granule trafficking in Toxoplasma gondii requires a unique class 27 myosin and actin filaments

    PubMed Central

    Heaslip, Aoife T.; Nelson, Shane R.; Warshaw, David M.

    2016-01-01

    The survival of Toxoplasma gondii within its host cell requires protein release from secretory vesicles, called dense granules, to maintain the parasite’s intracellular replicative niche. Despite the importance of DGs, nothing is known about the mechanisms underlying their transport. In higher eukaryotes, secretory vesicles are transported to the plasma membrane by molecular motors moving on their respective cytoskeletal tracks (i.e., microtubules and actin). Because the organization of these cytoskeletal structures differs substantially in T. gondii, the molecular motor dependence of DG trafficking is far from certain. By imaging the motions of green fluorescent protein–tagged DGs in intracellular parasites with high temporal and spatial resolution, we show through a combination of molecular genetics and chemical perturbations that directed DG transport is independent of microtubules and presumably their kinesin/dynein motors. However, directed DG transport is dependent on filamentous actin and a unique class 27 myosin, TgMyoF, which has structural similarity to myosin V, the prototypical cargo transporter. Actomyosin DG transport was unexpected, since filamentous parasite actin has yet to be visualized in vivo due in part to the prevailing model that parasite actin forms short, unstable filaments. Thus our data uncover new critical roles for these essential proteins in the lytic cycle of this devastating pathogen. PMID:27146112

  15. Dense granule trafficking in Toxoplasma gondii requires a unique class 27 myosin and actin filaments.

    PubMed

    Heaslip, Aoife T; Nelson, Shane R; Warshaw, David M

    2016-07-01

    The survival of Toxoplasma gondii within its host cell requires protein release from secretory vesicles, called dense granules, to maintain the parasite's intracellular replicative niche. Despite the importance of DGs, nothing is known about the mechanisms underlying their transport. In higher eukaryotes, secretory vesicles are transported to the plasma membrane by molecular motors moving on their respective cytoskeletal tracks (i.e., microtubules and actin). Because the organization of these cytoskeletal structures differs substantially in T. gondii, the molecular motor dependence of DG trafficking is far from certain. By imaging the motions of green fluorescent protein-tagged DGs in intracellular parasites with high temporal and spatial resolution, we show through a combination of molecular genetics and chemical perturbations that directed DG transport is independent of microtubules and presumably their kinesin/dynein motors. However, directed DG transport is dependent on filamentous actin and a unique class 27 myosin, TgMyoF, which has structural similarity to myosin V, the prototypical cargo transporter. Actomyosin DG transport was unexpected, since filamentous parasite actin has yet to be visualized in vivo due in part to the prevailing model that parasite actin forms short, unstable filaments. Thus our data uncover new critical roles for these essential proteins in the lytic cycle of this devastating pathogen. PMID:27146112

  16. Myosin-10 and actin filaments are essential for mitotic spindle function

    PubMed Central

    Woolner, Sarah; O'Brien, Lori L.; Wiese, Christiane; Bement, William M.

    2008-01-01

    Mitotic spindles are microtubule-based structures responsible for chromosome partitioning during cell division. Although the roles of microtubules and microtubule-based motors in mitotic spindles are well established, whether or not actin filaments (F-actin) and F-actin–based motors (myosins) are required components of mitotic spindles has long been controversial. Based on the demonstration that myosin-10 (Myo10) is important for assembly of meiotic spindles, we assessed the role of this unconventional myosin, as well as F-actin, in mitotic spindles. We find that Myo10 localizes to mitotic spindle poles and is essential for proper spindle anchoring, normal spindle length, spindle pole integrity, and progression through metaphase. Furthermore, we show that F-actin localizes to mitotic spindles in dynamic cables that surround the spindle and extend between the spindle and the cortex. Remarkably, although proper anchoring depends on both F-actin and Myo10, the requirement for Myo10 in spindle pole integrity is F-actin independent, whereas F-actin and Myo10 actually play antagonistic roles in maintenance of spindle length. PMID:18606852

  17. Emerging roles of sumoylation in the regulation of actin, microtubules, intermediate filaments, and septins

    PubMed Central

    Alonso, Annabel; Greenlee, Matt; Matts, Jessica; Kline, Jake; Davis, Kayla J.

    2015-01-01

    Sumoylation is a powerful regulatory system that controls many of the critical processes in the cell, including DNA repair, transcriptional regulation, nuclear transport, and DNA replication. Recently, new functions for SUMO have begun to emerge. SUMO is covalently attached to components of each of the four major cytoskeletal networks, including microtubule‐associated proteins, septins, and intermediate filaments, in addition to nuclear actin and actin‐regulatory proteins. However, knowledge of the mechanisms by which this signal transduction system controls the cytoskeleton is still in its infancy. One story that is beginning to unfold is that SUMO may regulate the microtubule motor protein dynein by modification of its adaptor Lis1. In other instances, cytoskeletal elements can both bind to SUMO non‐covalently and also be conjugated by it. The molecular mechanisms for many of these new functions are not yet clear, but are under active investigation. One emerging model links the function of MAP sumoylation to protein degradation through SUMO‐targeted ubiquitin ligases, also known as STUbL enzymes. Other possible functions for cytoskeletal sumoylation are also discussed. © 2015 The Authors. Cytoskeleton Published by Wiley Periodicals, Inc. PMID:26033929

  18. Drosophila cyfip regulates synaptic development and endocytosis by suppressing filamentous actin assembly.

    PubMed

    Zhao, Lu; Wang, Dan; Wang, Qifu; Rodal, Avital A; Zhang, Yong Q

    2013-04-01

    The formation of synapses and the proper construction of neural circuits depend on signaling pathways that regulate cytoskeletal structure and dynamics. After the mutual recognition of a growing axon and its target, multiple signaling pathways are activated that regulate cytoskeletal dynamics to determine the morphology and strength of the connection. By analyzing Drosophila mutations in the cytoplasmic FMRP interacting protein Cyfip, we demonstrate that this component of the WAVE complex inhibits the assembly of filamentous actin (F-actin) and thereby regulates key aspects of synaptogenesis. Cyfip regulates the distribution of F-actin filaments in presynaptic neuromuscular junction (NMJ) terminals. At cyfip mutant NMJs, F-actin assembly was accelerated, resulting in shorter NMJs, more numerous satellite boutons, and reduced quantal content. Increased synaptic vesicle size and failure to maintain excitatory junctional potential amplitudes under high-frequency stimulation in cyfip mutants indicated an endocytic defect. cyfip mutants exhibited upregulated bone morphogenetic protein (BMP) signaling, a major growth-promoting pathway known to be attenuated by endocytosis at the Drosophila NMJ. We propose that Cyfip regulates synapse development and endocytosis by inhibiting actin assembly.

  19. Mechanical force mobilizes zyxin from focal adhesions to actin filaments and regulates cytoskeletal reinforcement.

    PubMed

    Yoshigi, Masaaki; Hoffman, Laura M; Jensen, Christopher C; Yost, H Joseph; Beckerle, Mary C

    2005-10-24

    Organs and tissues adapt to acute or chronic mechanical stress by remodeling their actin cytoskeletons. Cells that are stimulated by cyclic stretch or shear stress in vitro undergo bimodal cytoskeletal responses that include rapid reinforcement and gradual reorientation of actin stress fibers; however, the mechanism by which cells respond to mechanical cues has been obscure. We report that the application of either unidirectional cyclic stretch or shear stress to cells results in robust mobilization of zyxin from focal adhesions to actin filaments, whereas many other focal adhesion proteins and zyxin family members remain at focal adhesions. Mechanical stress also induces the rapid zyxin-dependent mobilization of vasodilator-stimulated phosphoprotein from focal adhesions to actin filaments. Thickening of actin stress fibers reflects a cellular adaptation to mechanical stress; this cytoskeletal reinforcement coincides with zyxin mobilization and is abrogated in zyxin-null cells. Our findings identify zyxin as a mechanosensitive protein and provide mechanistic insight into how cells respond to mechanical cues. PMID:16247023

  20. AUTOMATED ACTIN FILAMENT SEGMENTATION, TRACKING AND TIP ELONGATION MEASUREMENTS BASED ON OPEN ACTIVE CONTOUR MODELS.

    PubMed

    Li, Hongsheng; Shen, Tian; Smith, Matthew B; Fujiwara, Ikuko; Vavylonis, Dimitrios; Huang, Xiaolei

    2009-06-28

    This paper presents an automated method for actin filament segmentation and tracking for measuring tip elongation rates in Total Internal Reflection Fluorescence Microscopy (TIRFM) images. The main contributions of the paper are: (i) we use a novel open active contour model for filament segmentation and tracking, which is fast and robust against noise; (ii) different strategies are proposed to solve the filament intersection problem, which is shown to be the main difficulty in filament tracking; and (iii) this fully automated method avoids the need of human interaction and thus reduces required time for the entire elongation measurement process on an image sequence. Application to experimental results demonstrated the robustness and effectiveness of this method.

  1. A three-dimensional FRET analysis to construct an atomic model of the actin-tropomyosin-troponin core domain complex on a muscle thin filament.

    PubMed

    Miki, Masao; Makimura, Satoshi; Sugahara, Yasuyuki; Yamada, Ryuta; Bunya, Masashi; Saitoh, Takahiro; Tobita, Hidetaka

    2012-06-29

    It is essential to know the detailed structure of the thin filament to understand the regulation mechanism of striated muscle contraction. Fluorescence resonance energy transfer (FRET) was used to construct an atomic model of the actin-tropomyosin (Tm)-troponin (Tn) core domain complex. We generated single-cysteine mutants in the 167-195 region of Tm and in TnC, TnI, and the β-TnT 25-kDa fragment, and each was attached with an energy donor probe. An energy acceptor probe was located at actin Gln41, actin Cys374, or the actin nucleotide-binding site. From these donor-acceptor pairs, FRET efficiencies were determined with and without Ca(2+). Using the atomic coordinates for F-actin, Tm, and the Tn core domain, we searched all possible arrangements for Tm or the Tn core domain on F-actin to calculate the FRET efficiency for each donor-acceptor pair in each arrangement. By minimizing the squared sum of deviations for the calculated FRET efficiencies from the observed FRET efficiencies, we determined the location of Tm segment 167-195 and the Tn core domain on F-actin with and without Ca(2+). The bulk of the Tn core domain is located near actin subdomains 3 and 4. The central helix of TnC is nearly perpendicular to the F-actin axis, directing the N-terminal domain of TnC toward the actin outer domain. The C-terminal region in the I-T arm forms a four-helix-bundle structure with the Tm 175-185 region. After Ca(2+) release, the Tn core domain moves toward the actin outer domain and closer to the center of the F-actin axis.

  2. Endothelial actin-binding proteins and actin dynamics in leukocyte transendothelial migration.

    PubMed

    Schnoor, Michael

    2015-04-15

    The endothelium is the first barrier that leukocytes have to overcome during recruitment to sites of inflamed tissues. The leukocyte extravasation cascade is a complex multistep process that requires the activation of various adhesion molecules and signaling pathways, as well as actin remodeling, in both leukocytes and endothelial cells. Endothelial adhesion molecules, such as E-selectin or ICAM-1, are connected to the actin cytoskeleton via actin-binding proteins (ABPs). Although the contribution of receptor-ligand interactions to leukocyte extravasation has been studied extensively, the contribution of endothelial ABPs to the regulation of leukocyte adhesion and transendothelial migration remains poorly understood. This review focuses on recently published evidence that endothelial ABPs, such as cortactin, myosin, or α-actinin, regulate leukocyte extravasation by controlling actin dynamics, biomechanical properties of endothelia, and signaling pathways, such as GTPase activation, during inflammation. Thus, ABPs may serve as targets for novel treatment strategies for disorders characterized by excessive leukocyte recruitment.

  3. Actions of cytochalasins on the organization of actin filaments and microtubules in a neuronal growth cone

    PubMed Central

    1988-01-01

    Actions of cytochalasin B (CB) on cytoskeletons and motility of growth cones from cultured Aplysia neurons were studied using a rapid flow perfusion chamber and digital video light microscopy. Living growth cones were observed using differential interference contrast optics and were also fixed at various time points to assay actin filament (F- actin) and microtubule distributions. Treatment with CB reversibly blocked motility and eliminated most of the phalloidin-stainable F- actin from the leading lamella. The loss of F-actin was nearly complete within 2-3 min of CB application and was largely reversed within 5-6 min of CB removal. The loss and recovery of F-actin were found to occur with a very distinctive spatial organization. Within 20-30 s of CB application, F-actin networks receded from the entire peripheral margin of the lamella forming a band devoid of F-actin. This band widened as F- actin receded at rates of 3-6 microns/min. Upon removal of CB, F-actin began to reappear within 20-30 s. The initial reappearance of F-actin took two forms: a coarse isotropic matrix of F-actin bundles throughout the lamella, and a denser matrix along the peripheral margin. The denser peripheral matrix then expanded in width, extending centrally to replace the coarse matrix at rates again between 3-6 microns/min. These results suggest that actin normally polymerizes at the leading edge and then flows rearward at a rate between 3-6 microns/min. CB treatment was also observed to alter the distribution of microtubules, assayed by antitubulin antibody staining. Normally, microtubules are restricted to the neurite shaft and a central growth cone domain. Within approximately 5 min after CB application, however, microtubules began extending into the lamellar region, often reaching the peripheral margin. Upon removal of CB, the microtubules were restored to their former central localization. The timing of these microtubule redistributions is consistent with their being secondary to

  4. Design, synthesis, and biological evaluation of simplified side chain hybrids of the potent actin binding polyketides rhizopodin and bistramide.

    PubMed

    Herkommer, Daniel; Dreisigacker, Sandra; Sergeev, Galina; Sasse, Florenz; Gohlke, Holger; Menche, Dirk

    2015-03-01

    The natural products rhizopodin and bistramide belong to an elite class of highly potent actin binding agents. They show powerful antiproliferative activities against a range of tumor cell lines, with IC50 values in the low-nanomolar range. At the molecular level they disrupt the actin cytoskeleton by binding specifically to a few critical sites of G-actin, resulting in actin filament stabilization. The important biological properties of rhizopodin and bistramide, coupled with their unique and intriguing molecular architectures, render them attractive compounds for further development. However, this is severely hampered by the structural complexity of these metabolites. We initiated an interdisciplinary approach at the interface between molecular modeling, organic synthesis, and chemical biology to support further biological applications. We also wanted to expand structure-activity relationship studies with the goal of accessing simplified analogues with potent biological properties. We report computational analyses of actin-inhibitor interactions involving molecular docking, validated on known actin binding ligands, that show a close match between the crystal and modeled structures. Based on these results, the ligand shape was simplified, and more readily accessible rhizopodin-bistramide mimetics were designed. A flexible and modular strategy was applied for the synthesis of these compounds, enabling diverse access to dramatically simplified rhizopodin-bistramide hybrids. This novel analogue class was analyzed for its antiproliferative and actin binding properties. PMID:25641798

  5. C-terminal fragment of amebin promotes actin filament bundling, inhibits acto-myosin ATPase activity and is essential for amoeba migration.

    PubMed

    Jóźwiak, Jolanta; Rzhepetskyy, Yuriy; Sobczak, Magdalena; Kocik, Elżbieta; Skórzewski, Radosław; Kłopocka, Wanda; Rędowicz, Maria Jolanta

    2011-02-01

    Amebin [formerly termed as ApABP-FI; Sobczak et al. (2007) Biochem. Cell Biol. 85] is encoded in Amoeba proteus by two transcripts, 2672-nt and 1125-nt. A product of the shorter transcript (termed as C-amebin), comprising C-terminal 375 amino-acid-residue fragment of amebin, has been expressed and purified as the recombinant GST-fusion protein. GST-C-amebin bound both to monomeric and filamentous actin. The binding was Ca(2+)-independent and promoted filament bundling, as revealed with the transmission electron microscopy. GST-C-amebin significantly decreased MgATPase activity of rabbit skeletal muscle acto-S1. Removal with endoproteinase ArgC of a positively charged C-terminal region of GST-amebin containing KLASMWEQ sequence abolished actin-binding and bundling as well as the ATPase-inhibitory effect of C-amebin, indicating that this protein region was involved in the interaction with actin. Microinjection of amoebae with antibody against C-terminus of amebin significantly affected amoebae morphology, disturbed cell polarization and transport of cytoplasmic granules as well as blocked migration. These data indicate that amebin may be one of key regulators of the actin-cytoskeleton dynamics and actin-dependent motility in A. proteus.

  6. Early events of fertilization in sea urchin eggs are sensitive to actin-binding organic molecules.

    PubMed

    Chun, Jong T; Limatola, Nunzia; Vasilev, Filip; Santella, Luigia

    2014-08-01

    We previously demonstrated that many aspects of the intracellular Ca(2+) increase in fertilized eggs of starfish are significantly influenced by the state of the actin cytoskeleton. In addition, the actin cytoskeleton appeared to play comprehensive roles in modulating cortical granules exocytosis and sperm entry during the early phase of fertilization. In the present communication, we have extended our work to sea urchin which is believed to have bifurcated from the common ancestor in the phylogenetic tree some 500 million years ago. To corroborate our earlier findings in starfish, we have tested how the early events of fertilization in sea urchin eggs are influenced by four different actin-binding drugs that promote either depolymerization or stabilization of actin filaments. We found that all the actin drugs commonly blocked sperm entry in high doses and significantly reduced the speed of the Ca(2+) wave. At low doses, however, cytochalasin B and phalloidin increased the rate of polyspermy. Overall, certain aspects of Ca(2+) signaling in these eggs were in line with the morphological changes induced by the actin drugs. That is, the time interval between the cortical flash and the first Ca(2+) spot at the sperm interaction site (the latent period) was significantly prolonged in the eggs pretreated with cytochalasin B or latrunculin A, whereas the Ca(2+) decay kinetics after the peak was specifically attenuated in the eggs pretreated with jasplakinolide or phalloidin. In addition, the sperm interacting with the eggs pretreated with actin drugs often generated multiple Ca(2+) waves, but tended to fail to enter the egg. Thus, our results indicated that generation of massive Ca(2+) waves is neither indicative of sperm entry nor sufficient for cortical granules exocytosis in the inseminated sea urchin eggs, whereas the structure and functionality of the actin cytoskeleton are the major determining factors in the two processes.

  7. Actin filament turnover drives leading edge growth during myelin sheath formation in the central nervous system

    PubMed Central

    Schmitt, Sebastian; Snaidero, Nicolas; Mitkovski, Mišo; Velte, Caroline; Brückner, Bastian R.; Alexopoulos, Ioannis; Czopka, Tim; Jung, Sang Y.; Rhee, Jeong S.; Janshoff, Andreas; Witke, Walter; Schaap, Iwan A.T.; Lyons, David A.; Simons, Mikael

    2016-01-01

    Summary During central nervous system development, oligodendrocytes wrap their plasma membrane around axons to generate multi-lamellar myelin sheaths. To drive growth at the leading edge of myelin at the interface with the axon, mechanical forces are necessary, but the underlying mechanisms are not known. Using an interdisciplinary approach that combines morphological, genetic and biophysical analyses, we identified a key role for actin filament network turnover in myelin growth. At the onset of myelin biogenesis, F-actin is redistributed to the leading edge, where its polymerization-based forces push out non-adhesive and motile protrusions. F-actin disassembly converts protrusions into sheets by reducing surface tension and in turn inducing membrane spreading and adhesion. We identified the actin depolymerizing factor ADF/Cofilin1, which mediates high F-actin turnover rates, as essential factor in this process. We propose that F-actin turnover is the driving force in myelin wrapping by regulating repetitive cycles of leading edge protrusion and spreading. PMID:26166299

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

    NASA Astrophysics Data System (ADS)

    Stam, Samantha; Gardel, Margaret

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

  9. Microtubules and actin filaments are not critically involved in the biogenesis of epithelial cell surface polarity.

    PubMed

    Salas, P J; Misek, D E; Vega-Salas, D E; Gundersen, D; Cereijido, M; Rodriguez-Boulan, E

    1986-05-01

    We have studied the role of microtubules and actin filaments in the biogenesis of epithelial cell surface polarity, using influenza hemagglutinin and vesicular stomatitis G protein as model apical and basolateral proteins in infected Madin-Darby canine kidney cells. Addition of colchicine or nocodazole to confluent monolayers at concentrations sufficient to completely disassemble microtubules did not affect the asymmetric budding of influenza or vesicular stomatitis virus and only slightly reduced the typical asymmetric surface distribution of their envelope proteins, despite extensive cytoplasmic redistribution of the Golgi apparatus. Alteration of microtubular function by taxol or dissociation of actin filaments by cytochalasin D also failed to have a significant effect. Furthermore, neither colchicine nor cytochalasin D pretreatment blocked the ability of subconfluent Madin-Darby canine kidney cells to sustain polarized budding of influenza virus a few hours after attachment to the substrate. Our results indicate that domain-specific microtubule or actin filament "tracks" are not responsible for the vectorial delivery of apically or basolaterally directed transport vesicles. In conjunction with currently available evidence, they are compatible with a model in which receptors in the cytoplasmic aspect of apical or basolateral regions provide vectoriality to the transport of vesicles carrying plasma membrane proteins to their final surface localization. PMID:2871031

  10. Mechanical output of myosin II motors is regulated by myosin filament size and actin network mechanics

    NASA Astrophysics Data System (ADS)

    Stam, Samantha; Alberts, Jonathan; Gardel, Margaret; Munro, Edwin

    2013-03-01

    The interactions of bipolar myosin II filaments with actin arrays are a predominate means of generating forces in numerous physiological processes including muscle contraction and cell migration. However, how the spatiotemporal regulation of these forces depends on motor mechanochemistry, bipolar filament size, and local actin mechanics is unknown. Here, we simulate myosin II motors with an agent-based model in which the motors have been benchmarked against experimental measurements. Force generation occurs in two distinct regimes characterized either by stable tension maintenance or by stochastic buildup and release; transitions between these regimes occur by changes to duty ratio and myosin filament size. The time required for building force to stall scales inversely with the stiffness of a network and the actin gliding speed of a motor. Finally, myosin motors are predicted to contract a network toward stiffer regions, which is consistent with experimental observations. Our representation of myosin motors can be used to understand how their mechanical and biochemical properties influence their observed behavior in a variety of in vitro and in vivo contexts.

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

    PubMed

    van der Honing, Hannie S; van Bezouwen, Laura S; Emons, Anne Mie C; Ketelaar, Tijs

    2011-10-01

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

  12. Structural Studies of Interactions between Cardiac Troponin I and Actin in Regulated Thin Filament using Förster Resonance Energy Transfer

    PubMed Central

    Xing, Jun; Chinnaraj, Mathivanan; Zhang, Zhihong; Cheung, Herbert C.; Dong, Wen-Ji

    2008-01-01

    The Ca2+-induced interaction between cardiac troponin I (cTnI) and actin plays a key role in the regulation of cardiac muscle contraction and relaxation. In this report we investigated changes of this interaction in response to strong crossbridge formation between myosin S1 and actin and PKA phosphorylation of cTnI within reconstituted thin filament. The interaction was monitored by measuring Förster resonance energy transfer (FRET) between the fluorescent donor 5-(iodoacetamidoethyl)aminonaphthalene-1-sulfonic acid (AEDANS) attached to the residues 131, 151, 160 167, 188 and 210 of cTnI and the nonfluorescent acceptor 4-dimethylaminophenylazophenyl-4′-maleimide (DABM) attached to cysteine 374 of actin. The FRET distance measurements showed that bound Ca2+ induced large increases in the distances from actin to the cTnI sites, indicating a Ca2+-triggered separation of actin from cTnI. Strongly bound myosin S1 induced additional increases in these distances in the presence of bound Ca2+. These two-step changes in the observed FRET distances provide a direct link of structural changes at the interface between cTnI and actin to the three-state model of thin filament regulation of muscle contraction and relaxation. When cTnC was inactivated through mutations of key residues within the 12-residue Ca2+-binding loop, strongly bound S1 alone induced increases in the distances in spite of the fact that the filaments no longer bound regulatory Ca2+. These results suggest bound Ca2+ or strongly bound S1 alone can partially activate thin filament, but full activation requires both bound Ca2+ and strongly bound S1. The distributions of the FRET distances revealed different structural dynamics associated with different regions of cTnI in different biochemical states. The second actin-binding region appears more rigid than the inhibitory/regulatory region. In the Mg2+ state, the regulatory region appears more flexible than the inhibitory region, and in the Ca2+ state, the

  13. Activation of the cAMP Pathway Induces RACK1-Dependent Binding of β-Actin to BDNF Promoter

    PubMed Central

    Neasta, Jeremie; Fiorenza, Anna; He, Dao-Yao; Phamluong, Khanhky; Kiely, Patrick A.; Ron, Dorit

    2016-01-01

    RACK1 is a scaffolding protein that contributes to the specificity and propagation of several signaling cascades including the cAMP pathway. As such, RACK1 participates in numerous cellular functions ranging from cell migration and morphology to gene transcription. To obtain further insights on the mechanisms whereby RACK1 regulates cAMP-dependent processes, we set out to identify new binding partners of RACK1 during activation of the cAMP signaling using a proteomics strategy. We identified β-actin as a direct RACK1 binding partner and found that the association between β-actin and RACK1 is increased in response to the activation of the cAMP pathway. Furthermore, we show that cAMP-dependent increase in BDNF expression requires filamentous actin. We further report that β-actin associates with the BDNF promoter IV upon the activation of the cAMP pathway and present data to suggest that the association of β-actin with BDNF promoter IV is RACK1-dependent. Taken together, our data suggest that β-actin is a new RACK1 binding partner and that the RACK1 and β-actin association participate in the cAMP-dependent regulation of BDNF transcription. PMID:27505161

  14. Electric field modulation of the motility of actin filaments on myosin-functionalised surfaces

    NASA Astrophysics Data System (ADS)

    Ramsey, L. C.; Aveyard, J.; van Zalinge, H.; Persson, M.; Mânsson, A.; Nicolau, D. V.

    2013-02-01

    We investigated the difference in electrically guided acto-myosin motility on two surfaces. Rabbit skeletal muscle heavy meromyosin (HMM) was absorbed onto surfaces coated with Nitrocellulose (NC) and Poly(butyl methacrylate) (PBMA). A modified in vitro motility assay with sealed chambers for the insertion of electrodes allowed an electrical field to be applied across the flow cell. On all surfaces a small increase in velocity and general guidance of the actin filaments towards the positive electrode is seen at field strengths in the range of ~3000 - 4000Vm-1. A large increase in velocity was observed at ~5000Vm-1 and a significant change in the velocity of the actin filaments present in field strengths higher than this. NC supported the highest percentage of motile filaments and at a field of 8000Vm-1 reached ~66%. PBMA however supported the least percentage of motile filaments and irregular motility was observed even at higher fields where guidance was expected to be strong. The change in velocity in the range of fields tested varied significantly on the surfaces with NC displaying a 46% increase from 0 to 8000Vm-1 whereas on PBMA this value was just 37%.

  15. Actin Filament Tracking Based on Particle Filters and Stretching Open Active Contour Models

    PubMed Central

    Li, Hongsheng; Shen, Tian; Vavylonis, Dimitrios; Huang, Xiaolei

    2010-01-01

    We introduce a novel algorithm for actin filament tracking and elongation measurement. Particle Filters (PF) and Stretching Open Active Contours (SOAC) work cooperatively to simplify the modeling of PF in a one-dimensional state space while naturally integrating filament body constraints to tip estimation. Existing microtubule (MT) tracking methods track either MT tips or entire bodies in high-dimensional state spaces. In contrast, our algorithm reduces the PF state spaces to one-dimensional spaces by tracking filament bodies using SOAC and probabilistically estimating tip locations along the curve length of SOACs. Experimental evaluation on TIRFM image sequences with very low SNRs demonstrates the accuracy and robustness of the proposed approach. PMID:20426170

  16. On the properties of a bundle of flexible actin filaments in an optical trap

    NASA Astrophysics Data System (ADS)

    Perilli, Alessia; Pierleoni, Carlo; Ciccotti, Giovanni; Ryckaert, Jean-Paul

    2016-06-01

    We establish the statistical mechanics framework for a bundle of Nf living and uncrosslinked actin filaments in a supercritical solution of free monomers pressing against a mobile wall. The filaments are anchored normally to a fixed planar surface at one of their ends and, because of their limited flexibility, they grow almost parallel to each other. Their growing ends hit a moving obstacle, depicted as a second planar wall, parallel to the previous one and subjected to a harmonic compressive force. The force constant is denoted as the trap strength while the distance between the two walls as the trap length to make contact with the experimental optical trap apparatus. For an ideal solution of reactive filaments and free monomers at fixed free monomer chemical potential μ1, we obtain the general expression for the grand potential from which we derive averages and distributions of relevant physical quantities, namely, the obstacle position, the bundle polymerization force, and the number of filaments in direct contact with the wall. The grafted living filaments are modeled as discrete Wormlike chains, with F-actin persistence length ℓp, subject to discrete contour length variations ±d (the monomer size) to model single monomer (de)polymerization steps. Rigid filaments (ℓp = ∞), either isolated or in bundles, all provide average values of the stalling force in agreement with Hill's predictions Fs H = N f k B T ln ( ρ 1 / ρ 1 c) / d , independent of the average trap length. Here ρ1 is the density of free monomers in the solution and ρ1c its critical value at which the filament does not grow nor shrink in the absence of external forces. Flexible filaments (ℓp < ∞) instead, for values of the trap strength suitable to prevent their lateral escape, provide an average bundle force and an average trap length slightly larger than the corresponding rigid cases (few percents). Still the stalling force remains nearly independent on the average trap length, but

  17. Structural insights into de novo actin polymerization

    PubMed Central

    Dominguez, Roberto

    2010-01-01

    Summary Many cellular functions depend on rapid and localized actin polymerization/depolymerization. Yet, the de novo polymerization of actin in cells is kinetically unfavorable because of the instability of polymerization intermediates (small actin oligomers) and the actions of actin monomer binding proteins. Cells use filament nucleation and elongation factors to initiate and sustain polymerization. Structural biology is beginning to shed light on the diverse mechanisms by which these unrelated proteins initiate polymerization, undergo regulation, and mediate the transition of monomeric actin onto actin filaments. A prominent role is played by the W domain, which in some of these proteins occurs in tandem repeats that recruit multiple actin subunits. Pro-rich regions are also abundant and mediate the binding of profilin-actin complexes, which are the main source of polymerization competent actin in cells. Filament nucleation and elongation factors frequently interact with Rho family GTPases, which relay signals from membrane receptors to regulate actin cytoskeleton remodeling. PMID:20096561

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

    NASA Astrophysics Data System (ADS)

    Weichsel, Julian; Schwarz, Ulrich S.

    2013-03-01

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

  19. He-Ne laser influenced actin filaments alleviate the damage of UV-B in wheat

    NASA Astrophysics Data System (ADS)

    Chen, Huize; Han, Rong

    2015-01-01

    This work investigated the use of a He-Ne laser in alleviating the damaging effects of ultraviolet-B (UV-B) radiation on wheat seedlings by influenced actin filaments. Triticum aestivum seedlings were irradiated with either enhanced UV-B (10.08 KJ m-2 d-1) or a combination of UV-B light and the He-Ne laser. Plants were also exposed to the He-Ne laser alone. In order to compare the effect of the He-Ne laser, red light (same power and wavelength as the He-Ne laser) treatment and the combined UV-B and red light treatment were added. Moreover, wheat seedlings were treated with actin special drugs, including cytochalasin B (CB) and jasplakinolide (JAS). We analyzed the growth of the seedlings, the distribution of actin filaments (AFs), DNA laddering and ACTIN expression in the different groups. The results showed that enhanced UV-B produced negative effects on the growth of wheat seedlings while implementing the He-Ne laser partially alleviated the injury. With the red light treatment, there are no positive effects. The ACTIN expression stayed the same in the different treatments, while the distribution and the protein content are different. The Fourier transform infrared (FTIR) microspectroscopic results further established significant changes in the chemical composition of the wall material. These results suggested that the He-Ne laser alleviated the damaging effects of UV-B radiation in wheat seedlings by changing the characteristics of the AFs.

  20. Substrate, focal adhesions, and actin filaments: a mechanical unit with a weak spot for mechanosensitive proteins

    NASA Astrophysics Data System (ADS)

    Kirchenbüchler, David; Born, Simone; Kirchgeßner, Norbert; Houben, Sebastian; Hoffmann, Bernd; Merkel, Rudolf

    2010-05-01

    Mechanosensing is a vital prerequisite for dynamic remodeling of focal adhesions and cytoskeletal structures upon substrate deformation. For example, tissue formation, directed cell orientation or cell differentiation are regulated by such mechanosensing processes. Focal adhesions and the actin cytoskeleton are believed to be involved in these processes, but where mechanosensing molecules are located and how elastic substrate, focal adhesions and the cytoskeleton couple with each other upon substrate deformation still remains obscure. To approach these questions we have developed a sensitive method to apply defined spatially decaying deformation fields to cells cultivated on ultrasoft elastic substrates and to accurately quantify the resulting displacements of the actin cytoskeleton, focal adhesions, as well as the substrate. Displacement fields were recorded in live cell microscopy by tracking either signals from fluorescent proteins or marker particles in the substrate. As model cell type we used myofibroblasts. These cells are characterized by highly stable adhesion and force generating structures but are still able to detect mechanical signals with high sensitivity. We found a rigid connection between substrate and focal adhesions. Furthermore, stress fibers were found to be barely extendable almost over their whole lengths. Plastic deformation took place only at the very ends of actin filaments close to focal adhesions. As a result, this area became elongated without extension of existing actin filaments by polymerization. Both ends of the stress fibers were mechanically coupled with detectable plastic deformations on either site. Interestingly, traction force dependent substrate deformation fields remained mostly unaffected even when stress fiber elongations were released. These data argue for a location of mechanosensing proteins at the ends of actin stress fibers and describe, except for these domains, the whole system to be relatively rigid for tensile

  1. 40-kDa protein from thin filaments of the mussel Crenomytilus grayanus changes the conformation of F-actin during the ATPase cycle.

    PubMed

    Sirenko, V V; Simonyan, A H; Dobrzhanskaya, A V; Shelud'ko, N S; Borovikov, Y S

    2013-03-01

    Polarized fluorimetry was used to study in ghost muscle fibers the influence of a 40-kDa protein from the thin filaments of the mussel Crenomytilus grayanus on conformational changes of F-actin modified by the fluorescent probes 1,5-IAEDANS and FITC-phalloidin during myosin subfragment (S1) binding in the absence of nucleotides and in the presence of MgADP or MgATP. The fluorescence probes were rigidly bound with actin, which made the absorption and emission dipoles of the probes sensitive to changes in the orientation and mobility of both actin monomer and its subdomain-1 in thin filaments of the muscle fiber. On modeling different intermediate states of actomyosin, the orientation and mobility of oscillators of the dyes were changed discretely, which suggests multistep changes in the actin conformation during the cycle of ATP hydrolysis. The 40-kDa protein influenced the orientation and mobility of the fluorescent probes markedly, suppressing changes in their orientation and mobility in the absence of nucleotides and in the presence of MgADP, but enhancing these changes in the presence of MgATP. The calponin-like 40-kDa protein is supposed to prevent formation of the strong binding state of actomyosin in the absence of nucleotides and in the presence of MgADP but to activate formation of this state in the presence of MgATP.

  2. Convoluted Plasma Membrane Domains in the Green Alga Chara are Depleted of Microtubules and Actin Filaments.

    PubMed

    Sommer, Aniela; Hoeftberger, Margit; Hoepflinger, Marion C; Schmalbrock, Sarah; Bulychev, Alexander; Foissner, Ilse

    2015-10-01

    Charasomes are convoluted plasma membrane domains in the green alga Chara australis. They harbor H(+)-ATPases involved in acidification of the medium, which facilitates carbon uptake required for photosynthesis. In this study we investigated the distribution of cortical microtubules and cortical actin filaments in relation to the distribution of charasomes. We found that microtubules and actin filaments were largely lacking beneath the charasomes, suggesting the absence of nucleating and/or anchoring complexes or an inhibitory effect on polymerization. We also investigated the influence of cytoskeleton inhibitors on the light-dependent growth and the darkness-induced degradation of charasomes. Inhibition of cytoplasmic streaming by cytochalasin D significantly inhibited charasome growth and delayed charasome degradation, whereas depolymerization of microtubules by oryzalin or stabilization of microtubules by paclitaxel had no effect. Our data indicate that the membrane at the cytoplasmic surface of charasomes has different properties in comparison with the smooth plasma membrane. We show further that the actin cytoskeleton is necessary for charasome growth and facilitates charasome degradation presumably via trafficking of secretory and endocytic vesicles, respectively. However, microtubules are required neither for charasome growth nor for charasome degradation. PMID:26272553

  3. Convoluted Plasma Membrane Domains in the Green Alga Chara are Depleted of Microtubules and Actin Filaments.

    PubMed

    Sommer, Aniela; Hoeftberger, Margit; Hoepflinger, Marion C; Schmalbrock, Sarah; Bulychev, Alexander; Foissner, Ilse

    2015-10-01

    Charasomes are convoluted plasma membrane domains in the green alga Chara australis. They harbor H(+)-ATPases involved in acidification of the medium, which facilitates carbon uptake required for photosynthesis. In this study we investigated the distribution of cortical microtubules and cortical actin filaments in relation to the distribution of charasomes. We found that microtubules and actin filaments were largely lacking beneath the charasomes, suggesting the absence of nucleating and/or anchoring complexes or an inhibitory effect on polymerization. We also investigated the influence of cytoskeleton inhibitors on the light-dependent growth and the darkness-induced degradation of charasomes. Inhibition of cytoplasmic streaming by cytochalasin D significantly inhibited charasome growth and delayed charasome degradation, whereas depolymerization of microtubules by oryzalin or stabilization of microtubules by paclitaxel had no effect. Our data indicate that the membrane at the cytoplasmic surface of charasomes has different properties in comparison with the smooth plasma membrane. We show further that the actin cytoskeleton is necessary for charasome growth and facilitates charasome degradation presumably via trafficking of secretory and endocytic vesicles, respectively. However, microtubules are required neither for charasome growth nor for charasome degradation.

  4. Convoluted Plasma Membrane Domains in the Green Alga Chara are Depleted of Microtubules and Actin Filaments

    PubMed Central

    Sommer, Aniela; Hoeftberger, Margit; Hoepflinger, Marion C.; Schmalbrock, Sarah; Bulychev, Alexander; Foissner, Ilse

    2015-01-01

    Charasomes are convoluted plasma membrane domains in the green alga Chara australis. They harbor H+-ATPases involved in acidification of the medium, which facilitates carbon uptake required for photosynthesis. In this study we investigated the distribution of cortical microtubules and cortical actin filaments in relation to the distribution of charasomes. We found that microtubules and actin filaments were largely lacking beneath the charasomes, suggesting the absence of nucleating and/or anchoring complexes or an inhibitory effect on polymerization. We also investigated the influence of cytoskeleton inhibitors on the light-dependent growth and the darkness-induced degradation of charasomes. Inhibition of cytoplasmic streaming by cytochalasin D significantly inhibited charasome growth and delayed charasome degradation, whereas depolymerization of microtubules by oryzalin or stabilization of microtubules by paclitaxel had no effect. Our data indicate that the membrane at the cytoplasmic surface of charasomes has different properties in comparison with the smooth plasma membrane. We show further that the actin cytoskeleton is necessary for charasome growth and facilitates charasome degradation presumably via trafficking of secretory and endocytic vesicles, respectively. However, microtubules are required neither for charasome growth nor for charasome degradation. PMID:26272553

  5. Dynamic Filament Formation by a Divergent Bacterial Actin-Like ParM Protein

    PubMed Central

    Brzoska, Anthony J.; Jensen, Slade O.; Barton, Deborah A.; Davies, Danielle S.; Overall, Robyn L.; Skurray, Ronald A.; Firth, Neville

    2016-01-01

    Actin-like proteins (Alps) are a diverse family of proteins whose genes are abundant in the chromosomes and mobile genetic elements of many bacteria. The low-copy-number staphylococcal multiresistance plasmid pSK41 encodes ParM, an Alp involved in efficient plasmid partitioning. pSK41 ParM has previously been shown to form filaments in vitro that are structurally dissimilar to those formed by other bacterial Alps. The mechanistic implications of these differences are not known. In order to gain insights into the properties and behavior of the pSK41 ParM Alp in vivo, we reconstituted the parMRC system in the ectopic rod-shaped host, E. coli, which is larger and more genetically amenable than the native host, Staphylococcus aureus. Fluorescence microscopy showed a functional fusion protein, ParM-YFP, formed straight filaments in vivo when expressed in isolation. Strikingly, however, in the presence of ParR and parC, ParM-YFP adopted a dramatically different structure, instead forming axial curved filaments. Time-lapse imaging and selective photobleaching experiments revealed that, in the presence of all components of the parMRC system, ParM-YFP filaments were dynamic in nature. Finally, molecular dissection of the parMRC operon revealed that all components of the system are essential for the generation of dynamic filaments. PMID:27310470

  6. Effects of intermediate filaments on actin-based motility of Listeria monocytogenes.

    PubMed

    Giardini, P A; Theriot, J A

    2001-12-01

    How does subcellular architecture influence the intracellular movements of large organelles and macromolecular assemblies? To investigate the effects of mechanical changes in cytoplasmic structure on intracellular motility, we have characterized the actin-based motility of the intracellular bacterial pathogen Listeria monocytogenes in normal mouse fibroblasts and in fibroblasts lacking intermediate filaments. The apparent diffusion coefficient of L. monocytogenes was two-fold greater in vimentin-null fibroblasts than in wild-type fibroblasts, indicating that intermediate filaments significantly restrict the Brownian motion of bacteria. However, the mean speed of L. monocytogenes actin-based motility was statistically identical in vimentin-null and wild-type cells. Thus, environmental drag is not rate limiting for bacterial motility. Analysis of the temporal variations in speed measurements indicated that bacteria in vimentin-null cells displayed larger fluctuations in speed than did trajectories in wild-type cells. Similarly, the presence of the vimentin meshwork influenced the turning behavior of the bacteria; in the vimentin-null cells, bacteria made sharper turns than they did in wild-type cells. Taken together, these results suggest that a network of intermediate filaments constrains bacterial movement and operates over distances of several microns to reduce fluctuations in motile behavior.

  7. Mechanochemistry of protein 4.1's spectrin-actin-binding domain: ternary complex interactions, membrane binding, network integration, structural strengthening

    PubMed Central

    1995-01-01

    Mechanical strength of the red cell membrane is dependent on ternary interactions among the skeletal proteins, spectrin, actin, and protein 4.1. Protein 4.1's spectrin-actin-binding (SAB) domain is specified by an alternatively spliced exon encoding 21 amino acid (aa) and a constitutive exon encoding 59 aa. A series of truncated SAB peptides were engineered to define the sequences involved in spectrin-actin interactions, and also membrane strength. Analysis of in vitro supramolecular assemblies showed that gelation activity of SAB peptides correlates with their ability to recruit a critical amount of spectrin into the complex to cross-link actin filaments. Also, several SAB peptides appeared to exhibit a weak, cooperative actin-binding activity which mapped to the first 26 residues of the constitutive 59 aa. Fluorescence-imaged microdeformation was used to show SAB peptide integration into the elastic skeletal network of spectrin, actin, and protein 4.1. In situ membrane-binding and membrane-strengthening abilities of the SAB peptides correlated with their in vitro gelation activity. The findings imply that sites for strong spectrin binding include both the alternative 21-aa cassette and a conserved region near the middle of the 59 aa. However, it is shown that only weak SAB affinity is necessary for physiologically relevant action. Alternatively spliced exons can thus translate into strong modulation of specific protein interactions, economizing protein function in the cell without, in and of themselves, imparting unique function. PMID:7642705

  8. PLEKHG3 enhances polarized cell migration by activating actin filaments at the cell front.

    PubMed

    Nguyen, Trang Thi Thu; Park, Wei Sun; Park, Byung Ouk; Kim, Cha Yeon; Oh, Yohan; Kim, Jin Man; Choi, Hana; Kyung, Taeyoon; Kim, Cheol-Hee; Lee, Gabsang; Hahn, Klaus M; Meyer, Tobias; Heo, Won Do

    2016-09-01

    Cells migrate by directing Ras-related C3 botulinum toxin substrate 1 (Rac1) and cell division control protein 42 (Cdc42) activities and by polymerizing actin toward the leading edge of the cell. Previous studies have proposed that this polarization process requires a local positive feedback in the leading edge involving Rac small GTPase and actin polymerization with PI3K likely playing a coordinating role. Here, we show that the pleckstrin homology and RhoGEF domain containing G3 (PLEKHG3) is a PI3K-regulated Rho guanine nucleotide exchange factor (RhoGEF) for Rac1 and Cdc42 that selectively binds to newly polymerized actin at the leading edge of migrating fibroblasts. Optogenetic inactivation of PLEKHG3 showed that PLEKHG3 is indispensable both for inducing and for maintaining cell polarity. By selectively binding to newly polymerized actin, PLEKHG3 promotes local Rac1/Cdc42 activation to induce more local actin polymerization, which in turn promotes the recruitment of more PLEKHG3 to induce and maintain cell front. Thus, autocatalytic reinforcement of PLEKHG3 localization to the leading edge of the cell provides a molecular basis for the proposed positive feedback loop that is required for cell polarization and directed migration. PMID:27555588

  9. PLEKHG3 enhances polarized cell migration by activating actin filaments at the cell front

    PubMed Central

    Nguyen, Trang Thi Thu; Park, Wei Sun; Park, Byung Ouk; Kim, Cha Yeon; Oh, Yohan; Kim, Jin Man; Choi, Hana; Kyung, Taeyoon; Kim, Cheol-Hee; Lee, Gabsang; Hahn, Klaus M.; Meyer, Tobias; Heo, Won Do

    2016-01-01

    Cells migrate by directing Ras-related C3 botulinum toxin substrate 1 (Rac1) and cell division control protein 42 (Cdc42) activities and by polymerizing actin toward the leading edge of the cell. Previous studies have proposed that this polarization process requires a local positive feedback in the leading edge involving Rac small GTPase and actin polymerization with PI3K likely playing a coordinating role. Here, we show that the pleckstrin homology and RhoGEF domain containing G3 (PLEKHG3) is a PI3K-regulated Rho guanine nucleotide exchange factor (RhoGEF) for Rac1 and Cdc42 that selectively binds to newly polymerized actin at the leading edge of migrating fibroblasts. Optogenetic inactivation of PLEKHG3 showed that PLEKHG3 is indispensable both for inducing and for maintaining cell polarity. By selectively binding to newly polymerized actin, PLEKHG3 promotes local Rac1/Cdc42 activation to induce more local actin polymerization, which in turn promotes the recruitment of more PLEKHG3 to induce and maintain cell front. Thus, autocatalytic reinforcement of PLEKHG3 localization to the leading edge of the cell provides a molecular basis for the proposed positive feedback loop that is required for cell polarization and directed migration. PMID:27555588

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

    SciTech Connect

    Stall, Richard; Ramos, Joseph; Kent Fulcher, F.; Patel, Yashomati M.

    2014-03-10

    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 that 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.

  11. Kinetic Insights into the Elongation Reaction of Actin Filaments as a Function of Temperature, Pressure, and Macromolecular Crowding.

    PubMed

    Gao, Mimi; Winter, Roland

    2015-12-01

    Actin polymerization is an essential process in eukaryotic cells that provides a driving force for motility and mechanical resistance for cell shape. By using preformed gelsolin-actin nuclei and applying stopped-flow methodology, we quantitatively studied the elongation kinetics of actin filaments as a function of temperature and pressure in the presence of synthetic and protein crowding agents. We show that the association of actin monomers to the pointed end of double-stranded helical actin filaments (F-actin) proceeds via a transition state that requires an activation energy of 56 kJ mol(-1) for conformational and hydration rearrangements, but exhibits a negligible activation volume, pointing to a compact transition state that is devoid of packing defects. Macromolecular crowding causes acceleration of the F-actin elongation rate and counteracts the deteriorating effect of pressure. The results shed new light on the combined effect of these parameters on the polymerization process of actin, and help us understand the temperature and pressure sensitivity of actin polymerization under extreme conditions.

  12. Cellular target of weak magnetic fields: ionic conduction along actin filaments of microvilli.

    PubMed

    Gartzke, Joachim; Lange, Klaus

    2002-11-01

    The interaction of weak electromagnetic fields (EMF) with living cells is a most important but still unresolved biophysical problem. For this interaction, thermal and other types of noise appear to cause severe restrictions in the action of weak signals on relevant components of the cell. A recently presented general concept of regulation of ion and substrate pathways through microvilli provides a possible theoretical basis for the comprehension of physiological effects of even extremely low magnetic fields. The actin-based core of microfilaments in microvilli is proposed to represent a cellular interaction site for magnetic fields. Both the central role of F-actin in Ca2+ signaling and its polyelectrolyte nature eliciting specific ion conduction properties render the microvillar actin filament bundle an ideal interaction site for magnetic and electric fields. Ion channels at the tip of microvilli are connected with the cytoplasm by a bundle of microfilaments forming a diffusion barrier system. Because of its polyelectrolyte nature, the microfilament core of microvilli allows Ca2+ entry into the cytoplasm via nonlinear cable-like cation conduction through arrays of condensed ion clouds. The interaction of ion clouds with periodically applied EMFs and field-induced cation pumping through the cascade of potential barriers on the F-actin polyelectrolyte follows well-known physical principles of ion-magnetic field (MF) interaction and signal discrimination as described by the stochastic resonance and Brownian motor hypotheses. The proposed interaction mechanism is in accord with our present knowledge about Ca2+ signaling as the biological main target of MFs and the postulated extreme sensitivity for coherent excitation by very low field energies within specific amplitude and frequency windows. Microvillar F-actin bundles shielded by a lipid membrane appear to function like electronic integration devices for signal-to-noise enhancement; the influence of coherent signals

  13. Myosin-10 produces its power-stroke in two phases and moves processively along a single actin filament under low load

    PubMed Central

    Takagi, Yasuharu; Farrow, Rachel E.; Billington, Neil; Nagy, Attila; Batters, Christopher; Yang, Yi; Sellers, James R.; Molloy, Justin E.

    2014-01-01

    Myosin-10 is an actin-based molecular motor that participates in essential intracellular processes such as filopodia formation/extension, phagocytosis, cell migration, and mitotic spindle maintenance. To study this motor protein’s mechano-chemical properties, we used a recombinant, truncated form of myosin-10 consisting of the first 936 amino acids, followed by a GCN4 leucine zipper motif, to force dimerization. Negative-stain electron microscopy reveals that the majority of molecules are dimeric with a head-to-head contour distance of ∼50 nm. In vitro motility assays show that myosin-10 moves actin filaments smoothly with a velocity of ∼310 nm/s. Steady-state and transient kinetic analysis of the ATPase cycle shows that the ADP release rate (∼13 s−1) is similar to the maximum ATPase activity (∼12–14 s−1) and therefore contributes to rate limitation of the enzymatic cycle. Single molecule optical tweezers experiments show that under intermediate load (∼0.5 pN), myosin-10 interacts intermittently with actin and produces a power stroke of ∼17 nm, composed of an initial 15-nm and subsequent 2-nm movement. At low optical trap loads, we observed staircase-like processive movements of myosin-10 interacting with the actin filament, consisting of up to six ∼35-nm steps per binding interaction. We discuss the implications of this load-dependent processivity of myosin-10 as a filopodial transport motor. PMID:24753602

  14. [The effect of the functional electrostimulation of rat fast and slow muscles on the structural state of actin in the thin filaments of a ghost muscle fiber].

    PubMed

    Kirillina, V P; Borovikov, Iu S; Szczepanowska, J; Carraro, U

    1992-01-01

    The effect of electrostimulation of fast (EDL) and slow (SOL) rat muscles on the orientation and mobility of fluorescent probes rhodamine-phalloidine and 1.5-IAEDANS (N-iodoacetyl-N'-(5-sulpho-1-naphtyl)-ethylenediamine), located in various parts of actin molecule, has been studied by polarized microfluorimetry techniques. Muscles were stimulated at 20 Hz with the pulse width of 0.3 msec, some muscles were treated for 6 h during the first day, the other muscles for 6 h a day during the next 4 days before glycerinization. Then muscle fibres freed by the extraction of myosin, tropomyosin and troponin (ghost fibres) were used. It was shown that the binding of myosin subfragment 1 (S1) to actin induced the changes in polarized fluorescence of the fibres. The analysis of the obtained data showed that the formation of actomyosin complex in stimulated muscles resulted in increasing the angle between the thin filaments and the emission dipole of rhodamine-phalloidine, as well as in decreasing the mobility of this dye. In the experiments with the 1.5-IAEDANS label, the angle of the emission dipole decreased, while the label mobility increased. It was suggested that the orientation of domains in actomyosin complex changes following the electrostimulation to affect both the conformational state of F-actin in thin filaments of ghost fibres and actin-myosin interaction.

  15. FMRP regulates actin filament organization via the armadillo protein p0071

    PubMed Central

    Nolze, Alexander; Schneider, Jacqueline; Keil, René; Lederer, Marcell; Hüttelmaier, Stefan; Kessels, Michael M.; Qualmann, Britta; Hatzfeld, Mechthild

    2013-01-01

    Loss of fragile X mental retardation protein (FMRP) causes synaptic dysfunction and intellectual disability. FMRP is an RNA-binding protein that controls the translation or turnover of a subset of mRNAs. Identifying these target transcripts is an important step toward understanding the pathology of the disease. Here, we show that FMRP regulates actin organization and neurite outgrowth via the armadillo protein p0071. In mouse embryonic fibroblasts (MEFs) lacking FMRP (Fmr1−), the actin cytoskeleton was markedly reorganized with reduced stress fibers and F-actin/G-actin ratios compared to fibroblasts re-expressing the protein. FMRP interfered with the translation of the p0071 mRNA in a 3′-UTR-dependent manner. Accordingly, FMRP-depleted cells revealed elevated levels of p0071 protein. The knockdown of p0071 in Fmr1− fibroblasts restored stress fibers and an elongated cell shape, thus rescuing the Fmr1− phenotype, whereas overexpression of p0071 in Fmr1+ cells mimicked the Fmr1− phenotype. Moreover, p0071 and FMRP regulated neurite outgrowth and branching in a diametrically opposed way in agreement with the negative regulation of p0071 by FMRP. These results identify p0071 as an important and novel FMRP target and strongly suggest that impaired actin cytoskeletal functions mediated by an excess of p0071 are key aspects underlying the fragile X syndrome. PMID:24062571

  16. Characterization of Actin Filament Dynamics during Mitosis in Wheat Protoplasts under UV-B Radiation

    PubMed Central

    Chen, Huize; Han, Rong

    2016-01-01

    Enhanced ultraviolet-B (UV-B) radiation is caused by the thinning ozone and affects photosynthesis and crop yield. Recently, UV-B radiation has been considered as an environmental signal that regulates plant growth. Elucidating the downstream effectors in UV-B-triggered pathways is of particular interest. Previous studies have shown that actin filaments (AFs) play many roles during cell physiological processes. However, the underlying response of AFs to UV-B radiation remains unclear. In this study, wheat protoplasts were isolated from 7-d-old leaves. The dynamics of AFs during mitosis were observed under different treatments. The protoplasts were treated with UV-B radiation, cytochalasin B (CB) and jasplakinolide (JAS). Ph-FITC labelling results revealed typical actin filament structures in the control group; AFs were rearranged under UV-B radiation. AFs polymerized into bundles during interphase, the preprophase band (PPB) structure was destroyed during prophase, and the AFs gathered into plaques during metaphase in response to UV-B radiation. During anaphase and telophase, the distribution of AFs was dispersed. Pharmacologic experiments revealed that CB induced apoptosis and JAS induced nuclear division without cytokinesis in wheat protoplasts. These results indicated that AFs respond to UV-B radiation during mitosis, supplying evidence of UV-B signal transduction in plants. PMID:26823006

  17. Characterization of Actin Filament Dynamics during Mitosis in Wheat Protoplasts under UV-B Radiation.

    PubMed

    Chen, Huize; Han, Rong

    2016-01-01

    Enhanced ultraviolet-B (UV-B) radiation is caused by the thinning ozone and affects photosynthesis and crop yield. Recently, UV-B radiation has been considered as an environmental signal that regulates plant growth. Elucidating the downstream effectors in UV-B-triggered pathways is of particular interest. Previous studies have shown that actin filaments (AFs) play many roles during cell physiological processes. However, the underlying response of AFs to UV-B radiation remains unclear. In this study, wheat protoplasts were isolated from 7-d-old leaves. The dynamics of AFs during mitosis were observed under different treatments. The protoplasts were treated with UV-B radiation, cytochalasin B (CB) and jasplakinolide (JAS). Ph-FITC labelling results revealed typical actin filament structures in the control group; AFs were rearranged under UV-B radiation. AFs polymerized into bundles during interphase, the preprophase band (PPB) structure was destroyed during prophase, and the AFs gathered into plaques during metaphase in response to UV-B radiation. During anaphase and telophase, the distribution of AFs was dispersed. Pharmacologic experiments revealed that CB induced apoptosis and JAS induced nuclear division without cytokinesis in wheat protoplasts. These results indicated that AFs respond to UV-B radiation during mitosis, supplying evidence of UV-B signal transduction in plants. PMID:26823006

  18. Actin filaments are involved in the maintenance of Golgi cisternae morphology and intra-Golgi pH.

    PubMed

    Lázaro-Diéguez, Francisco; Jiménez, Nuria; Barth, Holger; Koster, Abraham J; Renau-Piqueras, Jaime; Llopis, Juan L; Burger, Koert N J; Egea, Gustavo

    2006-12-01

    Here we examine the contribution of actin dynamics to the architecture and pH of the Golgi complex. To this end, we have used toxins that depolymerize (cytochalasin D, latrunculin B, mycalolide B, and Clostridium botulinum C2 toxin) or stabilize (jasplakinolide) filamentous actin. When various clonal cell lines were examined by epifluorescence microscopy, all of these actin toxins induced compaction of the Golgi complex. However, ultrastructural analysis by transmission electron microscopy and electron tomography/three-dimensional modelling of the Golgi complex showed that F-actin depolymerization first induces perforation/fragmentation and severe swelling of Golgi cisternae, which leads to a completely disorganized structure. In contrast, F-actin stabilization results only in cisternae perforation/fragmentation. Concomitantly to actin depolymerization-induced cisternae swelling and disorganization, the intra-Golgi pH significantly increased. Similar ultrastructural and Golgi pH alkalinization were observed in cells treated with the vacuolar H+ -ATPases inhibitors bafilomycin A1 and concanamycin A. Overall, these results suggest that actin filaments are implicated in the preservation of the flattened shape of Golgi cisternae. This maintenance seems to be mediated by the regulation of the state of F-actin assembly on the Golgi pH homeostasis.

  19. Genetic deletion of ABP-120 alters the three-dimensional organization of actin filaments in Dictyostelium pseudopods

    PubMed Central

    1995-01-01

    This study extends the observations on the defects in pseudopod formation of ABP-120+ and ABP-120- cells by a detailed morphological and biochemical analysis of the actin based cytoskeleton. Both ABP-120+ and ABP-120- cells polymerize the same amount of F-actin in response to stimulation with cAMP. However, unlike ABP-120+ cells, ABP-120- cells do not incorporate actin into the Triton X-100-insoluble cytoskeleton at 30-50 s, the time when ABP-120 is incorporated into the cytoskeleton and when pseudopods are extended after cAMP stimulation in wild-type cells. By confocal and electron microscopy, pseudopods extended by ABP- 120- cells are not as large or thick as those produced by ABP-120+ cells and in the electron microscope, an altered filament network is found in pseudopods of ABP-120- cells when compared to pseudopods of ABP-120+ cells. The actin filaments found in areas of pseudopods in ABP- 120+ cells either before or after stimulation were long, straight, and arranged into space filling orthogonal networks. Protrusions of ABP-120- cells are less three-dimensional, denser, and filled with multiple foci of aggregated filaments consistent with collapse of the filament network due to the absence of ABP-120-mediated cross-linking activity. The different organization of actin filaments may account for the diminished size of protrusions observed in living and fixed ABP-120- cells compared to ABP-120+ cells and is consistent with the role of ABP- 120 in regulating pseudopod extension through its cross-linking of actin filaments. PMID:7876307

  20. Structural Polymorphism of the Actin-Espin System: A Prototypical System of Filaments and Linkers in Stereocilia

    SciTech Connect

    Purdy, Kirstin R.; Wong, Gerard C. L.; Bartles, James R.

    2007-02-02

    We examine the interaction between cytoskeletal F-actin and espin 3A, a prototypical actin bundling protein found in sensory cell microvilli, including ear cell stereocilia. Espin induces twist distortions in F-actin as well as facilitates bundle formation. Mutations in one of the two F-actin binding sites of espin, which have been implicated in deafness, can tune espin-actin interactions and radically transform the system's phase behavior. These results are compared to recent theoretical work on the general phase behavior linker-rod systems.

  1. Structural Polymorphism of the Actin-Espin System: A Prototypical System of Filaments and Linkers in Stereocilia

    NASA Astrophysics Data System (ADS)

    Purdy, Kirstin R.; Bartles, James R.; Wong, Gerard C. L.

    2007-02-01

    We examine the interaction between cytoskeletal F-actin and espin 3A, a prototypical actin bundling protein found in sensory cell microvilli, including ear cell stereocilia. Espin induces twist distortions in F-actin as well as facilitates bundle formation. Mutations in one of the two F-actin binding sites of espin, which have been implicated in deafness, can tune espin-actin interactions and radically transform the system’s phase behavior. These results are compared to recent theoretical work on the general phase behavior linker-rod systems.

  2. Nuclear F-actin enhances the transcriptional activity of β-catenin by increasing its nuclear localization and binding to chromatin.

    PubMed

    Yamazaki, Shota; Yamamoto, Koji; de Lanerolle, Primal; Harata, Masahiko

    2016-04-01

    Actin plays multiple roles both in the cytoplasm and in the nucleus. Cytoplasmic actin, in addition to its structural role in the cytoskeleton, also contributes to the subcellular localization of transcription factors by interacting with them or their partners. The transcriptional cofactor β-catenin, which acts as an intracellular transducer of canonical Wnt signaling, indirectly associates with the cytoplasmic filamentous actin (F-actin). Recently, it has been observed that F-actin is transiently formed within the nucleus in response to serum stimulation and integrin signaling, and also during gene reprogramming. Despite these earlier observations, information about the function of nuclear F-actin is poorly defined. Here, by facilitating the accumulation of nuclear actin artificially, we demonstrate that polymerizing nuclear actin enhanced the nuclear accumulation and transcriptional function of β-catenin. Our results also show that the nuclear F-actin colocalizes with β-catenin and enhances the binding of β-catenin to the downstream target genes of the Wnt/β-catenin signaling pathway, including the genes for the cell cycle regulators c-myc and cyclin D, and the OCT4 gene. Nuclear F-actin itself also associated with these genes. Since Wnt/β-catenin signaling has important roles in cell differentiation and pluripotency, our observations suggest that nuclear F-actin formed during these biological processes is involved in regulating Wnt/β-catenin signaling. PMID:26900020

  3. Nuclear F-actin enhances the transcriptional activity of β-catenin by increasing its nuclear localization and binding to chromatin.

    PubMed

    Yamazaki, Shota; Yamamoto, Koji; de Lanerolle, Primal; Harata, Masahiko

    2016-04-01

    Actin plays multiple roles both in the cytoplasm and in the nucleus. Cytoplasmic actin, in addition to its structural role in the cytoskeleton, also contributes to the subcellular localization of transcription factors by interacting with them or their partners. The transcriptional cofactor β-catenin, which acts as an intracellular transducer of canonical Wnt signaling, indirectly associates with the cytoplasmic filamentous actin (F-actin). Recently, it has been observed that F-actin is transiently formed within the nucleus in response to serum stimulation and integrin signaling, and also during gene reprogramming. Despite these earlier observations, information about the function of nuclear F-actin is poorly defined. Here, by facilitating the accumulation of nuclear actin artificially, we demonstrate that polymerizing nuclear actin enhanced the nuclear accumulation and transcriptional function of β-catenin. Our results also show that the nuclear F-actin colocalizes with β-catenin and enhances the binding of β-catenin to the downstream target genes of the Wnt/β-catenin signaling pathway, including the genes for the cell cycle regulators c-myc and cyclin D, and the OCT4 gene. Nuclear F-actin itself also associated with these genes. Since Wnt/β-catenin signaling has important roles in cell differentiation and pluripotency, our observations suggest that nuclear F-actin formed during these biological processes is involved in regulating Wnt/β-catenin signaling.

  4. Engineering Circular Gliding of Actin Filaments Along Myosin-Patterned DNA Nanotube Rings To Study Long-Term Actin-Myosin Behaviors.

    PubMed

    Hariadi, Rizal F; Appukutty, Abhinav J; Sivaramakrishnan, Sivaraj

    2016-09-27

    Nature has evolved molecular motors that are critical in cellular processes occurring over broad time scales, ranging from seconds to years. Despite the importance of the long-term behavior of molecular machines, topics such as enzymatic lifetime are underexplored due to the lack of a suitable approach for monitoring motor activity over long time periods. Here, we developed an "O"-shaped Myosin Empowered Gliding Assay (OMEGA) that utilizes engineered micron-scale DNA nanotube rings with precise arrangements of myosin VI to trap gliding actin filaments. This circular gliding assay platform allows the same individual actin filament to glide over the same myosin ensemble (50-1000 motors per ring) multiple times. First, we systematically characterized the formation of DNA nanotubes rings with 4, 6, 8, and 10 helix circumferences. Individual actin filaments glide along the nanotube rings with high processivity for up to 12.8 revolutions or 11 min in run time. We then show actin gliding speed is robust to variation in motor number and independent of ring curvature within our sample space (ring diameter of 0.5-4 μm). As a model application of OMEGA, we then analyze motor-based mechanical influence on "stop-and-go" gliding behavior of actin filaments, revealing that the stop-to-go transition probability is dependent on motor flexibility. Our circular gliding assay may provide a closed-loop platform for monitoring long-term behavior of broad classes of molecular motors and enable characterization of motor robustness and long time scale nanomechanical processes.

  5. Binding of the N-terminal fragment C0-C2 of cardiac MyBP-C to cardiac F-actin.

    PubMed

    Kensler, Robert W; Shaffer, Justin F; Harris, Samantha P

    2011-04-01

    Cardiac myosin-binding protein C (cMyBP-C), a major accessory protein of cardiac thick filaments, is thought to play a key role in the regulation of myocardial contraction. Although current models for the function of the protein focus on its binding to myosin S2, other evidence suggests that it may also bind to F-actin. We have previously shown that the N-terminal fragment C0-C2 of cardiac myosin-binding protein-C (cMyBP-C) bundles actin, providing evidence for interaction of cMyBP-C and actin. In this paper we directly examined the interaction between C0-C2 and F-actin at physiological ionic strength and pH by negative staining and electron microscopy. We incubated C0-C2 (5-30μM, in a buffer containing in mM: 180 KCl, 1 MgCl(2), 1 EDTA, 1 DTT, 20 imidazole, at pH 7.4) with F-actin (5μM) for 30min and examined negatively-stained samples of the solution by electron microscopy (EM). Examination of EM images revealed that C0-C2 bound to F-actin to form long helically-ordered complexes. Fourier transforms indicated that C0-C2 binds with the helical periodicity of actin with strong 1st and 6th layer lines. The results provide direct evidence that the N-terminus of cMyBP-C can bind to F-actin in a periodic complex. This interaction of cMyBP-C with F-actin supports the possibility that binding of cMyBP-C to F-actin may play a role in the regulation of cardiac contraction.

  6. The evolution of the actin binding NET superfamily

    PubMed Central

    Hawkins, Timothy J.; Deeks, Michael J.; Wang, Pengwei; Hussey, Patrick J.

    2014-01-01

    The Arabidopsis Networked (NET) superfamily are plant-specific actin binding proteins which specifically label different membrane compartments and identify specialized sites of interaction between actin and membranes unique to plants. There are 13 members of the superfamily in Arabidopsis, which group into four distinct clades or families. NET homologs are absent from the genomes of metazoa and fungi; furthermore, in plantae, NET sequences are also absent from the genome of mosses and more ancient extant plant clades. A single family of the NET proteins is found encoded in the club moss genome, an extant species of the earliest vascular plants. Gymnosperms have examples from families 4 and 3, with a hybrid form of NET1 and 2 which shows characteristics of both NET1 and NET2. In addition to NET3 and 4 families, the NET1 and pollen-expressed NET2 families are found only as independent sequences in Angiosperms. This is consistent with the divergence of reproductive actin. The four families are conserved across Monocots and Eudicots, with the numbers of members of each clade expanding at this point, due, in part, to regions of genome duplication. Since the emergence of the NET superfamily at the dawn of vascular plants, they have continued to develop and diversify in a manner which has mirrored the divergence and increasing complexity of land-plant species. PMID:24926301

  7. The evolution of the actin binding NET superfamily.

    PubMed

    Hawkins, Timothy J; Deeks, Michael J; Wang, Pengwei; Hussey, Patrick J

    2014-01-01

    The Arabidopsis Networked (NET) superfamily are plant-specific actin binding proteins which specifically label different membrane compartments and identify specialized sites of interaction between actin and membranes unique to plants. There are 13 members of the superfamily in Arabidopsis, which group into four distinct clades or families. NET homologs are absent from the genomes of metazoa and fungi; furthermore, in plantae, NET sequences are also absent from the genome of mosses and more ancient extant plant clades. A single family of the NET proteins is found encoded in the club moss genome, an extant species of the earliest vascular plants. Gymnosperms have examples from families 4 and 3, with a hybrid form of NET1 and 2 which shows characteristics of both NET1 and NET2. In addition to NET3 and 4 families, the NET1 and pollen-expressed NET2 families are found only as independent sequences in Angiosperms. This is consistent with the divergence of reproductive actin. The four families are conserved across Monocots and Eudicots, with the numbers of members of each clade expanding at this point, due, in part, to regions of genome duplication. Since the emergence of the NET superfamily at the dawn of vascular plants, they have continued to develop and diversify in a manner which has mirrored the divergence and increasing complexity of land-plant species.

  8. The evolution of the actin binding NET superfamily.

    PubMed

    Hawkins, Timothy J; Deeks, Michael J; Wang, Pengwei; Hussey, Patrick J

    2014-01-01

    The Arabidopsis Networked (NET) superfamily are plant-specific actin binding proteins which specifically label different membrane compartments and identify specialized sites of interaction between actin and membranes unique to plants. There are 13 members of the superfamily in Arabidopsis, which group into four distinct clades or families. NET homologs are absent from the genomes of metazoa and fungi; furthermore, in plantae, NET sequences are also absent from the genome of mosses and more ancient extant plant clades. A single family of the NET proteins is found encoded in the club moss genome, an extant species of the earliest vascular plants. Gymnosperms have examples from families 4 and 3, with a hybrid form of NET1 and 2 which shows characteristics of both NET1 and NET2. In addition to NET3 and 4 families, the NET1 and pollen-expressed NET2 families are found only as independent sequences in Angiosperms. This is consistent with the divergence of reproductive actin. The four families are conserved across Monocots and Eudicots, with the numbers of members of each clade expanding at this point, due, in part, to regions of genome duplication. Since the emergence of the NET superfamily at the dawn of vascular plants, they have continued to develop and diversify in a manner which has mirrored the divergence and increasing complexity of land-plant species. PMID:24926301

  9. A novel multitarget tracking algorithm for Myosin VI protein molecules on actin filaments in TIRFM sequences.

    PubMed

    Li, G; Sanchez, V; Nagaraj, P C S B; Khan, S; Rajpoot, N

    2015-12-01

    We propose a novel multitarget tracking framework for Myosin VI protein molecules in total internal reflection fluorescence microscopy sequences which integrates an extended Hungarian algorithm with an interacting multiple model filter. The extended Hungarian algorithm, which is a linear assignment problem based method, helps to solve measurement assignment and spot association problems commonly encountered when dealing with multiple targets, although a two-motion model interacting multiple model filter increases the tracking accuracy by modelling the nonlinear dynamics of Myosin VI protein molecules on actin filaments. The evaluation of our tracking framework is conducted on both real and synthetic total internal reflection fluorescence microscopy sequences. The results show that the framework achieves higher tracking accuracies compared to the state-of-the-art tracking methods, especially for sequences with high spot density. PMID:26259144

  10. Specific Transformation of Assembly with Actin Filaments and Molecular Motors in a Cell-Sized Self-Emerged Liposome

    NASA Astrophysics Data System (ADS)

    Takiguchi, Kingo; Negishi, Makiko; Tanaka-Takiguchi, Yohko; Hayashi, Masahito; Yoshikawa, Kenichi

    2014-12-01

    Eukaryotes, by the same combination of cytoskeleton and molecular motor, for example actin filament and myosin, can generate a variety of movements. For this diversity, the organization of biological machineries caused by the confinement and/or crowding effects of internal living cells, may play very important roles.

  11. Generation of contractile actomyosin bundles depends on mechanosensitive actin filament assembly and disassembly

    PubMed Central

    Tojkander, Sari; Gateva, Gergana; Husain, Amjad; Krishnan, Ramaswamy; Lappalainen, Pekka

    2015-01-01

    Adhesion and morphogenesis of many non-muscle cells are guided by contractile actomyosin bundles called ventral stress fibers. While it is well established that stress fibers are mechanosensitive structures, physical mechanisms by which they assemble, align, and mature have remained elusive. Here we show that arcs, which serve as precursors for ventral stress fibers, undergo lateral fusion during their centripetal flow to form thick actomyosin bundles that apply tension to focal adhesions at their ends. Importantly, this myosin II-derived force inhibits vectorial actin polymerization at focal adhesions through AMPK-mediated phosphorylation of VASP, and thereby halts stress fiber elongation and ensures their proper contractility. Stress fiber maturation additionally requires ADF/cofilin-mediated disassembly of non-contractile stress fibers, whereas contractile fibers are protected from severing. Taken together, these data reveal that myosin-derived tension precisely controls both actin filament assembly and disassembly to ensure generation and proper alignment of contractile stress fibers in migrating cells. DOI: http://dx.doi.org/10.7554/eLife.06126.001 PMID:26652273

  12. Generation of contractile actomyosin bundles depends on mechanosensitive actin filament assembly and disassembly.

    PubMed

    Tojkander, Sari; Gateva, Gergana; Husain, Amjad; Krishnan, Ramaswamy; Lappalainen, Pekka

    2015-01-01

    Adhesion and morphogenesis of many non-muscle cells are guided by contractile actomyosin bundles called ventral stress fibers. While it is well established that stress fibers are mechanosensitive structures, physical mechanisms by which they assemble, align, and mature have remained elusive. Here we show that arcs, which serve as precursors for ventral stress fibers, undergo lateral fusion during their centripetal flow to form thick actomyosin bundles that apply tension to focal adhesions at their ends. Importantly, this myosin II-derived force inhibits vectorial actin polymerization at focal adhesions through AMPK-mediated phosphorylation of VASP, and thereby halts stress fiber elongation and ensures their proper contractility. Stress fiber maturation additionally requires ADF/cofilin-mediated disassembly of non-contractile stress fibers, whereas contractile fibers are protected from severing. Taken together, these data reveal that myosin-derived tension precisely controls both actin filament assembly and disassembly to ensure generation and proper alignment of contractile stress fibers in migrating cells. PMID:26652273

  13. The effects of actin cytoskeleton perturbation on keratin intermediate filament formation in mesenchymal stem/stromal cells.

    PubMed

    Chang, Tzu-Hao; Huang, Hsien-Da; Ong, Wei-Kee; Fu, Yun-Ju; Lee, Oscar K; Chien, Shu; Ho, Jennifer H

    2014-04-01

    F-actin plays a crucial role in composing the three-dimensional cytoskeleton and F-actin depolymerization alters fate choice of mesenchymal stem/stromal cells (MSCs). Here, we investigated differential gene expression and subsequent physiological changes in response to F-actin perturbation by latrunculin B in MSCs. Nineteen genes were down-regulated and 27 genes were up-regulated in the first 15 min after F-actin depolymerization. Functional enrichment analysis revealed that five genes involved in keratin (KRT) intermediate filaments clustering in the chromosome 17q21.2 region, i.e., KRT14, KRT19, KRT34, KRT-associated protein (KRTAP) 1-5, and KRTAP2-3, were strongly up-regulated. Transcription factor prediction identified NKX2.5 as the potential transcription factor to control KRT19, KRT34, KRTAP1-5, and KRTAP2-3; and indeed, the protein level of NKX2.5 was markedly increased in the nuclear fraction within 15 min of F-actin depolymerization. The peak of keratin intermediate filament formation was 1 h after actin perturbation, and the morphological changes showed by decrease in the ratio of long-axis to short-axis diameter in MSCs was observed after 4 h. Together, F-actin depolymerization rapidly triggers keratin intermediate filament formation by turning on keratin-related genes on chromosome 17q21.2. Such findings offer new insight in lineage commitment of MSCs and further scaffold design in MSC-based tissue engineering.

  14. Spatial relationship between the nucleotide-binding site, Lys-61 and Cys-374 in actin and a conformational change induced by myosin subfragment-1 binding.

    PubMed

    Miki, M; dos Remedios, C G; Barden, J A

    1987-10-15

    The spatial relationship between Lys-61, the nucleotide binding site and Cys-374 was studied. Lys-61 was labelled with fluorescein-5-isothiocyanate as a resonance energy acceptor, the nucleotide-binding site was labelled with the fluorescent ATP analogues epsilon ATP or formycin-A 5'-triphosphate (FTP) and Cys-374 was labelled with 5-(2-[(iodoacetyl)amino]ethyl)aminonaphthalene-1-sulfonic acid (1,5-IAEDANS) as a resonance energy donor. The distances between the nucleotide binding site and Lys-61 or between Lys-61 and Cys-374 were calculated to be 3.5 +/- 0.3 nm and 4.60 +/- 0.03 nm, respectively. (The assumption has been made in calculating these distances that the energy donor and acceptor rotate rapidly relative to the fluorescence lifetime.) On the other hand, when doubly-labelled actin with 1,5-IAEDANS at Cys-374 and FITC at Lys-61 was polymerized in the presence of a twofold molar excess of phalloidin [Miki, M. (1987) Eur. J. Biochem. 164, 229-235], the fluorescence of 1,5-IAEDANS bound to actin was quenched significantly. This could be attributed to inter-monomer energy transfer. The inter-monomer distance between FITC attached to Lys-61 in a monomer and 1,5-IAEDANS attached to Cys-374 in its nearest-neighbour monomer in an F-actin filament was calculated to be 3.34 +/- 0.06 nm, assuming that the likely change in the intra-monomer distance does not change during polymerization by more than 0.4 nm. One possible spatial relationship between Lys-61, Cys-374 and the nucleotide binding site in an F-actin filament is proposed. The effect of myosin subfragment-1 (S1) binding on the energy transfer efficiency was studied. The fluorescence intensity of AEDANS-FITC-actin decreased by 30% upon interaction with S1. The fluorescence intensity of AEDANS-FITC-actin polymer in the presence of phalloidin increased by 21% upon interaction with S1. The addition of ATP led to the fluorescence intensity returning to the initial level. Assuming that the change of fluorescence

  15. Engagement of CD81 induces ezrin tyrosine phosphorylation and its cellular redistribution with filamentous actin

    SciTech Connect

    Coffey, Greg P.; Rajapaksa, Ranjani; Liu, Raymond; Sharpe, Orr; Kuo, Chiung-Chi; Wald Krauss, Sharon; Sagi, Yael; Davis, R. Eric; Staudt, Louis M.; Sharman, Jeff P.; Robinson, William H.; Levy, Shoshana

    2009-06-09

    CD81 is a tetraspanin family member involved in diverse cellular interactions in the immune and nervous systems and in cell fusion events. However, the mechanism of action of CD81 and of other tetraspanins has not been defined. We reasoned that identifying signaling molecules downstream of CD81 would provide mechanistic clues. We engaged CD81 on the surface of Blymphocytes and identified the induced tyrosine-phosphorylated proteins by mass spectrometry. This analysis showed that the most prominent tyrosine phosphorylated protein was ezrin, an actin binding protein and a member of the ezrin-radixin-moesin family. We also found that CD81 engagement induces spleen tyrosine kinase (Syk) and that Syk was involved in tyrosine phosphorylation of ezrin. Ezrin colocalized with CD81 and F-actin upon stimulation and this association was disrupted when Syk activation was blocked. Taken together, these studies suggest a model in which CD81 interfaces between the plasma membrane and the cytoskeleton by activating Syk, mobilizing ezrin, and recruiting F-actin to facilitate cytoskeletal reorganization and cell signaling. This may be a mechanism explaining the pleiotropic effects induced in response to stimulating cells by anti-CD81 antibodies or by the hepatitis C virus, which uses this molecule as its key receptor.

  16. Engagement of CD81 induces ezrin tyrosine phosphorylation and its cellular redistribution with filamentous actin

    PubMed Central

    Coffey, Greg P.; Rajapaksa, Ranjani; Liu, Raymond; Sharpe, Orr; Kuo, Chiung-Chi; Krauss, Sharon Wald; Sagi, Yael; Davis, R. Eric; Staudt, Louis M.; Sharman, Jeff P.; Robinson, William H.; Levy, Shoshana

    2009-01-01

    Summary CD81 is a tetraspanin family member involved in diverse cellular interactions in the immune and nervous systems and in cell fusion events. However, the mechanism of action of CD81 and of other tetraspanins has not been defined. We reasoned that identifying signaling molecules downstream of CD81 would provide mechanistic clues. We engaged CD81 on the surface of B-lymphocytes and identified the induced tyrosine-phosphorylated proteins by mass spectrometry. This analysis showed that the most prominent tyrosine phosphorylated protein was ezrin, an actin-binding protein and a member of the ezrin-radixin-moesin family. We also found that CD81 engagement induces spleen tyrosine kinase (Syk) and that Syk was involved in tyrosine phosphorylation of ezrin. After engagement of CD81, it colocalized with ezrin and F-actin, and this association was disrupted when Syk activation was blocked. Taken together, these studies suggest a model in which CD81 interfaces between the plasma membrane and the cytoskeleton by activating Syk, mobilizing ezrin, and recruiting F-actin to facilitate cytoskeletal reorganization and cell signaling. This mechanism might explain the pleiotropic effects induced in response to stimulation of cells by anti-CD81 antibodies or by the hepatitis C virus, which uses this molecule as its key receptor. PMID:19654214

  17. Single-Molecule Discrimination within Dendritic Spines of Discrete Perisynaptic Sites of Actin Filament Assembly Driving Postsynaptic Reorganization

    NASA Astrophysics Data System (ADS)

    Blanpied, Thomas A.

    2013-03-01

    In the brain, the strength of synaptic transmission between neurons is principally set by the organization of proteins within the receptive, postsynaptic cell. Synaptic strength at an individual site of contact can remain remarkably stable for months or years. However, it also can undergo diverse forms of plasticity which change the strength at that contact independent of changes to neighboring synapses. Such activity-triggered neural plasticity underlies memory storage and cognitive development, and is disrupted in pathological physiology such as addiction and schizophrenia. Much of the short-term regulation of synaptic plasticity occurs within the postsynaptic cell, in small subcompartments surrounding the synaptic contact. Biochemical subcompartmentalization necessary for synapse-specific plasticity is achieved in part by segregation of synapses to micron-sized protrusions from the cell called dendritic spines. Dendritic spines are heavily enriched in the actin cytoskeleton, and regulation of actin polymerization within dendritic spines controls both basal synaptic strength and many forms of synaptic plasticity. However, understanding the mechanism of this control has been difficult because the submicron dimensions of spines limit examination of actin dynamics in the spine interior by conventional confocal microscopy. To overcome this, we developed single-molecule tracking photoactivated localization microscopy (smtPALM) to measure the movement of individual actin molecules within living spines. This revealed inward actin flow from broad areas of the spine plasma membrane, as well as a dense central core of heterogeneous filament orientation. The velocity of single actin molecules along filaments was elevated in discrete regions within the spine, notably near the postsynaptic density but surprisingly not at the endocytic zone which is involved in some forms of plasticity. We conclude that actin polymerization is initiated at many well-separated foci within

  18. Dynamic actin structures stabilized by profilin.

    PubMed Central

    Finkel, T; Theriot, J A; Dise, K R; Tomaselli, G F; Goldschmidt-Clermont, P J

    1994-01-01

    We describe the production and analysis of clonal cell lines in which we have overexpressed human profilin, a small ubiquitous actin monomer binding protein, to assess the role of profilin on actin function in vivo. The concentration of filamentous actin is increased in cells with higher profilin levels, and actin filament half-life measured in these cells is directly proportional to the steady-state profilin concentration. The distribution of actin filaments is altered by profilin overexpression. While parallel actin bundles crossing the cells are virtually absent in cells overexpressing profilin, the submembranous actin network of these cells is denser than in control cells. These results suggest that in vivo profilin regulates the stability, and thereby distribution, of specific dynamic actin structures. Images PMID:8108438

  19. Modulation of actin structure and function by phosphorylation of Tyr-53 and profilin binding

    SciTech Connect

    Baek, Kyuwon; Liu, Xiong; Ferron, Francois; Shu, Shi; Korn, Edward D.; Dominguez, Roberto

    2008-08-27

    On starvation, Dictyostelium cells aggregate to form multicellular fruiting bodies containing spores that germinate when transferred to nutrient-rich medium. This developmental cycle correlates with the extent of actin phosphorylation at Tyr-53 (pY53-actin), which is low in vegetative cells but high in viable mature spores. Here we describe high-resolution crystal structures of pY53-actin and unphosphorylated actin in complexes with gelsolin segment 1 and profilin. In the structure of pY53-actin, the phosphate group on Tyr-53 makes hydrogen-bonding interactions with residues of the DNase I-binding loop (D-loop) of actin, resulting in a more stable conformation of the D-loop than in the unphosphorylated structures. A more rigidly folded D-loop may explain some of the previously described properties of pY53-actin, including its increased critical concentration for polymerization, reduced rates of nucleation and pointed end elongation, and weak affinity for DNase I. We show here that phosphorylation of Tyr-53 inhibits subtilisin cleavage of the D-loop and reduces the rate of nucleotide exchange on actin. The structure of profilin-Dictyostelium-actin is strikingly similar to previously determined structures of profilin-{beta}-actin and profilin-{alpha}-actin. By comparing this representative set of profilin-actin structures with other structures of actin, we highlight the effects of profilin on the actin conformation. In the profilin-actin complexes, subdomains 1 and 3 of actin close around profilin, producing a 4.7 deg. rotation of the two major domains of actin relative to each other. As a result, the nucleotide cleft becomes moderately more open in the profilin-actin complex, probably explaining the stimulation of nucleotide exchange on actin by profilin.

  20. Structural Characterization of the Binding of Myosin*ADP*Pi to Actin in Permeabilized Rabbit Psoas Muscle

    SciTech Connect

    Xu,S.; Gu, J.; Belknap, B.; White, H.; Yu, L.

    2006-01-01

    When myosin is attached to actin in a muscle cell, various structures in the filaments are formed. The two strongly bound states (A{center_dot}M{center_dot}ADP and A{center_dot}M) and the weakly bound A{center_dot}M{center_dot}ATP states are reasonably well understood. The orientation of the strongly bound myosin heads is uniform ('stereospecific' attachment), and the attached heads exhibit little spatial fluctuation. In the prehydrolysis weakly bound A{center_dot}M{center_dot}ATP state, the orientations of the attached myosin heads assume a wide range of azimuthal and axial angles, indicating considerable flexibility in the myosin head. The structure of the other weakly bound state, A{center_dot}M{center_dot}ADP{center_dot}P{sub i}, however, is poorly understood. This state is thought to be the critical pre-power-stroke state, poised to make the transition to the strongly binding, force-generating states, and hence it is of particular interest for understanding the mechanism of contraction. However, because of the low affinity between myosin and actin in the A{center_dot}M{center_dot}ADP{center_dot}P{sub i} state, the structure of this state has eluded determination both in isolated form and in muscle cells. With the knowledge recently gained in the structures of the weakly binding M{center_dot}ATP, M{center_dot}ADP{center_dot}P{sub i} states and the weakly attached A{center_dot}M{center_dot}ATP state in muscle fibers, it is now feasible to delineate the in vivo structure of the attached state of A{center_dot}M{center_dot}ADP{center_dot}P{sub i}. The series of experiments presented in this article were carried out under relaxing conditions at 25{sup o}C, where {approx}95% of the myosin heads in the skinned rabbit psoas muscle contain the hydrolysis products. The affinity for actin is enhanced by adding polyethylene glycol (PEG) or by lowering the ionic strength in the bathing solution. Solution kinetics and binding constants were determined in the presence and in

  1. RAB-10 Promotes EHBP-1 Bridging of Filamentous Actin and Tubular Recycling Endosomes.

    PubMed

    Wang, Peixiang; Liu, Hang; Wang, Yu; Liu, Ou; Zhang, Jing; Gleason, Adenrele; Yang, Zhenrong; Wang, Hui; Shi, Anbing; Grant, Barth D

    2016-06-01

    EHBP-1 (Ehbp1) is a conserved regulator of endocytic recycling, acting as an effector of small GTPases including RAB-10 (Rab10). Here we present evidence that EHBP-1 associates with tubular endosomal phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] enriched membranes through an N-terminal C2-like (NT-C2) domain, and define residues within the NT-C2 domain that mediate membrane interaction. Furthermore, our results indicate that the EHBP-1 central calponin homology (CH) domain binds to actin microfilaments in a reaction that is stimulated by RAB-10(GTP). Loss of any aspect of this RAB-10/EHBP-1 system in the C. elegans intestinal epithelium leads to retention of basolateral recycling cargo in endosomes that have lost their normal tubular endosomal network (TEN) organization. We propose a mechanism whereby RAB-10 promotes the ability of endosome-bound EHBP-1 to also bind to the actin cytoskeleton, thereby promoting endosomal tubulation. PMID:27272733

  2. RAB-10 Promotes EHBP-1 Bridging of Filamentous Actin and Tubular Recycling Endosomes

    PubMed Central

    Wang, Yu; Liu, Ou; Zhang, Jing; Gleason, Adenrele; Yang, Zhenrong; Wang, Hui; Shi, Anbing; Grant, Barth D.

    2016-01-01

    EHBP-1 (Ehbp1) is a conserved regulator of endocytic recycling, acting as an effector of small GTPases including RAB-10 (Rab10). Here we present evidence that EHBP-1 associates with tubular endosomal phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] enriched membranes through an N-terminal C2-like (NT-C2) domain, and define residues within the NT-C2 domain that mediate membrane interaction. Furthermore, our results indicate that the EHBP-1 central calponin homology (CH) domain binds to actin microfilaments in a reaction that is stimulated by RAB-10(GTP). Loss of any aspect of this RAB-10/EHBP-1 system in the C. elegans intestinal epithelium leads to retention of basolateral recycling cargo in endosomes that have lost their normal tubular endosomal network (TEN) organization. We propose a mechanism whereby RAB-10 promotes the ability of endosome-bound EHBP-1 to also bind to the actin cytoskeleton, thereby promoting endosomal tubulation. PMID:27272733

  3. BRCA2 BRC motifs bind RAD51-DNA filaments.

    PubMed

    Galkin, Vitold E; Esashi, Fumiko; Yu, Xiong; Yang, Shixin; West, Stephen C; Egelman, Edward H

    2005-06-14

    Germ-line mutations in BRCA2 account for approximately half the cases of autosomal dominant familial breast cancers. BRCA2 has been shown to interact directly with RAD51, an essential component of the cellular machinery for homologous recombination and the maintenance of genome stability. Interactions between BRCA2 and RAD51 take place by means of the conserved BRC repeat regions of BRCA2. Previously, it was shown that peptides corresponding to BRC3 or BRC4 bind RAD51 monomers and block RAD51-DNA filament formation. In this work, we further analyze these interactions and find that at lower molar ratios BRC3 or BRC4 actually bind and form stable complexes with RAD51-DNA nucleoprotein filaments. Only at high concentrations of the BRC repeats are filaments disrupted. The specific protein-protein contacts occur in the RAD51 filament by means of the N-terminal domain of RAD51 for BRC3 and the nucleotide-binding core of RAD51 for BRC4. These observations show that the BRC repeats bind distinct regions of RAD51 and are nonequivalent in their mode of interaction. The results provide insight into why mutation in just one of the eight BRC repeats would affect the way that BRCA2 protein interacts with the RAD51 filament. Disruption of a single RAD51 interaction site, one of several simultaneous interactions occurring throughout the BRC repeat-containing exon 11 of BRCA2, might modulate the ability of RAD51 to promote recombinational repair and lead to an increased risk of breast cancer.

  4. A variational approach to the growth dynamics of pre-stressed actin filament networks.

    PubMed

    John, Karin; Stöter, Thomas; Misbah, Chaouqi

    2016-09-21

    In order to model the growth dynamics of elastic bodies with residual stresses a thermodynamically consistent approach is needed such that the cross-coupling between growth and mechanics can be correctly described. In the present work we apply a variational principle to the formulation of the interfacial growth dynamics of dendritic actin filament networks growing from biomimetic beads, an experimentally well studied system, where the buildup of residual stresses governs the network growth. We first introduce the material model for the network via a strain energy density for an isotropic weakly nonlinear elastic material and then derive consistently from this model the dynamic equations for the interfaces, i.e. for a polymerizing internal interface in contact with the bead and a depolymerizing external interface directed towards the solvent. We show that (i) this approach automatically preserves thermodynamic symmetry-properties, which is not the case for the often cited 'rubber-band-model' (Sekimoto et al 2004 Eur. Phys. J. E 13 247-59, Plastino et al 2004 Eur. Biophys. J. 33 310-20) and (ii) leads to a robust morphological instability of the treadmilling network interfaces. The nature of the instability depends on the interplay of the two dynamic interfaces. Depending on the biochemical conditions the network envelope evolves into a comet-like shape (i.e. the actin envelope thins out at one side and thickens on the opposite side of the bead) via a varicose instability or it breaks the symmetry via higher order zigzag modes. We conclude that morphological instabilities due to mechano-chemical coupling mechanisms and the presences of mechancial pre-stresses can play a major role in locally organizing the cytoskeleton of living cells. PMID:27420637

  5. A variational approach to the growth dynamics of pre-stressed actin filament networks

    NASA Astrophysics Data System (ADS)

    John, Karin; Stöter, Thomas; Misbah, Chaouqi

    2016-09-01

    In order to model the growth dynamics of elastic bodies with residual stresses a thermodynamically consistent approach is needed such that the cross-coupling between growth and mechanics can be correctly described. In the present work we apply a variational principle to the formulation of the interfacial growth dynamics of dendritic actin filament networks growing from biomimetic beads, an experimentally well studied system, where the buildup of residual stresses governs the network growth. We first introduce the material model for the network via a strain energy density for an isotropic weakly nonlinear elastic material and then derive consistently from this model the dynamic equations for the interfaces, i.e. for a polymerizing internal interface in contact with the bead and a depolymerizing external interface directed towards the solvent. We show that (i) this approach automatically preserves thermodynamic symmetry-properties, which is not the case for the often cited ‘rubber-band-model’ (Sekimoto et al 2004 Eur. Phys. J. E 13 247–59, Plastino et al 2004 Eur. Biophys. J. 33 310–20) and (ii) leads to a robust morphological instability of the treadmilling network interfaces. The nature of the instability depends on the interplay of the two dynamic interfaces. Depending on the biochemical conditions the network envelope evolves into a comet-like shape (i.e. the actin envelope thins out at one side and thickens on the opposite side of the bead) via a varicose instability or it breaks the symmetry via higher order zigzag modes. We conclude that morphological instabilities due to mechano-chemical coupling mechanisms and the presences of mechancial pre-stresses can play a major role in locally organizing the cytoskeleton of living cells.

  6. A variational approach to the growth dynamics of pre-stressed actin filament networks

    NASA Astrophysics Data System (ADS)

    John, Karin; Stöter, Thomas; Misbah, Chaouqi

    2016-09-01

    In order to model the growth dynamics of elastic bodies with residual stresses a thermodynamically consistent approach is needed such that the cross-coupling between growth and mechanics can be correctly described. In the present work we apply a variational principle to the formulation of the interfacial growth dynamics of dendritic actin filament networks growing from biomimetic beads, an experimentally well studied system, where the buildup of residual stresses governs the network growth. We first introduce the material model for the network via a strain energy density for an isotropic weakly nonlinear elastic material and then derive consistently from this model the dynamic equations for the interfaces, i.e. for a polymerizing internal interface in contact with the bead and a depolymerizing external interface directed towards the solvent. We show that (i) this approach automatically preserves thermodynamic symmetry-properties, which is not the case for the often cited ‘rubber-band-model’ (Sekimoto et al 2004 Eur. Phys. J. E 13 247-59, Plastino et al 2004 Eur. Biophys. J. 33 310-20) and (ii) leads to a robust morphological instability of the treadmilling network interfaces. The nature of the instability depends on the interplay of the two dynamic interfaces. Depending on the biochemical conditions the network envelope evolves into a comet-like shape (i.e. the actin envelope thins out at one side and thickens on the opposite side of the bead) via a varicose instability or it breaks the symmetry via higher order zigzag modes. We conclude that morphological instabilities due to mechano-chemical coupling mechanisms and the presences of mechancial pre-stresses can play a major role in locally organizing the cytoskeleton of living cells.

  7. Reorganized actin filaments anchor chloroplasts along the anticlinal walls of Vallisneria epidermal cells under high-intensity blue light.

    PubMed

    Sakai, Yuuki; Takagi, Shingo

    2005-08-01

    In epidermal cells of the aquatic angiosperm Vallisneria gigantea Graebner, high-intensity blue light (BL) induces the avoidance response of chloroplasts. We examined simultaneous BL-induced changes in the configuration of actin filaments in the cytoplasmic layers that face the outer periclinal wall (P side) and the anticlinal wall (A side). The results clearly showed that dynamic reorganization of the actin cytoskeleton occurs on both sides. Upon BL irradiation, thick, long bundles of actin filaments appeared, concomitant with the directed migration of chloroplasts from the P side to the A side. After 15-20 min of BL irradiation, fine actin bundles on only the A side appeared to associate with chloroplasts that had migrated from the P side. To examine the role of the fine actin bundles, we evaluated the anchorage of chloroplasts by centrifuging living cells. Upon BL irradiation, the resistance of chloroplasts on both the P and A sides to the centrifugal force decreased remarkably. After 20 min of BL irradiation, the resistance of chloroplasts on the A side increased again, but chloroplasts on the P side could still be displaced. The BL-induced recovery of resistance of chloroplasts on the A side was sensitive to photosynthesis inhibitors but insensitive to an inhibitor of flavoproteins. The photosynthesis inhibitors also prevented the fine actin bundles from appearing on the A side under BL irradiation. These results strongly suggest that the BL-induced avoidance response of chloroplasts includes photosynthesis-dependent and actin-dependent anchorage of chloroplasts on the A side of epidermal cells. PMID:15809866

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

    PubMed

    van der Honing, Hannie S; Kieft, Henk; Emons, Anne Mie C; Ketelaar, Tijs

    2012-03-01

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

  9. The role of actin binding proteins in epithelial morphogenesis: models based upon Listeria movement.

    PubMed

    Golsteyn, R M; Louvard, D; Friederich, E

    1997-10-01

    We summarize recent findings on the organization of the protein actin in eucaryotic cells. In particular we focus on how actin can be used to generate a vectorial force that is required for cell movement. These forces arise from protein molecules that recruit actin to the plasma membrane in such a manner that actin filaments extend outward from the cell body. This type of actin dependent force generation has been described in a nucleation-release model, which is one of several models currently being tested to explain actin dependent cell movement. Data in support of this model has arisen unexpectedly from studies of an intracellular bacteria, Listeria monocytogenes. This bacteria uses actin to propel itself during infection of eucaryotic cells. By studying Listeria movement, the roles of several eucaryotic actin interacting proteins have been identified. One of these is zyxin, a human protein that shares important structural and possibly functional properties with ActA, an actin dependent force generating protein of Listeria. We intend to test the function of these and other actin interacting proteins in a simplified system that should facilitate precise measurement of their properties of force generation in vitro.

  10. LlSR28 is involved in pollen germination by affecting filamentous actin dynamics.

    PubMed

    Cao, Li-Juan; Zhao, Meng-Meng; Liu, Chang; Dong, Huai-Jian; Li, Wang-Cheng; Ren, Hai-Yun

    2013-07-01

    Alternative splicing plays important roles in gene regulation and contributes to protein complexity. Previous studies suggest that alternative splicing exists in members of the villin/gelsolin/fragmin superfamily. In this study, a serine/argine-rich (SR) protein cDNA with 28 kDa protein (LlSR28) was isolated from a lily (Lilium longiflorum) expression library. Protein domain analysis showed that LlSR28 had similar structures to Arabidopsis SR45 (AtSR45), and LlSR28 could complement the phenotype of loss of AtSR45 function. Therefore, overexpression of LlSR28 and AtSR45 mutant (atsr45-1) were used in the following experiments. Overexpression of LlSR28 in Arabidopsis completely inhibited pollen germination. In contrast, the pollen germination of atsr45-1 was earlier than that of wild-type. In addition, pollen of atsr45-1 contained less F-actin at the corresponding hydration stage during pollen germination compared to that of wild-type. Alternative splicing analysis showed that Arabidopsis villin1 (AtVLN1) transcript encoding the full-length protein was increased, and that encoding the truncated protein was decreased in atst45-1. Moreover, the mRNA expression level of other actin-binding proteins (ABPs) abundant in Arabidopsis pollen was also changed in atsr45-1. In conclusion, we hypothesize that LlSR28 alters F-actin dynamics probably through its alternative splicing activities to affect directly or indirectly the alternative splicing of AtVLN1 and the expression of different ABPs, which then affects the pollen germination. PMID:23741063

  11. Cortical actin filament organization in developing and functioning stomatal complexes of Zea mays and Triticum turgidum.

    PubMed

    Panteris, Emmanuel; Galatis, Basil; Quader, Hartmut; Apostolakos, Panagiotis

    2007-07-01

    Cortical actin filament (AF) organization was studied in detail in developing stomatal complexes of the grasses Zea mays and Triticum turgidum. AF arrays during the whole stomatal complex development are dynamic, partly following the pattern of cortical microtubule (MT) organization. They also exhibit particular patterns of organization, spatially and temporarily restricted. Among AF arrays, the radial ones that underlie young guard cell (GC) periclinal walls, those that line the bulbous GC ends and the AF ring at the junction between subsidiary cells (SCs) and GCs are described here for the first time. Although many similarities in cortical AF organization exist among the stomatal cells of both plants studied, considerable differences have also been observed between them. Our data reveal that the expanding areas of stomatal cell walls are lined by distinct cortical AF aggregations that probably protect the plasmalemma against mechanical stresses. Experimental AF disruption does not seem to affect detectably stomatal cell morphogenesis. Moreover, the structural and experimental data of this study revealed that, in contrast to the elliptical stomata, in the dumbbell-shaped ones the AFs and MTs seem not to be involved in the mechanism of opening and closing of the stomatal pore.

  12. Crystallization of fluorescent quantum dots within a three-dimensional bio-organic template of actin filaments and lipid membranes.

    PubMed

    Henry, Etienne; Dif, Aurélien; Schmutz, Marc; Legoff, Loic; Amblard, François; Marchi-Artzner, Valérie; Artzner, Franck

    2011-12-14

    Biological molecules and molecular self-assemblies are promising templates to organize well-defined inorganic nanostructures. We demonstrate the ability of a self-assembled three-dimensional crystal template of helical actin protein filaments and lipids bilayers to generate a hierarchical self-assembly of quantum dots. Functionnalized tricystein peptidic quantum dots (QDs) are incorporated during the dynamical self-assembly of this actin/lipid template resulting in the formation of crystalline fibers. The crystal parameters, 26.5×18.9×35.5 nm3, are imposed by the membrane thickness, the diameter, and the pitch of the actin self-assembly. This process ensures the high quality of the crystal and results in unexpected fluorescence properties. This method of preparation offers opportunities to generate crystals with new symmetries and a large range of distance parameters.

  13. Crystallization of fluorescent quantum dots within a three-dimensional bio-organic template of actin filaments and lipid membranes.

    PubMed

    Henry, Etienne; Dif, Aurélien; Schmutz, Marc; Legoff, Loic; Amblard, François; Marchi-Artzner, Valérie; Artzner, Franck

    2011-12-14

    Biological molecules and molecular self-assemblies are promising templates to organize well-defined inorganic nanostructures. We demonstrate the ability of a self-assembled three-dimensional crystal template of helical actin protein filaments and lipids bilayers to generate a hierarchical self-assembly of quantum dots. Functionnalized tricystein peptidic quantum dots (QDs) are incorporated during the dynamical self-assembly of this actin/lipid template resulting in the formation of crystalline fibers. The crystal parameters, 26.5×18.9×35.5 nm3, are imposed by the membrane thickness, the diameter, and the pitch of the actin self-assembly. This process ensures the high quality of the crystal and results in unexpected fluorescence properties. This method of preparation offers opportunities to generate crystals with new symmetries and a large range of distance parameters. PMID:22074314

  14. Actin filaments and microtubule dual-granule transport in human adhered platelets: the role of alpha-dystrobrevins.

    PubMed

    Cerecedo, Doris; Cisneros, Bulmaro; Mondragón, Ricardo; González, Sirenia; Galván, Iván J

    2010-04-01

    Upon activation with physiological stimuli, human platelets undergo morphological changes, centralizing their organelles and secreting effector molecules at the site of vascular injury. Previous studies have indicated that the actin filaments and microtubules of suspension-activated platelets play a critical role in granule movement and exocytosis; however, the participation of these cytoskeleton elements in adhered platelets remains unexplored. alpha- and beta-dystrobrevin members of the dystrophin-associated protein complex in muscle and non-muscle cells have been described as motor protein receptors that might participate in the transport of cellular components in neurons. Recently, we characterized the expression of dystrobrevins in platelets; however, their functional diversity within this cellular model had not been elucidated. The present study examined the contribution of actin filaments and microtubules in granule trafficking during the platelet adhesion process using cytoskeleton-disrupting drugs, quantification of soluble P-selectin, fluorescence resonance transfer energy analysis and immunoprecipitation assays. Likewise, we assessed the interaction of alpha-dystrobrevins with the ubiquitous kinesin heavy chain. Our results strongly suggest that microtubules and actin filaments participate in the transport of alpha and dense granules in the platelet adhesion process, during which alpha-dystrobrevins play the role of regulatory and adaptor proteins that govern trafficking events.

  15. [Tropomyosin and myosin subfragment 1 induce in thin muscle fiber filaments differing conformational changes in the C-terminal portion of the polypeptide chain of actin].

    PubMed

    Borovikov, Iu S; Dobrowolski, Z; Dabrowska, R

    1988-08-01

    Muscle fibres, free of myosin, troponin and tropomyosin, containing thin filaments reconstructed from G-actin and modified by fluorescent label 1,5-IAEDANS were used for polarized microfluorimetric studies of the effect of tropomyosin (TM) from smooth muscles, and of subfragment 1 (S1) from skeletal muscles on the structural state of F-actin. TM and S1 were shown to initiate different changes in polarized fluorescence of 1,5-IAEDANS of F-actin: TM increases, whereas S1 decreases fluorescent anisotropy. It was suggested that the structural state of F-actin may differ in the C-terminal of polypeptide chain of actin.

  16. Kv3.3 Channels Bind Hax-1 and Arp2/3 to Assemble a Stable Local Actin Network that Regulates Channel Gating.

    PubMed

    Zhang, Yalan; Zhang, Xiao-Feng; Fleming, Matthew R; Amiri, Anahita; El-Hassar, Lynda; Surguchev, Alexei A; Hyland, Callen; Jenkins, David P; Desai, Rooma; Brown, Maile R; Gazula, Valeswara-Rao; Waters, Michael F; Large, Charles H; Horvath, Tamas L; Navaratnam, Dhasakumar; Vaccarino, Flora M; Forscher, Paul; Kaczmarek, Leonard K

    2016-04-01

    Mutations in the Kv3.3 potassium channel (KCNC3) cause cerebellar neurodegeneration and impair auditory processing. The cytoplasmic C terminus of Kv3.3 contains a proline-rich domain conserved in proteins that activate actin nucleation through Arp2/3. We found that Kv3.3 recruits Arp2/3 to the plasma membrane, resulting in formation of a relatively stable cortical actin filament network resistant to cytochalasin D that inhibits fast barbed end actin assembly. These Kv3.3-associated actin structures are required to prevent very rapid N-type channel inactivation during short depolarizations of the plasma membrane. The effects of Kv3.3 on the actin cytoskeleton are mediated by the binding of the cytoplasmic C terminus of Kv3.3 to Hax-1, an anti-apoptotic protein that regulates actin nucleation through Arp2/3. A human Kv3.3 mutation within a conserved proline-rich domain produces channels that bind Hax-1 but are impaired in recruiting Arp2/3 to the plasma membrane, resulting in growth cones with deficient actin veils in stem cell-derived neurons.

  17. Kv3.3 Channels Bind Hax-1 and Arp2/3 to Assemble a Stable Local Actin Network that Regulates Channel Gating.

    PubMed

    Zhang, Yalan; Zhang, Xiao-Feng; Fleming, Matthew R; Amiri, Anahita; El-Hassar, Lynda; Surguchev, Alexei A; Hyland, Callen; Jenkins, David P; Desai, Rooma; Brown, Maile R; Gazula, Valeswara-Rao; Waters, Michael F; Large, Charles H; Horvath, Tamas L; Navaratnam, Dhasakumar; Vaccarino, Flora M; Forscher, Paul; Kaczmarek, Leonard K

    2016-04-01

    Mutations in the Kv3.3 potassium channel (KCNC3) cause cerebellar neurodegeneration and impair auditory processing. The cytoplasmic C terminus of Kv3.3 contains a proline-rich domain conserved in proteins that activate actin nucleation through Arp2/3. We found that Kv3.3 recruits Arp2/3 to the plasma membrane, resulting in formation of a relatively stable cortical actin filament network resistant to cytochalasin D that inhibits fast barbed end actin assembly. These Kv3.3-associated actin structures are required to prevent very rapid N-type channel inactivation during short depolarizations of the plasma membrane. The effects of Kv3.3 on the actin cytoskeleton are mediated by the binding of the cytoplasmic C terminus of Kv3.3 to Hax-1, an anti-apoptotic protein that regulates actin nucleation through Arp2/3. A human Kv3.3 mutation within a conserved proline-rich domain produces channels that bind Hax-1 but are impaired in recruiting Arp2/3 to the plasma membrane, resulting in growth cones with deficient actin veils in stem cell-derived neurons. PMID:26997484

  18. Guardians of the actin monomer.

    PubMed

    Xue, Bo; Robinson, Robert C

    2013-01-01

    Actin is a universal force provider in eukaryotic cells. Biological processes harness the pressure generated from actin polymerization through dictating the time, place and direction of filament growth. As such, polymerization is initiated and maintained via tightly controlled filament nucleation and elongation machineries. Biological systems integrate force into their activities through recruiting and activating these machineries. In order that actin function as a common force generating polymerization motor, cells must maintain a pool of active, polymerization-ready monomeric actin, and minimize extemporaneous polymerization. Maintenance of the active monomeric actin pool requires the recycling of actin filaments, through depolymerization, nucleotide exchange and reloading of the polymerization machineries, while the levels of monomers are constantly monitored and supplemented, when needed, via the access of a reserve pool of monomers and through gene expression. Throughout its monomeric life, actin needs to be protected against gratuitous nucleation events. Here, we review the proteins that act as custodians of monomeric actin. We estimate their levels on a tissue scale, and calculate the implied concentrations of each actin complex based on reported binding affinities. These estimations predict that monomeric actin is rarely, if ever, alone. Thus, the guardians keep the volatility of actin in check, so that its explosive power is only released in the controlled environments of the nucleation and polymerization machineries. PMID:24268205

  19. Microtubule-dependent transport of vimentin filament precursors is regulated by actin and by the concerted action of Rho- and p21-activated kinases.

    PubMed

    Robert, Amélie; Herrmann, Harald; Davidson, Michael W; Gelfand, Vladimir I

    2014-07-01

    Intermediate filaments (IFs) form a dense and dynamic network that is functionally associated with microtubules and actin filaments. We used the GFP-tagged vimentin mutant Y117L to study vimentin-cytoskeletal interactions and transport of vimentin filament precursors. This mutant preserves vimentin interaction with other components of the cytoskeleton, but its assembly is blocked at the unit-length filament (ULF) stage. ULFs are easy to track, and they allow a reliable and quantifiable analysis of movement. Our results show that in cultured human vimentin-negative SW13 cells, 2% of vimentin-ULFs move along microtubules bidirectionally, while the majority are stationary and tightly associated with actin filaments. Rapid motor-dependent transport of ULFs along microtubules is enhanced ≥ 5-fold by depolymerization of actin cytoskeleton with latrunculin B. The microtubule-dependent transport of vimentin ULFs is further regulated by Rho-kinase (ROCK) and p21-activated kinase (PAK): ROCK inhibits ULF transport, while PAK stimulates it. Both kinases act on microtubule transport independently of their effects on actin cytoskeleton. Our study demonstrates the importance of the actin cytoskeleton to restrict IF transport and reveals a new role for PAK and ROCK in the regulation of IF precursor transport.-Robert, A., Herrmann, H., Davidson, M. W., and Gelfand, V. I. Microtubule-dependent transport of vimentin filament precursors is regulated by actin and by the concerted action of Rho- and p21-activated kinases.

  20. Microtubule-dependent transport of vimentin filament precursors is regulated by actin and by the concerted action of Rho- and p21-activated kinases

    PubMed Central

    Robert, Amélie; Herrmann, Harald; Davidson, Michael W.; Gelfand, Vladimir I.

    2014-01-01

    Intermediate filaments (IFs) form a dense and dynamic network that is functionally associated with microtubules and actin filaments. We used the GFP-tagged vimentin mutant Y117L to study vimentin-cytoskeletal interactions and transport of vimentin filament precursors. This mutant preserves vimentin interaction with other components of the cytoskeleton, but its assembly is blocked at the unit-length filament (ULF) stage. ULFs are easy to track, and they allow a reliable and quantifiable analysis of movement. Our results show that in cultured human vimentin-negative SW13 cells, 2% of vimentin-ULFs move along microtubules bidirectionally, while the majority are stationary and tightly associated with actin filaments. Rapid motor-dependent transport of ULFs along microtubules is enhanced ≥5-fold by depolymerization of actin cytoskeleton with latrunculin B. The microtubule-dependent transport of vimentin ULFs is further regulated by Rho-kinase (ROCK) and p21-activated kinase (PAK): ROCK inhibits ULF transport, while PAK stimulates it. Both kinases act on microtubule transport independently of their effects on actin cytoskeleton. Our study demonstrates the importance of the actin cytoskeleton to restrict IF transport and reveals a new role for PAK and ROCK in the regulation of IF precursor transport.—Robert, A., Herrmann, H., Davidson, M. W., and Gelfand, V. I. Microtubule-dependent transport of vimentin filament precursors is regulated by actin and by the concerted action of Rho- and p21-activated kinases. PMID:24652946

  1. Phytoplasma infection in tomato is associated with re-organization of plasma membrane, ER stacks, and actin filaments in sieve elements

    PubMed Central

    Buxa, Stefanie V.; Degola, Francesca; Polizzotto, Rachele; De Marco, Federica; Loschi, Alberto; Kogel, Karl-Heinz; di Toppi, Luigi Sanità; van Bel, Aart J. E.; Musetti, Rita

    2015-01-01

    Phytoplasmas, biotrophic wall-less prokaryotes, only reside in sieve elements of their host plants. The essentials of the intimate interaction between phytoplasmas and their hosts are poorly understood, which calls for research on potential ultrastructural modifications. We investigated modifications of the sieve-element ultrastructure induced in tomato plants by ‘Candidatus Phytoplasma solani,’ the pathogen associated with the stolbur disease. Phytoplasma infection induces a drastic re-organization of sieve-element substructures including changes in plasma membrane surface and distortion of the sieve-element reticulum. Observations of healthy and stolbur-diseased plants provided evidence for the emergence of structural links between sieve-element plasma membrane and phytoplasmas. One-sided actin aggregates on the phytoplasma surface also inferred a connection between phytoplasma and sieve-element cytoskeleton. Actin filaments displaced from the sieve-element mictoplasm to the surface of the phytoplasmas in infected sieve elements. Western blot analysis revealed a decrease of actin and an increase of ER-resident chaperone luminal binding protein (BiP) in midribs of phytoplasma-infected plants. Collectively, the studies provided novel insights into ultrastructural responses of host sieve elements to phloem-restricted prokaryotes. PMID:26347766

  2. Using the peptide BP100 as a cell-penetrating tool for the chemical engineering of actin filaments within living plant cells.

    PubMed

    Eggenberger, Kai; Mink, Christian; Wadhwani, Parvesh; Ulrich, Anne S; Nick, Peter

    2011-01-01

    The delivery of externally applied macromolecules or nanoparticles into living cells still represents a critically limiting step before the full capabilities of chemical engineering can be explored. Molecular transporters such as cell-penetrating peptides, peptoids, and other mimetics can be used to carry cargo across the cellular membrane, but it is still difficult to find suitable sequences that operate efficiently for any particular type of cell. Here we report that BP100 (KKLFKKILKYL-amide), originally designed as an antimicrobial peptide against plant pathogens, can be employed as a fast and efficient cell-penetrating agent to transport fluorescent test cargoes into the cytosol of walled plant cells. The uptake of BP100 proceeds slightly more slowly than the endocytosis of fluorescent dextranes, but BP100 accumulates more efficiently and to much higher levels (by an order of magnitude). The entry of BP100 can be efficiently blocked by latrunculin B; this suggests that actin filaments are essential to the uptake mechanism. To test whether this novel transporter can also be used to deliver functional cargoes, we designed a fusion construct of BP100 with the actin-binding Lifeact peptide (MGVADLIKKFESISKEE). We demonstrated that the short BP100 could transport the attached 17-residue sequence quickly and efficiently into tobacco cells. The Lifeact construct retained its functionality as it successfully labeled the actin bundles that tether the nucleus in the cell center.

  3. Ultra-fast optical manipulation of single proteins binding to the actin cytoskeleton

    NASA Astrophysics Data System (ADS)

    Capitanio, Marco; Gardini, Lucia; Pavone, Francesco Saverio

    2014-02-01

    In the last decade, forces and mechanical stresses acting on biological systems are emerging as regulatory factors essential for cell life. Emerging evidences indicate that factors such as applied forces or the rigidity of the extracellular matrix (ECM) determine the shape and function of cells and organisms1. Classically, the regulation of biological systems is described through a series of biochemical signals and enzymatic reactions, which direct the processes and cell fate. However, mechanotransduction, i.e. the conversion of mechanical forces into biochemical and biomolecular signals, is at the basis of many biological processes fundamental for the development and differentiation of cells, for their correct function and for the development of pathologies. We recently developed an in vitro system that allows the investigation of force-dependence of the interaction of proteins binding the actin cytoskeleton, at the single molecule level. Our system displays a delay of only ~10 μs between formation of the molecular bond and application of the force and is capable of detecting interactions as short as 100 μs. Our assay allows direct measurements of load-dependence of lifetimes of single molecular bonds and conformational changes of single proteins and molecular motors. We demonstrate our technique on molecular motors, using myosin II from fast skeletal muscle and on protein-DNA interaction, specifically on Lactose repressor (LacI). The apparatus is stabilized to less than 1 nm with both passive and active stabilization, allowing resolving specific binding regions along the actin filament and DNA molecule. Our technique extends single-molecule force-clamp spectroscopy to molecular complexes that have been inaccessible up to now, opening new perspectives for the investigation of the effects of forces on biological processes.

  4. A tridimensional view of the organization of actin filaments in the central nervous system by use of fluorescent photooxidation.

    PubMed

    Capani, Francisco; Saraceno, Ezequiel; Boti, Valeria Romina; Aon-Bertolino, Laura; Fernández, Juan Carlos; Gato, Fernándo; Kruse, Maria Sol; Krause, Maria Sol; Giraldez, Lisandro; Ellisman, Mark H; Coirini, Héctor

    2008-04-01

    Cellular and subcellular organization and distribution of actin filaments have been studied with various techniques. The use of fluorescence photo-oxidation combined with phalloidin conjugates with eosin has allowed the examination of the precise cellular and subcellular location of F-actin. Correlative fluorescence light microscopy and transmission electron microscopy studies of F-actin distribution are facilitated with this method for morphological and physiological studies. Because phalloidin-eosin is smaller than other markers, this method allows the analysis of the three-dimensional location of F-actin with high-resolution light microscopy, three-d serial sections reconstructions, and electron tomography. The combination of selective staining and three-dimensional reconstructions provide a valuable tool for revealing aspects of the synaptic morphology that are not available when conventional electron microscopy is used. By applying this selective staining technique and three-dimensional imaging, we uncovered the structural organization of actin in the postsynaptic densities in physiological and pathological conditions. PMID:18669318

  5. Yeast translation elongation factor-1A binds vacuole-localized Rho1p to facilitate membrane integrity through F-actin remodeling.

    PubMed

    Bodman, James A R; Yang, Yang; Logan, Michael R; Eitzen, Gary

    2015-02-20

    Rho GTPases are molecular switches that modulate a variety of cellular processes, most notably those involving actin dynamics. We have previously shown that yeast vacuolar membrane fusion requires re-organization of actin filaments mediated by two Rho GTPases, Rho1p and Cdc42p. Cdc42p initiates actin polymerization to facilitate membrane tethering; Rho1p has a role in the late stages of vacuolar fusion, but its mode of action is unknown. Here, we identified eEF1A as a vacuolar Rho1p-interacting protein. eEF1A (encoded by the TEF1 and TEF2 genes in yeast) is an aminoacyl-tRNA transferase needed during protein translation. eEF1A also has a second function that is independent of translation; it binds and organizes actin filaments into ordered cable structures. Here, we report that eEF1A interacts with Rho1p via a C-terminal subdomain. This interaction occurs predominantly when both proteins are in the GDP-bound state. Therefore, eEF1A is an atypical downstream effector of Rho1p. eEF1A does not promote vacuolar fusion; however, overexpression of the Rho1p-interacting subdomain affects vacuolar morphology. Vacuoles were destabilized and prone to leakage when treated with the eEF1A inhibitor narciclasine. We propose a model whereby eEF1A binds to Rho1p-GDP on the vacuolar membrane; it is released upon Rho1p activation and then bundles actin filaments to stabilize fused vacuoles. Therefore, the Rho1p-eEF1A complex acts to spatially localize a pool of eEF1A to vacuoles where it can readily organize F-actin.

  6. Switch II mutants reveal coupling between the nucleotide- and actin-binding regions in myosin V.

    PubMed

    Trivedi, Darshan V; David, Charles; Jacobs, Donald J; Yengo, Christopher M

    2012-06-01

    Conserved active-site elements in myosins and other P-loop NTPases play critical roles in nucleotide binding and hydrolysis; however, the mechanisms of allosteric communication among these mechanoenzymes remain unresolved. In this work we introduced the E442A mutation, which abrogates a salt-bridge between switch I and switch II, and the G440A mutation, which abolishes a main-chain hydrogen bond associated with the interaction of switch II with the γ phosphate of ATP, into myosin V. We used fluorescence resonance energy transfer between mant-labeled nucleotides or IAEDANS-labeled actin and FlAsH-labeled myosin V to examine the conformation of the nucleotide- and actin-binding regions, respectively. We demonstrate that in the absence of actin, both the G440A and E442A mutants bind ATP with similar affinity and result in only minor alterations in the conformation of the nucleotide-binding pocket (NBP). In the presence of ADP and actin, both switch II mutants disrupt the formation of a closed NBP actomyosin.ADP state. The G440A mutant also prevents ATP-induced opening of the actin-binding cleft. Our results indicate that the switch II region is critical for stabilizing the closed NBP conformation in the presence of actin, and is essential for communication between the active site and actin-binding region.

  7. Switch II Mutants Reveal Coupling between the Nucleotide- and Actin-Binding Regions in Myosin V

    PubMed Central

    Trivedi, Darshan V.; David, Charles; Jacobs, Donald J.; Yengo, Christopher M.

    2012-01-01

    Conserved active-site elements in myosins and other P-loop NTPases play critical roles in nucleotide binding and hydrolysis; however, the mechanisms of allosteric communication among these mechanoenzymes remain unresolved. In this work we introduced the E442A mutation, which abrogates a salt-bridge between switch I and switch II, and the G440A mutation, which abolishes a main-chain hydrogen bond associated with the interaction of switch II with the γ phosphate of ATP, into myosin V. We used fluorescence resonance energy transfer between mant-labeled nucleotides or IAEDANS-labeled actin and FlAsH-labeled myosin V to examine the conformation of the nucleotide- and actin-binding regions, respectively. We demonstrate that in the absence of actin, both the G440A and E442A mutants bind ATP with similar affinity and result in only minor alterations in the conformation of the nucleotide-binding pocket (NBP). In the presence of ADP and actin, both switch II mutants disrupt the formation of a closed NBP actomyosin.ADP state. The G440A mutant also prevents ATP-induced opening of the actin-binding cleft. Our results indicate that the switch II region is critical for stabilizing the closed NBP conformation in the presence of actin, and is essential for communication between the active site and actin-binding region. PMID:22713570

  8. F-actin binding regions on the androgen receptor and huntingtin increase aggregation and alter aggregate characteristics.

    PubMed

    Angeli, Suzanne; Shao, Jieya; Diamond, Marc I

    2010-01-01

    Protein aggregation is associated with neurodegeneration. Polyglutamine expansion diseases such as spinobulbar muscular atrophy and Huntington disease feature proteins that are destabilized by an expanded polyglutamine tract in their N-termini. It has previously been reported that intracellular aggregation of these target proteins, the androgen receptor (AR) and huntingtin (Htt), is modulated by actin-regulatory pathways. Sequences that flank the polyglutamine tract of AR and Htt might influence protein aggregation and toxicity through protein-protein interactions, but this has not been studied in detail. Here we have evaluated an N-terminal 127 amino acid fragment of AR and Htt exon 1. The first 50 amino acids of ARN127 and the first 14 amino acids of Htt exon 1 mediate binding to filamentous actin in vitro. Deletion of these actin-binding regions renders the polyglutamine-expanded forms of ARN127 and Htt exon 1 less aggregation-prone, and increases the SDS-solubility of aggregates that do form. These regions thus appear to alter the aggregation frequency and type of polyglutamine-induced aggregation. These findings highlight the importance of flanking sequences in determining the propensity of unstable proteins to misfold. PMID:20140226

  9. Effects of Roundup and glyphosate formulations on intracellular transport, microtubules and actin filaments in Xenopus laevis melanophores.

    PubMed

    Hedberg, Daniel; Wallin, Margareta

    2010-04-01

    Glyphosate containing herbicides, such as Roundup, are commonly used and generally considered to be safe. However, some toxic effects are found on amphibians in vivo and human and mouse cells in vitro. In this study the effects of Roundup, glyphosate, glyphosateisopropylamine and isopropylamine were studied on intracellular transport by measuring aggregation capacity in Xenopus laevis melanophores. The chemicals inhibited retrograde transport of melanosomes in the range of 0.5-5mM. Cellular morphology and localization of microtubules and actin filaments were affected as determined by immunocytochemistry. Both glyphosate and Roundup decreased pH in the media. Acidic pH inhibited melanosome transport and altered microtubule and actin morphology in the absence of chemicals, while transport inhibiting concentrations of glyphosate, Roundup and glyphosateisopropylamine disassembled both microtubules and actin filaments. At physiological pH the effects of Roundup decreased whereas glyphosate failed to inhibit transport. Physiological pH decreases glyphosate lipophilicity and its diffusion into the cytoplasm. The Roundup formulation contains surfactants, such as POEA (polyetylated tallow amine) that increases membrane permeability allowing cellular uptake at physiological pH. Our results show that the effects of glyphosate containing compounds are pH-dependent and that they inhibit intracellular transport through disassembly of the cytoskeleton possibly by interfering with intracellular Ca(2+)-balance.

  10. Tau co-organizes dynamic microtubule and actin networks

    PubMed Central

    Elie, Auréliane; Prezel, Elea; Guérin, Christophe; Denarier, Eric; Ramirez-Rios, Sacnicte; Serre, Laurence; Andrieux, Annie; Fourest-Lieuvin, Anne; Blanchoin, Laurent; Arnal, Isabelle

    2015-01-01

    The crosstalk between microtubules and actin is essential for cellular functions. However, mechanisms underlying the microtubule-actin organization by cross-linkers remain largely unexplored. Here, we report that tau, a neuronal microtubule-associated protein, binds to microtubules and actin simultaneously, promoting in vitro co-organization and coupled growth of both networks. By developing an original assay to visualize concomitant microtubule and actin assembly, we show that tau can induce guided polymerization of actin filaments along microtubule tracks and growth of single microtubules along actin filament bundles. Importantly, tau mediates microtubule-actin co-alignment without changing polymer growth properties. Mutagenesis studies further reveal that at least two of the four tau repeated motifs, primarily identified as tubulin-binding sites, are required to connect microtubules and actin. Tau thus represents a molecular linker between microtubule and actin networks, enabling a coordination of the two cytoskeletons that might be essential in various neuronal contexts. PMID:25944224

  11. Drosophila Homologues of Adenomatous Polyposis Coli (APC) and the Formin Diaphanous Collaborate by a Conserved Mechanism to Stimulate Actin Filament Assembly*

    PubMed Central

    Jaiswal, Richa; Stepanik, Vince; Rankova, Aneliya; Molinar, Olivia; Goode, Bruce L.; McCartney, Brooke M.

    2013-01-01

    Adenomatous polyposis coli (APC) is a large multidomain protein that regulates the cytoskeleton. Recently, it was shown that vertebrate APC through its Basic domain directly collaborates with the formin mDia1 to stimulate actin filament assembly in the presence of nucleation barriers. However, it has been unclear whether these activities extend to homologues of APC and Dia in other organisms. Drosophila APC and Dia are each required to promote actin furrow formation in the syncytial embryo, suggesting a potential collaboration in actin assembly, but low sequence homology between the Basic domains of Drosophila and vertebrate APC has left their functional and mechanistic parallels uncertain. To address this question, we purified Drosophila APC1 and Dia and determined their individual and combined effects on actin assembly using both bulk fluorescence assays and total internal reflection fluorescence microscopy. Our data show that APC1, similar to its vertebrate homologue, bound to actin monomers and nucleated and bundled filaments. Further, Drosophila Dia nucleated actin assembly and protected growing filament barbed ends from capping protein. Drosophila APC1 and Dia directly interacted and collaborated to promote actin assembly in the combined presence of profilin and capping protein. Thus, despite limited sequence homology, Drosophila and vertebrate APCs exhibit highly related activities and mechanisms and directly collaborate with formins. These results suggest that APC-Dia interactions in actin assembly are conserved and may underlie important in vivo functions in a broad range of animal phyla. PMID:23558679

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

    PubMed

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

    2015-08-10

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

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

    PubMed

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

    2015-08-10

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

  14. Arabidopsis RIC1 Severs Actin Filaments at the Apex to Regulate Pollen Tube Growth

    PubMed Central

    Zhou, Zhenzhen; Shi, Haifan; Chen, Binqing; Zhang, Ruihui; Huang, Shanjin; Fu, Ying

    2015-01-01

    Pollen tubes deliver sperms to the ovule for fertilization via tip growth. The rapid turnover of F-actin in pollen tube tips plays an important role in this process. In this study, we demonstrate that Arabidopsis thaliana RIC1, a member of the ROP-interactive CRIB motif-containing protein family, regulates pollen tube growth via its F-actin severing activity. Knockout of RIC1 enhanced pollen tube elongation, while overexpression of RIC1 dramatically reduced tube growth. Pharmacological analysis indicated that RIC1 affected F-actin dynamics in pollen tubes. In vitro biochemical assays revealed that RIC1 directly bound and severed F-actin in the presence of Ca2+ in addition to interfering with F-actin turnover by capping F-actin at the barbed ends. In vivo, RIC1 localized primarily to the apical plasma membrane (PM) of pollen tubes. The level of RIC1 at the apical PM oscillated during pollen tube growth. The frequency of F-actin severing at the apex was notably decreased in ric1-1 pollen tubes but was increased in pollen tubes overexpressing RIC1. We propose that RIC1 regulates F-actin dynamics at the apical PM as well as the cytosol by severing F-actin and capping the barbed ends in the cytoplasm, establishing a novel mechanism that underlies the regulation of pollen tube growth. PMID:25804540

  15. Near-atomic resolution for one state of F-actin.

    PubMed

    Galkin, Vitold E; Orlova, Albina; Vos, Matthijn R; Schröder, Gunnar F; Egelman, Edward H

    2015-01-01

    Actin functions as a helical polymer, F-actin, but attempts to build an atomic model for this filament have been hampered by the fact that the filament cannot be crystallized and by structural heterogeneity. We have used a direct electron detector, cryo-electron microscopy, and the forces imposed on actin filaments in thin films to reconstruct one state of the filament at 4.7 Å resolution, which allows for building a reliable pseudo-atomic model of F-actin. We also report a different state of the filament where actin protomers adopt a conformation observed in the crystal structure of the G-actin-profilin complex with an open ATP-binding cleft. Comparison of the two structural states provides insights into ATP-hydrolysis and filament dynamics. The atomic model provides a framework for understanding why every buried residue in actin has been under intense selective pressure. PMID:25533486

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

    PubMed

    Bledzka, Kamila; Bialkowska, Katarzyna; Sossey-Alaoui, Khalid; Vaynberg, Julia; Pluskota, Elzbieta; Qin, Jun; Plow, Edward F

    2016-04-11

    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, LK(47)/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 LK(47)/AA mutation were equivalent in their ability to coactivate integrin αIIbβ3 in a CHO cell system when coexpressed with talin. However, K2-LK(47)/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

  17. Origins and evolution of the formin multigene family that is involved in the formation of actin filaments.

    PubMed

    Chalkia, Dimitra; Nikolaidis, Nikolas; Makalowski, Wojciech; Klein, Jan; Nei, Masatoshi

    2008-12-01

    In eukaryotes, the assembly and elongation of unbranched actin filaments is controlled by formins, which are long, multidomain proteins. These proteins are important for dynamic cellular processes such as determination of cell shape, cell division, and cellular interaction. Yet, no comprehensive study has been done about the origins and evolution of this gene family. We therefore performed extensive phylogenetic and motif analyses of the formin genes by examining 597 prokaryotic and 53 eukaryotic genomes. Additionally, we used three-dimensional protein structure data in an effort to uncover distantly related sequences. Our results suggest that the formin homology 2 (FH2) domain, which promotes the formation of actin filaments, is a eukaryotic innovation and apparently originated only once in eukaryotic evolution. Despite the high degree of FH2 domain sequence divergence, the FH2 domains of most eukaryotic formins are predicted to assume the same fold and thus have similar functions. The formin genes have experienced multiple taxon-specific duplications and followed the birth-and-death model of evolution. Additionally, the formin genes experienced taxon-specific genomic rearrangements that led to the acquisition of unrelated protein domains. The evolutionary diversification of formin genes apparently increased the number of formin's interacting molecules and consequently contributed to the development of a complex and precise actin assembly mechanism. The diversity of formin types is probably related to the range of actin-based cellular processes that different cells or organisms require. Our results indicate the importance of gene duplication and domain acquisition in the evolution of the eukaryotic cell and offer insights into how a complex system, such as the cytoskeleton, evolved.

  18. Origins and evolution of the formin multigene family that is involved in the formation of actin filaments.

    PubMed

    Chalkia, Dimitra; Nikolaidis, Nikolas; Makalowski, Wojciech; Klein, Jan; Nei, Masatoshi

    2008-12-01

    In eukaryotes, the assembly and elongation of unbranched actin filaments is controlled by formins, which are long, multidomain proteins. These proteins are important for dynamic cellular processes such as determination of cell shape, cell division, and cellular interaction. Yet, no comprehensive study has been done about the origins and evolution of this gene family. We therefore performed extensive phylogenetic and motif analyses of the formin genes by examining 597 prokaryotic and 53 eukaryotic genomes. Additionally, we used three-dimensional protein structure data in an effort to uncover distantly related sequences. Our results suggest that the formin homology 2 (FH2) domain, which promotes the formation of actin filaments, is a eukaryotic innovation and apparently originated only once in eukaryotic evolution. Despite the high degree of FH2 domain sequence divergence, the FH2 domains of most eukaryotic formins are predicted to assume the same fold and thus have similar functions. The formin genes have experienced multiple taxon-specific duplications and followed the birth-and-death model of evolution. Additionally, the formin genes experienced taxon-specific genomic rearrangements that led to the acquisition of unrelated protein domains. The evolutionary diversification of formin genes apparently increased the number of formin's interacting molecules and consequently contributed to the development of a complex and precise actin assembly mechanism. The diversity of formin types is probably related to the range of actin-based cellular processes that different cells or organisms require. Our results indicate the importance of gene duplication and domain acquisition in the evolution of the eukaryotic cell and offer insights into how a complex system, such as the cytoskeleton, evolved. PMID:18840602

  19. Feeling for Filaments: Quantification of the Cortical Actin Web in Live Vascular Endothelium

    PubMed Central

    Kronlage, Cornelius; Schäfer-Herte, Marco; Böning, Daniel; Oberleithner, Hans; Fels, Johannes

    2015-01-01

    Contact-mode atomic force microscopy (AFM) has been shown to reveal cortical actin structures. Using live endothelial cells, we visualized cortical actin dynamics simultaneously by AFM and confocal fluorescence microscopy. We present a method that quantifies dynamic changes in the mechanical ultrastructure of the cortical actin web. We argue that the commonly used, so-called error signal imaging in AFM allows a qualitative, but not quantitative, analysis of cortical actin dynamics. The approach we used comprises fast force-curve-based topography imaging and subsequent image processing that enhances local height differences. Dynamic changes in the organization of the cytoskeleton network can be observed and quantified by surface roughness calculations and automated morphometrics. Upon treatment with low concentrations of the actin-destabilizing agent cytochalasin D, the cortical cytoskeleton network is thinned out and the average mesh size increases. In contrast, jasplakinolide, a drug that enhances actin polymerization, consolidates the cytoskeleton network and reduces the average mesh area. In conclusion, cortical actin dynamics can be quantified in live cells. To our knowledge, this opens a new pathway for conducting quantitative structure-function analyses of the endothelial actin web just beneath the apical plasma membrane. PMID:26287621

  20. Feeling for Filaments: Quantification of the Cortical Actin Web in Live Vascular Endothelium.

    PubMed

    Kronlage, Cornelius; Schäfer-Herte, Marco; Böning, Daniel; Oberleithner, Hans; Fels, Johannes

    2015-08-18

    Contact-mode atomic force microscopy (AFM) has been shown to reveal cortical actin structures. Using live endothelial cells, we visualized cortical actin dynamics simultaneously by AFM and confocal fluorescence microscopy. We present a method that quantifies dynamic changes in the mechanical ultrastructure of the cortical actin web. We argue that the commonly used, so-called error signal imaging in AFM allows a qualitative, but not quantitative, analysis of cortical actin dynamics. The approach we used comprises fast force-curve-based topography imaging and subsequent image processing that enhances local height differences. Dynamic changes in the organization of the cytoskeleton network can be observed and quantified by surface roughness calculations and automated morphometrics. Upon treatment with low concentrations of the actin-destabilizing agent cytochalasin D, the cortical cytoskeleton network is thinned out and the average mesh size increases. In contrast, jasplakinolide, a drug that enhances actin polymerization, consolidates the cytoskeleton network and reduces the average mesh area. In conclusion, cortical actin dynamics can be quantified in live cells. To our knowledge, this opens a new pathway for conducting quantitative structure-function analyses of the endothelial actin web just beneath the apical plasma membrane.

  1. Serotonin binds specifically and saturably to an actin-like protein isolated from rat brain synaptosomes.

    PubMed Central

    Small, D H; Wurtman, R J

    1984-01-01

    A soluble serotonin-binding protein was identified in a high-speed supernatant fraction of an osmotically shocked rat brain synaptosome (P2) preparation. The binding of serotonin was saturable (Bmax = 6.0 nmol per mg of protein) and was specific for serotonin and a few structurally related compounds including dopamine and norepinephrine. Binding of serotonin (1 microM) was inhibited approximately equal to 40% by chlorpromazine (10 microM). The affinity of serotonin for the binding protein was low in the crude extract (Kd = 1.7 X 10(-3)M). However, on purification by chromatography on a column of phenothiazine agarose, a higher affinity (Kd = 10(-5) M) binding component was also observed. The purified protein was greatly enriched in a polypeptide of Mr of 43,000 that comigrated on polyacrylamide gel with skeletal muscle actin. Muscle actin also bound serotonin, and the binding to actin was similar to that of the purified protein in both the specificity of the binding and the affinity for serotonin. It is likely that the serotonin-binding protein is identical to cytoplasmic G-actin or an actin-like protein of similar molecular weight. PMID:6583691

  2. Light-Induced Movements of Chloroplasts and Nuclei Are Regulated in Both Cp-Actin-Filament-Dependent and -Independent Manners in Arabidopsis thaliana

    PubMed Central

    2016-01-01

    Light-induced chloroplast movement and attachment to the plasma membrane are dependent on actin filaments. In Arabidopsis thaliana, the short actin filaments on the chloroplast envelope, cp-actin filaments, are essential for chloroplast movement and positioning. Furthermore, cp-actin-filament-mediated chloroplast movement is necessary for the strong-light-induced nuclear avoidance response. The proteins CHLOROPLAST UNUSUAL POSITIONING 1 (CHUP1), KINESIN-LIKE PROTEIN FOR ACTIN-BASED CHLOROPLAST MOVEMENT 1 (KAC1) and KAC2 are required for the generation and/or maintenance of cp-actin filaments in Arabidopsis. In land plants, CHUP1 and KAC family proteins play pivotal roles in the proper movement of chloroplasts and their attachment to the plasma membrane. Here, we report similar but distinct phenotypes in chloroplast and nuclear photorelocation movements between chup1 and kac1kac2 mutants. Measurement of chloroplast photorelocation movement indicated that kac1kac2, but not chup1, exhibited a clear strong-light-induced increase in leaf transmittance changes. The chloroplast movement in kac1kac2 depended on phototropin 2, CHUP1 and two other regulators for cp-actin filaments, PLASTID MOVEMENT IMPAIRED 1 and THRUMIN 1. Furthermore, kac1kac2 retained a weak but significant nuclear avoidance response although chup1 displayed a severe defect in the nuclear avoidance response. The kac1kac2chup1 triple mutant was completely defective in both chloroplast and nuclear avoidance responses. These results indicate that CHUP1 and the KACs function somewhat independently, but interdependently mediate both chloroplast and nuclear photorelocation movements. PMID:27310016

  3. Light-Induced Movements of Chloroplasts and Nuclei Are Regulated in Both Cp-Actin-Filament-Dependent and -Independent Manners in Arabidopsis thaliana.

    PubMed

    Suetsugu, Noriyuki; Higa, Takeshi; Gotoh, Eiji; Wada, Masamitsu

    2016-01-01

    Light-induced chloroplast movement and attachment to the plasma membrane are dependent on actin filaments. In Arabidopsis thaliana, the short actin filaments on the chloroplast envelope, cp-actin filaments, are essential for chloroplast movement and positioning. Furthermore, cp-actin-filament-mediated chloroplast movement is necessary for the strong-light-induced nuclear avoidance response. The proteins CHLOROPLAST UNUSUAL POSITIONING 1 (CHUP1), KINESIN-LIKE PROTEIN FOR ACTIN-BASED CHLOROPLAST MOVEMENT 1 (KAC1) and KAC2 are required for the generation and/or maintenance of cp-actin filaments in Arabidopsis. In land plants, CHUP1 and KAC family proteins play pivotal roles in the proper movement of chloroplasts and their attachment to the plasma membrane. Here, we report similar but distinct phenotypes in chloroplast and nuclear photorelocation movements between chup1 and kac1kac2 mutants. Measurement of chloroplast photorelocation movement indicated that kac1kac2, but not chup1, exhibited a clear strong-light-induced increase in leaf transmittance changes. The chloroplast movement in kac1kac2 depended on phototropin 2, CHUP1 and two other regulators for cp-actin filaments, PLASTID MOVEMENT IMPAIRED 1 and THRUMIN 1. Furthermore, kac1kac2 retained a weak but significant nuclear avoidance response although chup1 displayed a severe defect in the nuclear avoidance response. The kac1kac2chup1 triple mutant was completely defective in both chloroplast and nuclear avoidance responses. These results indicate that CHUP1 and the KACs function somewhat independently, but interdependently mediate both chloroplast and nuclear photorelocation movements.

  4. The More the Tubular: Dynamic Bundling of Actin Filaments for Membrane Tube Formation

    PubMed Central

    Weichsel, Julian; Geissler, Phillip L.

    2016-01-01

    Tubular protrusions are a common feature of living cells, arising from polymerization of stiff protein filaments against a comparably soft membrane. Although this process involves many accessory proteins in cells, in vitro experiments indicate that similar tube-like structures can emerge without them, through spontaneous bundling of filaments mediated by the membrane. Using theory and simulation of physical models, we have elaborated how nonequilibrium fluctuations in growth kinetics and membrane shape can yield such protrusions. Enabled by a new grand canonical Monte Carlo method for membrane simulation, our work reveals a cascade of dynamical transitions from individually polymerizing filaments to highly cooperatively growing bundles as a dynamical bottleneck to tube formation. Filament network organization as well as adhesion points to the membrane, which bias filament bending and constrain membrane height fluctuations, screen the effective attractive interactions between filaments, significantly delaying bundling and tube formation. PMID:27384915

  5. Integration of the Rac1- and actin-binding properties of Coronin-1C

    PubMed Central

    Tilley, Frances C; Williamson, Rosalind C; Race, Paul R; Rendall, Thomas C; Bass, Mark D

    2015-01-01

    The coronin family of actin-binding proteins regulate actin branching by inhibiting Arp2/3. We recently reported 2 interactions that were unique to coronin-1C: binding of a Rac1 inhibitor, RCC2, to the unique linker region and Rac1 itself to the propeller domain in a manner that differs from that proposed for other coronins. Through these interactions coronin-1C redistributes Rac1 from the back of the cell to the leading edge for either activation or sequestration by the associated Rac1-inhibitor, RCC2. Here we investigate the relationship between the Rac1- and actin-binding properties of coronin-1C and find that, although actin appears to be involved in the retrafficking of Rac1, signaling by Rac1 lies upstream of the stress fiber-formation, for which the coronins were originally characterized. PMID:25862165

  6. Purification of recombinant human and Drosophila septin hexamers for TIRF assays of actin-septin filament assembly.

    PubMed

    Mavrakis, M; Tsai, F-C; Koenderink, G H

    2016-01-01

    Septins are guanine nucleotide-binding proteins that are conserved from fungi to humans. Septins assemble into heterooligomeric complexes and higher-order structures with key roles in various cellular functions including cell migration and division. The mechanisms by which septins assemble and interact with other cytoskeletal elements like actin remain elusive. A powerful approach to address this question is by cell-free reconstitution of purified cytoskeletal proteins combined with fluorescence microscopy. Here, we describe procedures for the purification of recombinant Drosophila and human septin hexamers from Escherichia coli and reconstitution of actin-septin coassembly. These procedures can be used to compare assembly of Drosophila and human septins and their coassembly with the actin cytoskeleton by total internal reflection fluorescence microscopy. PMID:27473911

  7. Purification of recombinant human and Drosophila septin hexamers for TIRF assays of actin-septin filament assembly.

    PubMed

    Mavrakis, M; Tsai, F-C; Koenderink, G H

    2016-01-01

    Septins are guanine nucleotide-binding proteins that are conserved from fungi to humans. Septins assemble into heterooligomeric complexes and higher-order structures with key roles in various cellular functions including cell migration and division. The mechanisms by which septins assemble and interact with other cytoskeletal elements like actin remain elusive. A powerful approach to address this question is by cell-free reconstitution of purified cytoskeletal proteins combined with fluorescence microscopy. Here, we describe procedures for the purification of recombinant Drosophila and human septin hexamers from Escherichia coli and reconstitution of actin-septin coassembly. These procedures can be used to compare assembly of Drosophila and human septins and their coassembly with the actin cytoskeleton by total internal reflection fluorescence microscopy.

  8. Yersinia effector YopO uses actin as bait to phosphorylate proteins that regulate actin polymerization

    PubMed Central

    Lee, Wei Lin; Grimes, Jonathan M; Robinson, Robert C

    2016-01-01

    Pathogenic Yersinia species evade host immune systems through the injection of Yersinia outer proteins (Yops) into phagocytic cells. One Yop, YopO, also known as YpkA, induces actin-filament disruption, impairing phagocytosis. Here we describe the X-ray structure of Yersinia enterocolitica YopO in complex with actin, which reveals that YopO binds to an actin monomer in a manner that blocks polymerization yet allows the bound actin to interact with host actin-regulating proteins. SILAC-MS and biochemical analyses confirm that actin-polymerization regulators such as VASP, EVL, WASP, gelsolin and the formin diaphanous 1 are directly sequestered and phosphorylated by YopO through formation of ternary complexes with actin. This leads to a model in which YopO at the membrane sequesters actin from polymerization while using the bound actin as bait to recruit, phosphorylate and misregulate host actin-regulating proteins to disrupt phagocytosis. PMID:25664724

  9. Yersinia effector YopO uses actin as bait to phosphorylate proteins that regulate actin polymerization.

    PubMed

    Lee, Wei Lin; Grimes, Jonathan M; Robinson, Robert C

    2015-03-01

    Pathogenic Yersinia species evade host immune systems through the injection of Yersinia outer proteins (Yops) into phagocytic cells. One Yop, YopO, also known as YpkA, induces actin-filament disruption, impairing phagocytosis. Here we describe the X-ray structure of Yersinia enterocolitica YopO in complex with actin, which reveals that YopO binds to an actin monomer in a manner that blocks polymerization yet allows the bound actin to interact with host actin-regulating proteins. SILAC-MS and biochemical analyses confirm that actin-polymerization regulators such as VASP, EVL, WASP, gelsolin and the formin diaphanous 1 are directly sequestered and phosphorylated by YopO through formation of ternary complexes with actin. This leads to a model in which YopO at the membrane sequesters actin from polymerization while using the bound actin as bait to recruit, phosphorylate and misregulate host actin-regulating proteins to disrupt phagocytosis.

  10. Short actin-based mechanism for light-directed chloroplast movement in Arabidopsis

    PubMed Central

    Kadota, Akeo; Yamada, Noboru; Suetsugu, Noriyuki; Hirose, Mana; Saito, Chieko; Shoda, Keiko; Ichikawa, Satoshi; Kagawa, Takatoshi; Nakano, Akihiko; Wada, Masamitsu

    2009-01-01

    Organelle movement is essential for proper function of living cells. In plants, these movements generally depend on actin filaments, but the underlying mechanism is unknown. Here, in Arabidopsis, we identify associations of short actin filaments along the chloroplast periphery on the plasma membrane side associated with chloroplast photorelocation and anchoring to the plasma membrane. We have termed these chloroplast-actin filaments (cp-actin filaments). Cp-actin filaments emerge from the chloroplast edge and exhibit rapid turnover. The presence of cp-actin filaments depends on an actin-binding protein, chloroplast unusual positioning1 (CHUP1), localized on the chloroplast envelope. chup1 mutant lacked cp-actin filaments but showed normal cytoplasmic actin filaments. When irradiated with blue light to induce chloroplast movement, cp-actin filaments relocalize to the leading edge of chloroplasts before and during photorelocation and are regulated by 2 phototropins, phot1 and phot2. Our findings suggest that plants evolved a unique actin-based mechanism for organelle movement. PMID:19620714

  11. Actin depolymerizing factor controls actin turnover and gliding motility in Toxoplasma gondii

    PubMed Central

    Mehta, Simren; Sibley, L. David

    2011-01-01

    Apicomplexan parasites rely on actin-based gliding motility to move across the substratum, cross biological barriers, and invade their host cells. Gliding motility depends on polymerization of parasite actin filaments, yet ∼98% of actin is nonfilamentous in resting parasites. Previous studies suggest that the lack of actin filaments in the parasite is due to inherent instability, leaving uncertain the role of actin-binding proteins in controlling dynamics. We have previously shown that the single allele of Toxoplasma gondii actin depolymerizing factor (TgADF) has strong actin monomer–sequestering and weak filament-severing activities in vitro. Here we used a conditional knockout strategy to investigate the role of TgADF in vivo. Suppression of TgADF led to accumulation of actin-rich filaments that were detected by immunofluorescence and electron microscopy. Parasites deficient in TgADF showed reduced speed of motility, increased aberrant patterns of motion, and inhibition of sustained helical gliding. Lack of TgADF also led to severe defects in entry and egress from host cells, thus blocking infection in vitro. These studies establish that the absence of stable actin structures in the parasite are not simply the result of intrinsic instability, but that TgADF is required for the rapid turnover of parasite actin filaments, gliding motility, and cell invasion. PMID:21346192

  12. Stochastic model of profilin-actin polymerization

    NASA Astrophysics Data System (ADS)

    Horan, Brandon; Vavylonis, Dimitrios

    A driving factor in cell motility and other processes that involve changes of cell shape is the rapid polymerization of actin subunits into long filaments. This process is regulated by profilin, a protein which binds to actin subunits and regulates elongation of actin filaments. Whether profilin stimulates polymerization by coupling to hydrolysis of ATP-bound actin is debated. Previous studies have proposed indirect coupling to ATP hydrolysis using rate equations, but did not include the effects of fluctuations that are important near the critical concentration. We developed stochastic simulations using the Gillespie algorithm to study single filament elongation at the barbed end in the presence of profilin. We used recently measured rate constants and estimated the rate of profilin binding to the barbed end such that detailed balance is satisfied. Fast phosphate release at the tip of the filament was accounted for. The elongation rate and length diffusivity as functions of profilin and actin concentration were calculated and used to extract the critical concentrations of free actin and of total actin. We show under what conditions profilin leads to an increase in the critical concentration of total actin but a decrease in the critical concentration of free actin.

  13. Redundant mechanisms for anaphase chromosome movements: crane-fly spermatocyte spindles normally use actin filaments but also can function without them.

    PubMed

    Fabian, Lacramioara; Forer, Arthur

    2005-10-01

    Actin inhibitors block or slow anaphase chromosome movements in crane-fly spermatocytes, but stopping of movement is only temporary; we assumed that cells adapt to loss of actin by switching to mechanism(s) involving only microtubules. To test this, we produced actin-filament-free spindles: we added latrunculin B during prometaphase, 9-80 min before anaphase, after which chromosomes generally moved normally during anaphase. We confirmed the absence of actin filaments by staining with fluorescent phalloidin and by showing that cytochalasin D had no effect on chromosome movement. Thus, actin filaments are involved in normal anaphase movements, but in vivo, spindles nonetheless can function normally without them. We tested whether chromosome movements in actin-filament-free spindles arise via microtubules by challenging such spindles with anti-myosin drugs. Y-27632 and BDM (2,3-butanedione monoxime), inhibitors that affect myosin at different regulatory levels, blocked chromosome movement in normal spindles and in actin-filament-free spindles. We tested whether BDM has side effects on microtubule motors. BDM had no effect on ciliary and sperm motility or on ATPase activity of isolated ciliary axonemes, and thus it does not directly block dynein. Nor does it block kinesin, assayed by a microtubule sliding assay. BDM could conceivably indirectly affect these microtubule motors, though it is unlikely that it would have the same side effect on the motors as Y-27632. Since BDM and Y-27632 both affect chromosome movement in the same way, it would seem that both affect spindle myosin; this suggests that spindle myosin interacts with kinetochore microtubules, either directly or via an intermediate component. PMID:16228898

  14. Preparation and Characterization of a Polyclonal Antibody against Human Actin Filament-Associated Protein-120 kD

    PubMed Central

    Chen, Yujian; Liu, Yong; Guo, Jiayu; Tang, Tao; Gao, Jian; Huang, Tao; Wang, Bin; Liu, Shaojun

    2016-01-01

    Actin filament-associated protein-120kD (AFAP-120) is an alternatively spliced isoform of actin filament-associated protein-110kD (AFAP-110) and contains an additional neuronal insert (NINS) fragment in addition to identical domains to the AFAP-110. Unlike AFAP-110 widely expressed in tissues, AFAP-120 is specifically expressed in the nervous system and plays a role in organizing dynamic actin structures during neuronal differentiation. However, anti-AFAP-120 antibody is still commercially unavailable, and this may hinder the function research for AFAP-120. In this study, we simultaneously used the ABCpred online server and the BepiPred 1.0 server to predict B-cell epitopes in the exclusive NINS sequence of human AFAP-120 protein, and found that a 16aa-peptide sequence was the consensus epitope predicted by both tools. This peptide was chemically synthesized and used as an immunogen to develop polyclonal antibody against AFAP-120 (anti-AFAP-120). The sensitivity and specificity of anti-AFAP-120 were analyzed with immunoblotting, immunoprecipitation, and immunofluorescence assays. Our results indicated that anti-AFAP-120 could react with over-expressed and endogenous human AFAP-120 protein under denatured condition, but not with human AFAP-110 protein. Moreover, native human AFAP-120 protein could also be recognized by the anti-AFAP-120 antibody. These results suggested that the prepared anit-AFAP-120 antibody would be a useful tool for studying the biochemical and biological functions of AFAP-120. PMID:27322249

  15. UNC-45/CRO1/She4p (UCS) protein forms elongated dimer and joins two myosin heads near their actin binding region

    PubMed Central

    Shi, Hang; Blobel, Günter

    2010-01-01

    UNC-45/CRO1/She4p (UCS) proteins have variously been proposed to affect the folding, stability, and ATPase activity of myosins. They are the only proteins known to interact directly with the motor domain. To gain more insight into UCS function, we determined the atomic structure of the yeast UCS protein, She4p, at 2.9 Å resolution. We found that 16 helical repeats are organized into an L-shaped superhelix with an amphipathic N-terminal helix dangling off the short arm of the L-shaped molecule. In the crystal, She4p forms a 193-Å-long, zigzag-shaped dimer through three distinct and evolutionary conserved interfaces. We have identified She4p’s C-terminal region as a ligand for a 27-residue-long epitope on the myosin motor domain. Remarkably, this region consists of two adjacent, but distinct, binding epitopes localized at the nucleotide-responsive cleft between the nucleotide- and actin-filament-binding sites. One epitope is situated inside the cleft, the other outside the cleft. After ATP hydrolysis and Pi ejection, the cleft narrows at its base from 20 to 12 Å thereby occluding the inside the cleft epitope, while leaving the adjacent, outside the cleft binding epitope accessible to UCS binding. Hence, one cycle of higher and lower binding affinity would accompany one ATP hydrolysis cycle and a single step in the walk on an actin filament rope. We propose that a UCS dimer links two myosins at their motor domains and thereby functions as one of the determinants for step size of myosin on actin filaments. PMID:21115842

  16. Structure of kinetochore fibres in crane-fly spermatocytes after irradiation with an ultraviolet microbeam: neither microtubules nor actin filaments remain in the irradiated region.

    PubMed

    Forer, Arthur; Spurck, Tim; Pickett-Heaps, Jeremy D; Wilson, Paula J

    2003-11-01

    We studied chromosome movement after kinetochore microtubules were severed. Severing a kinetochore fibre in living crane-fly spermatocytes with an ultraviolet microbeam creates a kinetochore stub, a birefringent remnant of the spindle fibre connected to the kinetochore and extending only to the edge of the irradiated region. After the irradiation, anaphase chromosomes either move poleward led by their stubs or temporarily stop moving. We examined actin and/or microtubules in irradiated cells by means of confocal fluorescence microscopy or serial-section reconstructions from electron microscopy. For each cell thus examined, chromosome movement had been recorded continuously until the moment of fixation. Kinetochore microtubules were completely severed by the ultraviolet microbeam in cells in which chromosomes continued to move poleward after the irradiation: none were seen in the irradiated regions. Similarly, actin filaments normally present in kinetochore fibres were severed by the ultraviolet microbeam irradiations: the irradiated regions contained no actin filaments and only local spots of non-filamentous actin. There was no difference in irradiated regions when the associated chromosomes continued to move versus when they stopped moving. Thus, one cannot explain motion with severed kinetochore microtubules in terms of either microtubules or actin-filaments bridging the irradiated region. The data seem to negate current models for anaphase chromosome movement and support a model in which poleward chromosome movement results from forces generated within the spindle matrix that propel kinetochore fibres or kinetochore stubs poleward. PMID:14569597

  17. Association of hepatitis C virus replication complexes with microtubules and actin filaments is dependent on the interaction of NS3 and NS5A.

    PubMed

    Lai, Chao-Kuen; Jeng, King-Song; Machida, Keigo; Lai, Michael M C

    2008-09-01

    The hepatitis C virus (HCV) RNA replication complex (RC), which is composed of viral nonstructural (NS) proteins and host cellular proteins, replicates the viral RNA genome in association with intracellular membranes. Two viral NS proteins, NS3 and NS5A, are essential elements of the RC. Here, by using immunoprecipitation and fluorescence resonance energy transfer assays, we demonstrated that NS3 and NS5A interact with tubulin and actin. Furthermore, immunofluorescence microscopy and electron microscopy revealed that HCV RCs were aligned along microtubules and actin filaments in both HCV replicon cells and HCV-infected cells. In addition, the movement of RCs was inhibited when microtubules or actin filaments were depolymerized by colchicine and cytochalasin B, respectively. Based on our observations, we propose that microtubules and actin filaments provide the tracks for the movement of HCV RCs to other regions in the cell, and the molecular interactions between RCs and microtubules, or RCs and actin filaments, are mediated by NS3 and NS5A. PMID:18562541

  18. Distinct sites on the G-actin molecule bind group-specific component and deoxyribonuclease I.

    PubMed Central

    Goldschmidt-Clermont, P J; Galbraith, R M; Emerson, D L; Marsot, F; Nel, A E; Arnaud, P

    1985-01-01

    Addition of group-specific component (Gc) to G-actin with or without deoxyribonuclease I (DNAase) led to formation of binary complexes (Gc-G-actin) and ternary complexes (Gc-G-actin-DNAase) respectively. The electrophoretic mobility of ternary complexes, as shown by crossed and rocket immunoelectrophoresis, was slower than that of binary complexes, although both were faster than native Gc. In gradient polyacrylamide-gel electrophoresis, such complexes could again be resolved, apparently on the basis of relative molecular size: Gc-G-actin-DNAase (Mr approx. 131000), Gc-G-actin (Mr approx. 98000) and Gc (Mr approx. 56000). In contrast, the pI of ternary complex was indistinguishable by isoelectric focusing from that of binary complex, even though both were clearly more acidic than native Gc. The affinity of Gc for G-actin (affinity constant, Ka, 1.9 X 10(8) M-1) was not significantly altered by additional interaction with DNAase (Ka, 1.5 X 10(8)M-1), and both binary and ternary complexes still bound 25-hydroxycholecalciferol. In addition, the inhibitory effect of G-actin on DNAase activity was not discernibly affected by interaction with Gc. These results demonstrate that the various molecular forms of Gc can be distinguished by physicochemical parameters, and that Gc and DNAase bind to distinct sites on G-actin and can interact both independently and contemporaneously with this molecule. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. PMID:4040363

  19. Capping Protein Increases the Rate of Actin-based Motility by Promoting Filament Nucleation by the Arp2/3 Complex

    PubMed Central

    Akin, Orkun; Mullins, R. Dyche

    2008-01-01

    Summary Capping protein is an integral component of Arp2/3-nucleated actin networks that drive amoeboid motility. Increasing the concentration of capping protein, which caps barbed ends of actin filaments and prevents elongation, increases the rate of actin-based motility in vivo and in vitro. We studied the synergy between capping protein and Arp2/3 using an in vitro actin-based motility system reconstituted from purified proteins. We find that capping protein increases the rate of motility by promoting more frequent filament nucleation by the Arp2/3 complex, and not by increasing the rate of filament elongation as previously suggested. One consequence of this coupling between capping and nucleation is that, while the rate of motility depends strongly on the concentration of capping protein and Arp2/3, the net rate of actin assembly is insensitive to changes in either factor. By reorganizing their architecture, dendritic actin networks harness the same assembly kinetics to drive different rates of motility. PMID:18510928

  20. MamK, a bacterial actin, forms dynamic filaments in vivo that are regulated by the acidic proteins MamJ and LimJ

    PubMed Central

    Draper, Olga; Byrne, Meghan E.; Li, Zhuo; Keyhani, Sepehr; Cueto Barrozo, Joyce; Jensen, Grant; Komeili, Arash

    2011-01-01

    SUMMARY Bacterial actins, in contrast to their eukaryotic counterparts, are highly divergent proteins whose wide-ranging functions are thought to correlate with their evolutionary diversity. One clade, represented by the MamK protein of magnetotactic bacteria, is required for the subcellular organization of magnetosomes, membrane-bound organelles that aid in navigation along the earth’s magnetic field. Using a fluorescence recovery after photobleaching assay in Magnetospirillum magneticum AMB-1, we find that, like traditional actins, MamK forms dynamic filaments that require an intact NTPase motif for their turnover in vivo. We also uncover two proteins, MamJ and LimJ, which perform a redundant function to promote the dynamic behavior of MamK filaments in wildtype cells. The absence of both MamJ and LimJ leads to static filaments, a disrupted magnetosome chain, and an anomalous build-up of cytoskeletal filaments between magnetosomes. Our results suggest that MamK filaments, like eukaryotic actins, are intrinsically stable and rely on regulators for their dynamic behavior, a feature that stands in contrast to some classes of bacterial actins characterized to date. PMID:21883528

  1. Statistical Thermodynamics for Actin-Myosin Binding: The Crucial Importance of Hydration Effects.

    PubMed

    Oshima, Hiraku; Hayashi, Tomohiko; Kinoshita, Masahiro

    2016-06-01

    Actomyosin is an important molecular motor, and the binding of actin and myosin is an essential research target in biophysics. Nevertheless, the physical factors driving or opposing the binding are still unclear. Here, we investigate the role of water in actin-myosin binding using the most reliable statistical-mechanical method currently available for assessing biomolecules immersed in water. This method is characterized as follows: water is treated not as a dielectric continuum but as an ensemble of molecules; the polyatomic structures of proteins are taken into consideration; and the binding free energy is decomposed into physically insightful entropic and energetic components by accounting for the hydration effect to its full extent. We find that the actin-myosin binding brings large gains of electrostatic and Lennard-Jones attractive interactions. However, these gains are accompanied by even larger losses of actin-water and myosin-water electrostatic and LJ attractive interactions. Although roughly half of the energy increase due to the losses is cancelled out by the energy decrease arising from structural reorganization of the water released upon binding, the remaining energy increase is still larger than the energy decrease brought by the gains mentioned above. Hence, the net change in system energy is positive, which opposes binding. Importantly, the binding is driven by a large gain of configurational entropy of water, which surpasses the positive change in system energy and the conformational entropy loss occurring for actin and myosin. The principal physical origin of the large water-entropy gain is as follows: the actin-myosin interface is closely packed with the achievement of high shape complementarity on the atomic level, leading to a large increase in the total volume available to the translational displacement of water molecules in the system and a resultant reduction of water crowding (i.e., entropic correlations among water molecules). PMID

  2. Drebrin Inhibits Cofilin-Induced Severing of F-Actin

    PubMed Central

    Grintsevich, Elena E.; Reisler, Emil

    2015-01-01

    Molecular cross-talk between neuronal drebrin A and cofilin is believed to be a part of the activity-dependent cytoskeleton-modulating pathway in dendritic spines. Impairments in this pathway are implicated also in synaptic dysfunction in Alzheimer’s disease, Down syndrome, epilepsy, and normal aging. However, up to now the molecular interplay between cofilin and drebrin has not been elucidated. TIRF microscopy and solution experiments revealed that full length drebrin A or its actin binding core (Drb1-300) inhibits, but do not abolish cofilin-induced severing of actin filaments. Cosedimentation experiments showed that F-actin can be fully occupied with combination of these two proteins. The dependence of cofilin binding on fractional saturation of actin filaments with drebrin suggests direct competition between these two proteins for F-actin binding. This implies that cofilin and drebrin can either overcome or reverse the allosteric changes in F-actin induced by the competitor’s binding. The ability of cofilin to displace drebrin from actin filaments is pH dependent and is facilitated at acidic pH (6.8). Pre-steady state kinetic experiments reveal that both binding and dissociation of drebrin to/from actin filaments is faster than that reported for cooperative binding of cofilin. We found, that drebrin displacement by cofilin is greatly inhibited when actin severing is abolished, which might be linked to the cooperativity of drebrin binding to actin filaments. Our results contribute to molecular understanding of the competitive interactions of drebrin and cofilin with actin filaments. PMID:25047716

  3. CF2 represses Actin 88F gene expression and maintains filament balance during indirect flight muscle development in Drosophila.

    PubMed

    Gajewski, Kathleen M; Schulz, Robert A

    2010-01-01

    The zinc finger protein CF2 is a characterized activator of muscle structural genes in the body wall muscles of the Drosophila larva. To investigate the function of CF2 in the indirect flight muscle (IFM), we examined the phenotypes of flies bearing five homozygous viable mutations. The gross structure of the IFM was not affected, but the stronger hypomorphic alleles caused an increase of up to 1.5X in the diameter of the myofibrils. This size increase did not cause any disruption of the hexameric arrangement of thick and thin filaments. RT-PCR analysis revealed an increase in the transcription of several structural genes. Ectopic overexpression of CF2 in the developing IFM disrupts muscle formation. While our results indicate a role for CF2 as a direct negative regulator of the thin filament protein gene Actin 88F (Act88F), effects on levels of transcripts of myosin heavy chain (mhc) appear to be indirect. This role is in direct contrast to that described in the larval muscles, where CF2 activates structural gene expression. The variation in myofibril phenotypes of CF2 mutants suggest the CF2 may have separate functions in fine-tuning expression of structural genes to insure proper filament stoichiometry, and monitoring and/or controlling the final myofibril size. PMID:20520827

  4. Purification and properties of a 90-kDa nuclear actin-binding protein.

    PubMed

    Yeoman, L C; Bremer, J W

    1986-04-01

    A 90 kDa actin-binding nuclear protein (ABNP) with a pI of 5.2 has been purified from the 0.7 M NaCl extracted residue fraction of chromatin prepared from Novikoff hepatoma cell nuclei. This residue fraction was previously shown to contain nuclear actin. Although twice the size, similar in pI, and similar in amino acid composition to actin, the tryptic peptide map for ABNP is distinct and contains the appropriate number of tyrosine-containing tryptic peptides for a protein of 90,000 molecular weight. A comparison of the amino acid composition of ABNP with those reported in the literature for gelsolin and villin, using a calculation of S delta Q as an indication of relatedness, results in values of 30 and 27, respectively. Actin-binding activity, however, was demonstrated for both crude and gel purified ABNP using a gel-overlay technique that employs 125I-G-actin to detect specific actin-binding proteins.

  5. Determination of the persistence length of actin filaments on microcontact printed myosin patterns

    NASA Astrophysics Data System (ADS)

    Hajne, Joanna; Hanson, Kristi L.; van Zalinge, Harm; Nicolau, Dan V.; Nicolau, Dan V.

    2015-03-01

    Protein molecular motors, which convert chemical energy into kinetic energy, are prime candidates for use in nanodevice in which active transport is required. To be able to design these devices it is essential that the properties of the cytoskeletal filaments propelled by the molecular motors are well established. Here we used micro-contact printed BSA to limit the amount of HMM that can adsorb creating a tightly confined pathway for the filaments to travel. Both the image and statistical analysis of the movement of the filaments through these structures have been used to new insights into the motility behaviour of actomyosin on topographically homogenous, but motor-heterogeneous planar systems. It will be shown that it is possible to determine the persistence length of the filaments and that it is related to the amount of locally adsorbed HMM. This provides a basis that can be used to optimize the design of future nanodevices incorporating the actomyosin system for the active transport.

  6. Multiple CaMKII Binding Modes to the Actin Cytoskeleton Revealed by Single-Molecule Imaging.

    PubMed

    Khan, Shahid; Conte, Ianina; Carter, Tom; Bayer, K Ulrich; Molloy, Justin E

    2016-07-26

    Localization of the Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) to dendritic spine synapses is determined in part by the actin cytoskeleton. We determined binding of GFP-tagged CaMKII to tag-RFP-labeled actin cytoskeleton within live cells using total internal reflection fluorescence microscopy and single-molecule tracking. Stepwise photobleaching showed that CaMKII formed oligomeric complexes. Photoactivation experiments demonstrated that diffusion out of the evanescent field determined the track lifetimes. Latrunculin treatment triggered a coupled loss of actin stress fibers and the colocalized, long-lived CaMKII tracks. The CaMKIIα (α) isoform, which was previously thought to lack F-actin interactions, also showed binding, but this was threefold weaker than that observed for CaMKIIβ (β). The βE' splice variant bound more weakly than α, showing that binding by β depends critically on the interdomain linker. The mutations βT287D and αT286D, which mimic autophosphorylation states, also abolished F-actin binding. Autophosphorylation triggers autonomous CaMKII activity, but does not impair GluN2B binding, another important synaptic protein interaction of CaMKII. The CaMKII inhibitor tatCN21 or CaMKII mutations that inhibit GluN2B association by blocking binding of ATP (βK43R and αK42M) or Ca(2+)/calmodulin (βA303R) had no effect on the interaction with F-actin. These results provide the first rationale for the reduced synaptic spine localization of the αT286D mutant, indicating that transient F-actin binding contributes to the synaptic localization of the CaMKIIα isoform. The track lifetime distributions had a stretched exponential form consistent with a heterogeneously diffusing population. This heterogeneity suggests that CaMKII adopts different F-actin binding modes, which is most easily rationalized by multiple subunit contacts between the CaMKII dodecamer and the F-actin cytoskeleton that stabilize the initial weak (micromolar

  7. A 45,000-mol-wt protein from unfertilized sea urchin eggs severs actin filaments in a calcium-dependent manner and increases the steady-state concentration of nonfilamentous actin.

    PubMed

    Wang, L L; Spudich, J A

    1984-09-01

    A 45,000-mol-wt protein has been purified from unfertilized sea urchin (Strongylocentrotus purpuratus) eggs. The isolation scheme includes DEAE cellulose ion-exchange chromatography, gel filtration, and hydroxylapatite chromatography. The homogeneity of the isolated protein is greater than 90% by SDS PAGE. The 45,000-mol-wt protein reduces the viscosity of actin filaments in a Ca2+-dependent manner. The free calcium concentration required for the activity of this protein is in the micromolar range. Electron microscopic studies reveal that the formation of short filaments parallels the decrease in viscosity. Energy transfer and sedimentation experiments indicate a net disassembly of actin filaments and an increase in the steady-state nonfilamentous actin concentration in the presence of Ca2+ ions and the 45,000-mol-wt protein. The increase in the steady-state nonfilamentous actin concentration is proportional to the amount of 45,000-mol-wt protein added. The actin molecules disassembled by the addition of the 45,000-mol-wt protein are capable of polymerization.

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

    PubMed

    Gladilin, Evgeny; Gonzalez, Paula; Eils, Roland

    2014-08-22

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

  9. Myosin Va bound to phagosomes binds to F-actin and delays microtubule-dependent motility.

    PubMed

    Al-Haddad, A; Shonn, M A; Redlich, B; Blocker, A; Burkhardt, J K; Yu, H; Hammer, J A; Weiss, D G; Steffen, W; Griffiths, G; Kuznetsov, S A

    2001-09-01

    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

  10. Binding of actin to thioglycolic acid modified superparamagnetic nanoparticles for antibody conjugation.

    PubMed

    Maltas, Esra; Ertekin, Betul

    2015-01-01

    Thioglycolic acid modified superparamagnetic iron oxide nanoparticles (TG-APTS-SPION) were synthesized by using (3-aminopropyl) triethoxysilane (APTS) and thioglycolic acid (TG). Actin was immobilized on the nanoparticle surfaces. Binding amount of the actin (Act) on TG-APTS-SPIONs was determined by using a calibration curve equation that was drawn using fluorescence spectra at 280 and 342 nm of excitation and emission wavelengths. Anti-Actin (anti-Act) was interacted with the actin immobilized TG-APTS-SPIONs as primary antibody. Horse radish peroxidase (HRP) was also interacted with antibody conjugated nanoparticles as secondary antibody. The binding capacity of primary and secondary antibodies was also estimated by fluorescence spectroscopy. Scanning electron microscopy (SEM), Infrared spectroscopy (FTIR) and energy dispersive X-ray (EDX) analysis were also clarified binding of the protein and antibodies to the nanoparticles' surfaces. Western blot analysis was also done for actin conjunction with anti Act antibody to confirm binding of the antibody to the protein. PMID:25451750

  11. Two-headed binding of a processive myosin to F-actin.

    PubMed

    Walker, M L; Burgess, S A; Sellers, J R; Wang, F; Hammer, J A; Trinick, J; Knight, P J

    2000-06-15

    Myosins are motor proteins in cells. They move along actin by changing shape after making stereospecific interactions with the actin subunits. As these are arranged helically, a succession of steps will follow a helical path. However, if the myosin heads are long enough to span the actin helical repeat (approximately 36 nm), linear motion is possible. Muscle myosin (myosin II) heads are about 16 nm long, which is insufficient to span the repeat. Myosin V, however, has heads of about 31 nm that could span 36 nm and thus allow single two-headed molecules to transport cargo by walking straight. Here we use electron microscopy to show that while working, myosin V spans the helical repeat. The heads are mostly 13 actin subunits apart, with values of 11 or 15 also found. Typically the structure is polar and one head is curved, the other straighter. Single particle processing reveals the polarity of the underlying actin filament, showing that the curved head is the leading one. The shape of the leading head may correspond to the beginning of the working stroke of the motor. We also observe molecules attached by one head in this conformation. PMID:10866203

  12. Sucrose Increases the Activation Energy Barrier for Actin-Myosin Strong Binding

    PubMed Central

    Jackson, Del R.; Webb, Milad; Stewart, Travis J.; Phillips, Travis; Carter, Michael; Cremo, Christine R.; Baker, Josh E.

    2014-01-01

    To determine the mechanism by which sucrose slows in vitro actin sliding velocities, V, we used stopped flow kinetics and a single molecule binding assay, SiMBA. We observed that in the absence of ATP, sucrose (880 mM) slowed the rate of actin-myosin (A-M) strong binding by 71 ± 8% with a smaller inhibitory effect observed on spontaneous rigor dissociation (21 ± 3%). Similarly, in the presence of ATP, sucrose slowed strong binding associated with Pi release by 85 ± 9% with a smaller inhibitory effect on ATP-induced A-M dissociation, kT (39 ± 2%). Sucrose had no noticeable effect on any other step in the ATPase reaction. In SiMBA, sucrose had a relatively small effect on the diffusion coefficient for actin fragments (25 ± 2%), and with stopped flow we showed that sucrose increased the activation energy barrier for A-M strong binding by 37 ± 3%, indicating that sucrose inhibits the rate of A-M strong binding by slowing bond formation more than diffusional searching. The inhibitory effects of sucrose on the rate of A-M rigor binding (71%) are comparable in magnitude to sucrose’s effects on both V (79 ± 33% decrease) and maximal actin-activated ATPase, kcat, (81 ± 16% decrease), indicating that the rate of A-M strong bond formation significantly influences both kcat and V. PMID:24370736

  13. A membrane cytoskeleton from Dictyostelium discoideum. I. Identification and partial characterization of an actin-binding activity

    PubMed Central

    1981-01-01

    Dictyostelium discoideum plasma membranes isolated by each of three procedures bind F-actin. The interactions between these membranes and actin are examined by a novel application of falling ball viscometry. Treating the membranes as multivalent actin-binding particles analogous to divalent actin-gelation factors, we observe large increases in viscosity (actin cross-linking) when membranes of depleted actin and myosin are incubated with rabbit skeletal muscle F-actin. Pre- extraction of peripheral membrane proteins with chaotropes or the inclusion of Triton X-100 during the assay does not appreciably diminish this actin cross-linking activity. Lipid vesicles, heat- denatured membranes, proteolyzed membranes, or membranes containing endogenous actin show minimal actin cross-linking activity. Heat- denatured, but not proteolyzed, membranes regain activity when assayed in the presence of Triton X-100. Thus, integral membrane proteins appear to be responsible for some or all of the actin cross-linking activity of D. discoideum membranes. In the absence of MgATP, Triton X- 100 extraction of isolated D. discoideum membranes results in a Triton- insoluble residue composed of actin, myosin, and associated membrane proteins. The inclusion of MgATP before and during Triton extraction greatly diminishes the amount of protein in the Triton-insoluble residue without appreciably altering its composition. Our results suggest the existence of a protein complex stabilized by actin and/or myosin (membrane cytoskeleton) associated with the D. discoideum plasma membrane. PMID:6894148

  14. Transcription factor binding and spacing constraints in the human beta-actin proximal promoter.

    PubMed Central

    Danilition, S L; Frederickson, R M; Taylor, C Y; Miyamoto, N G

    1991-01-01

    The human beta-actin promoter, including its 5' flanking region and 5' untranslated region, is ubiquitously active in mammalian cells in culture. In this report we investigated the transcriptional activity of, and the protein-DNA interactions that occur within, the proximal region of the human beta-actin promoter. Efficient beta-actin promoter activity in transfected human HeLa cells requires only 114bp of 5' flanking sequences. Two of the cis-actin regulatory elements within this region of the beta-actin promoter, the CCAAT box and proximal CCArGG box, are specific in vitro binding sites for the transcription factors, nuclear factor Y (NF-Y) and serum response factor (p67SRF), respectively. These two elements are required together to stimulate in vivo transcription from the homologous as well as a heterologous promoter. Finally, a particular spatial alignment between the CCAAT box and proximal CCArGG box is required for trans-activation in vivo. The above provides strong evidence for a functional interaction between NF-Y and p67SRF when bound to their respective binding sites in the beta-actin promoter. Images PMID:1762920

  15. Molecular analysis of insertion/deletion mutations in protein 4.1 in elliptocytosis. I. Biochemical identification of rearrangements in the spectrin/actin binding domain and functional characterizations.

    PubMed Central

    Marchesi, S L; Conboy, J; Agre, P; Letsinger, J T; Marchesi, V T; Speicher, D W; Mohandas, N

    1990-01-01

    Protein 4.1 (80 kD) interacts with spectrin and short actin filaments to form the erythrocyte membrane skeleton. Mutations of spectrin and protein 4.1 are associated with elliptocytosis or spherocytosis and anemia of varying severity. We analyzed two mutant protein 4.1 molecules associated with elliptocytosis: a high molecular weight 4.1 (95 kD) associated with mild elliptocytosis without anemia, and a low molecular weight 4.1 (two species at 68 and 65 kD) associated with moderate elliptocytosis and anemia. 4.1(95) was found to contain a approximately 15-kD insertion adjacent to the spectrin/actin binding domain comprised, at least in part, of repeated sequence. 4.1(68/65) was found to lack the entire spectrin-actin binding domain. The mechanical stability of erythrocyte membranes containing 4.1(95) was identical to that of normal membranes, consistent with the presence of an intact spectrin-actin binding domain in protein 4.1. In contrast, membranes containing 4.1(68/65) have markedly reduced mechanical stability as a result of deleting the spectrin-actin binding domain. The mechanical stability of these membranes was improved following reconstitution with normal 4.1. These studies have thus enabled us to establish the importance of the spectrin-actin binding domain in regulating the mechanical stability of the erythrocyte membrane. Images PMID:2384597

  16. A36-dependent actin filament nucleation promotes release of vaccinia virus.

    PubMed

    Horsington, Jacquelyn; Lynn, Helena; Turnbull, Lynne; Cheng, Delfine; Braet, Filip; Diefenbach, Russell J; Whitchurch, Cynthia B; Karupiah, Guna; Newsome, Timothy P

    2013-03-01

    Cell-to-cell transmission of vaccinia virus can be mediated by enveloped virions that remain attached to the outer surface of the cell or those released into the medium. During egress, the outer membrane of the double-enveloped virus fuses with the plasma membrane leaving extracellular virus attached to the cell surface via viral envelope proteins. Here we report that F-actin nucleation by the viral protein A36 promotes the disengagement of virus attachment and release of enveloped virus. Cells infected with the A36(YdF) virus, which has mutations at two critical tyrosine residues abrogating localised actin nucleation, displayed a 10-fold reduction in virus release. We examined A36(YdF) infected cells by transmission electron microscopy and observed that during release, virus appeared trapped in small invaginations at the plasma membrane. To further characterise the mechanism by which actin nucleation drives the dissociation of enveloped virus from the cell surface, we examined recombinant viruses by super-resolution microscopy. Fluorescently-tagged A36 was visualised at sub-viral resolution to image cell-virus attachment in mutant and parental backgrounds. We confirmed that A36(YdF) extracellular virus remained closely associated to the plasma membrane in small membrane pits. Virus-induced actin nucleation reduced the extent of association, thereby promoting the untethering of virus from the cell surface. Virus release can be enhanced via a point mutation in the luminal region of B5 (P189S), another virus envelope protein. We found that the B5(P189S) mutation led to reduced contact between extracellular virus and the host membrane during release, even in the absence of virus-induced actin nucleation. Our results posit that during release virus is tightly tethered to the host cell through interactions mediated by viral envelope proteins. Untethering of virus into the surrounding extracellular space requires these interactions be relieved, either through the force

  17. Hydrogen peroxide formation and actin filament reorganization by Cdc42 are essential for ethanol-induced in vitro angiogenesis.

    PubMed

    Qian, Yong; Luo, Jia; Leonard, Stephen S; Harris, Gabriel K; Millecchia, Lyndell; Flynn, Daniel C; Shi, Xianglin

    2003-05-01

    This report focuses on the identification of the molecular mechanisms of ethanol-induced in vitro angiogenesis. The manipulation of angiogenesis is an important therapeutic approach for the treatment of cancer, cardiovascular diseases, and chronic inflammation. Our results showed that ethanol stimulation altered the integrity of actin filaments and increased the formation of lamellipodia and filopodia in SVEC4-10 cells. Further experiments demonstrated that ethanol stimulation increased cell migration and invasion and induced in vitro angiogenesis in SVEC4-10 cells. Mechanistically, ethanol stimulation activated Cdc42 and produced H(2)O(2) a reactive oxygen species intermediate in SVEC4-10 cells. Measuring the time course of Cdc42 activation and H(2)O(2) production upon ethanol stimulation revealed that the Cdc42 activation and the increase of H(2)O(2) lasted more than 3 h, which indicates the mechanisms of the long duration effects of ethanol on the cells. Furthermore, either overexpression of a constitutive dominant negative Cdc42 or inhibition of H(2)O(2) production abrogated the effects of ethanol on SVEC4-10 cells, indicating that both the activation of Cdc42 and the production of H(2)O(2) are essential for the actions of ethanol. Interestingly, we also found that overexpression of a constitutive dominant positive Cdc42 itself was sufficient to produce H(2)O(2) and to induce in vitro angiogenesis. Taken together, our results suggest that ethanol stimulation can induce H(2)O(2) production through the activation of Cdc42, which results in reorganizing actin filaments and increasing cell motility and in vitro angiogenesis. PMID:12598535

  18. [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

  19. Structure of a Bud6/actin complex reveals a novel WH2-like actin monomer recruitment motif

    PubMed Central

    Park, Eunyoung; Graziano, Brian R.; Zheng, Wei; Garabedian, Mikael; Goode, Bruce L.; Eck, Michael J.

    2015-01-01

    SUMMARY In budding yeast, the actin-binding protein Bud6 cooperates with formins Bni1 and Bnr1 to catalyze the assembly of actin filaments. The nucleation-enhancing activity of Bud6 requires both a “core” domain that binds to the formin and a “flank” domain that binds monomeric actin. Here we describe the structure of the Bud6 flank domain in complex with actin. Two helices in Bud6flank interact with actin; one binds in a groove at the barbed-end of the actin monomer in a manner closely resembling the helix of WH2 domains, a motif found in many actin nucleation factors. The second helix rises along the face of actin. Mutational analysis verifies the importance of these Bud6-actin contacts for nucleation-enhancing activity. The Bud6 binding site on actin overlaps with that of the formin FH2 domain and is also incompatible with inter-subunit contacts in F-actin, suggesting that Bud6 interacts only transiently with actin monomers during filament nucleation. PMID:26118535

  20. Structure of a Bud6/Actin Complex Reveals a Novel WH2-like Actin Monomer Recruitment Motif.

    PubMed

    Park, Eunyoung; Graziano, Brian R; Zheng, Wei; Garabedian, Mikael; Goode, Bruce L; Eck, Michael J

    2015-08-01

    In budding yeast, the actin-binding protein Bud6 cooperates with formins Bni1 and Bnr1 to catalyze the assembly of actin filaments. The nucleation-enhancing activity of Bud6 requires both a "core" domain that binds to the formin and a "flank" domain that binds monomeric actin. Here, we describe the structure of the Bud6 flank domain in complex with actin. Two helices in Bud6(flank) interact with actin; one binds in a groove at the barbed end of the actin monomer in a manner closely resembling the helix of WH2 domains, a motif found in many actin nucleation factors. The second helix rises along the face of actin. Mutational analysis verifies the importance of these Bud6-actin contacts for nucleation-enhancing activity. The Bud6 binding site on actin overlaps with that of the formin FH2 domain and is also incompatible with inter-subunit contacts in F-actin, suggesting that Bud6 interacts only transiently with actin monomers during filament nucleation.

  1. Conformation of the ATP binding peptide in actin revealed by proton NMR spectroscopy

    SciTech Connect

    Barden, J.A.

    1987-09-22

    The actin peptide 106-124 exists in a completely conserved region of the sequence and binds strongly to both ATP and tripolyphosphate. Binding particularly affects residues 116 and 118 and generally affects the two segments 115-118 and 121-124. One-dimensional nuclear Overhauser enhancement difference spectroscopy was used to detect molecular interactions between both adjacent and nonadjacent residues. The N-terminal segment 106-112 was found to be largely extended. A sharp bend was detected between Pro-112 and Lys-113. The triphosphate moiety binds to the strongly hydrophilic central segment of the peptide. Evidence was obtained for a reverse turn involving residues 121-124. Amide proton temperature coefficients and coupling constants provide evidence for a type I ..beta..-turn. A model of the ATP binding site is proposed together with its relationship to other parts of the actin structure and to the phalloidin binding site.

  2. Actin filament barbed-end capping activity in neutrophil lysates: the role of capping protein-beta 2.

    PubMed

    DiNubile, M J; Cassimeris, L; Joyce, M; Zigmond, S H

    1995-12-01

    A barbed-end capping activity was found in high speed supernates of neutrophils lysed in submicromolar calcium. In dilute supernate (> or = 100-fold dilution of cytoplasm), this activity accounted for most of the inhibition of barbed-end elongation of pyrenyl-G-actin from spectrin-F-actin seeds. Pointed-end elongation from gelsolin-capped F-actin seeds was not inhibited at comparable concentrations of supernate, thus excluding actin monomer sequestration as a cause of the observed inhibition. Most of the capping activity was due to capping protein-beta 2 (a homologue of cap Z). Thus, while immunoadsorption of > or = 95% of the gelsolin in the supernate did not decrease capping activity, immunoadsorption of capping protein-beta 2 reduced capping activity proportionally to the amount of capping protein-beta 2 adsorbed. Depletion of > 90% of capping protein-beta 2 from the supernate removed 90% of its capping activity. The functional properties of the capping activity were defined. The dissociation constant for binding to barbed ends (determined by steady state and kinetic analyses) was approximately 1-2 nM; the on-rate of capping was between 7 x 10(5) and 5 x 10(6) M-1 s-1; and the off-rate was approximately 2 x 10(-3) s-1. The concentration of capper free in the intact cell (determined by adsorption of supernate with spectrin-actin seeds) was estimated to be approximately 1-2 microM. Thus, there appeared to be enough high affinity capper to cap all the barbed ends in vivo. Nevertheless, immediately after lysis with detergent, neutrophils contained sites that nucleate barbed-end elongation of pyrenyl-G-actin. These barbed ends subsequently become capped with a time course and concentration dependence similar to that of spectrin-F-actin seeds in high speed supernates. These observations suggest that, despite the excess of high affinity capper, some ends either are not capped in vivo or are transiently uncapped upon lysis and dilution. PMID:8590796

  3. Human RNASET2 derivatives as potential anti-angiogenic agents: actin binding sequence identification and characterization

    PubMed Central

    Nesiel-Nuttman, Liron; Doron, Shani; Schwartz, Betty; Shoseyov, Oded

    2015-01-01

    Human RNASET2 (hRNASET2) has been demonstrated to exert antiangiogenic and antitumorigenic effects independent of its ribonuclease capacity. We suggested that RNASET2 exerts its antiangiogenic and antitumorigenic activities via binding to actin and consequently inhibits cell motility. We focused herein on the identification of the actin binding site of hRNASET2 using defined sequences encountered within the whole hRNASET2 protein. For that purpose we designed 29 different hRNASET2-derived peptides. The 29 peptides were examined for their ability to bind immobilized actin. Two selected peptides-A103-Q159 consisting of 57 amino acids and peptide K108-K133 consisting of 26 amino acids were demonstrated to have the highest actin binding ability and concomitantly the most potent anti-angiogenic activity. Further analyses on the putative mechanisms associated with angiogenesis inhibition exerted by peptide K108-K133 involved its location during treatment within the HUVE cells. Peptide K108-K133 readily penetrates the cell membrane within 10 min of incubation. In addition, supplementation with angiogenin delays the entrance of peptide K108-K133 to the cell suggesting competition on the same cell internalization route. The peptide was demonstrated to co-localize with angiogenin, suggesting that both molecules bind analogous cellular epitopes, similar to our previously reported data for ACTIBIND and trT2-50. PMID:25815360

  4. A Structural Basis for Regulation of Actin Polymerization by Pectenotoxins

    PubMed Central

    Allingham, John S.; Miles, Christopher O.; Rayment, Ivan

    2007-01-01

    Pectenotoxins (PTXs) are polyether macrolides found in certain dinoflagellates, sponges and shellfish, and have been associated with diarrhetic shellfish poisoning. In addition to their in vivo toxicity, some PTXs are potently cytotoxic in human cancer cell lines. Recent studies have demonstrated that disruption of the actin cytoskeleton may be a key function of these compounds, although no clarification their mechanism of action at a molecular level was available. We have obtained an X-ray crystal structure of PTX-2 bound to actin which, in combination with analyses of the effect of PTX-2 on purified actin filament dynamics, provides a molecular explanation for its effects on actin. PTX-2 formed a 1:1 complex with actin and engaged a novel site between subdomains 1 and 3. Based on models of the actin filament, PTX binding would disrupt key lateral contacts between the PTX-bound actin monomer and the lower lateral actin monomer within the filament, thereby capping the barbed-end. The location of this binding position within the interior of the filament indicates that it may not be accessible once polymerization has occurred, a hypothesis supported by our observation that PTX-2 caused filament capping without inducing filament severing. This mode of action is unique, as other actin filament destabilizing toxins appear to exclusively disrupt longitudinal monomer contacts allowing many of them to sever filaments in addition to capping them. Examination of the PTX-binding site on actin provides a rationalization for the structure–activity relationships observed in vivo and in vitro, and may provide a basis for predicting toxicity of PTX analogues. PMID:17599353

  5. The actin-severing activity of cofilin is exerted by the interplay of three distinct sites on cofilin and essential for cell viability.

    PubMed Central

    Moriyama, Kenji; Yahara, Ichiro

    2002-01-01

    Cofilin/actin-depolymerizing factor is an essential and conserved modulator of actin dynamics. Cofilin binds to actin in either monomeric or filamentous form, severs and depolymerizes actin filaments, and speeds up their treadmilling. A high turnover rate of F-actin in actin-based motility seems driven largely by cofilin-mediated acceleration of directional subunit release, but little by fragmentation of the filaments. On the other hand, the filament-severing function of cofilin seems relevant for the healthy growth of cells. In this study, we have characterized three mutants of porcine cofilin to elucidate the molecular mechanism that underlies the filament-severing activity of cofilin. The first mutant could neither associate with actin filaments nor sever them, whereas it effectively accelerated their treadmilling and directional subunit release. The second mutant bound to actin filaments, but failed to sever them and to interfere with phalloidin binding to the filament. The third mutant could associate with actin filaments and sever them, although with a very reduced efficacy. Of these mutant proteins, only the last one was able to rescue Deltacof1 yeast cells and to induce thick actin bundles in mammalian cells upon overexpression. Therefore, the actin-severing activity of cofilin is an essential element in its vital function and suggested to be exerted by co-operation of at least three distinct sites of cofilin. PMID:12113256

  6. Identification of the plakoglobin-binding domain in desmoglein and its role in plaque assembly and intermediate filament anchorage

    PubMed Central

    1994-01-01

    The carboxyterminal cytoplasmic portions (tails) of desmosomal cadherins of both the desmoglein (Dsg) and desmocollin type are integral components of the desmosomal plaque and are involved in desmosome assembly and the anchorage of intermediate-sized filaments. When additional Dsg tails were introduced by cDNA transfection into cultured human epithelial cells, in the form of chimeras with the aminoterminal membrane insertion domain of rat connexin32 (Co32), the resulting stably transfected cells showed a dominant-negative defect specific for desmosomal junctions: despite the continual presence of all desmosomal proteins, the endogenous desmosomes disappeared and the formation of Co32-Dsg chimeric gap junctions was inhibited. Using cell transfection in combination with immunoprecipitation techniques, we have examined a series of deletion mutants of the Dsg1 tail in Co32-Dsg chimeras. We show that upon removal of the last 262 amino acids the truncated Dsg tail still effects the binding of plakoglobin but not of detectable amounts of any catenin and induces the dominant-negative phenotype. However, further truncation or excision of the next 41 amino acids, which correspond to the highly conserved carboxyterminus of the C-domain in other cadherins, abolishes plakoglobin binding and allows desmosomes to reform. Therefore, we conclude that this short segment provides a plakoglobin-binding site and is important for plaque assembly and the specific anchorage of either actin filaments in adherens junctions or IFs in desmosomes. PMID:7929560

  7. Disease causing mutations in inverted formin 2 regulate its binding to G-actin, F-actin capping protein (CapZ α-1) and profilin 2

    PubMed Central

    Rollason, Ruth; Wherlock, Matthew; Heath, Jenny A.; Heesom, Kate J.; Saleem, Moin A.; Welsh, Gavin I.

    2016-01-01

    Focal segmental glomerulosclerosis (FSGS) is a devastating form of nephrotic syndrome which ultimately leads to end stage renal failure (ESRF). Mutations in inverted formin 2 (INF2), a member of the formin family of actin-regulating proteins, have recently been associated with a familial cause of nephrotic syndrome characterized by FSGS. INF2 is a unique formin that can both polymerize and depolymerize actin filaments. How mutations in INF2 lead to disease is unknown. In the present study, we show that three mutations associated with FSGS, E184K, S186P and R218Q, reduce INF2 auto-inhibition and increase association with monomeric actin. Furthermore using a combination of GFP–INF2 expression in human podocytes and GFP-Trap purification coupled with MS we demonstrate that INF2 interacts with profilin 2 and the F-actin capping protein, CapZ α-1. These interactions are increased by the presence of the disease causing mutations. Since both these proteins are involved in the dynamic turnover and restructuring of the actin cytoskeleton these changes strengthen the evidence that aberrant regulation of actin dynamics underlies the pathogenesis of disease. PMID:26764407

  8. Reversible stress softening of actin networks

    NASA Astrophysics Data System (ADS)

    Chaudhuri, Ovijit; Parekh, Sapun H.; Fletcher, Daniel A.

    2007-01-01

    The mechanical properties of cells play an essential role in numerous physiological processes. Organized networks of semiflexible actin filaments determine cell stiffness and transmit force during mechanotransduction, cytokinesis, cell motility and other cellular shape changes. Although numerous actin-binding proteins have been identified that organize networks, the mechanical properties of actin networks with physiological architectures and concentrations have been difficult to measure quantitatively. Studies of mechanical properties in vitro have found that crosslinked networks of actin filaments formed in solution exhibit stress stiffening arising from the entropic elasticity of individual filaments or crosslinkers resisting extension. Here we report reversible stress-softening behaviour in actin networks reconstituted in vitro that suggests a critical role for filaments resisting compression. Using a modified atomic force microscope to probe dendritic actin networks (like those formed in the lamellipodia of motile cells), we observe stress stiffening followed by a regime of reversible stress softening at higher loads. This softening behaviour can be explained by elastic buckling of individual filaments under compression that avoids catastrophic fracture of the network. The observation of both stress stiffening and softening suggests a complex interplay between entropic and enthalpic elasticity in determining the mechanical properties of actin networks.

  9. Fimbrin phosphorylation by metaphase Cdk1 regulates actin cable dynamics in budding yeast.

    PubMed

    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.

  10. Fimbrin phosphorylation by metaphase Cdk1 regulates actin cable dynamics in budding yeast.

    PubMed

    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

  11. 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

  12. Direct binding of F actin to the cytoplasmic domain of the alpha 2 integrin chain in vitro

    NASA Technical Reports Server (NTRS)

    Kieffer, J. D.; Plopper, G.; Ingber, D. E.; Hartwig, J. H.; Kupper, T. S.

    1995-01-01

    The transmembrane integrins have been shown to interact with the cytoskeleton via noncovalent binding between cytoplasmic domains (CDs) of integrin beta chains and various actin binding proteins within the focal adhesion complex. Direct or indirect integrin alpha chain CD binding to the actin cytoskeleton has not been reported. We show here that actin, as an abundant constituent of focal adhesion complex proteins isolated from fibroblasts, binds strongly and specifically to alpha 2 CD, but not to alpha 1 CD peptide. Similar specific binding to alpha 2 CD peptide was seen for highly purified F actin, free of putative actin-binding proteins. The bound complex of actin and peptide was visualized directly by coprecipitation, and actin binding was abrogated by removal of a five amino acid sequence from the alpha 2 CD peptide. Our findings may explain the earlier observation that, while integrins alpha 2 beta 1 and alpha 1 beta 1 both bind to collagen, only alpha 2 beta 1 can mediate contraction of extracellular collagen matrices.

  13. Actin filaments and microtubules of Arabidopsis suspension cells show different responses to changing turgor pressure.

    PubMed

    Shi, Lanchun; Wang, Bochu; Gong, Wei; Zhang, Yungang; Zhu, Liqing; Yang, Xingyan

    2011-02-25

    Past decades have brought great advances in understanding the relationship between turgor pressure and plant cell growth. New studies have provided evidence that turgor pressure acts as a stimulus for cell growth, and is also a developmental cue for post-embryonic organogenesis. However, the subcellular mechanisms underlying plant cell turgor pressure sensing remain unclear. Here, using the relatively simple undifferentiated cells from suspension cultures, we report real-time in vivo observations of the reorganization of microtubules and actin microfilaments induced by turgor pressure changes. We found that these two cytoskeletal elements differed in their reorganization patterns. Our results will be useful in the understanding of the relationship between the cytoskeleton, turgor pressure, and stress in plant cell morphogenesis.

  14. Addition of Phenylboronic Acid to Malus domestica Pollen Tubes Alters Calcium Dynamics, Disrupts Actin Filaments and Affects Cell Wall Architecture.

    PubMed

    Fang, Kefeng; Gao, Sai; Zhang, Weiwei; Xing, Yu; Cao, Qingqin; Qin, Ling

    2016-01-01

    A key role of boron in plants is to cross-link the cell wall pectic polysaccharide rhamnogalacturonan-II (RG-II) through borate diester linkages. Phenylboronic acid (PBA) can form the same reversible ester bonds but cannot cross-link two molecules, so can be used as an antagonist to study the function of boron. This study aimed to evaluate the effect of PBA on apple (Malus domestica) pollen tube growth and the underlying regulatory mechanism. We observed that PBA caused an inhibition of pollen germination, tube growth and led to pollen tube morphological abnormalities. Fluorescent labeling, coupled with a scanning ion-selective electrode technique, revealed that PBA induced an increase in extracellular Ca2+ influx, thereby elevating the cytosolic Ca2+ concentration [Ca2+]c and disrupting the [Ca2+]c gradient, which is critical for pollen tube growth. Moreover the organization of actin filaments was severely perturbed by the PBA treatment. Immunolocalization studies and fluorescent labeling, together with Fourier-transform infrared analysis (FTIR) suggested that PBA caused an increase in the abundance of callose, de-esterified pectins and arabinogalactan proteins (AGPs) at the tip. However, it had no effect on the deposition of the wall polymers cellulose. These effects are similar to those of boron deficiency in roots and other organs, indicating that PBA can induce boron deficiency symptoms. The results provide new insights into the roles of boron in pollen tube development, which likely include regulating [Ca2+]c and the formation of the actin cytoskeleton, in addition to the synthesis and assembly of cell wall components. PMID:26886907

  15. Addition of Phenylboronic Acid to Malus domestica Pollen Tubes Alters Calcium Dynamics, Disrupts Actin Filaments and Affects Cell Wall Architecture.

    PubMed

    Fang, Kefeng; Gao, Sai; Zhang, Weiwei; Xing, Yu; Cao, Qingqin; Qin, Ling

    2016-01-01

    A key role of boron in plants is to cross-link the cell wall pectic polysaccharide rhamnogalacturonan-II (RG-II) through borate diester linkages. Phenylboronic acid (PBA) can form the same reversible ester bonds but cannot cross-link two molecules, so can be used as an antagonist to study the function of boron. This study aimed to evaluate the effect of PBA on apple (Malus domestica) pollen tube growth and the underlying regulatory mechanism. We observed that PBA caused an inhibition of pollen germination, tube growth and led to pollen tube morphological abnormalities. Fluorescent labeling, coupled with a scanning ion-selective electrode technique, revealed that PBA induced an increase in extracellular Ca2+ influx, thereby elevating the cytosolic Ca2+ concentration [Ca2+]c and disrupting the [Ca2+]c gradient, which is critical for pollen tube growth. Moreover the organization of actin filaments was severely perturbed by the PBA treatment. Immunolocalization studies and fluorescent labeling, together with Fourier-transform infrared analysis (FTIR) suggested that PBA caused an increase in the abundance of callose, de-esterified pectins and arabinogalactan proteins (AGPs) at the tip. However, it had no effect on the deposition of the wall polymers cellulose. These effects are similar to those of boron deficiency in roots and other organs, indicating that PBA can induce boron deficiency symptoms. The results provide new insights into the roles of boron in pollen tube development, which likely include regulating [Ca2+]c and the formation of the actin cytoskeleton, in addition to the synthesis and assembly of cell wall components.

  16. Addition of Phenylboronic Acid to Malus domestica Pollen Tubes Alters Calcium Dynamics, Disrupts Actin Filaments and Affects Cell Wall Architecture

    PubMed Central

    Fang, Kefeng; Gao, Sai; Zhang, Weiwei; Xing, Yu; Cao, Qingqin; Qin, Ling

    2016-01-01

    A key role of boron in plants is to cross-link the cell wall pectic polysaccharide rhamnogalacturonan-II (RG-II) through borate diester linkages. Phenylboronic acid (PBA) can form the same reversible ester bonds but cannot cross-link two molecules, so can be used as an antagonist to study the function of boron. This study aimed to evaluate the effect of PBA on apple (Malus domestica) pollen tube growth and the underlying regulatory mechanism. We observed that PBA caused an inhibition of pollen germination, tube growth and led to pollen tube morphological abnormalities. Fluorescent labeling, coupled with a scanning ion-selective electrode technique, revealed that PBA induced an increase in extracellular Ca2+ influx, thereby elevating the cytosolic Ca2+ concentration [Ca2+]c and disrupting the [Ca2+]c gradient, which is critical for pollen tube growth. Moreover the organization of actin filaments was severely perturbed by the PBA treatment. Immunolocalization studies and fluorescent labeling, together with Fourier-transform infrared analysis (FTIR) suggested that PBA caused an increase in the abundance of callose, de-esterified pectins and arabinogalactan proteins (AGPs) at the tip. However, it had no effect on the deposition of the wall polymers cellulose. These effects are similar to those of boron deficiency in roots and other organs, indicating that PBA can induce boron deficiency symptoms. The results provide new insights into the roles of boron in pollen tube development, which likely include regulating [Ca2+]c and the formation of the actin cytoskeleton, in addition to the synthesis and assembly of cell wall components. PMID:26886907

  17. Microtubule-associated Protein 2c Reorganizes Both Microtubules and Microfilaments into Distinct Cytological Structures in an Actin-binding Protein-280–deficient Melanoma Cell Line

    PubMed Central

    Cunningham, C. Casey; Leclerc, Nicole; Flanagan, Lisa A.; Lu, Mei; Janmey, Paul A.; Kosik, Kenneth S.

    1997-01-01

    The emergence of processes from cells often involves interactions between microtubules and microfilaments. Interactions between these two cytoskeletal systems are particularly apparent in neuronal growth cones. The juvenile isoform of the neuronal microtubule-associated protein 2 (MAP2c) is present in growth cones, where we hypothesize it mediates interactions between microfilaments and microtubules. To approach this problem in vivo, we used the human melanoma cell, M2, which lacks actin-binding protein-280 (ABP-280) and forms membrane blebs, which are not seen in wild-type or ABP-transfected cells. The microinjection of tau or mature MAP2 rescued the blebbing phenotype; MAP2c not only caused cessation of blebbing but also induced the formation of two distinct cellular structures. These were actin-rich lamellae, which often included membrane ruffles, and microtubule-bearing processes. The lamellae collapsed after treatment with cytochalasin D, and the processes retracted after treatment with colchicine. MAP2c was immunocytochemically visualized in zones of the cell that were devoid of tubulin, such as regions within the lamellae and in association with membrane ruffles. In vitro rheometry confirmed that MAP2c is an efficient actin gelation protein capable of organizing actin filaments into an isotropic array at very low concentrations; tau and mature MAP2 do not share this rheologic property. These results suggest that MAP2c engages in functionally specific interactions not only with microtubules but also with microfilaments. PMID:9049250

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

    PubMed Central

    Jensen, Mikkel Herholdt; Watt, James; Hodgkinson, Julie; 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 μm 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 suggest 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. PMID:22135101

  19. Modification of Cys-837 identifies an actin-binding site in the beta-propeller protein scruin.

    PubMed Central

    Sun, S; Footer, M; Matsudaira, P

    1997-01-01

    In the acrosomal process of Limulus sperm, the beta-propeller protein scruin cross-links actin into a crystalline bundle. To confirm that scruin has the topology of a beta-propeller protein and to understand how scruin binds actin, we compared the solvent accessibility of cysteine residues in scruin and the acrosomal process by chemical modification with (1,5-IAEDANS). In soluble scruin, the two most reactive cysteines of soluble scruin are C837 and C900, whereas C146, C333, and C683 are moderately reactive. This pattern of reactivity is consistent with the topology of a typical beta-propeller protein; all of the reactive cysteines map to putative loops and turns whereas the unreactive cysteines lie within the predicted interior of the protein. The chemical reactivities of cysteine in the acrosomal process implicate C837 at an actin-binding site. In contrast to soluble scruin, in the acrosomal process, C837 is completely unreactive while the other cysteines become less reactive. Binding studies of chemically modified scruin correlate the extent of modification at C837 with the extent of inhibition of actin binding. Furthermore, peptides corresponding to residues flanking C837 bind actin and narrow a possible actin-binding region to a KQK sequence. On the basis of these studies, our results suggest that an actin-binding site lies in the C-terminal domain of scruin and involves a putative loop defined by C837. Images PMID:9188095

  20. Interaction between microtubules and the Drosophila formin Cappuccino and its effect on actin assembly.

    PubMed

    Roth-Johnson, Elizabeth A; Vizcarra, Christina L; Bois, Justin S; Quinlan, Margot E

    2014-02-14

    Formin family actin nucleators are potential coordinators of the actin and microtubule cytoskeletons, as they can both nucleate actin filaments and bind microtubules in vitro. To gain a more detailed mechanistic understanding of formin-microtubule interactions and formin-mediated actin-microtubule cross-talk, we studied microtubule binding by Cappuccino (Capu), a formin involved in regulating actin and microtubule organization during Drosophila oogenesis. We found that two distinct domains within Capu, FH2 and tail, work together to promote high-affinity microtubule binding. The tail domain appears to bind microtubules through nonspecific charge-based interactions. In contrast, distinct residues within the FH2 domain are important for microtubule binding. We also report the first visualization of a formin polymerizing actin filaments in the presence of microtubules. Interestingly, microtubules are potent inhibitors of the actin nucleation activity of Capu but appear to have little effect on Capu once it is bound to the barbed end of an elongating filament. Because Capu does not simultaneously bind microtubules and assemble actin filaments in vitro, its actin assembly and microtubule binding activities likely require spatial and/or temporal regulation within the Drosophila oocyte.

  1. Actin-organising properties of the muscular dystrophy protein myotilin.

    PubMed

    von Nandelstadh, Pernilla; Grönholm, Mikaela; Moza, Monica; Lamberg, Arja; Savilahti, Harri; Carpén, Olli

    2005-10-15

    Myotilin is a sarcomeric Z-disc protein that binds F-actin directly and bundles actin filaments, although it does not contain a conventional actin-binding domain. Expression of mutant myotilin leads to sarcomeric alterations in the dominantly inherited limb-girdle muscular dystrophy 1A and in myofibrillar myopathy/desmin-related myopathy. Together, with previous in vitro studies, this indicates that myotilin has an important function in the assembly and maintenance of Z-discs. This study characterises further the interaction between myotilin and actin. Functionally important regions in myotilin were identified by actin pull-down and yeast two-hybrid assays and with a novel strategy that combines in vitro DNA transposition-based peptide insertion mutagenesis with phenotype analysis in yeast cells. The shortest fragment to bind actin was the second Ig domain together with a short C-terminal sequence. Concerted action of the first and second Ig domain was, however, necessary for the functional activity of myotilin, as verified by analysis of transposon mutants, actin binding and phenotypic effect in mammalian cells. Furthermore, the Ig domains flanked with N- and C-terminal regions were needed for actin-bundling, indicating that the mere actin-binding sequence was insufficient for the actin-regulating activity. None of the four known disease-associated mutations altered the actin-organising ability. These results, together with previous studies in titin and kettin, identify the Ig domain as an actin-binding unit.

  2. Regulation of Actin by Ion-Linked Equilibria

    PubMed Central

    Kang, Hyeran; Bradley, Michael J.; Elam, W. Austin; De La Cruz, Enrique M.

    2013-01-01

    Actin assembly, filament mechanical properties, and interactions with regulatory proteins depend on the types and concentrations of salts in solution. Salts modulate actin through both nonspecific electrostatic effects and specific binding to discrete sites. Multiple cation-binding site classes spanning a broad range of affinities (nanomolar to millimolar) have been identified on actin monomers and filaments. This review focuses on discrete, low-affinity cation-binding interactions that drive polymerization, regulate filament-bending mechanics, and modulate interactions with regulatory proteins. Cation binding may be perturbed by actin post-translational modifications and linked equilibria. Partial cation occupancy under physiological and commonly used in vitro solution conditions likely contribute to filament mechanical heterogeneity and structural polymorphism. Site-specific cation-binding residues are conserved in Arp2 and Arp3, and may play a role in Arp2/3 complex activation and actin-filament branching activity. Actin-salt interactions demonstrate the relevance of ion-linked equilibria in the operation and regulation of complex biological systems. PMID:24359734

  3. Role of Actin Filaments in Correlating Nuclear Shape and Cell Spreading

    PubMed Central

    Vishavkarma, Renu; Raghavan, Swetavalli; Kuyyamudi, Chandrashekar; Majumder, Abhijit; Dhawan, Jyotsna; Pullarkat, Pramod A.

    2014-01-01

    It is well known that substrate properties like stiffness and adhesivity influence stem cell morphology and differentiation. Recent experiments show that cell morphology influences nuclear geometry and hence gene expression profile. The mechanism by which surface properties regulate cell and nuclear properties is only beginning to be understood. Direct transmission of forces as well as chemical signalling are involved in this process. Here, we investigate the formal aspect by studying the correlation between cell spreading and nuclear deformation using Mesenchymal stem cells under a wide variety of conditions. It is observed that a robust quantitative relation holds between the cell and nuclear projected areas, irrespective of how the cell area is modified or when various cytoskeletal or nuclear components are perturbed. By studying the role of actin stress fibers in compressing the nucleus we propose that nuclear compression by stress fibers can lead to enhanced cell spreading due to an interplay between elastic and adhesion factors. The significance of myosin-II in regulating this process is also explored. We demonstrate this effect using a simple technique to apply external compressive loads on the nucleus. PMID:25251154

  4. Crystal structure of a nuclear actin ternary complex.

    PubMed

    Cao, Tingting; Sun, Lingfei; Jiang, Yuxiang; Huang, Shanjin; Wang, Jiawei; Chen, Zhucheng

    2016-08-01

    Actin polymerizes and forms filamentous structures (F-actin) in the cytoplasm of eukaryotic cells. It also exists in the nucleus and regulates various nucleic acid transactions, particularly through its incorporation into multiple chromatin-remodeling complexes. However, the specific structure of actin and the mechanisms that regulate its polymeric nature inside the nucleus remain unknown. Here, we report the crystal structure of nuclear actin (N-actin) complexed with actin-related protein 4 (Arp4) and the helicase-SANT-associated (HSA) domain of the chromatin remodeler Swr1. The inner face and barbed end of N-actin are sequestered by interactions with Arp4 and the HSA domain, respectively, which prevents N-actin from polymerization and binding to many actin regulators. The two major domains of N-actin are more twisted than those of globular actin (G-actin), and its nucleotide-binding pocket is occluded, freeing N-actin from binding to and regulation by ATP. These findings revealed the salient structural features of N-actin that distinguish it from its cytoplasmic counterpart and provide a rational basis for its functions and regulation inside the nucleus. PMID:27457955

  5. Profilin Regulates Apical Actin Polymerization to Control Polarized Pollen Tube Growth.

    PubMed

    Liu, Xiaonan; Qu, Xiaolu; Jiang, Yuxiang; Chang, Ming; Zhang, Ruihui; Wu, Youjun; Fu, Ying; Huang, Shanjin

    2015-12-01

    Pollen tube growth is an essential step during flowering plant reproduction, whose growth depends on a population of dynamic apical actin filaments. Apical actin filaments were thought to be involved in the regulation of vesicle fusion and targeting in the pollen tube. However, the molecular mechanisms that regulate the construction of apical actin structures in the pollen tube remain largely unclear. Here, we identify profilin as an important player in the regulation of actin polymerization at the apical membrane in the pollen tube. Downregulation of profilin decreased the amount of filamentous actin and induced disorganization of apical actin filaments, and reduced tip-directed vesicle transport and accumulation in the pollen tube. Direct visualization of actin dynamics revealed that the elongation of actin filaments originating at the apical membrane decreased in profilin mutant pollen tubes. Mutant profilin that is defective in binding poly-L-proline only partially rescues the actin polymerization defect in profilin mutant pollen tubes, although it fully rescues the actin turnover phenotype. We propose that profilin controls the construction of actin structures at the pollen tube tip, presumably by favoring formin-mediated actin polymerization at the apical membrane.

  6. Activation of 5-HT7 receptor stimulates neurite elongation through mTOR, Cdc42 and actin filaments dynamics

    PubMed Central

    Speranza, Luisa; Giuliano, Teresa; Volpicelli, Floriana; De Stefano, M. Egle; Lombardi, Loredana; Chambery, Angela; Lacivita, Enza; Leopoldo, Marcello; Bellenchi, Gian C.; di Porzio, Umberto; Crispino, Marianna; Perrone-Capano, Carla

    2015-01-01

    Recent studies have indicated that the serotonin receptor subtype 7 (5-HT7R) plays a crucial role in shaping neuronal morphology during embryonic and early postnatal life. Here we show that pharmacological stimulation of 5-HT7R using a highly selective agonist, LP-211, enhances neurite outgrowth in neuronal primary cultures from the cortex, hippocampus and striatal complex of embryonic mouse brain, through multiple signal transduction pathways. All these signaling systems, involving mTOR, the Rho GTPase Cdc42, Cdk5, and ERK, are known to converge on the reorganization of cytoskeletal proteins that subserve neurite outgrowth. Indeed, our data indicate that neurite elongation stimulated by 5-HT7R is modulated by drugs affecting actin polymerization. In addition, we show, by 2D Western blot analyses, that treatment of neuronal cultures with LP-211 alters the expression profile of cofilin, an actin binding protein involved in microfilaments dynamics. Furthermore, by using microfluidic chambers that physically separate axons from the soma and dendrites, we demonstrate that agonist-dependent activation of 5-HT7R stimulates axonal elongation. Our results identify for the first time several signal transduction pathways, activated by stimulation of 5-HT7R, that converge to promote cytoskeleton reorganization and consequent modulation of axonal elongation. Therefore, the activation of 5-HT7R might represent one of the key elements regulating CNS connectivity and plasticity during development. PMID:25814944

  7. Activation of 5-HT7 receptor stimulates neurite elongation through mTOR, Cdc42 and actin filaments dynamics.

    PubMed

    Speranza, Luisa; Giuliano, Teresa; Volpicelli, Floriana; De Stefano, M Egle; Lombardi, Loredana; Chambery, Angela; Lacivita, Enza; Leopoldo, Marcello; Bellenchi, Gian C; di Porzio, Umberto; Crispino, Marianna; Perrone-Capano, Carla

    2015-01-01

    Recent studies have indicated that the serotonin receptor subtype 7 (5-HT7R) plays a crucial role in shaping neuronal morphology during embryonic and early postnatal life. Here we show that pharmacological stimulation of 5-HT7R using a highly selective agonist, LP-211, enhances neurite outgrowth in neuronal primary cultures from the cortex, hippocampus and striatal complex of embryonic mouse brain, through multiple signal transduction pathways. All these signaling systems, involving mTOR, the Rho GTPase Cdc42, Cdk5, and ERK, are known to converge on the reorganization of cytoskeletal proteins that subserve neurite outgrowth. Indeed, our data indicate that neurite elongation stimulated by 5-HT7R is modulated by drugs affecting actin polymerization. In addition, we show, by 2D Western blot analyses, that treatment of neuronal cultures with LP-211 alters the expression profile of cofilin, an actin binding protein involved in microfilaments dynamics. Furthermore, by using microfluidic chambers that physically separate axons from the soma and dendrites, we demonstrate that agonist-dependent activation of 5-HT7R stimulates axonal elongation. Our results identify for the first time several signal transduction pathways, activated by stimulation of 5-HT7R, that converge to promote cytoskeleton reorganization and consequent modulation of axonal elongation. Therefore, the activation of 5-HT7R might represent one of the key elements regulating CNS connectivity and plasticity during development.

  8. Effect of disruption of actin filaments by Clostridium botulinum C2 toxin on insulin secretion in HIT-T15 cells and pancreatic islets.

    PubMed Central

    Li, G; Rungger-Brändle, E; Just, I; Jonas, J C; Aktories, K; Wollheim, C B

    1994-01-01

    To examine their role in insulin secretion, actin filaments (AFs) were disrupted by Clostridium botulinum C2 toxin that ADP-ribosylates G-actin. Ribosylation also prevents polymerization of G-actin to F-actin and inhibits AF assembly by capping the fast-growing end of F-actin. Pretreatment of HIT-T15 cells with the toxin inhibited stimulated insulin secretion in a time- and dose-dependent manner. The toxin did not affect cellular insulin content or nonstimulated secretion. In static incubation, toxin treatment caused 45-50% inhibition of secretion induced by nutrients alone (10 mM glucose + 5 mM glutamine + 5 mM leucine) or combined with bombesin (phospholipase C-activator) and 20% reduction of that potentiated by forskolin (stimulator of adenylyl cyclase). In perifusion, the stimulated secretion during the first phase was marginally diminished, whereas the second phase was inhibited by approximately 80%. Pretreatment of HIT cells with wartmannin, a myosin light chain kinase inhibitor, caused a similar pattern of inhibition of the biphasic insulin release as C2 toxin. Nutrient metabolism and bombesin-evoked rise in cytosolic free Ca2+ were not affected by C2 toxin, indicating that nutrient recognition and the coupling between receptor activation and second messenger generation was not changed. In the toxin-treated cells, the AF web beneath the plasma membrane and the diffuse cytoplasmic F-actin fibers disappeared, as shown both by staining with an antibody against G- and F-actin and by staining F-actin with fluorescent phallacidin. C2 toxin dose-dependently reduced cellular F-actin content. Stimulation of insulin secretion was not associated with changes in F-actin content and organization. Treatment of cells with cytochalasin E and B, which shorten AFs, inhibited the stimulated insulin release by 30-50% although differing in their effects on F-actin content. In contrast to HIT-T15 cells, insulin secretion was potentiated in isolated rat islets after disruption of

  9. Wide-ranging effects of eight cytochalasins and latrunculin A and B on intracellular motility and actin filament reorganization in characean internodal cells.

    PubMed

    Foissner, Ilse; Wasteneys, Geoffrey O

    2007-04-01

    Numerous forms of cytochalasins have been identified and, although they share common biological activity, they may differ considerably in potency. We investigated the effects of cytochalasins A, B, C, D, E, H and J and dihydrocytochalasin B in an ideal experimental system for cell motility, the giant internodal cells of the characean alga Nitella pseudoflabellata. Cytochalasins D (60 microM) and H (30 microM) were found to be most suited for fast and reversible inhibition of actin-based motility, while cytochalasins A and E arrested streaming at lower concentrations but irreversibly. We observed no clear correlation between the ability of cytochalasins to inhibit motility and the actual disruption of the subcortical actin bundle tracks on which myosin-dependent motility occurs. Indeed, the actin bundles remained intact at the time of streaming cessation and disassembled only after one to several days' treatment. Even when applied at concentrations lower than that required to inhibit cytoplasmic streaming, all of the cytochalasins induced reorganization of the more labile cortical actin filaments into actin patches, swirling clusters or short rods. Latrunculins A and B arrested streaming only after disrupting the subcortical actin bundles, a process requiring relatively high concentrations (200 microM) and very long treatment periods of >1 d. Latrunculins, however, worked synergistically with cytochalasins. A 1 h treatment with 15 nM latrunculin A and 4 microM cytochalasin D induced reversible fragmentation of subcortical actin bundles and arrested cytoplasmic streaming. Our findings provide insights into the mechanisms by which cytochalasins and latrunculins interfere with characean actin to inhibit motility.

  10. F-actin dismantling through a redox-driven synergy between Mical and cofilin.

    PubMed

    Grintsevich, Elena E; Yesilyurt, Hunkar Gizem; Rich, Shannon K; Hung, Ruei-Jiun; Terman, Jonathan R; Reisler, Emil

    2016-08-01

    Numerous cellular functions depend on actin filament (F-actin) disassembly. The best-characterized disassembly proteins, the ADF (actin-depolymerizing factor)/cofilins (encoded by the twinstar gene in Drosophila), sever filaments and recycle monomers to promote actin assembly. Cofilin is also a relatively weak actin disassembler, posing questions about mechanisms of cellular F-actin destabilization. Here we uncover a key link to targeted F-actin disassembly by finding that F-actin is efficiently dismantled through a post-translational-mediated synergism between cofilin and the actin-oxidizing enzyme Mical. We find that Mical-mediated oxidation of actin improves cofilin binding to filaments, where their combined effect dramatically accelerates F-actin disassembly compared with either effector alone. This synergism is also necessary and sufficient for F-actin disassembly in vivo, magnifying the effects of both Mical and cofilin on cellular remodelling, axon guidance and Semaphorin-Plexin repulsion. Mical and cofilin, therefore, form a redox-dependent synergistic pair that promotes F-actin instability by rapidly dismantling F-actin and generating post-translationally modified actin that has altered assembly properties. PMID:27454820

  11. Cloning and sequencing of a gene coding for an actin binding protein of Saccharomyces exiguus.

    PubMed

    Lange, U; Steiner, S; Grolig, F; Wagner, G; Philippsen, P

    1994-03-01

    The actin binding protein Abp1p of the yeast Saccharomyces cervisiae is thought to be involved in the spatial organisation of cell surface growth. It contains a potential actin binding domain and an SH-3 region, a common motif of many signal transduction proteins [1]. We have cloned and sequenced an ABP1 homologous gene of Saccharomyces exiguus, a yeast which is only distantly related to S. cerevisiae. The protein encoded by this gene is slightly larger than the respective S. cerevisiae protein (617 versus 592 amino acids). The two genes are 67.4% identical and the deduced amino acid sequences share an overall identity of 59.8%. The most conserved regions are the 148 N-terminal amino acids containing the potential actin binding site and the 58 C-terminal amino acids including the SH3 domain. In addition, both proteins contain a repeated motif of unknown function which is rich in glutamic acids with the sequence EEEEEEEAPAPSLPSR in the S. exiguus Abp1p. PMID:8110838

  12. A semi-flexible model prediction for the polymerization force exerted by a living F-actin filament on a fixed wall.

    PubMed

    Pierleoni, Carlo; Ciccotti, Giovanni; Ryckaert, Jean-Paul

    2015-10-14

    distance L is in practice L independent and very close to the value of the stalling force Fs (H)=(kBT/d)ln(ρˆ1) predicted by Hill, this expression being strictly valid in the rigid filament limit. The average filament force results from the product of the cumulative size fraction x=x(L,ℓp,ρˆ1), where the filament is in contact with the wall, times the buckling force on a filament of size Lc ≈ L, namely, Fs (H)=xfb(L;ℓp). The observed L independence of Fs (H) implies that x ∝ L(-2) for given (ℓp,ρˆ1) and x∝lnρˆ1 for given (ℓp, L). At fixed (L,ρˆ1), one also has x∝ℓp (-1) which indicates that the rigid filament limit ℓp → ∞ is a singular limit in which an infinite force has zero weight. Finally, we derive the physically relevant threshold for filament escaping in the case of actin filaments. PMID:26472399

  13. Profilin Binding to Poly-l-Proline and Actin Monomers along with Ability to Catalyze Actin Nucleotide Exchange Is Required for Viability of Fission Yeast

    PubMed Central

    Lu, Jia; Pollard, Thomas D.

    2001-01-01

    We tested the ability of 87 profilin point mutations to complement temperature-sensitive and null mutations of the single profilin gene of the fission yeast Schizosaccharomyces pombe. We compared the biochemical properties of 13 stable noncomplementing profilins with an equal number of complementing profilin mutants. A large quantitative database revealed the following: 1) in a profilin null background fission yeast grow normally with profilin mutations having >10% of wild-type affinity for actin or poly-l-proline, but lower affinity for either ligand is incompatible with life; 2) in the cdc3-124 profilin ts background, fission yeast function with profilin having only 2–5% wild-type affinity for actin or poly-l-proline; and 3) special mutations show that the ability of profilin to catalyze nucleotide exchange by actin is an essential function. Thus, poly-l-proline binding, actin binding, and actin nucleotide exchange are each independent requirements for profilin function in fission yeast. PMID:11294914

  14. C0 and C1 N-terminal Ig domains of myosin binding protein C exert different effects on thin filament activation.

    PubMed

    Harris, Samantha P; Belknap, Betty; Van Sciver, Robert E; White, Howard D; Galkin, Vitold E

    2016-02-01

    Mutations in genes encoding myosin, the molecular motor that powers cardiac muscle contraction, and its accessory protein, cardiac myosin binding protein C (cMyBP-C), are the two most common causes of hypertrophic cardiomyopathy (HCM). Recent studies established that the N-terminal domains (NTDs) of cMyBP-C (e.g., C0, C1, M, and C2) can bind to and activate or inhibit the thin filament (TF). However, the molecular mechanism(s) by which NTDs modulate interaction of myosin with the TF remains unknown and the contribution of each individual NTD to TF activation/inhibition is unclear. Here we used an integrated structure-function approach using cryoelectron microscopy, biochemical kinetics, and force measurements to reveal how the first two Ig-like domains of cMyPB-C (C0 and C1) interact with the TF. Results demonstrate that despite being structural homologs, C0 and C1 exhibit different patterns of binding on the surface of F-actin. Importantly, C1 but not C0 binds in a position to activate the TF by shifting tropomyosin (Tm) to the "open" structural state. We further show that C1 directly interacts with Tm and traps Tm in the open position on the surface of F-actin. Both C0 and C1 compete with myosin subfragment 1 for binding to F-actin and effectively inhibit actomyosin interactions when present at high ratios of NTDs to F-actin. Finally, we show that in contracting sarcomeres, the activating effect of C1 is apparent only once low levels of Ca(2+) have been achieved. We suggest that Ca(2+) modulates the interaction of cMyBP-C with the TF in the sarcomere. PMID:26831109

  15. C0 and C1 N-terminal Ig domains of myosin binding protein C exert different effects on thin filament activation

    PubMed Central

    Harris, Samantha P.; Belknap, Betty; Van Sciver, Robert E.; White, Howard D.; Galkin, Vitold E.

    2016-01-01

    Mutations in genes encoding myosin, the molecular motor that powers cardiac muscle contraction, and its accessory protein, cardiac myosin binding protein C (cMyBP-C), are the two most common causes of hypertrophic cardiomyopathy (HCM). Recent studies established that the N-terminal domains (NTDs) of cMyBP-C (e.g., C0, C1, M, and C2) can bind to and activate or inhibit the thin filament (TF). However, the molecular mechanism(s) by which NTDs modulate interaction of myosin with the TF remains unknown and the contribution of each individual NTD to TF activation/inhibition is unclear. Here we used an integrated structure–function approach using cryoelectron microscopy, biochemical kinetics, and force measurements to reveal how the first two Ig-like domains of cMyPB-C (C0 and C1) interact with the TF. Results demonstrate that despite being structural homologs, C0 and C1 exhibit different patterns of binding on the surface of F-actin. Importantly, C1 but not C0 binds in a position to activate the TF by shifting tropomyosin (Tm) to the “open” structural state. We further show that C1 directly interacts with Tm and traps Tm in the open position on the surface of F-actin. Both C0 and C1 compete with myosin subfragment 1 for binding to F-actin and effectively inhibit actomyosin interactions when present at high ratios of NTDs to F-actin. Finally, we show that in contracting sarcomeres, the activating effect of C1 is apparent only once low levels of Ca2+ have been achieved. We suggest that Ca2+ modulates the interaction of cMyBP-C with the TF in the sarcomere. PMID:26831109

  16. C0 and C1 N-terminal Ig domains of myosin binding protein C exert different effects on thin filament activation.

    PubMed

    Harris, Samantha P; Belknap, Betty; Van Sciver, Robert E; White, Howard D; Galkin, Vitold E

    2016-02-01

    Mutations in genes encoding myosin, the molecular motor that powers cardiac muscle contraction, and its accessory protein, cardiac myosin binding protein C (cMyBP-C), are the two most common causes of hypertrophic cardiomyopathy (HCM). Recent studies established that the N-terminal domains (NTDs) of cMyBP-C (e.g., C0, C1, M, and C2) can bind to and activate or inhibit the thin filament (TF). However, the molecular mechanism(s) by which NTDs modulate interaction of myosin with the TF remains unknown and the contribution of each individual NTD to TF activation/inhibition is unclear. Here we used an integrated structure-function approach using cryoelectron microscopy, biochemical kinetics, and force measurements to reveal how the first two Ig-like domains of cMyPB-C (C0 and C1) interact with the TF. Results demonstrate that despite being structural homologs, C0 and C1 exhibit different patterns of binding on the surface of F-actin. Importantly, C1 but not C0 binds in a position to activate the TF by shifting tropomyosin (Tm) to the "open" structural state. We further show that C1 directly interacts with Tm and traps Tm in the open position on the surface of F-actin. Both C0 and C1 compete with myosin subfragment 1 for binding to F-actin and effectively inhibit actomyosin interactions when present at high ratios of NTDs to F-actin. Finally, we show that in contracting sarcomeres, the activating effect of C1 is apparent only once low levels of Ca(2+) have been achieved. We suggest that Ca(2+) modulates the interaction of cMyBP-C with the TF in the sarcomere.

  17. An explicitly solvated full atomistic model of the cardiac thin filament and application on the calcium binding affinity effects from familial hypertrophic cardiomyopathy linked mutations

    NASA Astrophysics Data System (ADS)

    Williams, Michael; Schwartz, Steven

    2015-03-01

    The previous version of our cardiac thin filament (CTF) model consisted of the troponin complex (cTn), two coiled-coil dimers of tropomyosin (Tm), and 29 actin units. We now present the newest revision of the model to include explicit solvation. The model was developed to continue our study of genetic mutations in the CTF proteins which are linked to familial hypertrophic cardiomyopathies. Binding of calcium to the cTnC subunit causes subtle conformational changes to propagate through the cTnC to the cTnI subunit which then detaches from actin. Conformational changes propagate through to the cTnT subunit, which allows Tm to move into the open position along actin, leading to muscle contraction. Calcium disassociation allows for the reverse to occur, which results in muscle relaxation. The inclusion of explicit TIP3 water solvation allows for the model to get better individual local solvent to protein interactions; which are important when observing the N-lobe calcium binding pocket of the cTnC. We are able to compare in silica and in vitro experimental results to better understand the physiological effects from mutants, such as the R92L/W and F110V/I of the cTnT, on the calcium binding affinity compared to the wild type.

  18. 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

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

    PubMed

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

    2014-09-15

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

  20. Covisualization in living onion cells of putative integrin, putative spectrin, actin, putative intermediate filaments, and other proteins at the cell membrane and in an endomembrane sheath

    NASA Technical Reports Server (NTRS)

    Reuzeau, C.; Doolittle, K. W.; McNally, J. G.; Pickard, B. G.; Evans, M. L. (Principal Investigator)

    1997-01-01

    Covisualizations with wide-field computational optical-sectioning microscopy of living epidermal cells of the onion bulb scale have evidenced two major new cellular features. First, a sheath of cytoskeletal elements clads the endomembrane system. Similar elements clad the inner faces of punctate plasmalemmal sites interpreted as plasmalemmal control centers. One component of the endomembrane sheath and plasmalemmal control center cladding is anti-genicity-recognized by two injected antibodies against animal spectrin. Immunoblots of separated epidermal protein also showed bands recognized by these antibodies. Injected phalloidin identified F-actin with the same cellular distribution pattern, as did antibodies against intermediate-filament protein and other cytoskeletal elements known from animal cells. Injection of general protein stains demonstrated the abundance of endomembrane sheath protein. Second, the endomembrane system, like the plasmalemmal puncta, contains antigen recognized by an anti-beta 1 integrin injected into the cytoplasm. Previously, immunoblots of separated epidermal protein were shown to have a major band recognized both by this antibody prepared against a peptide representing the cytosolic region of beta 1 integrin and an antibody against the matrix region of beta 1 integrin. The latter antiboby also identified puncta at the external face of protoplasts. It is proposed that integrin and associated transmembrane proteins secure the endomembrane sheath and transmit signals between it and the lumen or matrix of the endoplasmic reticulum and organellar matrices. This function is comparable to that proposed for such transmembrane linkers in the plasmalemmal control centers, which also appear to bind cytoskeleton and a host of related molecules and transmit signals between them and the wall matrix. It is at the plasmalemmal control centers that the endoplasmic reticulum, a major component of the endomembrane system, attaches to the plasma membrane.

  1. Covisualization in living onion cells of putative integrin, putative spectrin, actin, putative intermediate filaments, and other proteins at the cell membrane and in an endomembrane sheath.

    PubMed

    Reuzeau, C; Doolittle, K W; McNally, J G; Pickard, B G

    1997-01-01

    Covisualizations with wide-field computational optical-sectioning microscopy of living epidermal cells of the onion bulb scale have evidenced two major new cellular features. First, a sheath of cytoskeletal elements clads the endomembrane system. Similar elements clad the inner faces of punctate plasmalemmal sites interpreted as plasmalemmal control centers. One component of the endomembrane sheath and plasmalemmal control center cladding is anti-genicity-recognized by two injected antibodies against animal spectrin. Immunoblots of separated epidermal protein also showed bands recognized by these antibodies. Injected phalloidin identified F-actin with the same cellular distribution pattern, as did antibodies against intermediate-filament protein and other cytoskeletal elements known from animal cells. Injection of general protein stains demonstrated the abundance of endomembrane sheath protein. Second, the endomembrane system, like the plasmalemmal puncta, contains antigen recognized by an anti-beta 1 integrin injected into the cytoplasm. Previously, immunoblots of separated epidermal protein were shown to have a major band recognized both by this antibody prepared against a peptide representing the cytosolic region of beta 1 integrin and an antibody against the matrix region of beta 1 integrin. The latter antiboby also identified puncta at the external face of protoplasts. It is proposed that integrin and associated transmembrane proteins secure the endomembrane sheath and transmit signals between it and the lumen or matrix of the endoplasmic reticulum and organellar matrices. This function is comparable to that proposed for such transmembrane linkers in the plasmalemmal control centers, which also appear to bind cytoskeleton and a host of related molecules and transmit signals between them and the wall matrix. It is at the plasmalemmal control centers that the endoplasmic reticulum, a major component of the endomembrane system, attaches to the plasma membrane

  2. EhNCABP166: a nucleocytoplasmic actin-binding protein from Entamoeba histolytica.

    PubMed

    Campos-Parra, A D; Hernández-Cuevas, N A; Hernandez-Rivas, R; Vargas, M

    2010-07-01

    The actin cytoskeleton consists of multiple actin binding proteins (ABPs) that participate cooperatively in different cellular functions such as the maintenance of polarity and cell motility as well as the invasion of target cells and regulation of gene expression, among others. Due to the important role of ABPs in the pathogenesis of Entamoeba histolytica, the role of a new nucleocytoplasmic ABP from E. histolytica named EhNCABP166 was investigated. The EhNCABP166 gene encodes a protein with an estimated molecular weight of 166kDa. Structurally, this peptide is composed of two CH domains arranged in tandem at the N-terminus of the protein, followed by an alpha-helical region containing a number of different domains with a low level of homology. Two (Bin1/Amphiphysin/Rvs167) (BAR) domains, one GTPase-binding/formin 3 homology (GBD/FH3) domain, three Bcl2-associated athanogene (BAG) domains, one basic-leucine zipper (bZIP) domain and one poly(A)-binding protein C-terminal (PABC) domain were also present. Molecular and biochemical studies showed that the EhNCABP166 protein is transcribed and translated in trophozoites of E. histolytica. It was also shown that the CH domains are functional and bind to F-actin, whereas the BAR and GBD/FH3 domains interact in vitro and in vivo with different families of GTPases such as Rho and Ras, and with different phosphoinositides. These findings suggest that these domains have the conserved functional properties described in other eukaryotic systems. These domains also interacted with additional GTPase and lipid targets that have not been previously described. Finally, cellular studies showed that EhNCABP166 is localized to the cytoplasm and nucleus of E. histolytica and that it has an important role in phagocytosis, proliferation, and motility of E. histolytica.

  3. The actin cytoskeleton as a sensor and mediator of apoptosis

    PubMed Central

    Desouza, Melissa; Gunning, Peter W.; Stehn, Justine R.

    2012-01-01

    Apoptosis is an important biological process required for the removal of unwanted or damaged cells. Mounting evidence implicates the actin cytoskeleton as both a sensor and mediator of apoptosis. Studies also suggest that actin binding proteins (ABPs) significantly contribute to apoptosis and that actin dynamics play a key role in regulating apoptosis signaling. Changes in the organization of the actin cytoskeleton has been attributed to the process of malignant transformation and it is hypothesized that remodeling of the actin cytoskeleton may enable tumor cells to evade normal apoptotic signaling. This review aims to illuminate the role of the actin cytoskeleton in apoptosis by systematically analyzing how actin and ABPs regulate different apoptosis pathways and to also highlight the potential for developing novel compounds that target tumor-specific actin filaments. PMID:22880146

  4. Calcium binding to an elastic portion of connectin/titin filaments.

    PubMed

    Tatsumi, R; Maeda, K; Hattori, A; Takahashi, K

    2001-01-01

    Alpha-connectin/titin-1 exists as an elastic filament that links a thick filament with the Z-disk, keeping thick filaments centered within the sarcomere during force generation. We have shown that the connectin filament has an affinity for calcium ions and its binding site(s) is restricted to the beta-connectin/titin-2 portion. We now report the localization and the characterization of calcium-binding sites on beta-connectin. Purified beta-connectin was digested by trypsin into 1700- and 400-kDa fragments. which were then subjected to fluorescence calcium-binding assays. The 400-kDa fragment possesses calcium-binding activity; the binding constant was 1.0 x 10(7) M(-1) and the molar ratio of bound calcium ions to the 400-kDa fragment reached a maximum of 12 at a free calcium ion concentration of approximately 1.0 microM. Antibodies against the 400-kDa fragment formed a sharp dense stripe at the boundary of the A and the I bands, indicating that the calcium-binding domain constitutes the N-terminal region of beta-connectin, that is, the elastic portion of connectin filaments. Furthermore, we estimated the N-terminal location of beta-connectin of various origins (n = 26). Myofibrils were treated with a solution containing 0.1 mM CaCl2 and 70 microM leupeptin to split connectin filaments into beta-connectin and a subfragment, and chain weights of these polypeptides were estimated according to their mobility in 2% polyacrylamide slab gels. The subfragment exhibited a similar chain weight of 1200+/-33 kDa (mean+/-SD), while alpha- and beta-connectins were variable in size according to their origin. These results suggest that the apparent length of the 1200-kDa subfragment portion is almost constant in all instances, about 0.34 microm at the slack condition, therefore that the C-terminus of the 1200-kDa subfragment, that is, the N-terminus of the calcium-binding domain, is at the N2 line region of parent filaments in situ. Because the secondary structure of the 400-k

  5. Differential effects of caldesmon on the intermediate conformational states of polymerizing actin.

    PubMed

    Huang, Renjian; Grabarek, Zenon; Wang, Chih-Lueh Albert

    2010-01-01

    The actin-binding protein caldesmon (CaD) reversibly inhibits smooth muscle contraction. In non-muscle cells, a shorter CaD isoform co-exists with microfilaments in the stress fibers at the quiescent state, but the phosphorylated CaD is found at the leading edge of migrating cells where dynamic actin filament remodeling occurs. We have studied the effect of a C-terminal fragment of CaD (H32K) on the kinetics of the in vitro actin polymerization by monitoring the fluorescence of pyrene-labeled actin. Addition of H32K or its phosphorylated form either attenuated or accelerated the pyrene emission enhancement, depending on whether it was added at the early or the late phase of actin polymerization. However, the CaD fragment had no effect on the yield of sedimentable actin, nor did it affect the actin ATPase activity. Our findings can be explained by a model in which nascent actin filaments undergo a maturation process that involves at least two intermediate conformational states. If present at early stages of actin polymerization, CaD stabilizes one of the intermediate states and blocks the subsequent filament maturation. Addition of CaD at a later phase accelerates F-actin formation. The fact that CaD is capable of inhibiting actin filament maturation provides a novel function for CaD and suggests an active role in the dynamic reorganization of the actin cytoskeleton.

  6. MARCKS is a natively unfolded protein with an inaccessible actin-binding site: evidence for long-range intramolecular interactions.

    PubMed

    Tapp, Hazel; Al-Naggar, Iman M; Yarmola, Elena G; Harrison, Alexis; Shaw, Gerry; Edison, Arthur S; Bubb, Michael R

    2005-03-18

    Myristoylated alanine-rich C kinase substrate (MARCKS) is an unfolded protein that contains well characterized actin-binding sites within the phosphorylation site domain (PSD), yet paradoxically, we now find that intact MARCKS does not bind to actin. Intact MARCKS also does not bind as well to calmodulin as does the PSD alone. Myristoylation at the N terminus alters how calmodulin binds to MARCKS, implying that, despite its unfolded state, the distant N terminus influences binding events at the PSD. We show that the free PSD binds with site specificity to MARCKS, suggesting that long-range intramolecular interactions within MARCKS are also possible. Because of the unusual primary sequence of MARCKS with an overall isoelectric point of 4.2 yet a very basic PSD (overall charge of +13), we speculated that ionic interactions between oppositely charged domains of MARCKS were responsible for long-range interactions within MARCKS that sterically influence binding events at the PSD and that explain the observed differences between properties of the PSD and MARCKS. Consistent with this hypothesis, chemical modifications of MARCKS that neutralize negatively charged residues outside of the PSD allow the PSD to bind to actin and increase the affinity of MARCKS for calmodulin. Similarly, both myristoylation of MARCKS and cleavage of MARCKS by calpain are shown to increase the availability of the PSD so as to activate its actin-binding activity. Because abundant evidence supports the conclusion that MARCKS is an important protein in regulating actin dynamics, our data imply that post-translational modifications of MARCKS are necessary and sufficient to regulate actin-binding activity. PMID:15640140

  7. High-Resolution Crystal Structures of Villin Headpiece nad Mutants with Reduced F-Actin Binding Activity

    SciTech Connect

    Meng,J.; Vardar, D.; Wang, Y.; Guo, H.; Head, J.; McKnight, C.

    2005-01-01

    Villin-type headpiece domains are approximately 70 amino acid modular motifs found at the C terminus of a variety of actin cytoskeleton-associated proteins. The headpiece domain of villin, a protein found in the actin bundles of the brush border epithelium, is of interest both as a compact F-actin binding domain and as a model folded protein. We have determined the high-resolution crystal structures of chicken villin headpiece (HP67) at 1.4 Angstrom resolution as well as two mutants, R37A and W64Y, at 1.45 and 1.5 Angstrom resolution, respectively. Replacement of R37 causes a 5-fold reduction in F-actin binding affinity in sedimentation assays. Replacement of W64 results in a much more drastic reduction in F-actin binding affinity without significant changes in headpiece structure or stability. The detailed comparison of these crystal structures with each other and to our previously determined NMR structures of HP67 and the 35-residue autonomously folding subdomain in villin headpiece, HP35, provides the details of the headpiece fold and further defines the F-actin binding site of villin-type headpiece domains.

  8. 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

  9. Redox-sensitive residue in the actin-binding interface of myosin.

    PubMed

    Moen, Rebecca J; Cornea, Sinziana; Oseid, Daniel E; Binder, Benjamin P; Klein, Jennifer C; Thomas, David D

    2014-10-24

    We have examined the chemical and functional reversibility of oxidative modification in myosin. Redox regulation has emerged as a crucial modulator of protein function, with particular relevance to aging. We previously identified a single methionine residue in Dictyostelium discoideum (Dicty) myosin II (M394, near the myosin cardiomyopathy loop in the actin-binding interface) that is functionally sensitive to oxidation. We now show that oxidation of M394 is reversible by methionine sulfoxide reductase (Msr), restoring actin-activated ATPase activity. Sequence alignment reveals that M394 of Dicty myosin II is a cysteine residue in all human isoforms of skeletal and cardiac myosin. Using Dicty myosin II as a model for site-specific redox sensitivity of this Cys residue, the M394C mutant can be glutathionylated in vitro, resulting in reversible inhibition of actin-activated ATPase activity, with effects similar to those of methionine oxidation at this site. This work illustrates the potential for myosin to function as a redox sensor in both non-muscle and muscle cells, modulating motility/contractility in response to oxidative stress. PMID:25264102

  10. Concomitant binding of Afadin to LGN and F-actin directs planar spindle orientation.

    PubMed

    Carminati, Manuel; Gallini, Sara; Pirovano, Laura; Alfieri, Andrea; Bisi, Sara; Mapelli, Marina

    2016-02-01

    Polarized epithelia form by oriented cell divisions in which the mitotic spindle aligns parallel to the epithelial plane. To orient the mitotic spindle, cortical cues trigger the recruitment of NuMA-dynein-based motors, which pull on astral microtubules via the protein LGN. We demonstrate that the junctional protein Afadin is required for spindle orientation and correct epithelial morphogenesis of Caco-2 cysts. Molecularly, Afadin binds directly and concomitantly to F-actin and to LGN. We determined the crystallographic structure of human Afadin in complex with LGN and show that it resembles the LGN-NuMA complex. In mitosis, Afadin is necessary for cortical accumulation of LGN and NuMA above the spindle poles, in an F-actin-dependent manner. Collectively, our results depict Afadin as a molecular hub governing the enrichment of LGN and NuMA at the cortex. To our knowledge, Afadin is the first-described mechanical anchor between dynein and cortical F-actin. PMID:26751642

  11. Redox-sensitive residue in the actin-binding interface of myosin

    PubMed Central

    Moen, Rebecca J.; Cornea, Sinziana; Oseid, Daniel E.; Binder, Benjamin P.; Klein, Jennifer C.; Thomas, David D.

    2014-01-01

    We have examined the chemical and functional reversibility of oxidative modification in myosin. Redox regulation has emerged as a crucial modulator of protein function, with particular relevance to aging. We previously identified a single methionine residue in Dictyostelium discoideum (Dicty) myosin II (M394, near the myosin cardiomyopathy loop in the actin-binding interface) that is functionally sensitive to oxidation. We now show that oxidation of M394 is reversible by methionine sulfoxide reductase (Msr), restoring actin-activated ATPase activity. Sequence alignment reveals that M394 of Dicty myosin II is a cysteine residue in all human isoforms of skeletal and cardiac myosin. Using Dicty myosin II as a model for site-specific redox sensitivity of this Cys residue, the M394C mutant can be glutathionylated in vitro, resulting in reversible inhibition of actin-activated ATPase activity, with effects similar to those of methionine oxidation at this site. This work illustrates the potential for myosin to function as a redox sensor in both non-muscle and muscle cells, modulating motility/contractility in response to oxidative stress. PMID:25264102

  12. Actin Filaments Are Involved in the Coupling of V0-V1 Domains of Vacuolar H+-ATPase at the Golgi Complex.

    PubMed

    Serra-Peinado, Carla; Sicart, Adrià; Llopis, Juan; Egea, Gustavo

    2016-04-01

    We previously reported that actin-depolymerizing agents promote the alkalization of the Golgi stack and thetrans-Golgi network. The main determinant of acidic pH at the Golgi is the vacuolar-type H(+)-translocating ATPase (V-ATPase), whose V1domain subunitsBandCbind actin. We have generated a GFP-tagged subunitB2construct (GFP-B2) that is incorporated into the V1domain, which in turn is coupled to the V0sector. GFP-B2 subunit is enriched at distal Golgi compartments in HeLa cells. Subcellular fractionation, immunoprecipitation, and inversal FRAP experiments show that the actin depolymerization promotes the dissociation of V1-V0domains, which entails subunitB2translocation from Golgi membranes to the cytosol. Moreover, molecular interaction between subunitsB2andC1and actin were detected. In addition, Golgi membrane lipid order disruption byd-ceramide-C6 causes Golgi pH alkalization. We conclude that actin regulates the Golgi pH homeostasis maintaining the coupling of V1-V0domains of V-ATPase through the binding of microfilaments to subunitsBandCand preserving the integrity of detergent-resistant membrane organization. These results establish the Golgi-associated V-ATPase activity as the molecular link between actin and the Golgi pH. PMID:26872971

  13. Actin Filaments Are Involved in the Coupling of V0-V1 Domains of Vacuolar H+-ATPase at the Golgi Complex.

    PubMed

    Serra-Peinado, Carla; Sicart, Adrià; Llopis, Juan; Egea, Gustavo

    2016-04-01

    We previously reported that actin-depolymerizing agents promote the alkalization of the Golgi stack and thetrans-Golgi network. The main determinant of acidic pH at the Golgi is the vacuolar-type H(+)-translocating ATPase (V-ATPase), whose V1domain subunitsBandCbind actin. We have generated a GFP-tagged subunitB2construct (GFP-B2) that is incorporated into the V1domain, which in turn is coupled to the V0sector. GFP-B2 subunit is enriched at distal Golgi compartments in HeLa cells. Subcellular fractionation, immunoprecipitation, and inversal FRAP experiments show that the actin depolymerization promotes the dissociation of V1-V0domains, which entails subunitB2translocation from Golgi membranes to the cytosol. Moreover, molecular interaction between subunitsB2andC1and actin were detected. In addition, Golgi membrane lipid order disruption byd-ceramide-C6 causes Golgi pH alkalization. We conclude that actin regulates the Golgi pH homeostasis maintaining the coupling of V1-V0domains of V-ATPase through the binding of microfilaments to subunitsBandCand preserving the integrity of detergent-resistant membrane organization. These results establish the Golgi-associated V-ATPase activity as the molecular link between actin and the Golgi pH.

  14. Functional Analysis of Actin-Binding Proteins in the Central Nervous System of Drosophila.

    PubMed

    He, Qi; Roblodowski, Christopher

    2016-01-01

    Using Drosophila actin-binding protein Dunc-115 as model system, this chapter describes a MARCM (mosaic analysis with a repressible cell marker)-based method for analyzing cytoskeletal components for their functions in the nervous system. Following a concise description about the principle, a step-by-step protocol is provided for generating the needed stocks and for histological analysis. Additional details and explanations have been given in the accompanying notes. Together, this should form a practical and sufficient recipe for performing at the single-cell-level loss-of-function and gain-of-function analyses of proteins associated with the cytoskeleton.

  15. Ligand electronic properties modulate tau filament binding site density

    PubMed Central

    Cisek, Katryna; Jensen, Jordan R.; Honson, Nicolette S.; Schafer, Kelsey N.; Cooper, Grace L.; Kuret, Jeff

    2012-01-01

    Small molecules that bind tau-bearing neurofibrillary lesions are being sought for premortem diagnosis, staging, and treatment of Alzheimer’s disease and other tauopathic neurodegenerative diseases. The utility of these agents will depend on both their binding affinity and binding site density (Bmax). Previously we identified polarizability as a descriptor of protein aggregate binding affinity. To examine its contribution to binding site density, we investigated the ability of two closely related benzothiazole derivatives ((E)-2-[[4-(dimethylamino)phenyl]azo]-6-methoxybenzothiazole) and ((E)-2-[2-[4-(dimethylamino)phenyl]ethenyl]-6-methoxybenzothiazole)) that differed in polarizability to displace probes of high (Thioflavin S) and low (radiolabeled (E,E)-1-iodo-2,5-bis(3-hydroxycarbonyl-4-methoxy)styrylbenzene; IMSB) density sites. Consistent with their site densities, Thioflavin S completely displaced radiolabeled IMSB, but IMSB was incapable of displacing Thioflavin S. Although both benzothiazoles displaced the low Bmax IMSB probe, only the highly polarizable analog displaced near saturating concentrations of the Thioflavin S probe. Quantum calculations showed that high polarizability reflected extensive pi-electron delocalization fostered by the presence of electron donating and accepting groups. These data suggest that electron delocalization promotes ligand binding at a subset of sites on tau aggregates that are present at high density, and that optimizing this aspect of ligand structure can yield tau-directed agents with superior diagnostic and therapeutic performance. PMID:23072817

  16. Temperature-enhanced association of proteins due to electrostatic interaction: a coarse-grained simulation of actin-myosin binding.

    PubMed

    Okazaki, Kei-ichi; Sato, Takato; Takano, Mitsunori

    2012-05-30

    Association of protein molecules constitutes the basis for the interaction network in a cell. Despite its fundamental importance, the thermodynamic aspect of protein-protein binding, particularly the issues relating to the entropy change upon binding, remains elusive. The binding of actin and myosin, which are vital proteins in motility, is a typical example, in which two different binding mechanisms have been argued: the binding affinity increases with increasing temperature and with decreasing salt-concentration, indicating the entropy-driven binding and the enthalpy-driven binding, respectively. How can these thermodynamically different binding mechanisms coexist? To address this question, which is of general importance in understanding protein-protein bindings, we conducted an in silico titration of the actin-myosin system by molecular dynamics simulation using a residue-level coarse-grained model, with particular focus on the role of the electrostatic interaction. We found a good agreement between in silico and in vitro experiments on the salt-concentration dependence and the temperature dependence of the binding affinity. We then figured out how the two binding mechanisms can coexist: the enthalpy (due to electrostatic interaction between actin and myosin) provides the basal binding affinity, and the entropy (due to the orientational disorder of water molecules) enhances it at higher temperatures. In addition, we analyzed the actin-myosin complex structures observed during the simulation and obtained a variety of weak-binding complex structures, among which were found an unusual binding mode suggested by an earlier experiment and precursor structures of the strong-binding complex proposed by electron microscopy. These results collectively indicate the potential capability of a residue-level coarse-grained model to simulate the association-dissociation dynamics (particularly for transient weak-bindings) exhibited by larger and more complicated systems, as in a

  17. Reconstitution and regulation of actin gel-sol transformation with purified filamin and villin.

    PubMed

    Nunnally, M H; Powell, L D; Craig, S W

    1981-03-10

    Gel-sol transformation of actin filaments, a process essential for cell motility, can be reconstituted in vitro and regulated in a predictable fashion by the combined action of villin and filamin. Measurements made in a low shear falling ball viscometer show that mixtures of actin, villin, and filamin exist either as a gel (yield point greater than or equal to 140 dynes/cm2) or as a low viscosity liquid depending on the relative ration of villin:actin. Filamin induces gelation of F-actin by forming stable cross-links between actin filaments. Villin inhibits filamin-induced F-actin gelation, but the effect can be overcome by increasing the amount of filamin. Sedimentation assays show that villin does not inhibit gelation of actin by preventing filamin from binding to F-actin. Results from viscosity measurements and filament length determinations show that villin increases actin filament number by reducing the average filament length without altering the total amount of polymer. Because the gel point of a fixed amount of polymer is sharply dependent on the ratio of cross-links to number of polymers, the solation effect of villin might be explained by its effect on filament number. Based on the network theory of gel formation, calculations of the amount of additional cross-linker required to overcome the effect of a known increase in the number of actin filaments agree reasonably well with experimental findings. These results document the existence of cellular proteins which could regulate gel-sol transformation in vivo by their effect on actin polymer length and, therefore, on actin filament number.

  18. Myosin Head Configuration in Relaxed Insect Flight Muscle: X-Ray Modeled Resting Cross-Bridges in a Pre-Powerstroke State Are Poised for Actin Binding

    PubMed Central

    AL-Khayat, Hind A.; Hudson, Liam; Reedy, Michael K.; Irving, Thomas C.; Squire, John M.

    2003-01-01

    Low-angle x-ray diffraction patterns from relaxed insect flight muscle recorded on the BioCAT beamline at the Argonne APS have been modeled to 6.5 nm resolution (R-factor 9.7%, 65 reflections) using the known myosin head atomic coordinates, a hinge between the motor (catalytic) domain and the light chain-binding (neck) region (lever arm), together with a simulated annealing procedure. The best head conformation angles around the hinge gave a head shape that was close to that typical of relaxed M•ADP•Pi heads, a head shape never before demonstrated in intact muscle. The best packing constrained the eight heads per crown within a compact crown shelf projecting at ∼90° to the filament axis. The two heads of each myosin molecule assume nonequivalent positions, one head projecting outward while the other curves round the thick filament surface to nose against the proximal neck of the projecting head of the neighboring molecule. The projecting heads immediately suggest a possible cross-bridge cycle. The relaxed projecting head, oriented almost as needed for actin attachment, will attach, then release Pi followed by ADP, as the lever arm with a purely axial change in tilt drives ∼10 nm of actin filament sliding on the way to the nucleotide-free limit of its working stroke. The overall arrangement appears well designed to support precision cycling for the myogenic oscillatory mode of contraction with its enhanced stretch-activation response used in flight by insects equipped with asynchronous fibrillar flight muscles. PMID:12885653

  19. Role and structural mechanism of WASP-triggered conformational changes in branched actin filament nucleation by Arp2/3 complex.

    PubMed

    Rodnick-Smith, Max; Luan, Qing; Liu, Su-Ling; Nolen, Brad J

    2016-07-01

    The Arp2/3 (Actin-related proteins 2/3) complex is activated by WASP (Wiskott-Aldrich syndrome protein) family proteins to nucleate branched actin filaments that are important for cellular motility. WASP recruits actin monomers to the complex and stimulates movement of Arp2 and Arp3 into a "short-pitch" conformation that mimics the arrangement of actin subunits within filaments. The relative contribution of these functions in Arp2/3 complex activation and the mechanism by which WASP stimulates the conformational change have been unknown. We purified budding yeast Arp2/3 complex held in or near the short-pitch conformation by an engineered covalent cross-link to determine if the WASP-induced conformational change is sufficient for activity. Remarkably, cross-linked Arp2/3 complex bypasses the need for WASP in activation and is more active than WASP-activated Arp2/3 complex. These data indicate that stimulation of the short-pitch conformation is the critical activating function of WASP and that monomer delivery is not a fundamental requirement for nucleation but is a specific requirement for WASP-mediated activation. During activation, WASP limits nucleation rates by releasing slowly from nascent branches. The cross-linked complex is inhibited by WASP's CA region, even though CA potently stimulates cross-linking, suggesting that slow WASP detachment masks the activating potential of the short-pitch conformational switch. We use structure-based mutations and WASP-Arp fusion chimeras to determine how WASP stimulates movement toward the short-pitch conformation. Our data indicate that WASP displaces the autoinhibitory Arp3 C-terminal tail from a hydrophobic groove at Arp3's barbed end to destabilize the inactive state, providing a mechanism by which WASP stimulates the short-pitch conformation and activates Arp2/3 complex. PMID:27325766

  20. DNA Binding Properties of the Actin-Related Protein Arp8 and Its Role in DNA Repair

    PubMed Central

    Murakami, Hirokazu; Otawa, Kenji; Tachiwana, Hiroaki; Oma, Yukako; Nishijima, Hitoshi; Shibahara, Kei-ich; Kurumizaka, Hitoshi; Harata, Masahiko

    2014-01-01

    Actin and actin-related proteins (Arps), which are members of the actin family, are essential components of many of these remodeling complexes. Actin, Arp4, Arp5, and Arp8 are found to be evolutionarily conserved components of the INO80 chromatin remodeling complex, which is involved in transcriptional regulation, DNA replication, and DNA repair. A recent report showed that Arp8 forms a module in the INO80 complex and this module can directly capture a nucleosome. In the present study, we showed that recombinant human Arp8 binds to DNAs, and preferentially binds to single-stranded DNA. Analysis of the binding of adenine nucleotides to Arp8 mutants suggested that the ATP-binding pocket, located in the evolutionarily conserved actin fold, plays a regulatory role in the binding of Arp8 to DNA. To determine the cellular function of Arp8, we derived tetracycline-inducible Arp8 knockout cells from a cultured human cell line. Analysis of results obtained after treating these cells with aphidicolin and camptothecin revealed that Arp8 is involved in DNA repair. Together with the previous observation that Arp8, but not γ-H2AX, is indispensable for recruiting INO80 complex to DSB in human, results of our study suggest an individual role for Arp8 in DNA repair. PMID:25299602

  1. The actin-binding proteins eps8 and gelsolin have complementary roles in regulating the growth and stability of mechanosensory hair bundles of mammalian cochlear outer hair cells.

    PubMed

    Olt, Jennifer; Mburu, Philomena; Johnson, Stuart L; Parker, Andy; Kuhn, Stephanie; Bowl, Mike; Marcotti, Walter; Brown, Steve D M

    2014-01-01

    Sound transduction depends upon mechanosensitive channels localized on the hair-like bundles that project from the apical surface of cochlear hair cells. Hair bundles show a stair-case structure composed of rows of stereocilia, and each stereocilium contains a core of tightly-packed and uniformly-polarized actin filaments. The growth and maintenance of the stereociliary actin core are dynamically regulated. Recently, it was shown that the actin-binding protein gelsolin is expressed in the stereocilia of outer hair cells (OHCs) and in its absence they become long and straggly. Gelsolin is part of a whirlin scaffolding protein complex at the stereocilia tip, which has been shown to interact with other actin regulatory molecules such as Eps8. Here we investigated the physiological effects associated with the absence of gelsolin and its possible overlapping role with Eps8. We found that, in contrast to Eps8, gelsolin does not affect mechanoelectrical transduction during immature stages of development. Moreover, OHCs from gelsolin knockout mice were able to mature into fully functional sensory receptors as judged by the normal resting membrane potential and basolateral membrane currents. Mechanoelectrical transducer current in gelsolin-Eps8 double knockout mice showed a profile similar to that observed in the single mutants for Eps8. We propose that gelsolin has a non-overlapping role with Eps8. While Eps8 is mainly involved in the initial growth of stereocilia in both inner hair cells (IHCs) and OHCs, gelsolin is required for the maintenance of mature hair bundles of low-frequency OHCs after the onset of hearing. PMID:24475274

  2. Comparative genome analysis of cortactin and HS1: the significance of the F-actin binding repeat domain

    PubMed Central

    van Rossum, Agnes GSH; Schuuring-Scholtes, Ellen; Seggelen, Vera van Buuren-van; Kluin, Philip M; Schuuring, Ed

    2005-01-01

    Background In human carcinomas, overexpression of cortactin correlates with poor prognosis. Cortactin is an F-actin-binding protein involved in cytoskeletal rearrangements and cell migration by promoting actin-related protein (Arp)2/3 mediated actin polymerization. It shares a high amino acid sequence and structural similarity to hematopoietic lineage cell-specific protein 1 (HS1) although their functions differ considerable. In this manuscript we describe the genomic organization of these two genes in a variety of species by a combination of cloning and database searches. Based on our analysis, we predict the genesis of the actin-binding repeat domain during evolution. Results Cortactin homologues exist in sponges, worms, shrimps, insects, urochordates, fishes, amphibians, birds and mammalians, whereas HS1 exists in vertebrates only, suggesting that both genes have been derived from an ancestor cortactin gene by duplication. In agreement with this, comparative genome analysis revealed very similar exon-intron structures and sequence homologies, especially over the regions that encode the characteristic highly conserved F-actin-binding repeat domain. Cortactin splice variants affecting this F-actin-binding domain were identified not only in mammalians, but also in amphibians, fishes and birds. In mammalians, cortactin is ubiquitously expressed except in hematopoietic cells, whereas HS1 is mainly expressed in hematopoietic cells. In accordance with their distinct tissue specificity, the putative promoter region of cortactin is different from HS1. Conclusions Comparative analysis of the genomic organization and amino acid sequences of cortactin and HS1 provides inside into their origin and evolution. Our analysis shows that both genes originated from a gene duplication event and subsequently HS1 lost two repeats, whereas cortactin gained one repeat. Our analysis genetically underscores the significance of the F-actin binding domain in cytoskeletal remodeling, which

  3. Structural and biochemical studies of actin in complex with synthetic macrolide tail analogs

    PubMed Central

    Pereira, Jose H.; Petchprayoon, Chutima; Hoepker, Alexander C.; Moriarty, Nigel; Fink, Sarah J.; Cecere, Giuseppe; Paterson, Ian; Adams, Paul D.; Marriott, Gerard

    2014-01-01

    The actin filament-binding and filament-severing activities of the aplyronine, kabiramide and reidispongiolide families of marine macrolides are located within the hydrophobic tail region of the molecule. Two synthetic tail analogs of aplyronine C (SF-01 and GC-04) are shown to bind to G-actin with kd values of 285 +/−33 nM and 132 +/−13 nM, respectively. The crystal structures of actin complexes with GC-04, SF-01 and kabiramide C reveal a conserved mode of tail binding within the cleft that forms between sub-domains (SD) 1 and 3. Our studies support the view that filament severing is brought about by specific binding of the tail region to the SD1/3 cleft on the upper protomer, which displaces loop-D from the lower protomer on the same half-filament. With previous studies showing that GC-04 analog can sever actin filaments, it is argued that the shorter complex lifetime of tail analogs with F-actin would make them more effective at severing filaments compared with plasma gelsolin. Structure-based analyses are used to suggest more reactive or targetable forms of GC-04 and SF-01, which may serve to boost the capacity of the serum actin scavenging system, to generate antibody conjugates against tumor cell antigens, and to reduce sputum viscosity in children with cystic fibrosis. PMID:25047814

  4. Structural and Biochemical Studies of Actin in Complex with Synthetic Macrolide Tail Analogues

    DOE PAGES

    Pereira, Jose H.; Petchprayoon, Chutima; Hoepker, Alexander C.; Moriarty, Nigel W.; Fink, Sarah J.; Cecere, Giuseppe; Paterson, Ian; Adams, Paul D.; Marriott, Gerard

    2014-07-22

    The actin filament-binding and filament-severing activities of the aplyronine, kabiramide, and reidispongiolide families of marine macrolides are located within the hydrophobic tail region of the molecule. Two synthetic tail analogues of aplyronine C (SF-01 and GC-04) are shown to bind to G-actin with dissociation constants of (285±33) and (132±13) nM, respectively. The crystal structures of actin complexes with GC-04, SF-01, and kabiramide C reveal a conserved mode of tail binding within the cleft that forms between subdomains (SD) 1 and 3. Our studies support the view that filament severing is brought about by specific binding of the tail region tomore » the SD1/SD3 cleft on the upper protomer, which displaces loop-D from the lower protomer on the same half-filament. With previous studies showing that the GC-04 analogue can sever actin filaments, it is argued that the shorter complex lifetime of tail analogues with F-actin would make them more effective at severing filaments compared with plasma gelsolin. In conclusion, structure-based analyses are used to suggest more reactive or targetable forms of GC-04 and SF-01, which may serve to boost the capacity of the serum actin scavenging system, to generate antibody conjugates against tumor cell antigens, and to decrease sputum viscosity in children with cystic fibrosis.« less

  5. Structural and Biochemical Studies of Actin in Complex with Synthetic Macrolide Tail Analogues

    SciTech Connect

    Pereira, Jose H.; Petchprayoon, Chutima; Hoepker, Alexander C.; Moriarty, Nigel W.; Fink, Sarah J.; Cecere, Giuseppe; Paterson, Ian; Adams, Paul D.; Marriott, Gerard

    2014-07-22

    The actin filament-binding and filament-severing activities of the aplyronine, kabiramide, and reidispongiolide families of marine macrolides are located within the hydrophobic tail region of the molecule. Two synthetic tail analogues of aplyronine C (SF-01 and GC-04) are shown to bind to G-actin with dissociation constants of (285±33) and (132±13) nM, respectively. The crystal structures of actin complexes with GC-04, SF-01, and kabiramide C reveal a conserved mode of tail binding within the cleft that forms between subdomains (SD) 1 and 3. Our studies support the view that filament severing is brought about by specific binding of the tail region to the SD1/SD3 cleft on the upper protomer, which displaces loop-D from the lower protomer on the same half-filament. With previous studies showing that the GC-04 analogue can sever actin filaments, it is argued that the shorter complex lifetime of tail analogues with F-actin would make them more effective at severing filaments compared with plasma gelsolin. In conclusion, structure-based analyses are used to suggest more reactive or targetable forms of GC-04 and SF-01, which may serve to boost the capacity of the serum actin scavenging system, to generate antibody conjugates against tumor cell antigens, and to decrease sputum viscosity in children with cystic fibrosis.

  6. Docking, molecular dynamics and QM/MM studies to delineate the mode of binding of CucurbitacinE to F-actin.

    PubMed

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

    2016-01-01

    CucurbitacinE (CurE) has been known to bind covalently to F-actin and inhibit depolymerization. However, the mode of binding of CurE to F-actin and the consequent changes in the F-actin dynamics have not been studied. Through quantum mechanical/molecular mechanical (QM/MM) and density function theory (DFT) simulations after the molecular dynamics (MD) simulations of the docked complex of F-actin and CurE, a detailed transition state (TS) model for the Michael reaction is proposed. The TS model shows nucleophilic attack of the sulphur of Cys257 at the β-carbon of Michael Acceptor of CurE producing an enol intermediate that forms a covalent bond with CurE. The MD results show a clear difference between the structure of the F-actin in free form and F-actin complexed with CurE. CurE affects the conformation of the nucleotide binding pocket increasing the binding affinity between F-actin and ADP, which in turn could affect the nucleotide exchange. CurE binding also limits the correlated displacement of the relatively flexible domain 1 of F-actin causing the protein to retain a flat structure and to transform into a stable "tense" state. This structural transition could inhibit depolymerization of F-actin. In conclusion, CurE allosterically modulates ADP and stabilizes F-actin structure, thereby affecting nucleotide exchange and depolymerization of F-actin.

  7. The actin cytoskeleton may control the polar distribution of an auxin transport protein

    NASA Technical Reports Server (NTRS)

    Muday, G. K.; Hu, S.; Brady, S. R.; Davies, E. (Principal Investigator)

    2000-01-01

    The gravitropic bending of plants has long been linked to the changes in the transport of the plant hormone auxin. To understand the mechanism by which gravity alters auxin movement, it is critical to know how polar auxin transport is initially established. In shoots, polar auxin transport is basipetal (i.e., from the shoot apex toward the base). It is driven by the basal localization of the auxin efflux carrier complex. One mechanism for localizing this efflux carrier complex to the basal membrane may be through attachment to the actin cytoskeleton. The efflux carrier protein complex is believed to consist of several polypeptides, including a regulatory subunit that binds auxin transport inhibitors, such as naphthylphthalamic acid (NPA). Several lines of experimentation have been used to determine if the NPA binding protein interacts with actin filaments. The NPA binding protein has been shown to partition with the actin cytoskeleton during detergent extraction. Agents that specifically alter the polymerization state of the actin cytoskeleton change the amount of NPA binding protein and actin recovered in these cytoskeletal pellets. Actin-affinity columns were prepared with polymers of actin purified from zucchini hypocotyl tissue. NPA binding activity was eluted in a single peak from the actin filament column. Cytochalasin D, which fragments the actin cytoskeleton, was shown to reduce polar auxin transport in zucchini hypocotyls. The interaction of the NPA binding protein with the actin cytoskeleton may localize it in one plane of the plasma membrane, and thereby control the polarity of auxin transport.

  8. RecA-ssDNA filaments supercoil in the presence of single-stranded DNA-binding protein

    SciTech Connect

    Shi Weixian; Larson, Ronald G. . E-mail: rlarson@umich.edu

    2007-06-08

    Using atomic force microscopy (AFM), we find that RecA-single-stranded DNA (RecA-ssDNA) filaments, in the presence of single-stranded DNA-binding (SSB) protein, organize into left-handed bundles, which differ from the previously reported disordered aggregates formed when SSB is excluded from the reaction. In addition, we see both left- and right-handedness on bundles of two filaments. These two-filament supercoils, individual filaments, and other smaller bundles further organize into more complicated bundles, showing overall left-handedness which cannot be explained by earlier arguments that presumed supercoiling is absent in RecA-ssDNA filaments. This novel finding and our previous results regarding supercoiling of RecA-double-stranded DNA (RecA-dsDNA) filaments are, however, consistent with each other and can possibly be explained by the intrinsic tendency of RecA-DNA filaments, in their fully coated form, to order themselves into helical bundles, independent of the DNA inside the filaments (ssDNA or dsDNA). RecA-RecA interactions may dominate the bundling process, while the original conformation of DNA inside filaments and other factors (mechanical properties of filaments, concentration of filaments, and Mg{sup 2+} concentration) could contribute to the variation in the appearance and pitch of supercoils. The tendency of RecA-DNA filaments to form ordered supercoils and their presence during strand exchange suggest a possible biological importance of supercoiled filaments.

  9. NAC1 is an actin-binding protein that is essential for effective cytokinesis in cancer cells.

    PubMed

    Yap, Kai Lee; Fraley, Stephanie I; Thiaville, Michelle M; Jinawath, Natini; Nakayama, Kentaro; Wang, Jianlong; Wang, Tian-Li; Wirtz, Denis; Shih, Ie-Ming

    2012-08-15

    NAC1 is a transcriptional corepressor protein that is essential to sustain cancer cell proliferation and migration. However, the underlying molecular mechanisms of NAC1 function in cancer cells remain unknown. In this study, we show that NAC1 functions as an actin monomer-binding protein. The conserved BTB protein interaction domain in NAC1 is the minimal region for actin binding. Disrupting NAC1 complex function by dominant-negative or siRNA strategies reduced cell retraction and abscission during late-stage cytokinesis, causing multinucleation in cancer cells. In Nac1-deficient murine fibroblasts, restoring NAC1 expression was sufficient to partially avert multinucleation. We found that siRNA-mediated silencing of the actin-binding protein profilin-1 in cancer cells caused a similar multinucleation phenotype and that NAC1 modulated the binding of actin to profillin-1. Taken together, our results indicate that the NAC1/actin/profilin-1 complex is crucial for cancer cell cytokinesis, with a variety of important biologic and clinical implications.

  10. Fluorescent labelling of the actin cytoskeleton in plants using a cameloid antibody

    PubMed Central

    2014-01-01

    Background Certain members of the Camelidae family produce a special type of antibody with only one heavy chain. The antigen binding domains are the smallest functional fragments of these heavy-chain only antibodies and as a consequence have been termed nanobodies. Discovery of these nanobodies has allowed the development of a number of therapeutic proteins and tools. In this study a class of nanobodies fused to fluorescent proteins (chromobodies), and therefore allowing antigen-binding and visualisation by fluorescence, have been used. Such chromobodies can be expressed in living cells and used as genetically encoded immunocytochemical markers. Results