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

PubMed

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

2004-05-10

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

Microtubule Actin Cross-linking Factor 1 regulates cardiomyocyte microtubule distribution and adaptation to hemodynamic overload.

PubMed

Fassett, John T; Xu, Xin; Kwak, Dongmin; Wang, Huan; Liu, Xiaoyu; Hu, Xinli; Bache, Robert J; Chen, Yingjie

2013-01-01

Aberrant cardiomyocyte microtubule growth is a feature of pressure overload induced cardiac hypertrophy believed to contribute to left ventricular (LV) dysfunction. Microtubule Actin Cross-linking Factor 1 (MACF1/Acf7) is a 600 kd spectraplakin that stabilizes and guides microtubule growth along actin filaments. MACF1 is expressed in the heart, but its impact on cardiac microtubules, and how this influences cardiac structure, function, and adaptation to hemodynamic overload is unknown. Here we used inducible cardiac-specific MACF1 knockout mice (MACF1 KO) to determine the impact of MACF1 on cardiac microtubules and adaptation to pressure overload (transverse aortic constriction (TAC).In adult mouse hearts, MACF1 expression was low under basal conditions, but increased significantly in response to TAC. While MACF1 KO had no observable effect on heart size or function under basal conditions, MACF1 KO exacerbated TAC induced LV hypertrophy, LV dilation and contractile dysfunction. Interestingly, subcellular fractionation of ventricular lysates revealed that MACF1 KO altered microtubule distribution in response to TAC, so that more tubulin was associated with the cell membrane fraction. Moreover, TAC induced microtubule redistribution into this cell membrane fraction in both WT and MACF1 KO mice correlated strikingly with the level of contractile dysfunction (r(2) = 0.786, p<.001). MACF1 disruption also resulted in reduction of membrane caveolin 3 levels, and increased levels of membrane PKCα and β1 integrin after TAC, suggesting MACF1 function is important for spatial regulation of several physiologically relevant signaling proteins during hypertrophy. Together, these data identify for the first time, a role for MACF1 in cardiomyocyte microtubule distribution and in adaptation to hemodynamic overload.

Mechanically tunable actin networks using programmable DNA based cross-linkers

NASA Astrophysics Data System (ADS)

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

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

Microtubule Actin Cross-Linking Factor 1 Regulates Cardiomyocyte Microtubule Distribution and Adaptation to Hemodynamic Overload

PubMed Central

Kwak, Dongmin; Wang, Huan; Liu, Xiaoyu; Hu, Xinli; Bache, Robert J.; Chen, Yingjie

2013-01-01

Aberrant cardiomyocyte microtubule growth is a feature of pressure overload induced cardiac hypertrophy believed to contribute to left ventricular (LV) dysfunction. Microtubule Actin Cross-linking Factor 1 (MACF1/Acf7) is a 600 kd spectraplakin that stabilizes and guides microtubule growth along actin filaments. MACF1 is expressed in the heart, but its impact on cardiac microtubules, and how this influences cardiac structure, function, and adaptation to hemodynamic overload is unknown. Here we used inducible cardiac-specific MACF1 knockout mice (MACF1 KO) to determine the impact of MACF1 on cardiac microtubules and adaptation to pressure overload (transverse aortic constriction (TAC).In adult mouse hearts, MACF1 expression was low under basal conditions, but increased significantly in response to TAC. While MACF1 KO had no observable effect on heart size or function under basal conditions, MACF1 KO exacerbated TAC induced LV hypertrophy, LV dilation and contractile dysfunction. Interestingly, subcellular fractionation of ventricular lysates revealed that MACF1 KO altered microtubule distribution in response to TAC, so that more tubulin was associated with the cell membrane fraction. Moreover, TAC induced microtubule redistribution into this cell membrane fraction in both WT and MACF1 KO mice correlated strikingly with the level of contractile dysfunction (r2 = 0.786, p<.001). MACF1 disruption also resulted in reduction of membrane caveolin 3 levels, and increased levels of membrane PKCα and β1 integrin after TAC, suggesting MACF1 function is important for spatial regulation of several physiologically relevant signaling proteins during hypertrophy. Together, these data identify for the first time, a role for MACF1 in cardiomyocyte microtubule distribution and in adaptation to hemodynamic overload. PMID:24086300

Novel alternative splicings of BPAG1 (bullous pemphigoid antigen 1) including the domain structure closely related to MACF (microtubule actin cross-linking factor).

PubMed

Okumura, Masayo; Yamakawa, Hisashi; Ohara, Osamu; Owaribe, Katsushi

2002-02-22

BPAG1 (bullous pemphigoid antigen 1) was originally identified as a 230-kDa hemidesmosomal protein and belongs to the plakin family, because it consists of a plakin domain, a coiled-coil rod domain and a COOH-terminal intermediate filament binding domain. To date, alternatively spliced products of BPAG1, BPAG1e, and BPAG1n are known. BPAG1e is expressed in epithelial tissues and localized to hemidesmosomes, on the other hand, BPAG1n is expressed in neural tissues and muscles and has an actin binding domain at the NH(2)-terminal of BPAG1e. BPAG1 is also known as a gene responsible for Dystonia musculorum (dt) neurodegeneration syndrome of the mouse. Another plakin family protein MACF (microtubule actin cross-linking factor) has also an actin binding domain and the plakin domain at the NH(2)-terminal. However, in contrast to its high homology with BPAG1 at the NH(2)-terminal, the COOH-terminal structure of MACF, including a microtubule binding domain, resembles dystrophin rather than plakins. Here, we investigated RNAs and proteins expressed from the BPAG1 locus and suggest novel alternative splicing variants, which include one consisting of the COOH-terminal domain structure homologous to MACF. The results indicate that BPAG1 has three kinds of cytoskeletal binding domains and seems to play an important role in linking the different types of cytoskeletons.

Genetic Variants of Microtubule Actin Cross-linking Factor 1 (MACF1) Confer Risk for Parkinson's Disease.

PubMed

Wang, Xin; Li, Nuomin; Xiong, Nian; You, Qi; Li, Jie; Yu, Jinlong; Qing, Hong; Wang, Tao; Cordell, Heather J; Isacson, Ole; Vance, Jeffery M; Martin, Eden R; Zhao, Ying; Cohen, Bruce M; Buttner, Edgar A; Lin, Zhicheng

2017-05-01

The cytoskeleton not only provides structure, it is an active component of cell function, and in several neurodegenerative disorders, there is evidence of cytoskeletal collapse. Cytoskeletal proteins have been specifically implicated in the pathogenesis of Parkinson's disease (PD), where degeneration of dopaminergic (DA) neurons is the hallmark, but in which many factors may determine the resilience of DA neurons during aging and stress. Here we report that the human Microtubule Actin Cross-linking Factor 1 gene (MACF1), a downstream target of PD biochemical pathways, was significantly associated with PD in 713 nuclear families. A significant allelic association between PD and rs12118033, with P = 0.0098, was observed, and a P < 0.03 was observed in the association analysis by both a trend test and an allelic test. We further observed that it is the MACF1b isoform, not the MACF1a isoform, which is expressed in DA neurons from six human postmortem brains. In a Caenorhabditis elegans system, used to explore the effect of altered MACF1b on neurons, knockdown or knockout of the MACF1b orthologue vab-10 resulted in the selective loss of DA neurons, which validated MACF1's risk candidacy in PD. These findings strongly suggest that MACF1b may contribute to the genetic etiology and mechanistic causation of PD.

The role of microtubule actin cross-linking factor 1 (MACF1) in the Wnt signaling pathway.

PubMed

Chen, Hui-Jye; Lin, Chung-Ming; Lin, Chyuan-Sheng; Perez-Olle, Raul; Leung, Conrad L; Liem, Ronald K H

2006-07-15

MACF1 (microtubule actin cross-linking factor 1) is a multidomain protein that can associate with microfilaments and microtubules. We found that MACF1 was highly expressed in neuronal tissues and the foregut of embryonic day 8.5 (E8.5) embryos and the head fold and primitive streak of E7.5 embryos. MACF1(-/-) mice died at the gastrulation stage and displayed developmental retardation at E7.5 with defects in the formation of the primitive streak, node, and mesoderm. This phenotype was similar to Wnt-3(-/-) and LRP5/6 double-knockout embryos. In the absence of Wnt, MACF1 associated with a complex that contained Axin, beta-catenin, GSK3beta, and APC. Upon Wnt stimulation, MACF1 appeared to be involved in the translocation and subsequent binding of the Axin complex to LRP6 at the cell membrane. Reduction of MACF1 with small interfering RNA decreased the amount of beta-catenin in the nucleus, and led to an inhibition of Wnt-induced TCF/beta-catenin-dependent transcriptional activation. Similar results were obtained with a dominant-negative MACF1 construct that contained the Axin-binding region. Reduction of MACF1 in Wnt-1-expressing P19 cells resulted in decreased T (Brachyury) gene expression, a DNA-binding transcription factor that is a direct target of Wnt/beta-catenin signaling and required for mesoderm formation. These results suggest a new role of MACF1 in the Wnt signaling pathway.

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

NASA Astrophysics Data System (ADS)

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

2006-02-01

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

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

PubMed

Unterberger, Michael J; Holzapfel, Gerhard A

2014-11-01

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

The role of microtubule actin cross-linking factor 1 (MACF1) in the Wnt signaling pathway

PubMed Central

Chen, Hui-Jye; Lin, Chung-Ming; Lin, Chyuan-Sheng; Perez-Olle, Raul; Leung, Conrad L.; Liem, Ronald K.H.

2006-01-01

MACF1 (microtubule actin cross-linking factor 1) is a multidomain protein that can associate with microfilaments and microtubules. We found that MACF1 was highly expressed in neuronal tissues and the foregut of embryonic day 8.5 (E8.5) embryos and the head fold and primitive streak of E7.5 embryos. MACF1−/− mice died at the gastrulation stage and displayed developmental retardation at E7.5 with defects in the formation of the primitive streak, node, and mesoderm. This phenotype was similar to Wnt-3−/− and LRP5/6 double-knockout embryos. In the absence of Wnt, MACF1 associated with a complex that contained Axin, β-catenin, GSK3β, and APC. Upon Wnt stimulation, MACF1 appeared to be involved in the translocation and subsequent binding of the Axin complex to LRP6 at the cell membrane. Reduction of MACF1 with small interfering RNA decreased the amount of β-catenin in the nucleus, and led to an inhibition of Wnt-induced TCF/β-catenin-dependent transcriptional activation. Similar results were obtained with a dominant-negative MACF1 construct that contained the Axin-binding region. Reduction of MACF1 in Wnt-1-expressing P19 cells resulted in decreased T (Brachyury) gene expression, a DNA-binding transcription factor that is a direct target of Wnt/β-catenin signaling and required for mesoderm formation. These results suggest a new role of MACF1 in the Wnt signaling pathway. PMID:16815997

F-actin cross-linking enhances the stability of force generation in disordered actomyosin networks

NASA Astrophysics Data System (ADS)

Jung, Wonyeong; Murrell, Michael P.; Kim, Taeyoon

2015-12-01

Myosin molecular motors and actin cross-linking proteins (ACPs) are known to mediate the generation and transmission of mechanical forces within the cortical F-actin cytoskeleton that drive major cellular processes such as cell division and migration. However, how motors and ACPs interact collectively over diverse timescales to modulate the time-dependent mechanical properties of the cytoskeleton remains unclear. In this study, we present a three-dimensional agent-based computational model of the cortical actomyosin network to quantitatively determine the effects of motor activity and the density and kinetics of ACPs on the accumulation and maintenance of mechanical tension within a disordered actomyosin network. We found that motors accumulate large stress quickly by behaving as temporary cross-linkers although this stress is relaxed over time unless there are sufficient passive ACPs to stabilize the network. Stabilization by ACPs helps motors to generate forces up to their maximum potential, leading to significant enhancement of the efficiency and stability of stress generation. Thus, we demonstrated that the force-dependent kinetics of ACP dissociation plays a critical role for the accumulation and sustainment of stress and the structural remodeling of networks.

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

PubMed Central

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

2014-01-01

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

Formation of Hirano Bodies Induced by Expression of an Actin Cross-Linking Protein with a Gain-of-Function Mutation

PubMed Central

Maselli, Andrew; Furukawa, Ruth; Thomson, Susanne A. M.; Davis, Richard C.; Fechheimer, Marcus

2003-01-01

Hirano bodies are paracrystalline actin filament-containing structures reported to be associated with a variety of neurodegenerative diseases. However, the biological function of Hirano bodies remains poorly understood, since nearly all prior studies of these structures were done with postmortem samples of tissue. In the present study, we generated a full-length form of a Dictyostelium 34-kDa actin cross-linking protein with point mutations in the first putative EF hand, termed 34-kDa ΔEF1. The 34-kDa ΔEF1 protein binds calcium normally but has activated actin binding that is unregulated by calcium. The expression of the 34-kDa ΔEF1 protein in Dictyostelium induces the formation of Hirano bodies, as assessed by both fluorescence microscopy and transmission electron microscopy. Dictyostelium cells bearing Hirano bodies grow normally, indicating that Hirano bodies are not associated with cell death and are not deleterious to cell growth. Moreover, the expression of the 34-kDa ΔEF1 protein rescues the phenotypes of cells lacking the 34-kDa protein and cells lacking both the 34-kDa protein and α-actinin. Finally, the expression of the 34-kDa ΔEF1 protein also initiates the formation of Hirano bodies in cultured mouse fibroblasts. These results show that the failure to regulate the activity and/or affinity of an actin cross-linking protein can provide a signal for the formation of Hirano bodies. More generally, the formation of Hirano bodies is a cellular response to or a consequence of aberrant function of the actin cytoskeleton. PMID:12912897

Mena–GRASP65 interaction couples actin polymerization to Golgi ribbon linking

PubMed Central

Tang, Danming; Zhang, Xiaoyan; Huang, Shijiao; Yuan, Hebao; Li, Jie; Wang, Yanzhuang

2016-01-01

In mammalian cells, the Golgi reassembly stacking protein 65 (GRASP65) has been implicated in both Golgi stacking and ribbon linking by forming trans-oligomers through the N-terminal GRASP domain. Because the GRASP domain is globular and relatively small, but the gaps between stacks are large and heterogeneous, it remains puzzling how GRASP65 physically links Golgi stacks into a ribbon. To explore the possibility that other proteins may help GRASP65 in ribbon linking, we used biochemical methods and identified the actin elongation factor Mena as a novel GRASP65-binding protein. Mena is recruited onto the Golgi membranes through interaction with GRASP65. Depleting Mena or disrupting actin polymerization resulted in Golgi fragmentation. In cells, Mena and actin were required for Golgi ribbon formation after nocodazole washout; in vitro, Mena and microfilaments enhanced GRASP65 oligomerization and Golgi membrane fusion. Thus Mena interacts with GRASP65 to promote local actin polymerization, which facilitates Golgi ribbon linking. PMID:26538023

Actin assembly factors regulate the gelation kinetics and architecture of F-actin networks.

PubMed

Falzone, Tobias T; Oakes, Patrick W; Sees, Jennifer; Kovar, David R; Gardel, Margaret L

2013-04-16

Dynamic regulation of the actin cytoskeleton is required for diverse cellular processes. Proteins regulating the assembly kinetics of the cytoskeletal biopolymer F-actin are known to impact the architecture of actin cytoskeletal networks in vivo, but the underlying mechanisms are not well understood. Here, we demonstrate that changes to actin assembly kinetics with physiologically relevant proteins profilin and formin (mDia1 and Cdc12) have dramatic consequences on the architecture and gelation kinetics of otherwise biochemically identical cross-linked F-actin networks. Reduced F-actin nucleation rates promote the formation of a sparse network of thick bundles, whereas increased nucleation rates result in a denser network of thinner bundles. Changes to F-actin elongation rates also have marked consequences. At low elongation rates, gelation ceases and a solution of rigid bundles is formed. By contrast, rapid filament elongation accelerates dynamic arrest and promotes gelation with minimal F-actin density. These results are consistent with a recently developed model of how kinetic constraints regulate network architecture and underscore how molecular control of polymer assembly is exploited to modulate cytoskeletal architecture and material properties. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Determinants of fluidlike behavior and effective viscosity in cross-linked actin networks.

PubMed

Kim, Taeyoon; Gardel, Margaret L; Munro, Ed

2014-02-04

The actin cortex has a well-documented ability to rapidly remodel and flow while maintaining long-range connectivity, but how this is achieved remains poorly understood. Here, we use computer simulations to explore how stress relaxation in cross-linked actin networks subjected to extensional stress depends on the interplay between network architecture and turnover. We characterize a regime in which a network response is nonaffine and stress relaxation is governed by the continuous dissipation of elastic energy via cyclic formation, elongation, and turnover of tension-bearing elements. Within this regime, for a wide range of network parameters, we observe a constant deformation (creep) rate that is linearly proportional to the rate of filament turnover, leading to a constant effective viscosity that is inversely proportional to turnover rate. Significantly, we observe a biphasic dependence of the creep rate on applied stress: below a critical stress threshold, the creep rate increases linearly with applied stress; above that threshold, the creep rate becomes independent of applied stress. We show that this biphasic stress dependence can be understood in terms of the nonlinear force-extension behavior of individual force-transmitting network elements. These results have important implications for understanding the origins and control of viscous flows both in the cortex of living cells and in other polymer networks. Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Actin Assembly Factors Regulate the Gelation Kinetics and Architecture of F-actin Networks

PubMed Central

Falzone, Tobias T.; Oakes, Patrick W.; Sees, Jennifer; Kovar, David R.; Gardel, Margaret L.

2013-01-01

Dynamic regulation of the actin cytoskeleton is required for diverse cellular processes. Proteins regulating the assembly kinetics of the cytoskeletal biopolymer F-actin are known to impact the architecture of actin cytoskeletal networks in vivo, but the underlying mechanisms are not well understood. Here, we demonstrate that changes to actin assembly kinetics with physiologically relevant proteins profilin and formin (mDia1 and Cdc12) have dramatic consequences on the architecture and gelation kinetics of otherwise biochemically identical cross-linked F-actin networks. Reduced F-actin nucleation rates promote the formation of a sparse network of thick bundles, whereas increased nucleation rates result in a denser network of thinner bundles. Changes to F-actin elongation rates also have marked consequences. At low elongation rates, gelation ceases and a solution of rigid bundles is formed. By contrast, rapid filament elongation accelerates dynamic arrest and promotes gelation with minimal F-actin density. These results are consistent with a recently developed model of how kinetic constraints regulate network architecture and underscore how molecular control of polymer assembly is exploited to modulate cytoskeletal architecture and material properties. PMID:23601318

Mena-GRASP65 interaction couples actin polymerization to Golgi ribbon linking.

PubMed

Tang, Danming; Zhang, Xiaoyan; Huang, Shijiao; Yuan, Hebao; Li, Jie; Wang, Yanzhuang

2016-01-01

In mammalian cells, the Golgi reassembly stacking protein 65 (GRASP65) has been implicated in both Golgi stacking and ribbon linking by forming trans-oligomers through the N-terminal GRASP domain. Because the GRASP domain is globular and relatively small, but the gaps between stacks are large and heterogeneous, it remains puzzling how GRASP65 physically links Golgi stacks into a ribbon. To explore the possibility that other proteins may help GRASP65 in ribbon linking, we used biochemical methods and identified the actin elongation factor Mena as a novel GRASP65-binding protein. Mena is recruited onto the Golgi membranes through interaction with GRASP65. Depleting Mena or disrupting actin polymerization resulted in Golgi fragmentation. In cells, Mena and actin were required for Golgi ribbon formation after nocodazole washout; in vitro, Mena and microfilaments enhanced GRASP65 oligomerization and Golgi membrane fusion. Thus Mena interacts with GRASP65 to promote local actin polymerization, which facilitates Golgi ribbon linking. © 2016 Tang et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

A structural study of F-actin - filamin networks

NASA Astrophysics Data System (ADS)

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

2010-03-01

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

Cross-Linking Molecules Modify Composite Actin Networks Independently

NASA Astrophysics Data System (ADS)

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

2008-09-01

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

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

PubMed

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

2014-07-15

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

A single charge in the actin binding domain of fascin can independently tune the linear and non-linear response of an actin bundle network.

PubMed

Maier, M; Müller, K W; Heussinger, C; Köhler, S; Wall, W A; Bausch, A R; Lieleg, O

2015-05-01

Actin binding proteins (ABPs) not only set the structure of actin filament assemblies but also mediate the frequency-dependent viscoelastic moduli of cross-linked and bundled actin networks. Point mutations in the actin binding domain of those ABPs can tune the association and dissociation dynamics of the actin/ABP bond and thus modulate the network mechanics both in the linear and non-linear response regime. We here demonstrate how the exchange of a single charged amino acid in the actin binding domain of the ABP fascin triggers such a modulation of the network rheology. Whereas the overall structure of the bundle networks is conserved, the transition point from strain-hardening to strain-weakening sensitively depends on the cross-linker off-rate and the applied shear rate. Our experimental results are consistent both with numerical simulations of a cross-linked bundle network and a theoretical description of the bundle network mechanics which is based on non-affine bending deformations and force-dependent cross-link dynamics.

Force Exertion and Transmission in Cross-Linked Actin Networks

NASA Astrophysics Data System (ADS)

Stam, Samantha

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

Diversification of caldesmon-linked actin cytoskeleton in cell motility

PubMed Central

Mayanagi, Taira

2011-01-01

The actin cytoskeleton plays a key role in regulating cell motility. Caldesmon (CaD) is an actin-linked regulatory protein found in smooth muscle and non-muscle cells that is conserved among a variety of vertebrates. It binds and stabilizes actin filaments, as well as regulating actin-myosin interaction in a calcium (Ca2+)/calmodulin (CaM)- and/or phosphorylation-dependent manner. CaD function is regulated qualitatively by Ca2+/CaM and by its phosphorylation state and quantitatively at the mRNA level, by three different transcriptional regulation of the CALD1 gene. CaD has numerous functions in cell motility, such as migration, invasion and proliferation, exerted via the reorganization of the actin cytoskeleton. Here we will outline recent findings regarding CaD's structural features and functions. PMID:21350330

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

PubMed Central

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

2013-01-01

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

Polycation induced actin bundles.

PubMed

Muhlrad, Andras; Grintsevich, Elena E; Reisler, Emil

2011-04-01

Three polycations, polylysine, the polyamine spermine and the polycationic protein lysozyme were used to study the formation, structure, ionic strength sensitivity and dissociation of polycation-induced actin bundles. Bundles form fast, simultaneously with the polymerization of MgATP-G-actins, upon the addition of polycations to solutions of actins at low ionic strength conditions. This indicates that nuclei and/or nascent filaments bundle due to attractive, electrostatic effect of polycations and the neutralization of repulsive interactions of negative charges on actin. The attractive forces between the filaments are strong, as shown by the low (in nanomolar range) critical concentration of their bundling at low ionic strength. These bundles are sensitive to ionic strength and disassemble partially in 100 mM NaCl, but both the dissociation and ionic strength sensitivity can be countered by higher polycation concentrations. Cys374 residues of actin monomers residing on neighboring filaments in the bundles can be cross-linked by the short span (5.4Å) MTS-1 (1,1-methanedyl bismethanethiosulfonate) cross-linker, which indicates a tight packing of filaments in the bundles. The interfilament cross-links, which connect monomers located on oppositely oriented filaments, prevent disassembly of bundles at high ionic strength. Cofilin and the polysaccharide polyanion heparin disassemble lysozyme induced actin bundles more effectively than the polylysine-induced bundles. The actin-lysozyme bundles are pathologically significant as both proteins are found in the pulmonary airways of cystic fibrosis patients. Their bundles contribute to the formation of viscous mucus, which is the main cause of breathing difficulties and eventual death in this disorder. Copyright © 2011 Elsevier B.V. All rights reserved.

WHAMM links actin assembly via the Arp2/3 complex to autophagy.

PubMed

Kast, David J; Dominguez, Roberto

2015-01-01

Macroautophagy (hereafter autophagy) is the process by which cytosolic material destined for degradation is enclosed inside a double-membrane cisterna known as the autophagosome and processed for secretion and/or recycling. This process requires a large collection of proteins that converge on certain sites of the ER membrane to generate the autophagosome membrane. Recently, it was shown that actin accumulates around autophagosome precursors and could play a role in this process, but the mechanism and role of actin polymerization in autophagy were unknown. Here, we discuss our recent finding that the nucleation-promoting factor (NPF) WHAMM recruits and activates the Arp2/3 complex for actin assembly at sites of autophagosome formation on the ER. Using high-resolution, live-cell imaging, we showed that WHAMM forms dynamic puncta on the ER that comigrate with several autophagy markers, and propels the spiral movement of these puncta by an Arp2/3 complex-dependent actin comet tail mechanism. In starved cells, WHAMM accumulates at the interface between neighboring autophagosomes, whose number and size increases with WHAMM expression. Conversely, knocking down WHAMM, inhibiting the Arp2/3 complex or interfering with actin polymerization reduces the size and number of autophagosomes. These findings establish a link between Arp2/3 complex-mediated actin assembly and autophagy.

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

NASA Astrophysics Data System (ADS)

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

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

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

PubMed Central

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

2011-01-01

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

Actin stress in cell reprogramming

PubMed Central

Guo, Jun; Wang, Yuexiu; Sachs, Frederick; Meng, Fanjie

2014-01-01

Cell mechanics plays a role in stem cell reprogramming and differentiation. To understand this process better, we created a genetically encoded optical probe, named actin–cpstFRET–actin (AcpA), to report forces in actin in living cells in real time. We showed that stemness was associated with increased force in actin. We reprogrammed HEK-293 cells into stem-like cells using no transcription factors but simply by softening the substrate. However, Madin-Darby canine kidney (MDCK) cell reprogramming required, in addition to a soft substrate, Harvey rat sarcoma viral oncogene homolog expression. Replating the stem-like cells on glass led to redifferentiation and reduced force in actin. The actin force probe was a FRET sensor, called cpstFRET (circularly permuted stretch sensitive FRET), flanked by g-actin subunits. The labeled actin expressed efficiently in HEK, MDCK, 3T3, and bovine aortic endothelial cells and in multiple stable cell lines created from those cells. The viability of the cell lines demonstrated that labeled actin did not significantly affect cell physiology. The labeled actin distribution was similar to that observed with GFP-tagged actin. We also examined the stress in the actin cross-linker actinin. Actinin force was not always correlated with actin force, emphasizing the need for addressing protein specificity when discussing forces. Because actin is a primary structural protein in animal cells, understanding its force distribution is central to understanding animal cell physiology and the many linked reactions such as stress-induced gene expression. This new probe permits measuring actin forces in a wide range of experiments on preparations ranging from isolated proteins to transgenic animals. PMID:25422450

Identification of mammalian proteins cross-linked to DNA by ionizing radiation.

PubMed

Barker, Sharon; Weinfeld, Michael; Zheng, Jing; Li, Liang; Murray, David

2005-10-07

Ionizing radiation (IR) is an important environmental risk factor for various cancers and also a major therapeutic agent for cancer treatment. Exposure of mammalian cells to IR induces several types of damage to DNA, including double- and single-strand breaks, base and sugar damage, as well as DNA-DNA and DNA-protein cross-links (DPCs). Little is known regarding the biological consequences of DPCs. Identifying the proteins that become cross-linked to DNA by IR would be an important first step in this regard. We have therefore undertaken a proteomics study to isolate and identify proteins involved in IR-induced DPCs. DPCs were induced in AA8 Chinese hamster ovary or GM00637 human fibroblast cells using 0-4 gray of gamma-rays under either aerated or hypoxic conditions. DPCs were isolated using a recently developed method, and proteins were identified by mass spectrometry. We identified 29 proteins as being cross-linked to DNA by IR under aerated and/or hypoxic conditions. The identified proteins include structural proteins, actin-associated proteins, transcription regulators, RNA-splicing components, stress-response proteins, cell cycle regulatory proteins, and GDP/GTP-binding proteins. The involvement of several proteins (actin, histone H2B, and others) in DPCs was confirmed by using Western blot analysis. The dose responsiveness of DPC induction was examined by staining one-dimensional SDS-polyacrylamide gels with SYPRO Tangerine followed by analysis using fluorescence imaging. Quantitation of the fluorescence signal indicated no significant difference in total yields of IR-induced DPCs generated under aerated or hypoxic conditions, although differences were observed for several individual protein bands.

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

PubMed

Yamashiro, Sawako; Watanabe, Naoki

2014-11-01

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

Drosophila Spire is an actin nucleation factor.

PubMed

Quinlan, Margot E; Heuser, John E; Kerkhoff, Eugen; Mullins, R Dyche

2005-01-27

The actin cytoskeleton is essential for many cellular functions including shape determination, intracellular transport and locomotion. Previous work has identified two factors--the Arp2/3 complex and the formin family of proteins--that nucleate new actin filaments via different mechanisms. Here we show that the Drosophila protein Spire represents a third class of actin nucleation factor. In vitro, Spire nucleates new filaments at a rate that is similar to that of the formin family of proteins but slower than in the activated Arp2/3 complex, and it remains associated with the slow-growing pointed end of the new filament. Spire contains a cluster of four WASP homology 2 (WH2) domains, each of which binds an actin monomer. Maximal nucleation activity requires all four WH2 domains along with an additional actin-binding motif, conserved among Spire proteins. Spire itself is conserved among metazoans and, together with the formin Cappuccino, is required for axis specification in oocytes and embryos, suggesting that multiple actin nucleation factors collaborate to construct essential cytoskeletal structures.

A systems-biology approach to yeast actin cables.

PubMed

Drake, Tyler; Yusuf, Eddy; Vavylonis, Dimitrios

2012-01-01

We focus on actin cables in yeast as a model system for understanding cytoskeletal organization and the workings of actin itself. In particular, we highlight quantitative approaches on the kinetics of actin-cable assembly and methods of measuring their morphology by image analysis. Actin cables described by these studies can span greater lengths than a thousand end-to-end actin-monomers. Because of this difference in length scales, control of the actin-cable system constitutes a junction between short-range interactions - among actin-monomers and nucleating, polymerization-facilitating, side-binding, severing, and cross-linking proteins - and the emergence of cell-scale physical form as embodied by the actin cables themselves.

The effect of intact talin and talin tail fragment on actin filament dynamics and structure depends on pH and ionic strength.

PubMed

Goldmann, W H; Hess, D; Isenberg, G

1999-03-01

We employed quasi-elastic light scattering and electron microscopy to investigate the influence of intact talin and talin tail fragment on actin filament dynamics and network structure. Using these methods, we confirm previous reports that intact talin induces cross-linking as well as filament shortening on actin networks. We now show that the effect of intact talin as well as talin tail fragment on actin networks is controlled by pH and ionic strength. At pH 7.5, actin filament dynamics in the presence of intact talin and talin tail fragment are characterized by a rapid decay of the dynamic structure factor and by a square root power law for the stretched exponential decay which is in contrast with the theory for pure actin solutions. At pH 6 and low ionic strength, intact talin cross-links actin filaments more tightly than talin tail fragment. Talin head fragment showed no effect on actin networks, indicating that the actin binding sites reside probably exclusively within the tail domain.

A Systems-Biology Approach to Yeast Actin Cables

PubMed Central

Drake, Tyler; Yusuf, Eddy; Vavylonis, Dimitrios

2011-01-01

We focus on actin cables in yeast as a model system for understanding cytoskeletal organization and the workings of actin itself. In particular, we highlight quantitative approaches on the kinetics of actin cable assembly and methods of measuring their morphology by image analysis. Actin cables described by these studies can span greater lengths than a thousand end-to-end actin monomers. Because of this difference in length scales, control of the actin-cable system constitutes a junction between short-range interactions—among actin monomers and nucleating, polymerization-facilitating, side-binding, severing, and cross-linking proteins—and the emergence of cell-scale physical form as embodied by the actin cables themselves. PMID:22161338

Interaction of aldolase with actin-containing filaments. Structural studies.

PubMed Central

Stewart, M; Morton, D J; Clarke, F M

1980-01-01

Electron micrographs of the paracrystals formed when fructose bisphosphate aldolase (EC 4.1.2.13) is added to actin-containing filaments were analysed by computer methods so that ultrastructural changes could be correlated with the various stoicheiometries of binding determined in the preceding paper [Walsh, Winzor, Clarke, Masters & Morton (1980) Biochem. J. 186, 89-98]. Paracrystals formed with aldolase and either F-actin or F-actin-tropomyosin have a single light transverse band every 38 nm, which is due to aldolase molecules cross-linking the filaments. In contrast, the paracrystals formed between aldolase and F-actin-tropomyosin-troponin filaments show two transverse bands every 38 nm: a major band, interpreted as aldolase binding to troponin, and a minor band, interpreted as aldolase cross-linking the filaments. The intensity of the minor band varies with Ca2+ concentration, being greatest when the Ca2+ concentration is low. A model for the different paracrystal structures which relates the various patterns and binding stoicheiometries to structural changes in the actin-containing filaments is proposed. Images PLATE 1 PMID:6892771

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

PubMed

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

2016-01-01

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

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

PubMed Central

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

2016-01-01

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

Soft Listeria: actin-based propulsion of liquid drops.

PubMed

Boukellal, Hakim; Campás, Otger; Joanny, Jean-François; Prost, Jacques; Sykes, Cécile

2004-06-01

We study the motion of oil drops propelled by actin polymerization in cell extracts. Drops deform and acquire a pearlike shape under the action of the elastic stresses exerted by the actin comet, a tail of cross-linked actin filaments. We solve this free boundary problem and calculate the drop shape taking into account the elasticity of the actin gel and the variation of the polymerization velocity with normal stress. The pressure balance on the liquid drop imposes a zero propulsive force if gradients in surface tension or internal pressure are not taken into account. Quantitative parameters of actin polymerization are obtained by fitting theory to experiment.

The yeast actin cytoskeleton.

PubMed

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

2014-03-01

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

Single-Molecule Studies of Actin Assembly and Disassembly Factors

PubMed Central

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

2014-01-01

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

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.

Modes of caldesmon binding to actin: sites of caldesmon contact and modulation of interactions by phosphorylation.

PubMed

Foster, D Brian; Huang, Renjian; Hatch, Victoria; Craig, Roger; Graceffa, Philip; Lehman, William; Wang, C-L Albert

2004-12-17

Smooth muscle caldesmon binds actin and inhibits actomyosin ATPase activity. Phosphorylation of caldesmon by extracellular signal-regulated kinase (ERK) reverses this inhibitory effect and weakens actin binding. To better understand this function, we have examined the phosphorylation-dependent contact sites of caldesmon on actin by low dose electron microscopy and three-dimensional reconstruction of actin filaments decorated with a C-terminal fragment, hH32K, of human caldesmon containing the principal actin-binding domains. Helical reconstruction of negatively stained filaments demonstrated that hH32K is located on the inner portion of actin subdomain 1, traversing its upper surface toward the C-terminal segment of actin, and forms a bridge to the neighboring actin monomer of the adjacent long pitch helical strand by connecting to its subdomain 3. Such lateral binding was supported by cross-linking experiments using a mutant isoform, which was capable of cross-linking actin subunits. Upon ERK phosphorylation, however, the mutant no longer cross-linked actin to polymers. Three-dimensional reconstruction of ERK-phosphorylated hH32K indeed indicated loss of the interstrand connectivity. These results, together with fluorescence quenching data, are consistent with a phosphorylation-dependent conformational change that moves the C-terminal end segment of caldesmon near the phosphorylation site but not the upstream region around Cys(595), away from F-actin, thus neutralizing its inhibitory effect on actomyosin interactions. The binding pattern of hH32K suggests a mechanism by which unphosphorylated, but not ERK-phosphorylated, caldesmon could stabilize actin filaments and resist F-actin severing or depolymerization in both smooth muscle and nonmuscle cells.

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

PubMed

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

2017-03-06

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

Steady-state nuclear actin levels are determined by export competent actin pool.

PubMed

Skarp, Kari-Pekka; Huet, Guillaume; Vartiainen, Maria K

2013-10-01

A number of studies in the last decade have irrevocably promoted actin into a fully fledged member of the nuclear compartment, where it, among other crucial tasks, facilitates transcription and chromatin remodeling. Changes in nuclear actin levels have been linked to different cellular processes: decreased nuclear actin to quiescence and increased nuclear actin to differentiation. Importin 9 and exportin 6 transport factors are responsible for the continuous nucleocytoplasmic shuttling of actin, but the mechanisms, which result in modulated actin levels, have not been characterized. We find that in cells growing under normal growth conditions, the levels of nuclear actin vary considerably from cell to cell. To understand the basis for this, we have extensively quantified several cellular parameters while at the same time recording the import and export rates of green fluorescent protein (GFP)-tagged actin. Surprisingly, our dataset shows that the ratio of nuclear to cytoplasmic fluorescence intensity, but not nuclear shape, size, cytoplasm size, or their ratio, correlates negatively with both import and export rate of actin. This suggests that high-nuclear actin content is maintained by both diminished import and export. The high nuclear actin containing cells still show high mobility of actin, but it is not export competent, suggesting increased binding of actin to nuclear complexes. Creation of such export incompetent actin pool would ensure enough actin is retained in the nucleus and make it available for the various nuclear functions described for actin. Copyright © 2013 Wiley Periodicals, Inc.

Actin, actin-binding proteins, and actin-related proteins in the nucleus.

PubMed

Kristó, Ildikó; Bajusz, Izabella; Bajusz, Csaba; Borkúti, Péter; Vilmos, Péter

2016-04-01

Extensive research in the past decade has significantly broadened our view about the role actin plays in the life of the cell and added novel aspects to actin research. One of these new aspects is the discovery of the existence of nuclear actin which became evident only recently. Nuclear activities including transcriptional activation in the case of all three RNA polymerases, editing and nuclear export of mRNAs, and chromatin remodeling all depend on actin. It also became clear that there is a fine-tuned equilibrium between cytoplasmic and nuclear actin pools and that this balance is ensured by an export-import system dedicated to actin. After over half a century of research on conventional actin and its organizing partners in the cytoplasm, it was also an unexpected finding that the nucleus contains more than 30 actin-binding proteins and new classes of actin-related proteins which are not able to form filaments but had evolved nuclear-specific functions. The actin-binding and actin-related proteins in the nucleus have been linked to RNA transcription and processing, nuclear transport, and chromatin remodeling. In this paper, we attempt to provide an overview of the wide range of information that is now available about actin, actin-binding, and actin-related proteins in the nucleus.

Rheology of Membrane-Attached Minimal Actin Cortices.

PubMed

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

2018-04-26

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

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

PubMed

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

2016-03-04

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

Actin-Binding Protein Requirement for Cortical Stability and Efficient Locomotion

NASA Astrophysics Data System (ADS)

Cunningham, C. Casey; Gorlin, Jed B.; Kwiatkowski, David J.; Hartwig, John H.; Janmey, Paul A.; Randolph Byers, H.; Stossel, Thomas P.

1992-01-01

Three unrelated tumor cell lines derived from human malignant melanomas lack actin-binding protein (ABP), which cross-links actin filaments in vitro and connects these filaments to plasma membrane glycoproteins. The ABP-deficient cells have impaired locomotion and display circumferential blebbing of the plasma membrane. Expression of ABP in one of the lines after transfection restored translocational motility and reduced membrane blebbing. These findings establish that ABP functions to stabilize cortical actin in vivo and is required for efficient cell locomotion.

Maleimidobenzoyl-G-actin: Structural properties and interaction with skeletal myosin subfragment-1

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

Bettache, N.; Bertrand, R.; Kassab, R.

1990-09-25

The authors have investigated various structural and interaction properties of maleimidobenzoyl-G-actin (MBS-actin), a new, internally cross-linked G-actin derivative that does not exhibit, at moderate protein concentration, the salt-and myosin subfragment 1 (S-1)--induced polymerizations of G-actin and reacts reversibly and covalently in solution with S-1 at or near the F-actin binding region of the heavy chain. The far-ultraviolet CD spectrum and {alpha}-helix content of the MBS-actin were identical with those displayed by native G-actin. {sup 45}Ca{sup 2+} measurements showed the same content of tightly bound Ca{sup 2+} in MBS-actin as in G-actin and the EDTA treatment of the modified protein promotedmore » the same red shift of the intrinsic fluorescence spectrum as observed with native G-actin. Incubation of concentrated MBS-actin solutions with 100 mM KCl+5 mM MgCl{sub 2} led to the polymerization of the actin derivative when the critical monomer concentration reached 1.6mg/mL, at 25{degree}C, pH 8.0. The MBS-F-actin formed activated the Mg{sup 2+}-ATPase of S-1 to the same extent as native F-actin. The MBS-G-actin exhibited a DNase I inhibitor activity very close to that found with native G-actin and was to be at all affected by its specific covalent conjugation to S-1. This finding led them to isolate, for the first time, by gel filtration, a ternary complex comprising DNase I tightly bound to MBS-actin cross-linked to the S-1 heavy chain, demonstrating that S-1 and DNase I bind at distinct sites on G-actin. Collectively, the data illustrate further the nativeness of the MBS-G-actin and its potential use in solution studies of the actin-myosin head interactions.« less

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

PubMed

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

2013-02-01

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

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

The Non-catalytic B Subunit of Coagulation Factor XIII Accelerates Fibrin Cross-linking*

PubMed Central

Souri, Masayoshi; Osaki, Tsukasa; Ichinose, Akitada

2015-01-01

Covalent cross-linking of fibrin chains is required for stable blood clot formation, which is catalyzed by coagulation factor XIII (FXIII), a proenzyme of plasma transglutaminase consisting of catalytic A (FXIII-A) and non-catalytic B subunits (FXIII-B). Herein, we demonstrate that FXIII-B accelerates fibrin cross-linking. Depletion of FXIII-B from normal plasma supplemented with a physiological level of recombinant FXIII-A resulted in delayed fibrin cross-linking, reduced incorporation of FXIII-A into fibrin clots, and impaired activation peptide cleavage by thrombin; the addition of recombinant FXIII-B restored normal fibrin cross-linking, FXIII-A incorporation into fibrin clots, and activation peptide cleavage by thrombin. Immunoprecipitation with an anti-fibrinogen antibody revealed an interaction between the FXIII heterotetramer and fibrinogen mediated by FXIII-B and not FXIII-A. FXIII-B probably binds the γ-chain of fibrinogen with its D-domain, which is near the fibrin polymerization pockets, and dissociates from fibrin during or after cross-linking between γ-chains. Thus, FXIII-B plays important roles in the formation of a ternary complex between proenzyme FXIII, prosubstrate fibrinogen, and activator thrombin. Accordingly, congenital or acquired FXIII-B deficiency may result in increased bleeding tendency through impaired fibrin stabilization due to decreased FXIII-A activation by thrombin and secondary FXIII-A deficiency arising from enhanced circulatory clearance. PMID:25809477

Requirement of Nck adaptors for actin dynamics and cell migration stimulated by platelet-derived growth factor B.

PubMed

Rivera, G M; Antoku, S; Gelkop, S; Shin, N Y; Hanks, S K; Pawson, T; Mayer, B J

2006-06-20

The Nck family of Src homology (SH) 2/SH3 domain adaptors functions to link tyrosine phosphorylation induced by extracellular signals with downstream regulators of actin dynamics. We investigated the role of mammalian Nck adaptors in signaling from the activated platelet-derived growth factor (PDGF) receptor (PDGFbetaR) to the actin cytoskeleton. We report here that Nck adaptors are required for cytoskeletal reorganization and chemotaxis stimulated by PDGF-B. Analysis of tyrosine-phosphorylated proteins demonstrated that Crk-associated substrate (p130(Cas)), not the activated PDGFbetaR itself, is the major Nck SH2 domain-binding protein in PDGF-B-stimulated cells. Both Nck- and p130(Cas)-deficient cells fail to display cytoskeletal rearrangements, including the formation of membrane ruffles and the disassembly of actin bundles, typically shown by their WT counterparts in response to PDGF-B. Furthermore, Nck and p130(Cas) colocalize in phosphotyrosine-enriched membrane ruffles induced by PDGF-B in NIH 3T3 cells. These results suggest that Nck adaptors play an essential role in linking the activated PDGFbetaR with actin dynamics through a pathway that involves p130(Cas).

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

PubMed Central

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

1997-01-01

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

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

PubMed

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

2016-02-01

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

The actin filament cross-linker L-plastin confers resistance to TNF-α in MCF-7 breast cancer cells in a phosphorylation-dependent manner

PubMed Central

Janji, Bassam; Vallar, Laurent; Tanoury, Ziad Al; Bernardin, François; Vetter, Guillaume; Schaffner-Reckinger, Elisabeth; Berchem, Guy; Friederich, Evelyne; Chouaib, Salem

2010-01-01

Abstract We used a tumour necrosis factor (TNF)-α resistant breast adenocarcinoma MCF-7 cell line to investigate the involvement of the actin cytoskeleton in the mechanism of cell resistance to this cytokine. We found that TNF resistance correlates with the loss of cell epithelial properties and the gain of a mesenchymal phenotype, reminiscent of an epithelial-to-mesenchymal transition (EMT). Morphological changes were associated with a profound reorganization of the actin cytoskeleton and with a change in the repertoire of expressed actin cytoskeleton genes and EMT markers, as revealed by DNA microarray-based expression profiling. L-plastin, an F-actin cross-linking and stabilizing protein, was identified as one of the most significantly up-regulated genes in TNF-resistant cells. Knockdown of L-plastin in these cells revealed its crucial role in conferring TNF resistance. Importantly, overexpression of wild-type L-plastin in TNF-sensitive MCF-7 cells was sufficient to protect them against TNF-mediated cell death. Furthermore, we found that this effect is dependent on serine-5 phosphorylation of L-plastin and that non-conventional protein kinase C isoforms and the ceramide pathway may regulate its phosphorylation state. The protective role of L-plastin was not restricted to TNF-α resistant MCF-7 cells because a correlation between the expression of L-plastin and the resistance to TNF-α was observed in other breast cancer cell lines. Together, our study discloses a novel unexpected role of the actin bundling protein L-plastin as a cell protective protein against TNF-cytotoxicity. PMID:19799649

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

PubMed

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

2009-08-01

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

Regulation of the Pollen-Specific Actin-Depolymerizing Factor LlADF1

PubMed Central

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

2002-01-01

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

Actin-induced dimerization of palladin promotes actin-bundling

PubMed Central

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

2015-01-01

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

To Cross-Link or Not to Cross-Link? Cross-Linking Associated Foreign Body Response of Collagen-Based Devices

PubMed Central

Delgado, Luis M.; Bayon, Yves; Pandit, Abhay

2015-01-01

Collagen-based devices, in various physical conformations, are extensively used for tissue engineering and regenerative medicine applications. Given that the natural cross-linking pathway of collagen does not occur in vitro, chemical, physical, and biological cross-linking methods have been assessed over the years to control mechanical stability, degradation rate, and immunogenicity of the device upon implantation. Although in vitro data demonstrate that mechanical properties and degradation rate can be accurately controlled as a function of the cross-linking method utilized, preclinical and clinical data indicate that cross-linking methods employed may have adverse effects on host response, especially when potent cross-linking methods are employed. Experimental data suggest that more suitable cross-linking methods should be developed to achieve a balance between stability and functional remodeling. PMID:25517923

Dual effect of pseudorabies virus growth factor (PRGF) displayed on actin cytoskeleton.

PubMed

Urbancíková, M; Vozárová, G; Lesko, J; Golais, F

1999-10-01

Pseudorabies virus growth factor (PRGF) was shown to possess transforming activity as well as transformation repressing activity in in vitro systems. In order to better understand these phenomena we studied actin cytoskeleton and its alterations induced by PRGF using normal human fibroblasts VH-10 and transformed cell line HeLa. For specific detection of filamentous actin cells were stained with phalloidin conjugated with fluorescein isothiocyanate (FITC)-phalloidin. PRGF was applied to VH-10 cells for various length of time from 10 min up to 48 h. The effect was very fast and changes in actin filament composition could be detected already after 10 min. In comparison to untreated cells the staining of treated cells was more diffuse and a number of actin microfilaments in individual stress fibers became reduced. After 30 min thick short actin bundles appeared in the perinuclear region. A 24-h exposure resulted in a large reduction of actin bundles. After additional 24 h a partial restoration of actin cytoskeleton in cells was observed. In transformed HeLa cells PRGF induced opposite process than in normal cells: the number of filamentous actin structures increased. We hypothesise that PRGF may act as a transcription-like factor and may initiate changes in gene expression which consequently result in actin cytoskeleton alterations.

Light-induced cross-linking and post-cross-linking modification of polyglycidol.

PubMed

Marquardt, F; Bruns, M; Keul, H; Yagci, Y; Möller, M

2018-02-08

The photoinduced radical generation process has received renewed interest due to its economic and ecological appeal. Herein the light-induced cross-linking of functional polyglycidol and its post-cross-linking modification are presented. Linear polyglycidol was first functionalized with a tertiary amine in a two-step reaction. Dimethylaminopropyl functional polyglycidol was cross-linked in a UV-light mediated reaction with camphorquinone as a type II photoinitiator. The cross-linked polyglycidol was further functionalized by quaternization with various organoiodine compounds. Aqueous dispersions of the cross-linked polymers were investigated by means of DLS and zeta potential measurements. Polymer films were evaluated by DSC and XPS.

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

PubMed Central

Mattila, Pieta K.; Batista, Facundo D.

2016-01-01

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

Nitric oxide-induced interstrand cross-links in DNA.

PubMed

Caulfield, Jennifer L; Wishnok, John S; Tannenbaum, Steven R

2003-05-01

The DNA damaging effects of nitrous acid have been extensively studied, and the formation of interstrand cross-links have been observed. The potential for this cross-linking to occur through a common nitrosating intermediate derived from nitric oxide is investigated here. Using a HPLC laser-induced fluorescence (LIF) system, the amount of interstrand cross-link formed on nitric oxide treatment of the 5'-fluorescein-labeled oligomer ATATCGATCGATAT was determined. This self-complimentary sequence contains two 5'-CG sequences, which is the preferred site for nitrous acid-induced cross-linking. Nitric oxide was delivered to an 0.5 mM oligomer solution at 15 nmol/mL/min to give a final nitrite concentration of 652 microM. The resulting concentration of the deamination product, xanthine, in this sample was found to be 211 +/- 39 nM, using GC/MS, and the amount of interstrand cross-link was determined to be 13 +/- 2.5 nM. Therefore, upon nitric oxide treatment, the cross-link is found at approximately 6% of the amount of the deamination product. Using this system, detection of the cross-link is also possible for significantly lower doses of nitric oxide, as demonstrated by treatment of the same oligomer with NO at a rate of 18 nmol/mL/min resulting in a final nitrite concentration of 126 microM. The concentration of interstrand cross-link was determined to be 3.6 +/- 0.1 nM in this sample. Therefore, using the same dose rate, when the total nitric oxide concentration delivered drops by a factor of approximately 5, the concentration of cross-link drops by a factor of about 4-indicating a qausi-linear response. It may now be possible to predict the number of cross-links in a small genome based on the number of CpG sequences and the yield of xanthine derived from nitrosative deamination.

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

PubMed Central

Deshpande, Siddharth; Pfohl, Thomas

2012-01-01

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

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

PubMed Central

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

2008-01-01

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

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

PubMed

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

2005-07-01

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

Curvature and torsion in growing actin networks

NASA Astrophysics Data System (ADS)

Shaevitz, Joshua W.; Fletcher, Daniel A.

2008-06-01

Intracellular pathogens such as Listeria monocytogenes and Rickettsia rickettsii move within a host cell by polymerizing a comet-tail of actin fibers that ultimately pushes the cell forward. This dense network of cross-linked actin polymers typically exhibits a striking curvature that causes bacteria to move in gently looping paths. Theoretically, tail curvature has been linked to details of motility by considering force and torque balances from a finite number of polymerizing filaments. Here we track beads coated with a prokaryotic activator of actin polymerization in three dimensions to directly quantify the curvature and torsion of bead motility paths. We find that bead paths are more likely to have low rather than high curvature at any given time. Furthermore, path curvature changes very slowly in time, with an autocorrelation decay time of 200 s. Paths with a small radius of curvature, therefore, remain so for an extended period resulting in loops when confined to two dimensions. When allowed to explore a three-dimensional (3D) space, path loops are less evident. Finally, we quantify the torsion in the bead paths and show that beads do not exhibit a significant left- or right-handed bias to their motion in 3D. These results suggest that paths of actin-propelled objects may be attributed to slow changes in curvature, possibly associated with filament debranching, rather than a fixed torque.

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.

Dimerization and actin-bundling properties of villin and its role in the assembly of epithelial cell brush borders.

PubMed

George, Sudeep P; Wang, Yaohong; Mathew, Sijo; Srinivasan, Kamalakkannan; Khurana, Seema

2007-09-07

Villin is a major actin-bundling protein in the brush border of epithelial cells. In this study we demonstrate for the first time that villin can bundle actin filaments using a single F-actin binding site, because it has the ability to self-associate. Using fluorescence resonance energy transfer, we demonstrate villin self-association in living cells in microvilli and in growth factor-stimulated cells in membrane ruffles and lamellipodia. Using sucrose density gradient, size-exclusion chromatography, and matrix-assisted laser desorption ionization time-of-flight, the majority of villin was identified as a monomer or dimer. Villin dimers were also identified in Caco-2 cells, which endogenously express villin and Madin-Darby canine kidney cells that ectopically express villin. Using truncation mutants of villin, site-directed mutagenesis, and fluorescence resonance energy transfer, an amino-terminal dimerization site was identified that regulated villin self-association in parallel conformation as well as actin bundling by villin. This detailed analysis describes for the first time microvillus assembly by villin, redefines the actin-bundling function of villin, and provides a molecular mechanism for actin bundling by villin, which could have wider implications for other actin cross-linking proteins that share a villin-like headpiece domain. Our study also provides a molecular basis to separate the morphologically distinct actin-severing and actin-bundling properties of villin.

DNA damage induces nuclear actin filament assembly by Formin -2 and Spire-½ that promotes efficient DNA repair. [corrected].

PubMed

Belin, Brittany J; Lee, Terri; Mullins, R Dyche

2015-08-19

Actin filaments assemble inside the nucleus in response to multiple cellular perturbations, including heat shock, protein misfolding, integrin engagement, and serum stimulation. We find that DNA damage also generates nuclear actin filaments-detectable by phalloidin and live-cell actin probes-with three characteristic morphologies: (i) long, nucleoplasmic filaments; (ii) short, nucleolus-associated filaments; and (iii) dense, nucleoplasmic clusters. This DNA damage-induced nuclear actin assembly requires two biologically and physically linked nucleation factors: Formin-2 and Spire-1/Spire-2. Formin-2 accumulates in the nucleus after DNA damage, and depletion of either Formin-2 or actin's nuclear import factor, importin-9, increases the number of DNA double-strand breaks (DSBs), linking nuclear actin filaments to efficient DSB clearance. Nuclear actin filaments are also required for nuclear oxidation induced by acute genotoxic stress. Our results reveal a previously unknown role for nuclear actin filaments in DNA repair and identify the molecular mechanisms creating these nuclear filaments.

Cofilin-Linked Changes in Actin Filament Flexibility Promote Severing

PubMed Central

McCullough, Brannon R.; Grintsevich, Elena E.; Chen, Christine K.; Kang, Hyeran; Hutchison, Alan L.; Henn, Arnon; Cao, Wenxiang; Suarez, Cristian; Martiel, Jean-Louis; Blanchoin, Laurent; Reisler, Emil; De La Cruz, Enrique M.

2011-01-01

The actin regulatory protein, cofilin, increases the bending and twisting elasticity of actin filaments and severs them. It has been proposed that filaments partially decorated with cofilin accumulate stress from thermally driven shape fluctuations at bare (stiff) and decorated (compliant) boundaries, thereby promoting severing. This mechanics-based severing model predicts that changes in actin filament compliance due to cofilin binding affect severing activity. Here, we test this prediction by evaluating how the severing activities of vertebrate and yeast cofilactin scale with the flexural rigidities determined from analysis of shape fluctuations. Yeast actin filaments are more compliant in bending than vertebrate actin filaments. Severing activities of cofilactin isoforms correlate with changes in filament flexibility. Vertebrate cofilin binds but does not increase the yeast actin filament flexibility, and does not sever them. Imaging of filament thermal fluctuations reveals that severing events are associated with local bending and fragmentation when deformations attain a critical angle. The critical severing angle at boundaries between bare and cofilin-decorated segments is smaller than in bare or fully decorated filaments. These measurements support a cofilin-severing mechanism in which mechanical asymmetry promotes local stress accumulation and fragmentation at boundaries of bare and cofilin-decorated segments, analogous to failure of some nonprotein materials. PMID:21723825

Myocardin-Related Transcription Factor A Activation by Competition with WH2 Domain Proteins for Actin Binding.

PubMed

Weissbach, Julia; Schikora, Franziska; Weber, Anja; Kessels, Michael; Posern, Guido

2016-05-15

The myocardin-related transcription factors (MRTFs) are coactivators of serum response factor (SRF)-mediated gene expression. Activation of MRTF-A occurs in response to alterations in actin dynamics and critically requires the dissociation of repressive G-actin-MRTF-A complexes. However, the mechanism leading to the release of MRTF-A remains unclear. Here we show that WH2 domains compete directly with MRTF-A for actin binding. Actin nucleation-promoting factors, such as N-WASP and WAVE2, as well as isolated WH2 domains, including those of Spire2 and Cobl, activate MRTF-A independently of changes in actin dynamics. Simultaneous inhibition of Arp2-Arp3 or mutation of the CA region only partially reduces MRTF-A activation by N-WASP and WAVE2. Recombinant WH2 domains and the RPEL domain of MRTF-A bind mutually exclusively to cellular and purified G-actin in vitro The competition by different WH2 domains correlates with MRTF-SRF activation. Following serum stimulation, nonpolymerizable actin dissociates from MRTF-A, and de novo formation of the G-actin-RPEL complex is impaired by a transferable factor. Our work demonstrates that WH2 domains activate MRTF-A and contribute to target gene regulation by a competitive mechanism, independently of their role in actin filament formation. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Levels of mature cross-links and advanced glycation end product cross-links in human vitreous.

PubMed

Matsumoto, Yukihiro; Takahashi, Masaaki; Chikuda, Makoto; Arai, Kiyomi

2002-01-01

To determine the levels of pyridinoline and deoxypyridinoline, two mature enzymatic cross-links, and pentosidine, an advanced glycation end product (AGE) cross-link, in the human vitreous, and to investigate the correlations among the cross-links and the effects of aging and diabetes mellitus (DM) on the levels of cross-links. Forty-five vitreous samples were collected from 32 patients (32 eyes) undergoing vitrectomy for diabetic retinopathy (DM group) and from 13 patients (13 eyes) (control group) who were age- and sex-matched patients with idiopathic macular hole or epiretinal membrane with no systemic conditions. The levels of the cross-links were determined using high-performance liquid chromatography after acid hydrolysis and pretreatment with SP-Sephadex. The levels of pentosidine, pyridinoline, and deoxypyridinoline were 27.3 +/- 23.1 (mean +/- SD) pmol/mL (detectable in 45 of 45 specimens), 79.0 +/- 40.2 ng/mL (43 of 45 specimens), and 54.0 +/- 9.5 (32 of 45 specimens) ng/mL, respectively. When the vitreous samples from the DM and the control groups were compared, a significant difference (P <.05) was found in the pentosidine level but not in the levels of pyridinoline or deoxypyridinoline. No significant correlations were found between age and the cross-links. Significant correlations (P <.01) were found among the cross-links. The results indicate that mature cross-link substances exist in the human vitreous. The results also suggest that glycation may occur in the vitreous after mature cross-links form and result in the formation of AGE cross-links. In human vitreous from patients with DM, increased levels of AGE cross-links may stabilize the formation of mature cross-links, but they did not increase the mature cross-links.

Pollen specific expression of maize genes encoding actin depolymerizing factor-like proteins.

PubMed Central

Lopez, I; Anthony, R G; Maciver, S K; Jiang, C J; Khan, S; Weeds, A G; Hussey, P J

1996-01-01

In pollen development, a dramatic reorganization of the actin cytoskeleton takes place during the passage of the pollen grain into dormancy and on activation of pollen tube growth. A role for actin-binding proteins is implicated and we report here the identification of a small gene family in maize that encodes actin depolymerizing factor (ADF)-like proteins. The ADF group of proteins are believed to control actin polymerization and depolymerization in response to both intracellular and extracellular signals. Two of the maize genes ZmABP1 and ZmABP2 are expressed specifically in pollen and germinating pollen suggesting that the protein products may be involved in pollen actin reorganization. A third gene, ZmABP3, encodes a protein only 56% and 58% identical to ZmABP1 and ZmABP2, respectively, and its expression is suppressed in pollen and germinated pollen. The fundamental biochemical characteristics of the ZmABP proteins has been elucidated using bacterially expressed ZmABP3 protein. This has the ability to bind monomeric actin (G-actin) and filamentous actin (F-actin). Moreover, it decreases the viscosity of polymerized actin solutions consistent with an ability to depolymerize filaments. These biochemical characteristics, taken together with the sequence comparisons, support the inclusion of the ZmABP proteins in the ADF group. Images Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8693008

Arabidopsis ACTIN-DEPOLYMERIZING FACTOR3 Is Required for Controlling Aphid Feeding from the Phloem1[OPEN

PubMed Central

Mondal, Hossain A.; Louis, Joe; Archer, Lani; Patel, Monika; Nalam, Vamsi J.; Sarowar, Sujon; Sivapalan, Vishala

2018-01-01

The actin cytoskeleton network has an important role in plant cell growth, division, and stress response. Actin-depolymerizing factors (ADFs) are a group of actin-binding proteins that contribute to reorganization of the actin network. Here, we show that the Arabidopsis (Arabidopsis thaliana) ADF3 is required in the phloem for controlling infestation by Myzus persicae Sülzer, commonly known as the green peach aphid (GPA), which is an important phloem sap-consuming pest of more than fifty plant families. In agreement with a role for the actin-depolymerizing function of ADF3 in defense against the GPA, we show that resistance in adf3 was restored by overexpression of the related ADF4 and the actin cytoskeleton destabilizers, cytochalasin D and latrunculin B. Electrical monitoring of the GPA feeding behavior indicates that the GPA stylets found sieve elements faster when feeding on the adf3 mutant compared to the wild-type plant. In addition, once they found the sieve elements, the GPA fed for a more prolonged period from sieve elements of adf3 compared to the wild-type plant. The longer feeding period correlated with an increase in fecundity and population size of the GPA and a parallel reduction in callose deposition in the adf3 mutant. The adf3-conferred susceptibility to GPA was overcome by expression of the ADF3 coding sequence from the phloem-specific SUC2 promoter, thus confirming the importance of ADF3 function in the phloem. We further demonstrate that the ADF3-dependent defense mechanism is linked to the transcriptional up-regulation of PHYTOALEXIN-DEFICIENT4, which is an important regulator of defenses against the GPA. PMID:29133373

Monoubiquitination Inhibits the Actin Bundling Activity of Fascin*

PubMed Central

Lin, Shengchen; Lu, Shuang; Mulaj, Mentor; Fang, Bin; Keeley, Tyler; Wan, Lixin; Hao, Jihui; Muschol, Martin; Sun, Jianwei; Yang, Shengyu

2016-01-01

Fascin is an actin bundling protein that cross-links individual actin filaments into straight, compact, and stiff bundles, which are crucial for the formation of filopodia, stereocillia, and other finger-like membrane protrusions. The dysregulation of fascin has been implicated in cancer metastasis, hearing loss, and blindness. Here we identified monoubiquitination as a novel mechanism that regulates fascin bundling activity and dynamics. The monoubiquitination sites were identified to be Lys247 and Lys250, two residues located in a positive charge patch at the actin binding site 2 of fascin. Using a chemical ubiquitination method, we synthesized chemically monoubiquitinated fascin and determined the effects of monoubiquitination on fascin bundling activity and dynamics. Our data demonstrated that monoubiquitination decreased the fascin bundling EC50, delayed the initiation of bundle assembly, and accelerated the disassembly of existing bundles. By analyzing the electrostatic properties on the solvent-accessible surface of fascin, we proposed that monoubiquitination introduced steric hindrance to interfere with the interaction between actin filaments and the positively charged patch at actin binding site 2. We also identified Smurf1 as a E3 ligase regulating the monoubiquitination of fascin. Our findings revealed a previously unidentified regulatory mechanism for fascin, which will have important implications for the understanding of actin bundle regulation under physiological and pathological conditions. PMID:27879315

Monoubiquitination Inhibits the Actin Bundling Activity of Fascin.

PubMed

Lin, Shengchen; Lu, Shuang; Mulaj, Mentor; Fang, Bin; Keeley, Tyler; Wan, Lixin; Hao, Jihui; Muschol, Martin; Sun, Jianwei; Yang, Shengyu

2016-12-30

Fascin is an actin bundling protein that cross-links individual actin filaments into straight, compact, and stiff bundles, which are crucial for the formation of filopodia, stereocillia, and other finger-like membrane protrusions. The dysregulation of fascin has been implicated in cancer metastasis, hearing loss, and blindness. Here we identified monoubiquitination as a novel mechanism that regulates fascin bundling activity and dynamics. The monoubiquitination sites were identified to be Lys 247 and Lys 250 , two residues located in a positive charge patch at the actin binding site 2 of fascin. Using a chemical ubiquitination method, we synthesized chemically monoubiquitinated fascin and determined the effects of monoubiquitination on fascin bundling activity and dynamics. Our data demonstrated that monoubiquitination decreased the fascin bundling EC 50 , delayed the initiation of bundle assembly, and accelerated the disassembly of existing bundles. By analyzing the electrostatic properties on the solvent-accessible surface of fascin, we proposed that monoubiquitination introduced steric hindrance to interfere with the interaction between actin filaments and the positively charged patch at actin binding site 2. We also identified Smurf1 as a E3 ligase regulating the monoubiquitination of fascin. Our findings revealed a previously unidentified regulatory mechanism for fascin, which will have important implications for the understanding of actin bundle regulation under physiological and pathological conditions. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

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

PubMed Central

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

2013-01-01

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

Porous Cross-Linked Polyimide Networks

NASA Technical Reports Server (NTRS)

Meador, Mary Ann B. (Inventor); Guo, Haiquan (Inventor)

2015-01-01

Porous cross-linked polyimide networks are provided. The networks comprise an anhydride end-capped polyamic acid oligomer. The oligomer (i) comprises a repeating unit of a dianhydride and a diamine and terminal anhydride groups, (ii) has an average degree of polymerization of 10 to 50, (iii) has been cross-linked via a cross-linking agent, comprising three or more amine groups, at a balanced stoichiometry of the amine groups to the terminal anhydride groups, and (iv) has been chemically imidized to yield the porous cross-linked polyimide network. Also provided are porous cross-linked polyimide aerogels comprising a cross-linked and imidized anhydride end-capped polyamic acid oligomer, wherein the oligomer comprises a repeating unit of a dianhydride and a diamine, and the aerogel has a density of 0.10 to 0.333 g/cm.sup.3 and a Young's modulus of 1.7 to 102 MPa. Also provided are thin films comprising aerogels, and methods of making porous cross-linked polyimide networks.

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

PubMed Central

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

2013-01-01

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

G-actin sequestering protein thymosin-β4 regulates the activity of myocardin-related transcription factor

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

Morita, Tsuyoshi, E-mail: tsuyo@nbiochem.med.osaka-u.ac.jp; Hayashi, Ken’ichiro

2013-08-02

Highlights: •Tβ4 competed with MRTF-A for G-actin binding. •Tβ4 activated the MRTF–SRF signaling pathway. •Tβ4 increased the endogenous expression of SRF-dependent genes. -- Abstract: Myocardin-related transcription factors (MRTFs) are robust coactivators of serum response factor (SRF). MRTFs contain three copies of the RPEL motif at their N-terminus, and they bind to monomeric globular actin (G-actin). Previous studies illustrate that G-actin binding inhibits MRTF activity by preventing the MRTFs nuclear accumulation. In the living cells, the majority of G-actin is sequestered by G-actin binding proteins that prevent spontaneous actin polymerization. Here, we demonstrate that the most abundant G-actin sequestering protein thymosin-β4more » (Tβ4) was involved in the regulation of subcellular localization and activity of MRTF-A. Tβ4 competed with MRTF-A for G-actin binding; thus, interfering with G-actin–MRTF-A complex formation. Tβ4 overexpression induced the MRTF-A nuclear accumulation and activation of MRTF–SRF signaling. The activation rate of MRTF-A by the Tβ4 mutant L17A, whose affinity for G-actin is very low, was lower than that by wild-type Tβ4. In contrast, the β-actin mutant 3DA, which has a lower affinity for Tβ4, more effectively suppressed MRTF-A activity than wild-type β-actin. Furthermore, ectopic Tβ4 increased the endogenous expression of SRF-dependent actin cytoskeletal genes. Thus, Tβ4 is an important MRTF regulator that controls the G-actin–MRTFs interaction.« less

Technical advance: identification of plant actin-binding proteins by F-actin affinity chromatography

NASA Technical Reports Server (NTRS)

Hu, S.; Brady, S. R.; Kovar, D. R.; Staiger, C. J.; Clark, G. B.; Roux, S. J.; Muday, G. K.

2000-01-01

Proteins that interact with the actin cytoskeleton often modulate the dynamics or organization of the cytoskeleton or use the cytoskeleton to control their localization. In plants, very few actin-binding proteins have been identified and most are thought to modulate cytoskeleton function. To identify actin-binding proteins that are unique to plants, the development of new biochemical procedures will be critical. Affinity columns using actin monomers (globular actin, G-actin) or actin filaments (filamentous actin, F-actin) have been used to identify actin-binding proteins from a wide variety of organisms. Monomeric actin from zucchini (Cucurbita pepo L.) hypocotyl tissue was purified to electrophoretic homogeneity and shown to be native and competent for polymerization to actin filaments. G-actin, F-actin and bovine serum albumin affinity columns were prepared and used to separate samples enriched in either soluble or membrane-associated actin-binding proteins. Extracts of soluble actin-binding proteins yield distinct patterns when eluted from the G-actin and F-actin columns, respectively, leading to the identification of a putative F-actin-binding protein of approximately 40 kDa. When plasma membrane-associated proteins were applied to these columns, two abundant polypeptides eluted selectively from the F-actin column and cross-reacted with antiserum against pea annexins. Additionally, a protein that binds auxin transport inhibitors, the naphthylphthalamic acid binding protein, which has been previously suggested to associate with the actin cytoskeleton, was eluted in a single peak from the F-actin column. These experiments provide a new approach that may help to identify novel actin-binding proteins from plants.

Technical advance: identification of plant actin-binding proteins by F-actin affinity chromatography.

PubMed

Hu, S; Brady, S R; Kovar, D R; Staiger, C J; Clark, G B; Roux, S J; Muday, G K

2000-10-01

Proteins that interact with the actin cytoskeleton often modulate the dynamics or organization of the cytoskeleton or use the cytoskeleton to control their localization. In plants, very few actin-binding proteins have been identified and most are thought to modulate cytoskeleton function. To identify actin-binding proteins that are unique to plants, the development of new biochemical procedures will be critical. Affinity columns using actin monomers (globular actin, G-actin) or actin filaments (filamentous actin, F-actin) have been used to identify actin-binding proteins from a wide variety of organisms. Monomeric actin from zucchini (Cucurbita pepo L.) hypocotyl tissue was purified to electrophoretic homogeneity and shown to be native and competent for polymerization to actin filaments. G-actin, F-actin and bovine serum albumin affinity columns were prepared and used to separate samples enriched in either soluble or membrane-associated actin-binding proteins. Extracts of soluble actin-binding proteins yield distinct patterns when eluted from the G-actin and F-actin columns, respectively, leading to the identification of a putative F-actin-binding protein of approximately 40 kDa. When plasma membrane-associated proteins were applied to these columns, two abundant polypeptides eluted selectively from the F-actin column and cross-reacted with antiserum against pea annexins. Additionally, a protein that binds auxin transport inhibitors, the naphthylphthalamic acid binding protein, which has been previously suggested to associate with the actin cytoskeleton, was eluted in a single peak from the F-actin column. These experiments provide a new approach that may help to identify novel actin-binding proteins from plants.

Role and structural mechanism of WASP-triggered conformational changes in branched actin filament nucleation by Arp2/3 complex

PubMed Central

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

2016-01-01

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

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

2016-07-05

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.

Cross-linking of type I collagen with microbial transglutaminase: identification of cross-linking sites.

PubMed

Stachel, Ines; Schwarzenbolz, Uwe; Henle, Thomas; Meyer, Michael

2010-03-08

Collagen is a popular biomaterial. To deal with its lack of thermal stability and its weak resistance to proteolytic degradation, collagen-based materials are stabilized via different cross-linking procedures. Regarding the potential toxicity of residual cross-linking agents, enzyme-mediated cross-linking would provide an alternative and nontoxic method for collagen stabilization. The results of this study show that type I collagen is a substrate for mTG. However, epsilon-(gamma-glutamyl)lysine cross-links are only incorporated at elevated temperatures when the protein is partially or completely denatured. A maximum number of 5.4 cross-links per collagen monomer were found for heat-denatured collagen. Labeling with the primary amine monodansylcadaverine revealed that at least half of the cross-links are located within the triple helical region of the collagen molecule. Because the triple helix is highly ordered in its native state, this finding might explain why the glutamine residues are inaccessible for mTG under nondenaturing conditions.

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.

Myocardin-Related Transcription Factor A Activation by Competition with WH2 Domain Proteins for Actin Binding

PubMed Central

Weissbach, Julia; Schikora, Franziska; Weber, Anja; Kessels, Michael

2016-01-01

The myocardin-related transcription factors (MRTFs) are coactivators of serum response factor (SRF)-mediated gene expression. Activation of MRTF-A occurs in response to alterations in actin dynamics and critically requires the dissociation of repressive G-actin–MRTF-A complexes. However, the mechanism leading to the release of MRTF-A remains unclear. Here we show that WH2 domains compete directly with MRTF-A for actin binding. Actin nucleation-promoting factors, such as N-WASP and WAVE2, as well as isolated WH2 domains, including those of Spire2 and Cobl, activate MRTF-A independently of changes in actin dynamics. Simultaneous inhibition of Arp2-Arp3 or mutation of the CA region only partially reduces MRTF-A activation by N-WASP and WAVE2. Recombinant WH2 domains and the RPEL domain of MRTF-A bind mutually exclusively to cellular and purified G-actin in vitro. The competition by different WH2 domains correlates with MRTF-SRF activation. Following serum stimulation, nonpolymerizable actin dissociates from MRTF-A, and de novo formation of the G-actin–RPEL complex is impaired by a transferable factor. Our work demonstrates that WH2 domains activate MRTF-A and contribute to target gene regulation by a competitive mechanism, independently of their role in actin filament formation. PMID:26976641

Elasticity in Physically Cross-Linked Amyloid Fibril Networks.

PubMed

Cao, Yiping; Bolisetty, Sreenath; Adamcik, Jozef; Mezzenga, Raffaele

2018-04-13

We provide a constitutive model of semiflexible and rigid amyloid fibril networks by combining the affine thermal model of network elasticity with the Derjaguin-Landau-Vervey-Overbeek (DLVO) theory of electrostatically charged colloids. When compared to rheological experiments on β-lactoglobulin and lysozyme amyloid networks, this approach provides the correct scaling of elasticity versus both concentration (G∼c^{2.2} and G∼c^{2.5} for semiflexible and rigid fibrils, respectively) and ionic strength (G∼I^{4.4} and G∼I^{3.8} for β-lactoglobulin and lysozyme, independent from fibril flexibility). The pivotal role played by the screening salt is to reduce the electrostatic barrier among amyloid fibrils, converting labile physical entanglements into long-lived cross-links. This gives a power-law behavior of G with I having exponents significantly larger than in other semiflexible polymer networks (e.g., actin) and carrying DLVO traits specific to the individual amyloid fibrils.

Radiation cross-linked collagen/dextran dermal scaffolds: effects of dextran on cross-linking and degradation.

PubMed

Zhang, Yaqing; Zhang, Xiangmei; Xu, Ling; Wei, Shicheng; Zhai, Maolin

2015-01-01

Ionizing radiation effectively cross-links collagen into network with enhanced anti-degradability and biocompatibility, while radiation-cross-linked collagen scaffold lacks flexibility, satisfactory surface appearance, and performs poor in cell penetration and ingrowth. To make the radiation-cross-linked collagen scaffold to serve as an ideal artificial dermis, dextran was incorporated into collagen. Scaffolds with the collagen/dextran (Col/Dex) ratios of 10/0, 7/3, and 5/5 were fabricated via (60)Co γ-irradiation cross-linking, followed by lyophilization. The morphology, microstructure, physicochemical, and biological properties were investigated. Compared with pure collagen, scaffolds with dextran demonstrated more porous appearance, enhanced hydrophilicity while the cross-linking density was lower with the consequence of larger pore size, higher water uptake, as well as reduced stiffness. Accelerated degradation was observed when dextran was incorporated in both the in vitro and in vivo assays, which led to earlier integration with cell and host tissue. The effect of dextran on degradation was ascribed to the decreased cross-linking density, looser microstructure, more porous and hydrophilic surface. Considering the better appearance, softness, moderate degradation rate due to controllable cross-linking degree and good biocompatibility as well, radiation-cross-linked collagen/dextran scaffolds are expected to serve as promising artificial dermal substitutes.

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

PubMed Central

Goldman, Robert D.; Louvard, Daniel

2010-01-01

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

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

NASA Astrophysics Data System (ADS)

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

2014-03-01

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

Z-disc-associated, Alternatively Spliced, PDZ Motif-containing Protein (ZASP) Mutations in the Actin-binding Domain Cause Disruption of Skeletal Muscle Actin Filaments in Myofibrillar Myopathy*

PubMed Central

Lin, Xiaoyan; Ruiz, Janelle; Bajraktari, Ilda; Ohman, Rachel; Banerjee, Soojay; Gribble, Katherine; Kaufman, Joshua D.; Wingfield, Paul T.; Griggs, Robert C.; Fischbeck, Kenneth H.; Mankodi, Ami

2014-01-01

The core of skeletal muscle Z-discs consists of actin filaments from adjacent sarcomeres that are cross-linked by α-actinin homodimers. Z-disc-associated, alternatively spliced, PDZ motif-containing protein (ZASP)/Cypher interacts with α-actinin, myotilin, and other Z-disc proteins via the PDZ domain. However, these interactions are not sufficient to maintain the Z-disc structure. We show that ZASP directly interacts with skeletal actin filaments. The actin-binding domain is between the modular PDZ and LIM domains. This ZASP region is alternatively spliced so that each isoform has unique actin-binding domains. All ZASP isoforms contain the exon 6-encoded ZASP-like motif that is mutated in zaspopathy, a myofibrillar myopathy (MFM), whereas the exon 8–11 junction-encoded peptide is exclusive to the postnatal long ZASP isoform (ZASP-LΔex10). MFM is characterized by disruption of skeletal muscle Z-discs and accumulation of myofibrillar degradation products. Wild-type and mutant ZASP interact with α-actin, α-actinin, and myotilin. Expression of mutant, but not wild-type, ZASP leads to Z-disc disruption and F-actin accumulation in mouse skeletal muscle, as in MFM. Mutations in the actin-binding domain of ZASP-LΔex10, but not other isoforms, cause disruption of the actin cytoskeleton in muscle cells. These isoform-specific mutation effects highlight the essential role of the ZASP-LΔex10 isoform in F-actin organization. Our results show that MFM-associated ZASP mutations in the actin-binding domain have deleterious effects on the core structure of the Z-discs in skeletal muscle. PMID:24668811

Structure, Subunit Topology, and Actin-binding Activity of the Arp2/3 Complex from Acanthamoeba

PubMed Central

Mullins, R. Dyche; Stafford, Walter F.; Pollard, Thomas D.

1997-01-01

The Arp2/3 complex, first isolated from Acanthamoeba castellani by affinity chromatography on profilin, consists of seven polypeptides; two actinrelated proteins, Arp2 and Arp3; and five apparently novel proteins, p40, p35, p19, p18, and p14 (Machesky et al., 1994). The complex is homogeneous by hydrodynamic criteria with a Stokes' radius of 5.3 nm by gel filtration, sedimentation coefficient of 8.7 S, and molecular mass of 197 kD by analytical ultracentrifugation. The stoichiometry of the subunits is 1:1:1:1:1:1:1, indicating the purified complex contains one copy each of seven polypeptides. In electron micrographs, the complex has a bilobed or horseshoe shape with outer dimensions of ∼13 × 10 nm, and mathematical models of such a shape and size are consistent with the measured hydrodynamic properties. Chemical cross-linking with a battery of cross-linkers of different spacer arm lengths and chemical reactivities identify the following nearest neighbors within the complex: Arp2 and p40; Arp2 and p35; Arp3 and p35; Arp3 and either p18 or p19; and p19 and p14. By fluorescent antibody staining with anti-p40 and -p35, the complex is concentrated in the cortex of the ameba, especially in linear structures, possibly actin filament bundles, that lie perpendicular to the leading edge. Purified Arp2/3 complex binds actin filaments with a K d of 2.3 μM and a stoichiometry of approximately one complex molecule per actin monomer. In electron micrographs of negatively stained samples, Arp2/3 complex decorates the sides of actin filaments. EDC/NHS cross-links actin to Arp3, p35, and a low molecular weight subunit, p19, p18, or p14. We propose structural and topological models for the Arp2/3 complex and suggest that affinity for actin filaments accounts for the localization of complex subunits to actinrich regions of Acanthamoeba. PMID:9015304

Elasticity in Physically Cross-Linked Amyloid Fibril Networks

NASA Astrophysics Data System (ADS)

Cao, Yiping; Bolisetty, Sreenath; Adamcik, Jozef; Mezzenga, Raffaele

2018-04-01

We provide a constitutive model of semiflexible and rigid amyloid fibril networks by combining the affine thermal model of network elasticity with the Derjaguin-Landau-Vervey-Overbeek (DLVO) theory of electrostatically charged colloids. When compared to rheological experiments on β -lactoglobulin and lysozyme amyloid networks, this approach provides the correct scaling of elasticity versus both concentration (G ˜c2.2 and G ˜c2.5 for semiflexible and rigid fibrils, respectively) and ionic strength (G ˜I4.4 and G ˜I3.8 for β -lactoglobulin and lysozyme, independent from fibril flexibility). The pivotal role played by the screening salt is to reduce the electrostatic barrier among amyloid fibrils, converting labile physical entanglements into long-lived cross-links. This gives a power-law behavior of G with I having exponents significantly larger than in other semiflexible polymer networks (e.g., actin) and carrying DLVO traits specific to the individual amyloid fibrils.

Microtubule-actin crosslinking factor 1 (Macf1) domain function in Balbiani body dissociation and nuclear positioning.

PubMed

Escobar-Aguirre, Matias; Zhang, Hong; Jamieson-Lucy, Allison; Mullins, Mary C

2017-09-01

Animal-vegetal (AV) polarity of most vertebrate eggs is established during early oogenesis through the formation and disassembly of the Balbiani Body (Bb). The Bb is a structure conserved from insects to humans that appears as a large granule, similar to a mRNP granule composed of mRNA and proteins, that in addition contains mitochondria, ER and Golgi. The components of the Bb, which have amyloid-like properties, include germ cell and axis determinants of the embryo that are anchored to the vegetal cortex upon Bb disassembly. Our lab discovered in zebrafish the only gene known to function in Bb disassembly, microtubule-actin crosslinking factor 1a (macf1a). Macf1 is a conserved, giant multi-domain cytoskeletal linker protein that can interact with microtubules (MTs), actin filaments (AF), and intermediate filaments (IF). In macf1a mutant oocytes the Bb fails to dissociate, the nucleus is acentric, and AV polarity of the oocyte and egg fails to form. The cytoskeleton-dependent mechanism by which Macf1a regulates Bb mRNP granule dissociation was unknown. We found that disruption of AFs phenocopies the macf1a mutant phenotype, while MT disruption does not. We determined that cytokeratins (CK), a type of IF, are enriched in the Bb. We found that Macf1a localizes to the Bb, indicating a direct function in regulating its dissociation. We thus tested if Macf1a functions via its actin binding domain (ABD) and plectin repeat domain (PRD) to integrate cortical actin and Bb CK, respectively, to mediate Bb dissociation at the oocyte cortex. We developed a CRISPR/Cas9 approach to delete the exons encoding these domains from the macf1a endogenous locus, while maintaining the open reading frame. Our analysis shows that Macf1a functions via its ABD to mediate Bb granule dissociation and nuclear positioning, while the PRD is dispensable. We propose that Macf1a does not function via its canonical mechanism of linking two cytoskeletal systems together in dissociating the Bb. Instead

Microtubule-actin crosslinking factor 1 (Macf1) domain function in Balbiani body dissociation and nuclear positioning

PubMed Central

Zhang, Hong; Jamieson-Lucy, Allison

2017-01-01

Animal-vegetal (AV) polarity of most vertebrate eggs is established during early oogenesis through the formation and disassembly of the Balbiani Body (Bb). The Bb is a structure conserved from insects to humans that appears as a large granule, similar to a mRNP granule composed of mRNA and proteins, that in addition contains mitochondria, ER and Golgi. The components of the Bb, which have amyloid-like properties, include germ cell and axis determinants of the embryo that are anchored to the vegetal cortex upon Bb disassembly. Our lab discovered in zebrafish the only gene known to function in Bb disassembly, microtubule-actin crosslinking factor 1a (macf1a). Macf1 is a conserved, giant multi-domain cytoskeletal linker protein that can interact with microtubules (MTs), actin filaments (AF), and intermediate filaments (IF). In macf1a mutant oocytes the Bb fails to dissociate, the nucleus is acentric, and AV polarity of the oocyte and egg fails to form. The cytoskeleton-dependent mechanism by which Macf1a regulates Bb mRNP granule dissociation was unknown. We found that disruption of AFs phenocopies the macf1a mutant phenotype, while MT disruption does not. We determined that cytokeratins (CK), a type of IF, are enriched in the Bb. We found that Macf1a localizes to the Bb, indicating a direct function in regulating its dissociation. We thus tested if Macf1a functions via its actin binding domain (ABD) and plectin repeat domain (PRD) to integrate cortical actin and Bb CK, respectively, to mediate Bb dissociation at the oocyte cortex. We developed a CRISPR/Cas9 approach to delete the exons encoding these domains from the macf1a endogenous locus, while maintaining the open reading frame. Our analysis shows that Macf1a functions via its ABD to mediate Bb granule dissociation and nuclear positioning, while the PRD is dispensable. We propose that Macf1a does not function via its canonical mechanism of linking two cytoskeletal systems together in dissociating the Bb. Instead

Di-isodityrosine is the intermolecular cross-link of isodityrosine-rich extensin analogs cross-linked in vitro.

PubMed

Held, Michael A; Tan, Li; Kamyab, Abdolreza; Hare, Michael; Shpak, Elena; Kieliszewski, Marcia J

2004-12-31

Extensins are cell wall hydroxyproline-rich glycoproteins that form covalent networks putatively involving tyrosyl and lysyl residues in cross-links catalyzed by one or more extensin peroxidases. The precise cross-links remain to be chemically identified both as network components in muro and as enzymic products generated in vitro with native extensin monomers as substrates. However, some extensin monomers contain variations within their putative cross-linking motifs that complicate cross-link identification. Other simpler extensins are recalcitrant to isolation including the ubiquitous P3-type extensin whose major repetitive motif, Hyp)(4)-Ser-Hyp-Ser-(Hyp)(4)-Tyr-Tyr-Tyr-Lys, is of particular interest, not least because its Tyr-Tyr-Tyr intramolecular isodityrosine cross-link motifs are also putative candidates for further intermolecular cross-linking to form di-isodityrosine. Therefore, we designed a set of extensin analogs encoding tandem repeats of the P3 motif, including Tyr --> Phe and Lys --> Leu variations. Expression of these P3 analogs in Nicotiana tabacum cells yielded glycoproteins with virtually all Pro residues hydroxylated and subsequently arabinosylated and with likely galactosylated Ser residues. This was consistent with earlier analyses of P3 glycopeptides isolated from cell wall digests and the predictions of the Hyp contiguity hypothesis. The tyrosine-rich P3 analogs also contained isodityrosine, formed in vivo. Significantly, these isodityrosine-containing analogs were further cross-linked in vitro by an extensin peroxidase to form the tetra-tyrosine intermolecular cross-link amino acid di-isodityrosine. This is the first identification of an inter-molecular cross-link amino acid in an extensin module and corroborates earlier suggestions that di-isodityrosine represents one mechanism for cross-linking extensins in muro.

Actin-Based Adhesion Modules Mediate Cell Interactions with the Extracellular Matrix and Neighboring Cells.

PubMed

Bachir, Alexia I; Horwitz, Alan Rick; Nelson, W James; Bianchini, Julie M

2017-07-05

Cell adhesions link cells to the extracellular matrix (ECM) and to each other and depend on interactions with the actin cytoskeleton. Both cell-ECM and cell-cell adhesion sites contain discrete, yet overlapping, functional modules. These modules establish physical associations with the actin cytoskeleton, locally modulate actin organization and dynamics, and trigger intracellular signaling pathways. Interplay between these modules generates distinct actin architectures that underlie different stages, types, and functions of cell-ECM and cell-cell adhesions. Actomyosin contractility is required to generate mature, stable adhesions, as well as to sense and translate the mechanical properties of the cellular environment into changes in cell organization and behavior. Here, we review the organization and function of different adhesion modules and how they interact with the actin cytoskeleton. We highlight the molecular mechanisms of mechanotransduction in adhesions and how adhesion molecules mediate cross talk between cell-ECM and cell-cell adhesion sites. Copyright © 2017 Cold Spring Harbor Laboratory Press; all rights reserved.

Ion exchange selectivity for cross-linked polyacrylic acid

NASA Technical Reports Server (NTRS)

May, C. E.; Philipp, W. H.

1983-01-01

The ion separation factors for 21 common metal ions with cross-linked polyacrylic acid were determined as a function of pH and the percent of the cross-linked polyacrylic acid neutralized. The calcium ion was used as a reference. At a pH of 5 the decreasing order of affinity of the ions for the cross-linked polyacrylic acid was found to be: Hg++, Fe+++, Pb++, Cr+++, Cu++, Cd++, Al+++, Ag+, Zn++, Ni++, Mn++, Co++, Ca++, Sr++, Ba++, Mg++, K+, Rb+, Cs+, Na+, and Li+. Members of a chemical family exhibited similar selectivities. The Hg++ ion appeared to be about a million times more strongly bound than the alkali metal ions. The relative binding of most of the metal ions varied with pH; the very tightly and very weakly bound ions showed the largest variations with pH. The calcium ion-hydrogen ion equilibrium was perturbed very little by the presence of the other ions. The separation factors and selectivity coefficients are discussed in terms of equilibrium and thermodynamic significance.

Adhesive F-actin Waves: A Novel Integrin-Mediated Adhesion Complex Coupled to Ventral Actin Polymerization

PubMed Central

Case, Lindsay B.; Waterman, Clare M.

2011-01-01

At the leading lamellipodium of migrating cells, protrusion of an Arp2/3-nucleated actin network is coupled to formation of integrin-based adhesions, suggesting that Arp2/3-mediated actin polymerization and integrin-dependent adhesion may be mechanistically linked. Arp2/3 also mediates actin polymerization in structures distinct from the lamellipodium, in “ventral F-actin waves” that propagate as spots and wavefronts along the ventral plasma membrane. Here we show that integrins engage the extracellular matrix downstream of ventral F-actin waves in several mammalian cell lines as well as in primary mouse embryonic fibroblasts. These “adhesive F-actin waves” require a cycle of integrin engagement and disengagement to the extracellular matrix for their formation and propagation, and exhibit morphometry and a hierarchical assembly and disassembly mechanism distinct from other integrin-containing structures. After Arp2/3-mediated actin polymerization, zyxin and VASP are co-recruited to adhesive F-actin waves, followed by paxillin and vinculin, and finally talin and integrin. Adhesive F-actin waves thus represent a previously uncharacterized integrin-based adhesion complex associated with Arp2/3-mediated actin polymerization. PMID:22069459

A genome-wide analysis reveals that the Drosophila transcription factor Lola promotes axon growth in part by suppressing expression of the actin nucleation factor Spire

PubMed Central

2011-01-01

Background The phylogenetically conserved transcription factor Lola is essential for many aspects of axon growth and guidance, synapse formation and neural circuit development in Drosophila. To date it has been difficult, however, to obtain an overall view of Lola functions and mechanisms. Results We use expression microarrays to identify the lola-dependent transcriptome in the Drosophila embryo. We find that lola regulates the expression of a large selection of genes that are known to affect each of several lola-dependent developmental processes. Among other loci, we find lola to be a negative regulator of spire, an actin nucleation factor that has been studied for its essential role in oogenesis. We show that spire is expressed in the nervous system and is required for a known lola-dependent axon guidance decision, growth of ISNb motor axons. We further show that reducing spire gene dosage suppresses this aspect of the lola phenotype, verifying that derepression of spire is an important contributor to the axon stalling phenotype of embryonic motor axons in lola mutants. Conclusions These data shed new light on the molecular mechanisms of many lola-dependent processes, and also identify several developmental processes not previously linked to lola that are apt to be regulated by this transcription factor. These data further demonstrate that excessive expression of the actin nucleation factor Spire is as deleterious for axon growth in vivo as is the loss of Spire, thus highlighting the need for a balance in the elementary steps of actin dynamics to achieve effective neuronal morphogenesis. PMID:22129300

A genome-wide analysis reveals that the Drosophila transcription factor Lola promotes axon growth in part by suppressing expression of the actin nucleation factor Spire.

PubMed

Gates, Michael A; Kannan, Ramakrishnan; Giniger, Edward

2011-11-30

The phylogenetically conserved transcription factor Lola is essential for many aspects of axon growth and guidance, synapse formation and neural circuit development in Drosophila. To date it has been difficult, however, to obtain an overall view of Lola functions and mechanisms. We use expression microarrays to identify the lola-dependent transcriptome in the Drosophila embryo. We find that lola regulates the expression of a large selection of genes that are known to affect each of several lola-dependent developmental processes. Among other loci, we find lola to be a negative regulator of spire, an actin nucleation factor that has been studied for its essential role in oogenesis. We show that spire is expressed in the nervous system and is required for a known lola-dependent axon guidance decision, growth of ISNb motor axons. We further show that reducing spire gene dosage suppresses this aspect of the lola phenotype, verifying that derepression of spire is an important contributor to the axon stalling phenotype of embryonic motor axons in lola mutants. These data shed new light on the molecular mechanisms of many lola-dependent processes, and also identify several developmental processes not previously linked to lola that are apt to be regulated by this transcription factor. These data further demonstrate that excessive expression of the actin nucleation factor Spire is as deleterious for axon growth in vivo as is the loss of Spire, thus highlighting the need for a balance in the elementary steps of actin dynamics to achieve effective neuronal morphogenesis.

Radiation-induced DNA-protein cross-links: Mechanisms and biological significance.

PubMed

Nakano, Toshiaki; Xu, Xu; Salem, Amir M H; Shoulkamy, Mahmoud I; Ide, Hiroshi

2017-06-01

Ionizing radiation produces various DNA lesions such as base damage, DNA single-strand breaks (SSBs), DNA double-strand breaks (DSBs), and DNA-protein cross-links (DPCs). Of these, the biological significance of DPCs remains elusive. In this article, we focus on radiation-induced DPCs and review the current understanding of their induction, properties, repair, and biological consequences. When cells are irradiated, the formation of base damage, SSBs, and DSBs are promoted in the presence of oxygen. Conversely, that of DPCs is promoted in the absence of oxygen, suggesting their importance in hypoxic cells, such as those present in tumors. DNA and protein radicals generated by hydroxyl radicals (i.e., indirect effect) are responsible for DPC formation. In addition, DPCs can also be formed from guanine radical cations generated by the direct effect. Actin, histones, and other proteins have been identified as cross-linked proteins. Also, covalent linkages between DNA and protein constituents such as thymine-lysine and guanine-lysine have been identified and their structures are proposed. In irradiated cells and tissues, DPCs are repaired in a biphasic manner, consisting of fast and slow components. The half-time for the fast component is 20min-2h and that for the slow component is 2-70h. Notably, radiation-induced DPCs are repaired more slowly than DSBs. Homologous recombination plays a pivotal role in the repair of radiation-induced DPCs as well as DSBs. Recently, a novel mechanism of DPC repair mediated by a DPC protease was reported, wherein the resulting DNA-peptide cross-links were bypassed by translesion synthesis. The replication and transcription of DPC-bearing reporter plasmids are inhibited in cells, suggesting that DPCs are potentially lethal lesions. However, whether DPCs are mutagenic and induce gross chromosomal alterations remains to be determined. Copyright © 2017 Elsevier Inc. All rights reserved.

Nucleation of actin polymerization by gelsolin.

PubMed

Ditsch, A; Wegner, A

1994-08-15

The time-course of assembly of actin with gelsolin was measured by the fluorescence increase of a fluorescent label covalently linked to actin. The actin concentrations ranged from values far below the critical concentration to values above the critical concentration of the pointed ends of actin filaments. If the concentration of actin was in the range of the critical monomer concentration (0.64 microM), the time-course of the concentration of actin assembled with gelsolin revealed a sigmoidal shape. At higher actin concentrations the time-course of association of actin with gelsolin approximated an exponential curve. The measured time-courses of assembly were quantitatively interpreted by kinetic rate equations. A poor fit was obtained if two actin molecules were assumed to bind to gelsolin to form a 1:2 gelsolin-actin complex and subsequently further actin molecules were assumed to polymerize onto the 1:2 gelsolin-actin complex toward the pointed end. A considerably better agreement between calculated and measured time-courses was achieved if additional creation of actin filaments by fast fragmentation of newly formed actin filaments by not yet consumed gelsolin was assumed to occur. This suggests that both polymerization of actin onto gelsolin and fragmentation of actin filaments contribute to formation of new actin filaments by gelsolin. Furthermore it could be demonstrated that below the critical monomer concentration appreciable amounts of actin are incorporated into gelsolin-actin oligomers.

Reconstitution of actin-based motility of Listeria and Shigella using pure proteins

NASA Astrophysics Data System (ADS)

Loisel, Thomas P.; Boujemaa, Rajaa; Pantaloni, Dominique; Carlier, Marie-France

1999-10-01

Actin polymerization is essential for cell locomotion and is thought to generate the force responsible for cellular protrusions. The Arp2/3 complex is required to stimulate actin assembly at the leading edge in response to signalling. The bacteria Listeria and Shigella bypass the signalling pathway and harness the Arp2/3 complex to induce actin assembly and to propel themselves in living cells. However, the Arp2/3 complex alone is insufficient to promote movement. Here we have used pure components of the actin cytoskeleton to reconstitute sustained movement in Listeria and Shigella in vitro. Actin-based propulsion is driven by the free energy released by ATP hydrolysis linked to actin polymerization, and does not require myosin. In addition to actin and activated Arp2/3 complex, actin depolymerizing factor (ADF, or cofilin) and capping protein are also required for motility as they maintain a high steady-state level of G-actin, which controls the rate of unidirectional growth of actin filaments at the surface of the bacterium. The movement is more effective when profilin, α-actinin and VASP (for Listeria) are also included. These results have implications for our understanding of the mechanism of actin-based motility in cells.

Co-transcriptional nuclear actin dynamics

PubMed Central

Percipalle, Piergiorgio

2013-01-01

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

Cross-Link Guided Molecular Modeling with ROSETTA

PubMed Central

Leitner, Alexander; Rosenberger, George; Aebersold, Ruedi; Malmström, Lars

2013-01-01

Chemical cross-links identified by mass spectrometry generate distance restraints that reveal low-resolution structural information on proteins and protein complexes. The technology to reliably generate such data has become mature and robust enough to shift the focus to the question of how these distance restraints can be best integrated into molecular modeling calculations. Here, we introduce three workflows for incorporating distance restraints generated by chemical cross-linking and mass spectrometry into ROSETTA protocols for comparative and de novo modeling and protein-protein docking. We demonstrate that the cross-link validation and visualization software Xwalk facilitates successful cross-link data integration. Besides the protocols we introduce XLdb, a database of chemical cross-links from 14 different publications with 506 intra-protein and 62 inter-protein cross-links, where each cross-link can be mapped on an experimental structure from the Protein Data Bank. Finally, we demonstrate on a protein-protein docking reference data set the impact of virtual cross-links on protein docking calculations and show that an inter-protein cross-link can reduce on average the RMSD of a docking prediction by 5.0 Å. The methods and results presented here provide guidelines for the effective integration of chemical cross-link data in molecular modeling calculations and should advance the structural analysis of particularly large and transient protein complexes via hybrid structural biology methods. PMID:24069194

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

PubMed Central

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

2015-01-01

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

Covalent cross-linking of insulin-like growth factor-1 to a specific inhibitor from human serum

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

Ooi, G.T.; Herington, A.C.

1986-05-29

Previous studies have shown that a specific inhibitor of insulin-like growth factor (IGF) action in vitro can be isolated from normal human serum and subsequently partially purified on an IGF-affinity column. The ability of the inhibitor to bind the IGFs has now been confirmed directly using covalent cross-linking techniques. When /sup 125/I-IGF-1 was cross-linked to inhibitor using disuccinimidyl suberate, five specifically labelled bands were seen on SDS-PAGE and autoradiography. Two bands (MW 21.5 K and 25.5 K) were intensely labelled, while the remaining three (MW 37 K, 34 K and 18 K) appeared as minor bands only. Inhibitor bioactivity, followingmore » further analysis by hydrophobic interaction chromatography or Con A-Sepharose affinity chromatography, was always associated with the presence of the 21.5 K and/or 25.5 K bands.« less

Treatment of Ras-induced cancers by the F-actin cappers tensin and chaetoglobosin K, in combination with the caspase-1 inhibitor N1445.

PubMed

Tikoo, A; Cutler, H; Lo, S H; Chen, L B; Maruta, H

1999-01-01

For transforming normal fibroblasts to malignant cells, oncogenic Ras mutants such as v-Ha-ras require Rho family GTPases (Rho, Rac, and CDC42) that are responsible for controlling actin-cytoskeleton organization. Ras activates Rac through a PI-3 kinase-mediated pathway. Rac causes uncapping of actin filaments (F-actin) at the plus-ends, through phosphatidylinositol 4,5 bisphosphate (PIP2), and eventually induces membrane ruffling. Several distinct F-actin/PIP2-binding proteins, such as gelsolin, which severs and caps the plus-ends of actin filaments, or HS1, which cross-links actin filaments, have been shown to suppress v-Ha-Ras-induced malignant transformation when they are overexpressed. Interestingly, an F-actin cross-linking drug (photosensitizer) called MKT-077 suppresses Ras transformation. Thus, an F-actin capping/severing drug might also have an anticancer potential. This study was conducted to determine first whether Ras-induced malignant phenotype (anchorage-independent growth) is suppressed by overexpression of the gene encoding a large plus-end F-actin capping protein called tensin and second to test the anti-Ras potential of a unique fungal antibiotic (small compound) called chaetoglobosin K (CK) that also caps the plus-ends of actin filaments. DNA transfection with a retroviral vector carrying the tensin cDNA was used to overexpress tensin in v-Ha-Ras-transformed NIH 3T3 cells. All stable tensin transfectants rarely formed colonies in soft agar, indicating that tensin suppresses the anchorage-independent growth. The anti-Ras action of CK was determined by incubating the Ras-transformants in the presence of CK in soft agar. Two microM CK almost completely inhibited their colony formation, indicating that CK also suppresses the malignant phenotype. However, unlike tensin, CK causes an apoptosis of Ras-transformed NIH 3T3 cells and, less effectively, of normal NIH 3T3 cells, indicating that CK has an F-actin capping-independent side effect(s). CK

FSGS3/CD2AP is a barbed-end capping protein that stabilizes actin and strengthens adherens junctions

PubMed Central

Brieher, William M.

2013-01-01

By combining in vitro reconstitution biochemistry with a cross-linking approach, we have identified focal segmental glomerulosclerosis 3/CD2-associated protein (FSGS3/CD2AP) as a novel actin barbed-end capping protein responsible for actin stability at the adherens junction. FSGS3/CD2AP colocalizes with E-cadherin and α-actinin-4 at the apical junction in polarized Madin-Darby canine kidney (MDCK) cells. Knockdown of FSGS3/CD2AP compromised actin stability and decreased actin accumulation at the adherens junction. Using a novel apparatus to apply mechanical stress to cell–cell junctions, we showed that knockdown of FSGS3/CD2AP compromised adhesive strength, resulting in tearing between cells and disruption of barrier function. Our results reveal a novel function of FSGS3/CD2AP and a previously unrecognized role of barbed-end capping in junctional actin dynamics. Our study underscores the complexity of actin regulation at cell–cell contacts that involves actin activators, inhibitors, and stabilizers to control adhesive strength, epithelial behavior, and permeability barrier integrity. PMID:24322428

Tip-localized actin polymerization and remodeling, reflected by the localization of ADF, profilin and villin, are fundamental for gravity-sensing and polar growth in characean rhizoids.

PubMed

Braun, Markus; Hauslage, Jens; Czogalla, Aleksander; Limbach, Christoph

2004-07-01

Polar organization and gravity-oriented, polarized growth of characean rhizoids are dependent on the actin cytoskeleton. In this report, we demonstrate that the prominent center of the Spitzenkörper serves as the apical actin polymerization site in the extending tip. After cytochalasin D-induced disruption of the actin cytoskeleton, the regeneration of actin microfilaments (MFs) starts with the reappearance of a flat, brightly fluorescing actin array in the outermost tip. The actin array rounds up, produces actin MFs that radiate in all directions and is then relocated into its original central position in the center of the Spitzenkörper. The emerging actin MFs rearrange and cross-link to form the delicate, subapical meshwork, which then controls the statolith positioning, re-establishes the tip-high calcium gradient and mediates the reorganization of the Spitzenkörper with its central ER aggregate and the accumulation of secretory vesicles. Tip growth and gravitropic sensing, which includes control of statolith positioning and gravity-induced sedimentation, are not resumed until the original polar actin organization is completely restored. Immunolocalization of the actin-binding proteins, actin-depolymerizing factor (ADF) and profilin, which both accumulate in the center of the Spitzenkörper, indicates high actin turnover and gives additional support for the actin-polymerizing function of this central, apical area. Association of villin immunofluorescence with two populations of thick undulating actin cables with uniform polarity underlying rotational cytoplasmic streaming in the basal region suggests that villin is the major actin-bundling protein in rhizoids. Our results provide evidence that the precise coordination of apical actin polymerization and dynamic remodeling of actin MFs by actin-binding proteins play a fundamental role in cell polarization, gravity sensing and gravity-oriented polarized growth of characean rhizoids.

Impact of branching on the elasticity of actin networks

PubMed Central

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

2012-01-01

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

N(4)C-ethyl-N(4)C cross-linked DNA: synthesis and characterization of duplexes with interstrand cross-links of different orientations.

PubMed

Noronha, Anne M; Noll, David M; Wilds, Christopher J; Miller, Paul S

2002-01-22

The preparation and physical properties of short DNA duplexes that contain a N(4)C-ethyl-N(4)C interstrand cross-link are described. Duplexes that contain an interstrand cross-link between mismatched C-C residues and duplexes in which the C residues of a -CG- or -GC- step are linked to give "staggered" interstrand cross-links were prepared using a novel N(4)C-ethyl-N(4)C phosphoramidite reagent. Duplexes with the C-C mismatch cross-link have UV thermal transition temperatures that are 25 degrees C higher than the melting temperatures of control duplexes in which the cross-link is replaced with a G-C base pair. It appears that this cross-link stabilizes adjacent base pairs and does not perturb the structure of the helix, a conclusion that is supported by the CD spectrum of this duplex and by molecular models. An even higher level of stabilization, 49 degrees C, is seen with the duplex that contains a -CG- staggered cross-link. Molecular models suggest that this cross-link may induce propeller twisting in the cross-linked base pairs, and the CD spectrum of this duplex exhibits an unusual negative band at 298 nm, although the remainder of the spectrum is similar to that of B-form DNA. Mismatched C-C or -CG- staggered cross-linked duplexes that have complementary overhanging ends can undergo self-ligation catalyzed by T4 DNA ligase. Analysis of the ligated oligomers by nondenaturing polyacrylamide gel electrophoresis shows that the resulting oligomers migrate in a manner similar to that of a mixture of non-cross-linked control oligomers and suggests that these cross-links do not result in significant bending of the helix. However, the orientation of the staggered cross-link can have a significant effect on the structure and stability of the cross-linked duplex. Thus, the thermal stability of the duplex that contains a -GC- staggered cross-link is 10 degrees C lower than the melting temperature of the control, non-cross-linked duplex. Unlike the -CG- staggered cross-link

Cross-linking of initiation factor IF3 to Escherichia coli 30S ribosomal subunit by trans-diamminedichloroplatinum(II): characterization of two cross-linking sites in 16S rRNA; a possible way of functioning for IF3.

PubMed Central

Ehresmann, C; Moine, H; Mougel, M; Dondon, J; Grunberg-Manago, M; Ebel, J P; Ehresmann, B

1986-01-01

The initiation factor IF3 is platinated with trans-diamminedichloroplatinum(II) and cross-linked to Escherichia coli 30S ribosomal subunit. Two cross-linking sites are unambiguously identified on the 16S rRNA: a major one, in the region 819-859 in the central domain, and a minor one, in the region 1506-1529 in the 3'-terminal domain. Specific features of these sequences together with their particular location within the 30S subunit lead us to postulate a role for IF3, that conciliates topographical and functional observations made so far. Images PMID:2425339

Actin dynamics involved in gravity perception in Arabidopsis inflorescense stem

NASA Astrophysics Data System (ADS)

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

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

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

PubMed Central

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

2013-01-01

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

From Cytoskeleton to Gene Expression: Actin in the Nucleus.

PubMed

Viita, Tiina; Vartiainen, Maria K

2017-01-01

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

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

PubMed Central

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

2017-01-01

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

Cross-linking Chemistry of Squid Beak*

PubMed Central

Miserez, Ali; Rubin, Daniel; Waite, J. Herbert

2010-01-01

In stark contrast to most aggressive predators, Dosidicus gigas (jumbo squids) do not use minerals in their powerful mouthparts known as beaks. Their beaks instead consist of a highly sclerotized chitinous composite with incremental hydration from the tip to the base. We previously reported l-3,4-dihydroxyphenylalanine (dopa)-histidine (dopa-His) as an important covalent cross-link providing mechanical strengthening to the beak material. Here, we present a more complete characterization of the sclerotization chemistry and describe additional cross-links from D. gigas beak. All cross-links presented in this report share common building blocks, a family of di-, tri-, and tetra-histidine-catecholic adducts, that were separated by affinity chromatography and high performance liquid chromatography (HPLC) and identified by tandem mass spectroscopy and proton nuclear magnetic resonance (1H NMR). The data provide additional insights into the unusually high cross-link density found in mature beaks. Furthermore, we propose both a low molecular weight catechol, and peptidyl-dopa, to be sclerotization agents of squid beak. This appears to represent a new strategy for forming hard tissue in animals. The interplay between covalent cross-linking and dehydration on the graded properties of the beaks is discussed. PMID:20870720

Cross-linking chemistry of squid beak.

PubMed

Miserez, Ali; Rubin, Daniel; Waite, J Herbert

2010-12-03

In stark contrast to most aggressive predators, Dosidicus gigas (jumbo squids) do not use minerals in their powerful mouthparts known as beaks. Their beaks instead consist of a highly sclerotized chitinous composite with incremental hydration from the tip to the base. We previously reported l-3,4-dihydroxyphenylalanine (dopa)-histidine (dopa-His) as an important covalent cross-link providing mechanical strengthening to the beak material. Here, we present a more complete characterization of the sclerotization chemistry and describe additional cross-links from D. gigas beak. All cross-links presented in this report share common building blocks, a family of di-, tri-, and tetra-histidine-catecholic adducts, that were separated by affinity chromatography and high performance liquid chromatography (HPLC) and identified by tandem mass spectroscopy and proton nuclear magnetic resonance ((1)H NMR). The data provide additional insights into the unusually high cross-link density found in mature beaks. Furthermore, we propose both a low molecular weight catechol, and peptidyl-dopa, to be sclerotization agents of squid beak. This appears to represent a new strategy for forming hard tissue in animals. The interplay between covalent cross-linking and dehydration on the graded properties of the beaks is discussed.

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

PubMed Central

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

1997-01-01

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

Actinous enigma or enigmatic actin

PubMed Central

Povarova, Olga I; Uversky, Vladimir N; Kuznetsova, Irina M; Turoverov, Konstantin K

2014-01-01

Being the most abundant protein of the eukaryotic cell, actin continues to keep its secrets for more than 60 years. Everything about this protein, its structure, functions, and folding, is mysteriously counterintuitive, and this review represents an attempt to solve some of the riddles and conundrums commonly found in the field of actin research. In fact, actin is a promiscuous binder with a wide spectrum of biological activities. It can exist in at least three structural forms, globular, fibrillar, and inactive (G-, F-, and I-actin, respectively). G-actin represents a thermodynamically instable, quasi-stationary state, which is formed in vivo as a result of the energy-intensive, complex posttranslational folding events controlled and driven by cellular folding machinery. The G-actin structure is dependent on the ATP and Mg2+ binding (which in vitro is typically substituted by Ca2+) and protein is easily converted to the I-actin by the removal of metal ions and by action of various denaturing agents (pH, temperature, and chemical denaturants). I-actin cannot be converted back to the G-form. Foldable and “natively folded” forms of actin are always involved in interactions either with the specific protein partners, such as Hsp70 chaperone, prefoldin, and the CCT chaperonin during the actin folding in vivo or with Mg2+ and ATP as it takes place in the G-form. We emphasize that the solutions for the mysteries of actin multifunctionality, multistructurality, and trapped unfolding can be found in the quasi-stationary nature of this enigmatic protein, which clearly possesses many features attributed to both globular and intrinsically disordered proteins. PMID:28232879

FERMT2 links cortical actin structures, plasma membrane tension and focal adhesion function to stabilize podocyte morphology.

PubMed

Yasuda-Yamahara, M; Rogg, M; Frimmel, J; Trachte, P; Helmstaedter, M; Schroder, P; Schiffer, M; Schell, C; Huber, T B

2018-01-11

Simplification and retraction of podocyte protrusions, generally termed as foot process effacement, is a uniform pathological pattern observed in the majority of glomerular disease, including focal segmental glomerulosclerosis. However, it is still incompletely understood how the interaction of cortical actin structures, actomyosin contractility and focal adhesions, is being orchestrated to control foot process morphology in health and disease. By uncovering the functional role of fermitin family member 2 (FERMT2 or kindlin-2) in podocytes, we provide now evidence, how cell-extracellular matrix (ECM) interactions modulate membrane tension and actomyosin contractility. A genetic modeling approach was applied by deleting FERMT2 in a set of in vivo systems as well as in CRISPR/Cas9 modified human podocytes. Loss of FERMT2 results in altered cortical actin composition, cell cortex destabilization associated with plasma membrane blebbing and a remodeling of focal adhesions. We further show that FERMT2 knockout podocytes have high levels of RhoA activation and concomitantly increased actomyosin contractility. Inhibition of actomyosin tension reverses the membrane blebbing phenotype. Thus, our findings establish a direct link between cell-matrix adhesions, cortical actin structures and plasma membrane tension allowing to better explain cell morphological changes in foot process effacement. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Nano and micro mechanical properties of uncross-linked and cross-linked chitosan films

PubMed Central

Aryaei, Ashkan; Jayatissa, Ahalapitiya H.; Jayasuriya, A. Champa

2016-01-01

The aim of this study is to determine the nano and micro mechanical properties for uncross-linked and cross-linked chitosan films. Specifically, we looked at nanoindentation hardness, microhardness, and elastic modulus. It is important to study the nano and microscale mechanical properties of chitosan since chitosan has been widely used for biomedical applications. Using the solvent-cast method, the chitosan films were prepared at room temperature on the cleaned glass plates. The chitosan solution was prepared by dissolving chitosan in acetic acid 1% (v/v). Tripolyphosphate (TPP) was used to create the cross-links between amine groups in chitosan and phosphate groups in TPP. In this study, atomic force microscopy was used to measure the nanoindentation hardness and surface topography of the uncross-linked and cross-linked chitosan films. Elastic modulus was then calculated from the nanoindentation results. The effective elastic modulus was determined by microhardness with some modifications to previous theories. The microhardness of the chitosan films were measured using Vicker’s hardness meter under three different loads. Our results show that the microhardness and elastic modulus for cross-linked chitosan films are higher than the uncross-linked films. However, the cross-linked chitosan films show increased brittleness when compared to uncross-linked films. By increasing the load magnitude, the microhardness increases for both uncross-linked and cross-linked chitosan films. PMID:22100082

A dynamic formin-dependent deep F-actin network in axons

PubMed Central

Ganguly, Archan; Tang, Yong; Wang, Lina; Ladt, Kelsey; Loi, Jonathan; Dargent, Bénédicte; Leterrier, Christophe

2015-01-01

Although actin at neuronal growth cones is well-studied, much less is known about actin organization and dynamics along axon shafts and presynaptic boutons. Using probes that selectively label filamentous-actin (F-actin), we found focal “actin hotspots” along axons—spaced ∼3–4 µm apart—where actin undergoes continuous assembly/disassembly. These foci are a nidus for vigorous actin polymerization, generating long filaments spurting bidirectionally along axons—a phenomenon we call “actin trails.” Super-resolution microscopy reveals intra-axonal deep actin filaments in addition to the subplasmalemmal “actin rings” described recently. F-actin hotspots colocalize with stationary axonal endosomes, and blocking vesicle transport diminishes the actin trails, suggesting mechanistic links between vesicles and F-actin kinetics. Actin trails are formin—but not Arp2/3—dependent and help enrich actin at presynaptic boutons. Finally, formin inhibition dramatically disrupts synaptic recycling. Collectively, available data suggest a two-tier F-actin organization in axons, with stable “actin rings” providing mechanical support to the plasma membrane and dynamic "actin trails" generating a flexible cytoskeletal network with putative physiological roles. PMID:26216902

Recent advances in corneal collagen cross-linking

PubMed Central

Sachdev, Gitansha Shreyas; Sachdev, Mahipal

2017-01-01

Corneal collagen cross-linking has become the preferred modality of treatment for corneal ectasia since its inception in late 1990s. Numerous studies have demonstrated the safety and efficacy of the conventional protocol. Our understanding of the cross-linking process is ever evolving, with its wide implications in the form of accelerated and pulsed protocols. Newer advancements in technology include various riboflavin formulations and the ability to deliver higher fluence protocols with customised irradiation patterns. A greater degree of customisation is likely the path forward, which will aim at achieving refractive improvements along with disease stability. The use of cross-linking for myopic correction is another avenue under exploration. Combination of half fluence cross-linking with refractive correction for high errors to prevent post LASIK regression is gaining interest. This review aims to highlight the various advancements in the cross-linking technology and its clinical applications. PMID:28905820

Drosophila melanogaster muscle LIM protein and alpha-actinin function together to stabilize muscle cytoarchitecture: a potential role for Mlp84B in actin-crosslinking.

PubMed

Clark, Kathleen A; Kadrmas, Julie L

2013-06-01

Stabilization of tissue architecture during development and growth is essential to maintain structural integrity. Because of its contractile nature, muscle is especially susceptible to physiological stresses, and has multiple mechanisms to maintain structural integrity. The Drosophila melanogaster Muscle LIM Protein (MLP), Mlp84B, participates in muscle maintenance, yet its precise mechanism of action is still controversial. Through a candidate approach, we identified α-actinin as a protein that functions with Mlp84B to ensure muscle integrity. α-actinin RNAi animals die primarily as pupae, and Mlp84B RNAi animals are adult viable. RNAi knockdown of Mlp84B and α-actinin together produces synergistic early larval lethality and destabilization of Z-line structures. We recapitulated these phenotypes using combinations of traditional loss-of-function alleles and single-gene RNAi. We observe that Mlp84B induces the formation of actin loops in muscle cell nuclei in the absence of nuclear α-actinin, suggesting Mlp84B has intrinsic actin cross-linking activity, which may complement α-actinin cross-linking activity at sites of actin filament anchorage. These results reveal a molecular mechanism for MLP stabilization of muscle and implicate reduced actin crosslinking as the primary destabilizing defect in MLP-associated cardiomyopathies. Our data support a model in which α-actinin and Mlp84B have important and overlapping functions at sites of actin filament anchorage to preserve muscle structure and function. Copyright © 2013 Wiley Periodicals, Inc.

Cytoplasmic γ-actin and tropomodulin isoforms link to the sarcoplasmic reticulum in skeletal muscle fibers

PubMed Central

Gokhin, David S.

2011-01-01

The sarcoplasmic reticulum (SR) serves as the Ca2+ reservoir for muscle contraction. Tropomodulins (Tmods) cap filamentous actin (F-actin) pointed ends, bind tropomyosins (Tms), and regulate F-actin organization. In this paper, we use a genetic targeting approach to examine the effect of Tmod1 deletion on the organization of cytoplasmic γ-actin (γcyto-actin) in the SR of skeletal muscle. In wild-type muscle fibers, γcyto-actin and Tmod3 defined an SR microdomain that was distinct from another Z line–flanking SR microdomain containing Tmod1 and Tmod4. The γcyto-actin/Tmod3 microdomain contained an M line complex composed of small ankyrin 1.5 (sAnk1.5), γcyto-actin, Tmod3, Tm4, and Tm5NM1. Tmod1 deletion caused Tmod3 to leave its SR compartment, leading to mislocalization and destabilization of the Tmod3–γcyto-actin–sAnk1.5 complex. This was accompanied by SR morphological defects, impaired Ca2+ release, and an age-dependent increase in sarcomere misalignment. Thus, Tmod3 regulates SR-associated γcyto-actin architecture, mechanically stabilizes the SR via a novel cytoskeletal linkage to sAnk1.5, and maintains the alignment of adjacent myofibrils. PMID:21727195

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

PubMed

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

2015-08-25

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

Structural interactions between retroviral Gag proteins examined by cysteine cross-linking.

PubMed Central

Hansen, M S; Barklis, E

1995-01-01

We have examined structural interactions between Gag proteins within Moloney murine leukemia virus (M-MuLV) particles by making use of the cysteine-specific cross-linking agents iodine and bis-maleimido hexane. Virion-associated wild-type M-MuLV Pr65Gag proteins in immature particles were intermolecularly cross-linked at cysteines to form Pr65Gag oligomers, from dimers to pentamers or hexamers. Following a systematic approach of cysteine-to-serine mutagenesis, we have shown that cross-linking of Pr65Gag occurred at cysteines of the nucleocapsid (NC) Cys-His motif, suggesting that the Cys-His motifs within virus particles are packed in close proximity. The M-MuLV Pr65Gag protein did not cross-link to the human immunodeficiency virus Pr55Gag protein when the two molecules were coexpressed, indicating either that they did not coassemble or that heterologous Gag proteins were not in close enough proximity to be cross-linked. Using an assembly-competent, protease-minus, cysteine-minus Pr65Gag protein as a template, novel cysteine residues were generated in the M-MuLV capsid domain major homology region (MHR). Cross-linking of proteins containing MHR cysteines showed above-background levels of Gag-Gag dimers but also identified a novel cellular factor, present in virions, that cross-linked to MHR residues. Although the NC cysteine mutation was compatible with M-MuLV particle assembly, deletions of the NC domain were not tolerated. These results suggest that the Cys-His motif is held in close proximity within immature M-MuLV particles by interactions between CA domains and/or non-Cys-His motif domains of the NC. PMID:7815493

Porous Cross-Linked Polyimide-Urea Networks

NASA Technical Reports Server (NTRS)

Meador, Mary Ann B. (Inventor); Nguyen, Baochau N. (Inventor)

2015-01-01

Porous cross-linked polyimide-urea networks are provided. The networks comprise a subunit comprising two anhydride end-capped polyamic acid oligomers in direct connection via a urea linkage. The oligomers (a) each comprise a repeating unit of a dianhydride and a diamine and a terminal anhydride group and (b) are formulated with 2 to 15 of the repeating units. The subunit was formed by reaction of the diamine and a diisocyanate to form a diamine-urea linkage-diamine group, followed by reaction of the diamine-urea linkage-diamine group with the dianhydride and the diamine to form the subunit. The subunit has been cross-linked via a cross-linking agent, comprising three or more amine groups, at a balanced stoichiometry of the amine groups to the terminal anhydride groups. The subunit has been chemically imidized to yield the porous cross-linked polyimide-urea network. Also provided are wet gels, aerogels, and thin films comprising the networks, and methods of making the networks.

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

21 CFR 177.1211 - Cross-linked polyacrylate copolymers.

Code of Federal Regulations, 2011 CFR

2011-04-01

... polyacrylate copolymers consist of: (1) The grafted copolymer of cross-linked sodium polyacrylate identified as... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Cross-linked polyacrylate copolymers. 177.1211... Basic Components of Single and Repeated Use Food Contact Surfaces § 177.1211 Cross-linked polyacrylate...

21 CFR 177.1211 - Cross-linked polyacrylate copolymers.

Code of Federal Regulations, 2010 CFR

2010-04-01

... polyacrylate copolymers consist of: (1) The grafted copolymer of cross-linked sodium polyacrylate identified as... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Cross-linked polyacrylate copolymers. 177.1211... Basic Components of Single and Repeated Use Food Contact Surfaces § 177.1211 Cross-linked polyacrylate...

Green fluorescent protein fusions to Arabidopsis fimbrin 1 for spatio-temporal imaging of F-actin dynamics in roots.

PubMed

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

2004-10-01

The visualization of green fluorescent protein (GFP) fusions with microtubule or actin filament (F-actin) binding proteins has provided new insights into the function of the cytoskeleton during plant development. For studies on actin, GFP fusions to talin have been the most generally used reporters. Although GFP-Talin has allowed in vivo F-actin imaging in a variety of plant cells, its utility in monitoring F-actin in stably transformed plants is limited particularly in developing roots where interesting actin dependent cell processes are occurring. In this study, we created a variety of GFP fusions to Arabidopsis Fimbrin 1 (AtFim1) to explore their utility for in vivo F-actin imaging in root cells and to better understand the actin binding properties of AtFim1 in living plant cells. Translational fusions of GFP to full-length AtFim1 or to some truncated variants of AtFim1 showed filamentous labeling in transient expression assays. One truncated fimbrin-GFP fusion was capable of labeling distinct filaments in stably transformed Arabidopsis roots. The filaments decorated by this construct were highly dynamic in growing root hairs and elongating root cells and were sensitive to actin disrupting drugs. Therefore, the fimbrin-GFP reporters we describe in this study provide additional tools for studying the actin cytoskeleton during root cell development. Moreover, the localization of AtFim1-GFP offers insights into the regulation of actin organization in developing roots by this class of actin cross-linking proteins. Copyright 2004 Wiley-Liss, Inc.

Computational investigation of kinetics of cross-linking reactions in proteins: importance in structure prediction.

PubMed

Bandyopadhyay, Pradipta; Kuntz, Irwin D

2009-01-01

The determination of protein structure using distance constraints is a new and promising field of study. One implementation involves attaching residues of a protein using a cross-linking agent, followed by protease digestion, analysis of the resulting peptides by mass spectroscopy, and finally sequence threading to detect the protein folds. In the present work, we carry out computational modeling of the kinetics of cross-linking reactions in proteins using the master equation approach. The rate constants of the cross-linking reactions are estimated using the pKas and the solvent-accessible surface areas of the residues involved. This model is tested with fibroblast growth factor (FGF) and cytochrome C. It is consistent with the initial experimental rate data for individual lysine residues for cytochrome C. Our model captures all observed cross-links for FGF and almost 90% of the observed cross-links for cytochrome C, although it also predicts cross-links that were not observed experimentally (false positives). However, the analysis of the false positive results is complicated by the fact that experimental detection of cross-links can be difficult and may depend on specific experimental conditions such as pH, ionic strength. Receiver operator characteristic plots showed that our model does a good job in predicting the observed cross-links. Molecular dynamics simulations showed that for cytochrome C, in general, the two lysines come closer for the observed cross-links as compared to the false positive ones. For FGF, no such clear pattern exists. The kinetic model and MD simulation can be used to study proposed cross-linking protocols.

Encoding mechano-memories in filamentous-actin networks

NASA Astrophysics Data System (ADS)

Majumdar, Sayantan; Foucard, Louis; Levine, Alex; Gardel, Margaret L.

History-dependent adaptation is a central feature of learning and memory. Incorporating such features into `adaptable materials' that can modify their mechanical properties in response to external cues, remains an outstanding challenge in materials science. Here, we study a novel mechanism of mechano-memory in cross-linked F-actin networks, the essential determinants of the mechanical behavior of eukaryotic cells. We find that the non-linear mechanical response of such networks can be reversibly programmed through induction of mechano-memories. In particular, the direction, magnitude, and duration of previously applied shear stresses can be encoded into the network architecture. The `memory' of the forcing history is long-lived, but it can be erased by force applied in the opposite direction. These results demonstrate that F-actin networks can encode analog read-write mechano-memories which can be used for adaptation to mechanical stimuli. We further show that the mechano-memory arises from changes in the nematic order of the constituent filaments. Our results suggest a new mechanism of mechanical sensing in eukaryotic cells and provide a strategy for designing a novel class of materials. S.M. acknowledges U. Chicago MRSEC for support through a Kadanoff-Rice fellowship.

The chain of cross-contamination: link-by-link.

PubMed

Schwartz, Jeanne

2002-01-01

Conscious efforts must be made to break the chain of cross-contamination--link-by-link. Pay attention to detail Avoid being careless Avoid touching objects while wearing soiled gloves Frequently wash hands (15-second hand washing) to remove pathogens--before and after gloving, and before handling food or drink Wear all PPE, and change accordingly.

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

PubMed Central

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

2015-01-01

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

Control of the actin cytoskeleton in root hair development.

PubMed

Pei, Weike; Du, Fei; Zhang, Yi; He, Tian; Ren, Haiyun

2012-05-01

The development of root hair includes four stages: bulge site selection, bulge formation, tip growth, and maturation. The actin cytoskeleton is involved in all of these stages and is organized into distinct arrangements in the different stages. In addition to the actin configuration, actin isoforms also play distinct roles in the different stages. The actin cytoskeleton is regulated by actin-binding proteins, such as formin, Arp2/3 complex, profilin, actin depolymerizing factor, and villin. Some upstream signals, i.e. calcium, phospholipids, and small GTPase regulate the activity of these actin-binding proteins to produce the proper actin configuration. We constructed a working model on how the actin cytoskeleton is controlled by actin-binding proteins and upstream signaling in root hair development based on the current literature: at the tip of hairs, actin polymerization appears to be facilitated by Arp2/3 complex that is activated by small GTPase, and profilin that is regulated by phosphatidylinositol 4,5-bisphosphate. Meanwhile, actin depolymerization and turnover are likely mediated by villin and actin depolymerizing factor, which are stimulated by calcium. At the shank, actin cables are produced by formin and villin. Under the complicated interaction, the actin cytoskeleton is controlled spatially and temporally during root hair development. © 2012 Elsevier Ireland Ltd. All rights reserved.

Synaptic Vesicle Mobility and Presynaptic F-Actin Are Disrupted in a N-ethylmaleimide–sensitive Factor Allele of Drosophila

PubMed Central

Nunes, Paula; Haines, Nicola; Kuppuswamy, Venkat; Fleet, David J.

2006-01-01

N-ethylmaleimide sensitive factor (NSF) can dissociate the soluble NSF attachment receptor (SNARE) complex, but NSF also participates in other intracellular trafficking functions by virtue of SNARE-independent activity. Drosophila that express a neural transgene encoding a dominant-negative form of NSF2 show an 80% reduction in the size of releasable synaptic vesicle pool, but no change in the number of vesicles in nerve terminal boutons. Here we tested the hypothesis that vesicles in the NSF2 mutant terminal are less mobile. Using a combination of genetics, pharmacology, and imaging we find a substantial reduction in vesicle mobility within the nerve terminal boutons of Drosophila NSF2 mutant larvae. Subsequent analysis revealed a decrease of filamentous actin in both NSF2 dominant-negative and loss-of-function mutants. Lastly, actin-filament disrupting drugs also decrease vesicle movement. We conclude that a factor contributing to the NSF mutant phenotype is a reduction in vesicle mobility, which is associated with decreased presynaptic F-actin. Our data are consistent with a model in which actin filaments promote vesicle mobility and suggest that NSF participates in establishing or maintaining this population of actin. PMID:16914524

Analysis of ligand-receptor cross-linked fragments by mass spectrometry

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

Son, C.D.; Sargsyan, H.; Hurst, Gregory

G-protein coupled receptors (GPCRs) are a class of integral membrane receptor proteins that are characterized by a signature seven-transmembrane (7-TM) configuration. The a-factor receptor (Ste2p) from Saccharomyces cerevisiae is a GPCR that, upon binding of a peptide ligand, transduces a signal to initiate a cascade of events leading to the mating of haploid yeast cells. This study summarizes the application of affinity purification and of matrix-assisted laser-desorption ionization time-of-flight (MALDI-TOF) experiments using biotinylated photoactivatable a-factor analogs. Affinity purification and enrichment of biotinylated peptides by monomeric avidin beads resulted in mass spectrometric detection of specific signals corresponding to crosslinked fragments ofmore » Ste2p. Data obtained from cyanogen bromide (CNBr) fragments of receptor cross-linked to an a-factor analog with the photoaffinity group p-benzoyl-L-phenylalanine on position 1 were in agreement with the previous results reported by our laboratory suggesting the cross-linking between position 1 of a-factor and a region of Ste2p covering residues 251 294.« less

Nckβ Adapter Regulates Actin Polymerization in NIH 3T3 Fibroblasts in Response to Platelet-Derived Growth Factor bb

PubMed Central

Chen, Min; She, Hongyun; Kim, Airie; Woodley, David T.; Li, Wei

2000-01-01

The SH3-SH3-SH3-SH2 adapter Nck represents a two-gene family that includes Nckα (Nck) and Nckβ (Grb4/Nck2), and it links receptor tyrosine kinases to intracellular signaling networks. The function of these mammalian Nck genes has not been established. We report here a specific role for Nckβ in platelet-derived growth factor (PDGF)-induced actin polymerization in NIH 3T3 cells. Overexpression of Nckβ but not Nckα blocks PDGF-stimulated membrane ruffling and formation of lamellipoda. Mutation in either the SH2 or the middle SH3 domain of Nckβ abolishes its interfering effect. Nckβ binds at Tyr-1009 in human PDGF receptor β (PDGFR-β) which is different from Nckα's binding site, Tyr-751, and does not compete with phosphatidylinositol-3 kinase for binding to PDGFR. Microinjection of an anti-Nckβ but not an anti-Nckα antibody inhibits PDGF-stimulated actin polymerization. Constitutively membrane-bound Nckβ but not Nckα blocks Rac1-L62-induced membrane ruffling and formation of lamellipodia, suggesting that Nckβ acts in parallel to or downstream of Rac1. This is the first report of Nckβ's role in receptor tyrosine kinase signaling to the actin cytoskeleton. PMID:11027258

EDC Cross-linking of Decellularized Tissue: A Promising Approach?

PubMed

Lehmann, Nadine; Christ, Torsten; Daugs, Aila; Bloch, Oliver; Holinski, Sebastian

2017-07-01

Decellularization of xenogenous cardiovascular structures is a promising approach to create scaffolds for tissue engineering. Unfortunately, handling and pliability of the unfixed tissue is challenging. N-(3-dimethylaminopropyl)-N9-ethylcarbodiimide (EDC) is an alternative cross-linking agent to glutaraldehyde (GA). Applied in native tissue, it provides biocompatibility and shows no potential for calcification. In addition, EDC can be used to link growth factors (GFs) to tissue scaffolds after decellularization. EDC cross-linking could thereby help to improve decellularized tissue without the toxicity of GA. Porcine aortic wall tissue specimens (TS) were decellularized, treated with EDC, and coated with fibroblast growth factor (FGF) or vascular endothelial growth factor (VEGF). Afterward, TS were subcutaneously implanted in 36 Lewis rats along with one decellularized TS without EDC treatment. After 2, 4, and 6 weeks TS were explanted from 12 rats, respectively. Vital cells were evaluated by RNA quantification, general cellular infiltration by hematoxylin and eosin staining (H&E), macrophage infiltration by CD68 staining, calcification by Von-Kossa staining, and tissue degradation by measurement of TS thickness. Quantification of vital cells showed reduced reseeding of EDC-treated TS compared to noncross-linked TS after 2 (p < 0.05) and 4 weeks (p < 0.05), while after 6 weeks only EDC+VEGF showed fewer viable cells (p < 0.01). Histological evaluation confirmed a reduced infiltration of EDC-treated TS. Macrophage infiltration decreased in all groups from 2 to 6 weeks, with the smallest population in EDC+VEGF-treated TS (p > 0.05). In EDC+FGF-treated TS, macrophages were reduced after 2 weeks compared to noncross-linked TS (p < 0.05), while after 4 and 6 weeks no significant difference was found (p > 0.05). Von-Kossa staining revealed no calcification in any of the specimens. Thickness of noncross-linked and EDC+FGF-treated TS was not

Translation elongation factor EF-Tu modulates filament formation of actin-like MreB protein in vitro.

PubMed

Defeu Soufo, Hervé Joël; Reimold, Christian; Breddermann, Hannes; Mannherz, Hans G; Graumann, Peter L

2015-04-24

EF-Tu has been shown to interact with actin-like protein MreB and to affect its localization in Escherichia coli and in Bacillus subtilis cells. We have purified YFP-MreB in an active form, which forms filaments on glass slides in vitro and was active in dynamic light-scattering assays, polymerizing in milliseconds after addition of magnesium. Purified EF-Tu enhanced the amount of MreB filaments, as seen by sedimentation assays, the speed of filament formation and the length of MreB filaments in vitro. EF-Tu had the strongest impact on MreB filaments in a 1:1 ratio, and EF-Tu co-sedimented with MreB filaments, revealing a stoichiometric interaction between both proteins. This was supported by cross-linking assays where 1:1 species were well detectable. When expressed in E. coli cells, B. subtilis MreB formed filaments and induced the formation of co-localizing B. subtilis EF-Tu structures, indicating that MreB can direct the positioning of EF-Tu structures in a heterologous cell system. Fluorescence recovery after photobleaching analysis showed that MreB filaments have a higher turnover in B. subtilis cells than in E. coli cells, indicating different filament kinetics in homologous or heterologous cell systems. The data show that MreB can direct the localization of EF-Tu in vivo, which in turn positively affects the formation and dynamics of MreB filaments. Thus, EF-Tu is a modulator of the activity of a bacterial actin-like protein. Copyright © 2015. Published by Elsevier Ltd.

Donor cross-linking for keratoplasty: a laboratory evaluation.

PubMed

Mukherjee, Achyut; Hayes, Sally; Aslanides, Ioannis; Lanchares, Elena; Meek, Keith M

2015-12-01

This laboratory-based investigation compares the topographic outcomes of conventional penetrating keratoplasty with that of a novel procedure in which donor corneas are cross-linked prior to keratoplasty. Penetrating keratoplasty procedures with continuous running sutures were carried out in a porcine whole globe model. Sixty eyes were randomly paired as 'donor' and 'host' tissue before being assigned to one of two groups. In the cross-linked group, donor corneas underwent riboflavin/UVA cross-linking prior to being trephined and sutured to untreated hosts. In the conventional keratoplasty group, both host and donor corneas remained untreated prior to keratoplasty. Topographic and corneal wavefront measurements were performed following surgery, and technical aspects of the procedure evaluated. Mean keratometric astigmatism was significantly lower in the cross-linked donor group at 3.67D (SD 1.8 D), vs. 8.43 D (SD 2.4 D) in the conventional keratoplasty group (p < 0.005). Mean wavefront astigmatism was also significantly reduced in the cross-linked donor group 4.71 D (SD 2.1) vs. 8.29D (SD 3.6) in the conventional keratoplasty group (p < 0.005). Mean RMS higher order aberration was significantly lower in the cross-linked donor group at 1.79 um (SD 0.98), vs. 3.05 um (SD 1.9) in the conventional keratoplasty group (P = 0.02). Qualitative analysis revealed less tissue distortion at the graft-host junction in the cross-linked group. Cross-linking of donor corneas prior to keratoplasty reduces intraoperative induced astigmatism and aberrations in an animal model. Further studies are indicated to evaluate the implications of this potential modification of keratoplasty surgery.

Microtubule-Actin Crosslinking Factor 1 and Plakins as Therapeutic Drug Targets.

PubMed

Quick, Quincy A

2018-01-26

Plakins are a family of seven cytoskeletal cross-linker proteins (microtubule-actin crosslinking factor 1 (MACF), bullous pemphigoid antigen (BPAG1) desmoplakin, envoplakin, periplakin, plectin, epiplakin) that network the three major filaments that comprise the cytoskeleton. Plakins have been found to be involved in disorders and diseases of the skin, heart, nervous system, and cancer that are attributed to autoimmune responses and genetic alterations of these macromolecules. Despite their role and involvement across a spectrum of several diseases, there are no current drugs or pharmacological agents that specifically target the members of this protein family. On the contrary, microtubules have traditionally been targeted by microtubule inhibiting agents, used for the treatment of diseases such as cancer, in spite of the deleterious toxicities associated with their clinical utility. The Research Collaboratory for Structural Bioinformatics (RCSB) was used here to identify therapeutic drugs targeting the plakin proteins, particularly the spectraplakins MACF1 and BPAG1, which contain microtubule-binding domains. RCSB analysis revealed that plakin proteins had 329 ligands, of which more than 50% were MACF1 and BPAG1 ligands and 10 were documented, clinically or experimentally, to have several therapeutic applications as anticancer, anti-inflammatory, and antibiotic agents.

Reverse actin sliding triggers strong myosin binding that moves tropomyosin

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

Bekyarova, T.I.; Reedy, M.C.; Baumann, B.A.J.

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 highmore » [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.« less

Quantification of the effect of cross-shear and applied nominal contact pressure on the wear of moderately cross-linked polyethylene.

PubMed

Abdelgaied, Abdellatif; Brockett, Claire L; Liu, Feng; Jennings, Louise M; Fisher, John; Jin, Zhongmin

2013-01-01

Polyethylene wear is a great concern in total joint replacement. It is now considered a major limiting factor to the long life of such prostheses. Cross-linking has been introduced to reduce the wear of ultra-high-molecular-weight polyethylene (UHMWPE). Computational models have been used extensively for wear prediction and optimization of artificial knee designs. However, in order to be independent and have general applicability and predictability, computational wear models should be based on inputs from independent experimentally determined wear parameters (wear factors or wear coefficients). The objective of this study was to investigate moderately cross-linked UHMWPE, using a multidirectional pin-on-plate wear test machine, under a wide range of applied nominal contact pressure (from 1 to 11 MPa) and under five different kinematic inputs, varying from a purely linear track to a maximum rotation of +/- 55 degrees. A computational model, based on a direct simulation of the multidirectional pin-on-plate wear tester, was developed to quantify the degree of cross-shear (CS) of the polyethylene pins articulating against the metallic plates. The moderately cross-linked UHMWPE showed wear factors less than half of that reported in the literature for, the conventional UHMWPE, under the same loading and kinematic inputs. In addition, under high applied nominal contact stress, the moderately crosslinked UHMWPE wear showed lower dependence on the degree of CS compared to that under low applied nominal contact stress. The calculated wear coefficients were found to be independent of the applied nominal contact stress, in contrast to the wear factors that were shown to be highly pressure dependent. This study provided independent wear data for inputs into computational models for moderately cross-linked polyethylene and supported the application of wear coefficient-based computational wear models.

Actin Turnover-Mediated Gravity Response in Maize Root Apices

PubMed Central

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

2006-01-01

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

Characterization of the Deoxyguanosine–Lysine Cross-Link of Methylglyoxal

PubMed Central

2015-01-01

Methylglyoxal is a mutagenic bis-electrophile that is produced endogenously from carbohydrate precursors. Methylglyoxal has been reported to induce DNA–protein cross-links (DPCs) in vitro and in cultured cells. Previous work suggests that these cross-links are formed between guanine and either lysine or cysteine side chains. However, the chemical nature of the methylglyoxal induced DPC have not been determined. We have examined the reaction of methylglyoxal, deoxyguanosine (dGuo), and Nα-acetyllysine (AcLys) and determined the structure of the cross-link to be the N2-ethyl-1-carboxamide with the lysine side chain amino group (1). The cross-link was identified by mass spectrometry and the structure confirmed by comparison to a synthetic sample. Further, the cross-link between methylglyoxal, dGuo, and a peptide (AcAVAGKAGAR) was also characterized. The mechanism of cross-link formation is likely to involve an Amadori rearrangement. PMID:24801980

Photocontrolled Cargo Release from Dual Cross-Linked Polymer Particles.

PubMed

Tan, Shereen; Cui, Jiwei; Fu, Qiang; Nam, Eunhyung; Ladewig, Katharina; Ren, Jing M; Wong, Edgar H H; Caruso, Frank; Blencowe, Anton; Qiao, Greg G

2016-03-09

Burst release of a payload from polymeric particles upon photoirradiation was engineered by altering the cross-linking density. This was achieved via a dual cross-linking concept whereby noncovalent cross-linking was provided by cyclodextrin host-guest interactions, and irreversible covalent cross-linking was mediated by continuous assembly of polymers (CAP). The dual cross-linked particles (DCPs) were efficiently infiltrated (∼80-93%) by the biomacromolecule dextran (molecular weight up to 500 kDa) to provide high loadings (70-75%). Upon short exposure (5 s) to UV light, the noncovalent cross-links were disrupted resulting in increased permeability and burst release of the cargo (50 mol % within 1 s) as visualized by time-lapse fluorescence microscopy. As sunlight contains UV light at low intensities, the particles can potentially be incorporated into systems used in agriculture, environmental control, and food packaging, whereby sunlight could control the release of nutrients and antimicrobial agents.

Cross-linked polyvinyl alcohol and method of making same

NASA Technical Reports Server (NTRS)

Hsu, L. C.; Sheibley, D. W.; Philipp, W. H. (Inventor)

1981-01-01

A film-forming polyvinyl alcohol polymer is mixed with a polyaldehyde-polysaccharide cross-linking agent having at least two monosaccharide units and a plurality of aldehyde groups per molecule, perferably an average of at least one aldehyde group per monosaccharide units. The cross-linking agent, such as a polydialdehyde starch, is used in an amount of about 2.5 to 20% of the theoretical amount required to cross-link all of the available hydroxyl groups of the polyvinyl alcohol polymer. Reaction between the polymer and cross-linking agent is effected in aqueous acidic solution to produce the cross-linked polymer. The polymer product has low electrical resistivity and other properties rendering it suitable for making separators for alkaline batteries.

Dermatological Feasibility of Multimodal Facial Color Imaging Modality for Cross-Evaluation of Facial Actinic Keratosis

PubMed Central

Bae, Youngwoo; Son, Taeyoon; Nelson, J. Stuart; Kim, Jae-Hong; Choi, Eung Ho; Jung, Byungjo

2010-01-01

Background/Purpose Digital color image analysis is currently considered as a routine procedure in dermatology. In our previous study, a multimodal facial color imaging modality (MFCIM), which provides a conventional, parallel- and cross-polarization, and fluorescent color image, was introduced for objective evaluation of various facial skin lesions. This study introduces a commercial version of MFCIM, DermaVision-PRO, for routine clinical use in dermatology and demonstrates its dermatological feasibility for cross-evaluation of skin lesions. Methods/Results Sample images of subjects with actinic keratosis or non-melanoma skin cancers were obtained at four different imaging modes. Various image analysis methods were applied to cross-evaluate the skin lesion and, finally, extract valuable diagnostic information. DermaVision-PRO is potentially a useful tool as an objective macroscopic imaging modality for quick prescreening and cross-evaluation of facial skin lesions. Conclusion DermaVision-PRO may be utilized as a useful tool for cross-evaluation of widely distributed facial skin lesions and an efficient database management of patient information. PMID:20923462

Prevalence and factors associated to actinic cheilitis in beach workers.

PubMed

de Souza Lucena, E E; Costa, D C B; da Silveira, E J D; Lima, K C

2012-09-01

Assess the prevalence and potential factors associated to actinic cheilitis (AC) in workers exposed to the sun. Workers assigned to urban beaches in a city in Northeastern Brazil completed a questionnaire containing personal and health data. Lips were examined to identify AC. A total of 362 workers, of which 15.5% exhibited AC, were examined. Among those with AC, there was a prevalence of males (86%), aged 37years or older (61.4%) and light-skinned (52.6%). They were undocumented (96.5%), exposed to the sun (84.2%), worked up to 6h daily (57.9%), five or more times per week (52.6%), and for more than 8years (54.4%). Photoprotection was used by 89.5%, including sunscreen (42.0%), lip protector (17.5%), and cap/hat (87.7%). Approximately 30% smoked and 29.8% consumed alcohol, 56.2% over two standard shots per day. Most (91.2%) ate healthy food five or more times per week. Being male, aged 37years or older, having up to 6years of schooling, being light-skinned, wearing a cap/hat, and using sunscreen were factors associated to AC. A high prevalence of actinic AC was observed. Special attention should be given to individuals chronically exposed to UV radiation, with the institution of educational, preventive, and curative measures. © 2012 John Wiley & Sons A/S.

Bacterial actin MreB forms antiparallel double filaments.

PubMed

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

2014-05-02

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. Copyright © 2014, van den Ent et al.

Actinic keratosis: a cross-sectional study of disease characteristics and treatment patterns in Danish dermatology clinics.

PubMed

Erlendsson, Andrés M; Egekvist, Henrik; Lorentzen, Henrik F; Philipsen, Peter A; Stausbøl-Grøn, Birgitte; Stender, Ida M; Haedersdal, Merete

2016-03-01

The incidence of actinic keratosis (AK) is increasing, and several treatment options are available. The aim of this study was to describe clinical characteristics and treatment patterns in patients with AK treated by Danish dermatologists. A multicenter, non-interventional, cross-sectional study was conducted. Three dermatology hospital departments and seven private dermatology clinics enrolled eligible AK patients consecutively during one week. A total of 312 patients were included. Non-melanoma skin cancer (NMSC) was previously reported in 51.0% of patients and currently suspected in 9.4% of AK-affected anatomical regions. Lesions of AK were located primarily on the face (38.6%), scalp (12.8%), and hands (11.2%). Actinic keratosis commonly presented with multiple AK lesions (38.6%) and field cancerization (38.5%). The treatments used most frequently were cryotherapy (57.7%) and photodynamic therapy (PDT) with methyl aminolevulinate (17.1%) and imiquimod (11.2%). The likelihood of receiving cryotherapy was higher for men (odds ratio [OR] 1.65, 95% confidence interval [CI] 1.10-2.47) and increased with age (2.2% per year, 0.4-4.0%). PDT represented the most frequently applied treatment for severe actinic damage and was more likely to be prescribed to women (OR 4.08, 95% CI 2.22-7.47) and young patients (OR 0.97 per year, 95% CI 0.95-0.99). The prevalence of severe actinic damage (17.3% versus 9.6%) and intake of immunosuppressive medication (29.0 versus 2.0) were higher among hospital patients compared with those treated in private practices (P < 0.0001). The majority of AK patients in Danish dermatology clinics have a history of skin cancer, and NMSC is suspected in almost 10% of AK-affected regions. Cryotherapy is the most frequently used treatment overall, except in instances of severe actinic damage, in which PDT is the first-choice treatment. © 2015 The International Society of Dermatology.

Cross-linking and the molecular packing of corneal collagen

NASA Technical Reports Server (NTRS)

Yamauchi, M.; Chandler, G. S.; Tanzawa, H.; Katz, E. P.

1996-01-01

We have quantitatively characterized, for the first time, the cross-linking in bovine cornea collagen as a function of age. The major iminium reducible cross-links were dehydro-hydroxylysinonorleucine (deH-HLNL) and dehydro-histidinohydroxymerodesmosine (deH-HHMD). The former rapidly diminished after birth; however, the latter persisted in mature animals at a level of 0.3 - 0.4 moles/mole of collagen. A nonreducible cross-link, histidinohydroxylysinonorleucine (HHL), previously found only in skin, was also found to be a major mature cross-link in cornea. The presence of HHL indicates that cornea fibrils have a molecular packing similar to skin collagen. However, like deH-HHMD, the HHL content in corneal fibrils only reaches a maximum value with time about half that of skin. These data suggest that the corneal fibrils are comprised of discrete filaments that are internally stabilized by HHL and deH-HHMD cross-links. This pattern of intermolecular cross-linking would facilitate the special collagen swelling property required for corneal transparency.

Fascin regulates nuclear actin during Drosophila oogenesis

PubMed Central

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

2016-01-01

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

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

PubMed

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

2016-04-01

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

Actin Polymerization is Stimulated by Actin Crosslinking Protein Palladin

PubMed Central

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

2016-01-01

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

Polarized Exocytosis Induces Compensatory Endocytosis by Sec4p-Regulated Cortical Actin Polymerization

PubMed Central

Johansen, Jesper; Alfaro, Gabriel; Beh, Christopher T.

2016-01-01

Polarized growth is maintained by both polarized exocytosis, which transports membrane components to specific locations on the cell cortex, and endocytosis, which retrieves these components before they can diffuse away. Despite functional links between these two transport pathways, they are generally considered to be separate events. Using live cell imaging, in vivo and in vitro protein binding assays, and in vitro pyrene-actin polymerization assays, we show that the yeast Rab GTPase Sec4p couples polarized exocytosis with cortical actin polymerization, which induces endocytosis. After polarized exocytosis to the plasma membrane, Sec4p binds Las17/Bee1p (yeast Wiskott—Aldrich Syndrome protein [WASp]) in a complex with Sla1p and Sla2p during actin patch assembly. Mutations that inactivate Sec4p, or its guanine nucleotide exchange factor (GEF) Sec2p, inhibit actin patch formation, whereas the activating sec4-Q79L mutation accelerates patch assembly. In vitro assays of Arp2/3-dependent actin polymerization established that GTPγS-Sec4p overrides Sla1p inhibition of Las17p-dependent actin nucleation. These results support a model in which Sec4p relocates along the plasma membrane from polarized sites of exocytic vesicle fusion to nascent sites of endocytosis. Activated Sec4p then promotes actin polymerization and triggers compensatory endocytosis, which controls surface expansion and kinetically refines cell polarization. PMID:27526190

Hydrogels Prepared from Cross-Linked Nanofibrillated Cellulose

Treesearch

Sandeep S. Nair; J.Y. Zhu; Yulin Deng; Arthur J. Ragauskas

2014-01-01

Nanocomposite hydrogels were developed by cross-linking nanofibrillated cellulose with poly(methyl vinyl ether-co-maleic acid) and polyethylene glycol. The cross-linked hydrogels showed enhanced water absorption and gel content with the addition of nanocellulose. In addition, the thermal stability, mechanical strength, and modulus increased with an increase in the...

DNA cross-linking by dehydromonocrotaline lacks apparent base sequence preference.

PubMed

Rieben, W Kurt; Coulombe, Roger A

2004-12-01

Pyrrolizidine alkaloids (PAs) are ubiquitous plant toxins, many of which, upon oxidation by hepatic mixed-function oxidases, become reactive bifunctional pyrrolic electrophiles that form DNA-DNA and DNA-protein cross-links. The anti-mitotic, toxic, and carcinogenic action of PAs is thought to be caused, at least in part, by these cross-links. We wished to determine whether the activated PA pyrrole dehydromonocrotaline (DHMO) exhibits base sequence preferences when cross-linked to a set of model duplex poly A-T 14-mer oligonucleotides with varying internal and/or end 5'-d(CG), 5'-d(GC), 5'-d(TA), 5'-d(CGCG), or 5'-d(GCGC) sequences. DHMO-DNA cross-links were assessed by electrophoretic mobility shift assay (EMSA) of 32P endlabeled oligonucleotides and by HPLC analysis of cross-linked DNAs enzymatically digested to their constituent deoxynucleosides. The degree of DNA cross-links depended upon the concentration of the pyrrole, but not on the base sequence of the oligonucleotide target. Likewise, HPLC chromatograms of cross-linked and digested DNAs showed no discernible sequence preference for any nucleotide. Added glutathione, tyrosine, cysteine, and aspartic acid, but not phenylalanine, threonine, serine, lysine, or methionine competed with DNA as alternate nucleophiles for cross-linking by DHMO. From these data it appears that DHMO exhibits no strong base preference when forming cross-links with DNA, and that some cellular nucleophiles can inhibit DNA cross-link formation.

Acting on Actin: Rac and Rho Played by Yersinia.

PubMed

Aepfelbacher, Martin; Wolters, Manuel

2017-01-01

Pathogenic bacteria of the genus Yersinia include Y. pestis-the agent of plaque-and two enteropathogens, Y. enterocolitica, and Y. pseudotuberculosis. These pathogens have developed an array of virulence factors aimed at manipulating Rho GTP-binding proteins and the actin cytoskeleton in host cells to cross the intestinal barrier and suppress the immune system. Yersinia virulence factors include outer membrane proteins triggering cell invasion by binding to integrins, effector proteins injected into host cells to manipulate Rho protein functions and a Rho protein-activating exotoxin. Here, we present an overview of how Yersinia and host factors are integrated in a regulatory network that orchestrates the subversion of host defense.

Mixed-Isotope Labeling with LC-IMS-MS for Characterization of Protein–Protein Interactions by Chemical Cross-Linking

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

Merkley, Eric D.; Baker, Erin S.; Crowell, Kevin L.

2013-02-20

Chemical cross-linking of proteins followed by proteolysis and mass spectrometric analysis of the resulting cross-linked peptides can provide insights into protein structure and protein-protein interactions. However, cross-linked peptides are by necessity of low stoichometry and have different physicochemical properties than linear peptides, routine unambiguous identification of the cross-linked peptides has remained difficult. To address this challenge, we demonstrated the use of liquid chromatography and ion mobility separations coupled with mass spectrometry in combination with a heavy-isotope labeling method. The combination of mixed-isotope cross-linking and ion mobility provided unique and easily interpretable spectral multiplet features for the intermolecular cross-linked peptides. Applicationmore » of the method to two different homodimeric proteins - SrfN, a virulence factor from Salmonella Typhimurium and SO_2176, a protein of unknown function from Shewanella oneidensis- revealed several cross-linked peptides from both proteins that were identified with a low false discovery rate (estimated using a decoy approach). A greater number of cross-linked peptides were identified using ion mobility drift time information in the analysis than when the data were summed across the drift time dimension before analysis. The identified cross-linked peptides migrated more quickly in the ion mobility drift tube than the unmodified peptides.« less

Electron Tomography and Simulation of Baculovirus Actin Comet Tails Support a Tethered Filament Model of Pathogen Propulsion

PubMed Central

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

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.

UV laser-induced cross-linking in peptides

PubMed Central

Leo, Gabriella; Altucci, Carlo; Bourgoin-Voillard, Sandrine; Gravagnuolo, Alfredo M.; Esposito, Rosario; Marino, Gennaro; Costello, Catherine E.; Velotta, Raffaele; Birolo, Leila

2013-01-01

RATIONALE The aim of this study was to demonstrate, and to characterize by high resolution mass spectrometry, that it is possible to preferentially induce covalent cross-links in peptides by using high energy femtosecond UV laser pulses. The cross-link is readily formed only when aromatic amino acids are present in the peptide sequence. METHODS Three peptides, xenopsin, angiotensin I, interleukin, individually or in combination, were exposed to high energy femtosecond UV laser pulses, either alone or in the presence of spin trapping molecules, the reaction products being characterized by high resolution mass spectrometry. RESULTS High resolution mass spectrometry and spin trapping strategies showed that cross-linking occurs readily, proceeds via a radical mechanism, and is the highly dominant reaction, proceeding without causing significant photo-damage in the investigated range of experimental parameters. CONCLUSIONS High energy femtosecond UV laser pulses can be used to induce covalent cross-links between aromatic amino acids in peptides, overcoming photo-oxidation processes, that predominate as the mean laser pulse intensity approaches illumination conditions achievable with conventional UV light sources. PMID:23754800

Multiscale mechanical effects of native collagen cross-linking in tendon.

PubMed

Eekhoff, Jeremy D; Fang, Fei; Lake, Spencer P

2018-06-06

The hierarchical structure of tendon allows for attenuation of mechanical strain down decreasing length scales. While reorganization of collagen fibers accounts for microscale strain attenuation, cross-linking between collagen molecules contributes to deformation mechanisms at the fibrillar and molecular scales. Divalent and trivalent enzymatic cross-links form during the development of collagen fibrils through the enzymatic activity of lysyl oxidase (LOX). By establishing connections between telopeptidyl and triple-helical domains of adjacent molecules within collagen fibrils, these cross-links stiffen the fibrils by resisting intermolecular sliding. Ultimately, greater enzymatic cross-linking leads to less compliant and stronger tendon as a result of stiffer fibrils. In contrast, nonenzymatic cross-links such as glucosepane and pentosidine are not produced during development but slowly accumulate through glycation of collagen. Therefore, these cross-links are only expected to be present in significant quantities in advanced age, where there has been sufficient time for glycation to occur, and in diabetes, where the presence of more free sugar in the extracellular matrix increases the rate of glycation. Unlike enzymatic cross-links, current evidence suggests that nonenzymatic cross-links are at least partially isolated to the surface of collagen fibers. As a result, glycation has been proposed to primarily impact tendon mechanics by altering molecular interactions at the fiber interface, thereby diminishing sliding between fibers. Thus, increased nonenzymatic cross-linking decreases microscale strain attenuation and the viscous response of tendon. In conclusion, enzymatic and nonenzymatic collagen cross-links have demonstrable and distinct effects on the mechanical properties of tendon across different length scales.

Nanoscale segregation of actin nucleation and elongation factors determines dendritic spine protrusion

PubMed Central

Chazeau, Anaël; Mehidi, Amine; Nair, Deepak; Gautier, Jérémie J; Leduc, Cécile; Chamma, Ingrid; Kage, Frieda; Kechkar, Adel; Thoumine, Olivier; Rottner, Klemens; Choquet, Daniel; Gautreau, Alexis; Sibarita, Jean-Baptiste; Giannone, Grégory

2014-01-01

Actin dynamics drive morphological remodeling of neuronal dendritic spines and changes in synaptic transmission. Yet, the spatiotemporal coordination of actin regulators in spines is unknown. Using single protein tracking and super-resolution imaging, we revealed the nanoscale organization and dynamics of branched F-actin regulators in spines. Branched F-actin nucleation occurs at the PSD vicinity, while elongation occurs at the tip of finger-like protrusions. This spatial segregation differs from lamellipodia where both branched F-actin nucleation and elongation occur at protrusion tips. The PSD is a persistent confinement zone for IRSp53 and the WAVE complex, an activator of the Arp2/3 complex. In contrast, filament elongators like VASP and formin-like protein-2 move outwards from the PSD with protrusion tips. Accordingly, Arp2/3 complexes associated with F-actin are immobile and surround the PSD. Arp2/3 and Rac1 GTPase converge to the PSD, respectively, by cytosolic and free-diffusion on the membrane. Enhanced Rac1 activation and Shank3 over-expression, both associated with spine enlargement, induce delocalization of the WAVE complex from the PSD. Thus, the specific localization of branched F-actin regulators in spines might be reorganized during spine morphological remodeling often associated with synaptic plasticity. PMID:25293574

Control of actin-based motility through localized actin binding

PubMed Central

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

2014-01-01

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

Modeling Protein Excited-state Structures from "Over-length" Chemical Cross-links.

PubMed

Ding, Yue-He; Gong, Zhou; Dong, Xu; Liu, Kan; Liu, Zhu; Liu, Chao; He, Si-Min; Dong, Meng-Qiu; Tang, Chun

2017-01-27

Chemical cross-linking coupled with mass spectroscopy (CXMS) provides proximity information for the cross-linked residues and is used increasingly for modeling protein structures. However, experimentally identified cross-links are sometimes incompatible with the known structure of a protein, as the distance calculated between the cross-linked residues far exceeds the maximum length of the cross-linker. The discrepancies may persist even after eliminating potentially false cross-links and excluding intermolecular ones. Thus the "over-length" cross-links may arise from alternative excited-state conformation of the protein. Here we present a method and associated software DynaXL for visualizing the ensemble structures of multidomain proteins based on intramolecular cross-links identified by mass spectrometry with high confidence. Representing the cross-linkers and cross-linking reactions explicitly, we show that the protein excited-state structure can be modeled with as few as two over-length cross-links. We demonstrate the generality of our method with three systems: calmodulin, enzyme I, and glutamine-binding protein, and we show that these proteins alternate between different conformations for interacting with other proteins and ligands. Taken together, the over-length chemical cross-links contain valuable information about protein dynamics, and our findings here illustrate the relationship between dynamic domain movement and protein function. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Prevalence of and risk factors for actinic cheilitis in Brazilian fishermen and women.

PubMed

de Oliveira Ribeiro, Artur; da Silva, Luiz Carlos Ferreira; Martins-Filho, Paulo Ricardo Saquete

2014-11-01

This study was conducted to investigate the prevalence of and risk factors for actinic cheilitis (AC) in Brazilian fishermen and women. A cross-sectional study was carried out in a representative sample (n = 210) of fishermen and women. Data related to age, skin color, cumulative and daily exposure to solar radiation, smoking, sun protection measures, and the presence of AC were collected. A Poisson regression model was created to estimate the prevalence ratio of variables. The prevalence of AC was 11.4% (95% confidence interval 7.8-16.4%). The disease was more prevalent in fishermen and women with fair skin and in those aged >50 years, those with cumulative exposure to solar radiation of >30 years, those with daily exposure of >4 hours, and those who did not use sunscreen. Brazilian fishermen and women constitute a population at risk for lip cancer. Public health programs that encourage the use of sunscreens and a lower daily exposure to solar radiation should be established. © 2014 The International Society of Dermatology.

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

PubMed Central

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

2018-01-01

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

Xilmass: A New Approach toward the Identification of Cross-Linked Peptides.

PubMed

Yılmaz, Şule; Drepper, Friedel; Hulstaert, Niels; Černič, Maša; Gevaert, Kris; Economou, Anastassios; Warscheid, Bettina; Martens, Lennart; Vandermarliere, Elien

2016-10-18

Chemical cross-linking coupled with mass spectrometry plays an important role in unravelling protein interactions, especially weak and transient ones. Moreover, cross-linking complements several structural determination approaches such as cryo-EM. Although several computational approaches are available for the annotation of spectra obtained from cross-linked peptides, there remains room for improvement. Here, we present Xilmass, a novel algorithm to identify cross-linked peptides that introduces two new concepts: (i) the cross-linked peptides are represented in the search database such that the cross-linking sites are explicitly encoded, and (ii) the scoring function derived from the Andromeda algorithm was adapted to score against a theoretical tandem mass spectrometry (MS/MS) spectrum that contains the peaks from all possible fragment ions of a cross-linked peptide pair. The performance of Xilmass was evaluated against the recently published Kojak and the popular pLink algorithms on a calmodulin-plectin complex data set, as well as three additional, published data sets. The results show that Xilmass typically had the highest number of identified distinct cross-linked sites and also the highest number of predicted cross-linked sites.

Infrared microspectroscopic determination of collagen cross-links in articular cartilage

NASA Astrophysics Data System (ADS)

Rieppo, Lassi; Kokkonen, Harri T.; Kulmala, Katariina A. M.; Kovanen, Vuokko; Lammi, Mikko J.; Töyräs, Juha; Saarakkala, Simo

2017-03-01

Collagen forms an organized network in articular cartilage to give tensile stiffness to the tissue. Due to its long half-life, collagen is susceptible to cross-links caused by advanced glycation end-products. The current standard method for determination of cross-link concentrations in tissues is the destructive high-performance liquid chromatography (HPLC). The aim of this study was to analyze the cross-link concentrations nondestructively from standard unstained histological articular cartilage sections by using Fourier transform infrared (FTIR) microspectroscopy. Half of the bovine articular cartilage samples (n=27) were treated with threose to increase the collagen cross-linking while the other half (n=27) served as a control group. Partial least squares (PLS) regression with variable selection algorithms was used to predict the cross-link concentrations from the measured average FTIR spectra of the samples, and HPLC was used as the reference method for cross-link concentrations. The correlation coefficients between the PLS regression models and the biochemical reference values were r=0.84 (p<0.001), r=0.87 (p<0.001) and r=0.92 (p<0.001) for hydroxylysyl pyridinoline (HP), lysyl pyridinoline (LP), and pentosidine (Pent) cross-links, respectively. The study demonstrated that FTIR microspectroscopy is a feasible method for investigating cross-link concentrations in articular cartilage.

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

PubMed

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

2012-01-01

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

Geometrical and Mechanical Properties Control Actin Filament Organization

PubMed Central

Ennomani, Hajer; Théry, Manuel; Nedelec, Francois; Blanchoin, Laurent

2015-01-01

The different actin structures governing eukaryotic cell shape and movement are not only determined by the properties of the actin filaments and associated proteins, but also by geometrical constraints. We recently demonstrated that limiting nucleation to specific regions was sufficient to obtain actin networks with different organization. To further investigate how spatially constrained actin nucleation determines the emergent actin organization, we performed detailed simulations of the actin filament system using Cytosim. We first calibrated the steric interaction between filaments, by matching, in simulations and experiments, the bundled actin organization observed with a rectangular bar of nucleating factor. We then studied the overall organization of actin filaments generated by more complex pattern geometries used experimentally. We found that the fraction of parallel versus antiparallel bundles is determined by the mechanical properties of actin filament or bundles and the efficiency of nucleation. Thus nucleation geometry, actin filaments local interactions, bundle rigidity, and nucleation efficiency are the key parameters controlling the emergent actin architecture. We finally simulated more complex nucleation patterns and performed the corresponding experiments to confirm the predictive capabilities of the model. PMID:26016478

Loss of glomerular foot processes is associated with uncoupling of podocalyxin from the actin cytoskeleton

PubMed Central

Takeda, Tetsuro; McQuistan, Tammie; Orlando, Robert A.; Farquhar, Marilyn G.

2001-01-01

Podocalyxin (PC), the major sialoprotein of glomerular epithelial cells (GECs), helps maintain the characteristic architecture of the foot processes and the patency of the filtration slits. PC associates with actin via ezrin, a member of the ERM family of cytoskeletal linker proteins. Here we show that PC is linked to ezrin and the actin cytoskeleton via Na+/H+-exchanger regulatory factor 2 (NHERF2), a scaffold protein containing two PDZ (PSD-95/Dlg/ZO-1) domains and an ERM-binding region. The cytoplasmic tail of PC contains a C-terminal PDZ-binding motif (DTHL) that binds to the second PDZ domain of NHERF2 in yeast two-hybrid and in vitro pull-down assays. By immunocytochemistry NHERF2 colocalizes with PC and ezrin along the apical domain of the GEC plasma membrane. NHERF2 and ezrin form a multimeric complex with PC, as they coimmunoprecipitate with PC. The PC/NHERF2/ezrin complex interacts with the actin cytoskeleton, and this interaction is disrupted in GECs from puromycin aminonucleoside–, protamine sulfate–, or sialidase-treated rats, which show a dramatic loss of foot processes, comparable to that seen in the nephrotic syndrome. Thus NHERF2 appears to function as a scaffold protein linking PC to ezrin and the actin cytoskeleton. PC/NHERF2/ezrin/actin interactions are disrupted in pathologic conditions associated with changes in GEC foot processes, indicating their importance for maintaining the unique organization of this epithelium. J. Clin. Invest. 108:289–301 (2001). DOI:10.1172/JCI200112539. PMID:11457882

Cross-linked polyvinyl alcohol films as alkaline battery separators

NASA Technical Reports Server (NTRS)

Sheibley, D. W.; Manzo, M. A.; Gonzalez-Sanabria, O. D.

1983-01-01

Cross-linking methods have been investigated to determine their effect on the performance of polyvinyl alcohol (PVA) films as alkaline battery separators. The following types of cross-linked PVA films are discussed: (1) PVA-dialdehyde blends post-treated with an acid or acid periodate solution (two-step method) and (2) PVA-dialdehyde blends cross-linked during film formation (drying) by using a reagent with both aldehyde and acid functionality (one-step method). Laboratory samples of each cross-linked type of film were prepared and evaluated in standard separator screening tests. Then pilot-plant batches of films were prepared and compared to measure differences due to the cross-linking method. The pilot-plant materials were then tested in nickel oxide-zinc cells to compare the two methods with respect to performance characteristics and cycle life. Cell test results are compared with those from tests with Celgard.

Cross-linked polyvinyl alcohol films as alkaline battery separators

NASA Technical Reports Server (NTRS)

Sheibley, D. W.; Manzo, M. A.; Gonzalez-Sanabria, O. D.

1982-01-01

Cross-linking methods were investigated to determine their effect on the performance of polyvinyl alcohol (PVA) films as alkaline battery separators. The following types of cross-linked PVA films are discussed: (1) PVA-dialdehyde blends post-treated with an acid or acid periodate solution (two-step method) and (2) PVA-dialdehyde blends cross-linked during film formation (drying) by using a reagent with both aldehyde and acid functionality (one-step method). Laboratory samples of each cross-linked type of film were prepared and evaluated in standard separator screening tests. The pilot-plant batches of films were prepared and compared to measure differences due to the cross-linking method. The pilot-plant materials were then tested in nickel oxide - zinc cells to compare the two methods with respect to performance characteristics and cycle life. Cell test results are compared with those from tests with Celgard.

Microtubule-Actin Crosslinking Factor 1 and Plakins as Therapeutic Drug Targets

PubMed Central

Quick, Quincy A.

2018-01-01

Plakins are a family of seven cytoskeletal cross-linker proteins (microtubule-actin crosslinking factor 1 (MACF), bullous pemphigoid antigen (BPAG1) desmoplakin, envoplakin, periplakin, plectin, epiplakin) that network the three major filaments that comprise the cytoskeleton. Plakins have been found to be involved in disorders and diseases of the skin, heart, nervous system, and cancer that are attributed to autoimmune responses and genetic alterations of these macromolecules. Despite their role and involvement across a spectrum of several diseases, there are no current drugs or pharmacological agents that specifically target the members of this protein family. On the contrary, microtubules have traditionally been targeted by microtubule inhibiting agents, used for the treatment of diseases such as cancer, in spite of the deleterious toxicities associated with their clinical utility. The Research Collaboratory for Structural Bioinformatics (RCSB) was used here to identify therapeutic drugs targeting the plakin proteins, particularly the spectraplakins MACF1 and BPAG1, which contain microtubule-binding domains. RCSB analysis revealed that plakin proteins had 329 ligands, of which more than 50% were MACF1 and BPAG1 ligands and 10 were documented, clinically or experimentally, to have several therapeutic applications as anticancer, anti-inflammatory, and antibiotic agents. PMID:29373494

Actin depolymerization enhances adipogenic differentiation in human stromal stem cells.

PubMed

Chen, Li; Hu, Huimin; Qiu, Weimin; Shi, Kaikai; Kassem, Moustapha

2018-05-01

Human stromal stem cells (hMSCs) differentiate into adipocytes that play a role in skeletal tissue homeostasis and whole body energy metabolism. During adipocyte differentiation, hMSCs exhibit significant changes in cell morphology suggesting changes in cytoskeletal organization. Here, we examined the effect of direct modulation of actin microfilament dynamics on adipocyte differentiation. Stabilizing actin filaments in hMSCs by siRNA-mediated knock down of the two main actin depolymerizing factors (ADFs): Cofilin 1 (CFL1) and Destrin (DSTN) or treating the cells by Phalloidin reduced adipocyte differentiation as evidenced by decreased number of mature adipocytes and decreased adipocyte specific gene expression (ADIPOQ, LPL, PPARG, FABP4). In contrast, disruption of actin cytoskeleton by Cytochalasin D enhanced adipocyte differentiation. Follow up studies revealed that the effects of CFL1 on adipocyte differentiation depended on the activity of LIM domain kinase 1 (LIMK1) which is the major upstream kinase of CFL1. Inhibiting LIMK by its specific chemical inhibitor LIMKi inhibited the phosphorylation of CFL1 and actin polymerization, and enhanced the adipocyte differentiation. Moreover, treating hMSCs by Cytochalasin D inhibited ERK and Smad2 signaling and this was associated with enhanced adipocyte differentiation. On the other hand, Phalloidin enhanced ERK and Smad2 signaling, but inhibited adipocyte differentiation which was rescued by ERK specific chemical inhibitor U0126. Our data provide a link between restructuring of hMSCs cytoskeleton and hMSCs lineage commitment and differentiation. Copyright © 2018 Elsevier B.V. All rights reserved.

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

PubMed

Wang, Pengwei; Hussey, Patrick J

2017-03-01

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

A nucleator arms race: cellular control of actin assembly.

PubMed

Campellone, Kenneth G; Welch, Matthew D

2010-04-01

For over a decade, the actin-related protein 2/3 (ARP2/3) complex, a handful of nucleation-promoting factors and formins were the only molecules known to directly nucleate actin filament formation de novo. However, the past several years have seen a surge in the discovery of mammalian proteins with roles in actin nucleation and dynamics. Newly recognized nucleation-promoting factors, such as WASP and SCAR homologue (WASH), WASP homologue associated with actin, membranes and microtubules (WHAMM), and junction-mediating regulatory protein (JMY), stimulate ARP2/3 activity at distinct cellular locations. Formin nucleators with additional biochemical and cellular activities have also been uncovered. Finally, the Spire, cordon-bleu and leiomodin nucleators have revealed new ways of overcoming the kinetic barriers to actin polymerization.

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

PubMed

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

2012-01-01

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

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

PubMed

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

2013-01-11

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

Durability improvements of two-dimensional metal nanoparticle sheets by molecular cross-linked structures between nanoparticles

NASA Astrophysics Data System (ADS)

Saito, Noboru; Ryuzaki, Sou; Wang, Pangpang; Park, Susie; Sakai, Nobuyuki; Tatsuma, Tetsu; Okamoto, Koichi; Tamada, Kaoru

2018-03-01

The durability of two-dimensional metal nanoparticle sheets is a crucial factor for realizing next-generation optoelectronic devices based on plasmonics such as organic light-emitting diodes. Here, we report improvements in the durability of Ag nanoparticle sheets by forming alkanedithiol (DT16) cross-linked structures between the nanoparticles. The cross-linked structures in a sheet were fabricated by the self-assembly of DT16-capped Ag nanoparticles with 10% coverage (AgDT16). The durabilities for thermal, organic solvent, and oxidation reactions of AgDT16 sheets were found to be improved owing to the cross-linked structures by comparing Ag nanoparticle sheets without the cross-linked structures. The absorbance spectra revealed that the Ag nanoparticle sheets without the structure are markedly damaged by each durability test, whereas the AgDT16 sheets remain. The molecular cross-linked structures between nanoparticles in two-dimansional metal nanoparticle sheets were found to have the potential to play a key role in the realization of plasmonic optoelectronic devices including metal nanoparticles.

Effects of alginate hydrogel cross-linking density on mechanical and biological behaviors for tissue engineering.

PubMed

Jang, Jinah; Seol, Young-Joon; Kim, Hyeon Ji; Kundu, Joydip; Kim, Sung Won; Cho, Dong-Woo

2014-09-01

An effective cross-linking of alginate gel was made through reaction with calcium carbonate (CaCO3). We used human chondrocytes as a model cell to study the effects of cross-linking density. Three different pore size ranges of cross-linked alginate hydrogels were fabricated. The morphological, mechanical, and rheological properties of various alginate hydrogels were characterized and responses of biosynthesis of cells encapsulated in each gel to the variation in cross-linking density were investigated. Desired outer shape of structure was maintained when the alginate solution was cross-linked with the applied method. The properties of alginate hydrogel could be tailored through applying various concentrations of CaCO3. The rate of synthesized GAGs and collagens was significantly higher in human chondrocytes encapsulated in the smaller pore structure than that in the larger pore structure. The expression of chondrogenic markers, including collagen type II and aggrecan, was enhanced in the smaller pore structure. It was found that proper structural morphology is a critical factor to enhance the performance and tissue regeneration. Copyright © 2014 Elsevier Ltd. All rights reserved.

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.

G-actin provides substrate-specificity to eukaryotic initiation factor 2α holophosphatases

PubMed Central

Chen, Ruming; Rato, Cláudia; Yan, Yahui; Crespillo-Casado, Ana; Clarke, Hanna J; Harding, Heather P; Marciniak, Stefan J; Read, Randy J; Ron, David

2015-01-01

Dephosphorylation of eukaryotic translation initiation factor 2a (eIF2a) restores protein synthesis at the waning of stress responses and requires a PP1 catalytic subunit and a regulatory subunit, PPP1R15A/GADD34 or PPP1R15B/CReP. Surprisingly, PPP1R15-PP1 binary complexes reconstituted in vitro lacked substrate selectivity. However, selectivity was restored by crude cell lysate or purified G-actin, which joined PPP1R15-PP1 to form a stable ternary complex. In crystal structures of the non-selective PPP1R15B-PP1G complex, the functional core of PPP1R15 made multiple surface contacts with PP1G, but at a distance from the active site, whereas in the substrate-selective ternary complex, actin contributes to one face of a platform encompassing the active site. Computational docking of the N-terminal lobe of eIF2a at this platform placed phosphorylated serine 51 near the active site. Mutagenesis of predicted surface-contacting residues enfeebled dephosphorylation, suggesting that avidity for the substrate plays an important role in imparting specificity on the PPP1R15B-PP1G-actin ternary complex. DOI: http://dx.doi.org/10.7554/eLife.04871.001 PMID:25774600

A multicomponent complex is required for the AAUAAA-dependent cross-linking of a 64-kilodalton protein to polyadenylation substrates.

PubMed Central

Wilusz, J; Shenk, T; Takagaki, Y; Manley, J L

1990-01-01

A 64-kilodalton (kDa) polypeptide that is cross-linked by UV light specifically to polyadenylation substrate RNAs containing a functional AAUAAA element has been identified previously. Fractionated HeLa nuclear components that can be combined to regenerate efficient and accurate polyadenylation in vitro have now been screened for the presence of the 64-kDa protein. None of the individual components contained an activity which could generate the 64-kDa species upon UV cross-linking in the presence of substrate RNA. It was necessary to mix two components, cleavage stimulation factor and specificity factor, to reconstitute 64-kDa protein-RNA cross-linking. The addition of cleavage factors to this mixture very efficiently reconstituted the AAUAAA-specific 64-kDa protein-RNA interaction. The 64-kDa protein, therefore, is present in highly purified, reconstituted polyadenylation reactions. However, it is necessary to form a multicomponent complex to efficiently cross-link the protein to a substrate RNA. Images PMID:2304466

Drug-Triggered and Cross-Linked Self-Assembling Nanofibrous Hydrogels

PubMed Central

Kumar, Vivek A.; Shi, Siyu; Wang, Benjamin K.; Li, I-Che; Jalan, Abhishek A.; Sarkar, Biplab; Wickremasinghe, Navindee C.; Hartgerink, Jeffrey D.

2015-01-01

Self-assembly of multidomain peptides (MDP) can be tailored to carry payloads that modulate the extracellular environment. Controlled release of growth factors, cytokines, and small-molecule drugs allows for unique control of in vitro and in vivo responses. In this study, we demonstrate this process of ionic cross-linking of peptides using multivalent drugs to create hydrogels for sustained long-term delivery of drugs. Using phosphate, heparin, clodronate, trypan, and suramin, we demonstrate the utility of this strategy. Although all multivalent anions result in good hydrogel formation, demonstrating the generality of this approach, suramin led to the formation of the best hydrogels per unit concentration and was studied in greater detail. Suramin ionically cross-linked MDP into a fibrous meshwork as determined by scanning and transmission electron microscopy. We measured material storage and loss modulus using rheometry and showed a distinct increase in G′ and G″ as a function of suramin concentration. Release of suramin from scaffolds was determined using UV spectroscopy and showed prolonged release over a 30 day period. Suramin bioavailability and function were demonstrated by attenuated M1 polarization of THP-1 cells compared to positive control. Overall, this design strategy has allowed for the development of a novel class of polymeric delivery vehicles with generally long-term release and, in the case of suramin, cross-linked hydrogels that can modulate cellular phenotype. PMID:25831137

Actin Bodies in Yeast Quiescent Cells: An Immediately Available Actin Reserve?

PubMed Central

Pinson, Benoît; Salin, Bénédicte; Daignan-Fornier, Bertrand

2006-01-01

Most eukaryotic cells spend most of their life in a quiescent state, poised to respond to specific signals to proliferate. In Saccharomyces cerevisiae, entry into and exit from quiescence are dependent only on the availability of nutrients in the environment. The transition from quiescence to proliferation requires not only drastic metabolic changes but also a complete remodeling of various cellular structures. Here, we describe an actin cytoskeleton organization specific of the yeast quiescent state. When cells cease to divide, actin is reorganized into structures that we named “actin bodies.” We show that actin bodies contain F-actin and several actin-binding proteins such as fimbrin and capping protein. Furthermore, by contrast to actin patches or cables, actin bodies are mostly immobile, and we could not detect any actin filament turnover. Finally, we show that upon cells refeeding, actin bodies rapidly disappear and actin cables and patches can be assembled in the absence of de novo protein synthesis. This led us to propose that actin bodies are a reserve of actin that can be immediately mobilized for actin cables and patches formation upon reentry into a proliferation cycle. PMID:16914523

Human spire interacts with the barbed end of the actin filament.

PubMed

Ito, Takuto; Narita, Akihiro; Hirayama, Tasuku; Taki, Masayasu; Iyoshi, Shohei; Yamamoto, Yukio; Maéda, Yuichiro; Oda, Toshiro

2011-04-22

Spire is an actin nucleator that initiates actin polymerization at a specific place in the cell. Similar to the Arp2/3 complex, spire was initially considered to bind to the pointed end of the actin filament when it generates a new actin filament. Subsequently, spire was reported to be associated with the barbed end (B-end); thus, there is still no consensus regarding the end with which spire interacts. Here, we report direct evidence that spire binds to the B-end of the actin filament, under conditions where spire accelerates actin polymerization. Using electron microscopy, we visualized the location of spire bound to the filament by gold nanoparticle labeling of the histidine-tagged spire, and the polarity of the actin filament was determined by image analysis. In addition, our results suggest that multiple spires, linked through one gold nanoparticle, enhance the acceleration of actin polymerization. The B-end binding of spire provides the basis for understanding its functional mechanism in the cell. Copyright © 2011 Elsevier Ltd. All rights reserved.

Cyclin-Dependent Kinase 5 Links Extracellular Cues to Actin Cytoskeleton During Dendritic Spine Development

PubMed Central

Fu, Amy KY

2007-01-01

Emerging evidence has indicated a regulatory role of cyclin-dependent kinase 5 (Cdk5) in synaptic plasticity as well as in higher brain functions, such as learning and memory. However, the molecular and cellular mechanisms underlying the actions of Cdk5 at synapses remain unclear. Recent findings demonstrate that Cdk5 regulates dendritic spine morphogenesis through modulating actin dynamics. Ephexin1 and WAVE-1, two important regulators of the actin cytoskeleton, have both been recently identified as substrates for Cdk5. Importantly, phosphorylation of these proteins by Cdk5 leads to dendritic spine loss, revealing a potential mechanism by which Cdk5 regulates synapse remodeling. Furthermore, Cdk5-dependent phosphorylation of ephexin1 is required for the ephrin-A1 mediated spine retraction, pointing to a critical role of Cdk5 in conveying signals from extracellular cues to actin cytoskeleton at synapses. Taken together, understanding the precise regulation of Cdk5 and its downstream targets at synapses would provide important insights into the multi-regulatory roles of Cdk5 in actin remodeling during dendritic spine development. PMID:19270534

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

Gamma interferon-induced guanylate binding protein 1 is a novel actin cytoskeleton remodeling factor.

PubMed

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; Stürzl, Michael

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.

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

PubMed Central

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

2010-01-01

demonstrate for the first time that L-plastin contributes to the fine-tuning of actin turn-over, an activity which is regulated by Ser5 phosphorylation promoting its high affinity binding to the cytoskeleton. In carcinoma cells, PKC-δ signaling pathways appear to link L-plastin phosphorylation to actin polymerization and invasion. PMID:20169155

Lipid-linked cell wall precursors regulate membrane association of bacterial actin MreB

PubMed Central

Schirner, Kathrin; Eun, Ye-Jin; Dion, Mike; Luo, Yun; Helmann, John D.; Garner, Ethan C.; Walker, Suzanne

2014-01-01

Summary The bacterial actin homolog MreB, which is critical for rod shape determination, forms filaments that rotate around the cell width on the inner surface of the cytoplasmic membrane. What determines filament association with the membranes or with other cell wall elongation proteins is not known. Using specific chemical and genetic perturbations while following MreB filament motion, we find that MreB membrane association is an actively regulated process that depends on the presence of lipid-linked peptidoglycan precursors. When precursors are depleted, MreB filaments disassemble into the cytoplasm and peptidoglycan synthesis becomes disorganized. In cells that lack wall teichoic acids, but continue to make peptidoglycan, dynamic MreB filaments are observed, although their presence is not sufficient to establish a rod shape. We propose that the cell regulates MreB filament association with the membrane, allowing rapid and reversible inactivation of cell wall enzyme complexes in response to the inhibition of cell wall synthesis. PMID:25402772

Lipid-linked cell wall precursors regulate membrane association of bacterial actin MreB.

PubMed

Schirner, Kathrin; Eun, Ye-Jin; Dion, Mike; Luo, Yun; Helmann, John D; Garner, Ethan C; Walker, Suzanne

2015-01-01

The bacterial actin homolog MreB, which is crucial for rod shape determination, forms filaments that rotate around the cell width on the inner surface of the cytoplasmic membrane. What determines filament association with the membranes or with other cell wall elongation proteins is not known. Using specific chemical and genetic perturbations while following MreB filament motion, we find that MreB membrane association is an actively regulated process that depends on the presence of lipid-linked peptidoglycan precursors. When precursors are depleted, MreB filaments disassemble into the cytoplasm, and peptidoglycan synthesis becomes disorganized. In cells that lack wall teichoic acids but continue to make peptidoglycan, dynamic MreB filaments are observed, although their presence is not sufficient to establish a rod shape. We propose that the cell regulates MreB filament association with the membrane, allowing rapid and reversible inactivation of cell wall enzyme complexes in response to the inhibition of cell wall synthesis.

In vivo oxidation in remelted highly cross-linked retrievals.

PubMed

Currier, B H; Van Citters, D W; Currier, J H; Collier, J P

2010-10-20

Elimination of free radicals to prevent oxidation has played a major role in the development and product differentiation of the latest generation of highly cross-linked ultra-high molecular weight polyethylene bearing materials. In the current study, we (1) examined oxidation in a series of retrieved remelted highly cross-linked ultra-high molecular weight polyethylene bearings from a number of device manufacturers and (2) compared the retrieval results with findings for shelf-stored control specimens. The hypothesis was that radiation-cross-linked remelted ultra-high molecular weight polyethylene would maintain oxidative stability in vivo comparable with the stability during shelf storage and in published laboratory aging tests. Fifty remelted highly cross-linked ultra-high molecular weight polyethylene acetabular liners and nineteen remelted highly cross-linked ultra-high molecular weight polyethylene tibial inserts were received after retrieval from twenty-one surgeons from across the U.S. Thirty-two of the retrievals had been in vivo for two years or more. Each was measured for oxidation with use of Fourier transform infrared spectroscopy. A control series of remelted highly cross-linked ultra-high molecular weight polyethylene acetabular liners from three manufacturers was analyzed with electron paramagnetic resonance spectroscopy to measure free radical content and with Fourier transform infrared spectroscopy to measure oxidation initially and after eight to nine years of shelf storage in air. The never-implanted, shelf-aged controls had no measurable free-radical content initially or after eight to nine years of shelf storage. The never-implanted controls showed no increase in oxidation during shelf storage. Oxidation measurements showed measurable oxidation in 22% of the retrieved remelted highly cross-linked liners and inserts after an average of two years in vivo. Because never-implanted remelted highly cross-linked ultra-high molecular weight

Cross-Linking Studies of Lysozyme Nucleation

NASA Technical Reports Server (NTRS)

Forsythe, Elizabeth; Pusey, Marc

2000-01-01

Tetragonal chicken egg white crystals consist of 4(sub 3) helices running in alternating directions, the helix rows having a two fold symmetry with each other. The unit cell consists of one complete tetrameric turn from each of two adjacent helices (an octamer). PBC analysis indicates that the helix intermolecular bonds are the strongest in the crystal, therefore likely formed first. AFM analysis of the (110) surface shows only complete helices, no half steps or bisected helices being found, while AFM line scans to measure the growth step increments show that they are multiples of the 4(sub 3) helix tetramer dimensions. This supports our thesis that the growth units are in fact multiples of the four molecule 4(sub 3) helix unit, the "average" growth unit size for the (110) face being an octamer (two turns about the helix) and the (101) growth unit averaging about the size of a hexamer. In an effort to better understand the species involved in the crystal nucleation and growth process, we have initiated an experimental program to study the species formed in solution compared to what is found in the crystal through covalent cross-linking studies. These experiments use the heterobifunctional cross-linking agent aminoethyl-4-azidonitroanaline (AEANA). An aliphatic amine at one end is covalently attached to the protein by a carbodiimide-mediated reaction, and a photo reactive group at the other can be used to initiate crosslinking. Modifications to the parent structure can be used to alter the distance between the two reactive groups and thus the cross-linking agents "reach". In practice, the cross-linking agent is first coupled to the asp101 side chain through the amine group. Asp101 lies within the active site cleft, and previous work with fluorescent probes had shown that derivatives at this site still crystallize in the tetragonal space group. This was also found to be the case with the AEANA derivative, which gave red tetragonal crystals. The protein now has a

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

PubMed

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

2018-05-08

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

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

PubMed Central

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

2011-01-01

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

Spectroscopic characterization of collagen cross-links in bone

NASA Technical Reports Server (NTRS)

Paschalis, E. P.; Verdelis, K.; Doty, S. B.; Boskey, A. L.; Mendelsohn, R.; Yamauchi, M.

2001-01-01

Collagen is the most abundant protein of the organic matrix in mineralizing tissues. One of its most critical properties is its cross-linking pattern. The intermolecular cross-linking provides the fibrillar matrices with mechanical properties such as tensile strength and viscoelasticity. In this study, Fourier transform infrared (FTIR) spectroscopy and FTIR imaging (FTIRI) analyses were performed in a series of biochemically characterized samples including purified collagen cross-linked peptides, demineralized bovine bone collagen from animals of different ages, collagen from vitamin B6-deficient chick homogenized bone and their age- and sex-matched controls, and histologically stained thin sections from normal human iliac crest biopsy specimens. One region of the FTIR spectrum of particular interest (the amide I spectral region) was resolved into its underlying components. Of these components, the relative percent area ratio of two subbands at approximately 1660 cm(-1) and approximately 1690 cm(-1) was related to collagen cross-links that are abundant in mineralized tissues (i.e., pyridinoline [Pyr] and dehydrodihydroxylysinonorleucine [deH-DHLNL]). This study shows that it is feasible to monitor Pyr and DHLNL collagen cross-links spatial distribution in mineralized tissues. The spectroscopic parameter established in this study may be used in FTIRI analyses, thus enabling the calculation of relative Pyr/DHLNL amounts in thin (approximately 5 microm) calcified tissue sections with a spatial resolution of approximately 7 microm.

Intra-molecular cross-linking of acidic residues for protein structure studies.

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

Kruppa, Gary Hermann; Young, Malin M.; Novak, Petr

2005-03-01

Intra-molecular cross-linking has been suggested as a method of obtaining distance constraints that would be useful in developing structural models of proteins. Recent work published on intra-molecular cross-linking for protein structural studies has employed commercially available primary amine selective reagents that can cross-link lysine residues to other lysine residues or the amino terminus. Previous work using these cross-linkers has shown that for several proteins of known structure, the number of cross-links that can be obtained experimentally may be small compared to what would be expected from the known structure, due to the relative reactivity, distribution, and solvent accessibility of themore » lysines in the protein sequence. To overcome these limitations we have investigated the use of cross-linking reagents that can react with other reactive sidechains in proteins. We used 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) to activate the carboxylic acid containing residues, aspartic acid (D), glutamic acid (E), and the carboxy terminus (O), for cross-linking reactions. Once activated, the DEO sidechains can react to form 'zero-length' cross-links with nearby primary amine containing resides, lysines (K) and the amino terminus (X), via the formation of a new amide bond. We also show that the EDC-activated DEO sidechains can be cross-linked to each other using dihydrazides, two hydrazide moieties connected by an alkyl cross-linker ann of variable length. Using these reagents, we have found three new 'zero-length' cross-links in ubiquitin consistent with its known structure (M1-E16, M1-E18, and K63-E64). Using the dihydrazide cross-linkers, we have identified 2 new cross-links (D21-D32 and E24-D32) unambiguously. Using a library of dihydrazide cross-linkers with varying arm length, we have shown that there is a minimum arm length required for the DEO-DEO cross-links of 5.8 angstroms. These results show that additional structural

Organization of photosystem I polypeptides examined by chemical cross-linking

NASA Technical Reports Server (NTRS)

Armbrust, T. S.; Chitnis, P. R.; Guikema, J. A.; Spooner, B. S. (Principal Investigator)

1996-01-01

Photosystem I from the cyanobacterium Synechocystis sp. PCC 6803 was examined using the chemical cross-linkers glutaraldehyde and N-ethyl-1-3-[3-(dimethylamino)propyl]carbodiimide to investigate the organization of the polypeptide subunits. Thylakoid membranes and photosystem I, which was isolated by Triton X-100 fractionation, were treated with cross-linking reagents and were resolved using a Tricine/urea low-molecular-weight resolution gel system. Subunit-specific antibodies and western blotting analysis were used to identify the components of cross-linked species. These analyses identified glutaraldehyde-dependent cross-linking products composed of small amounts of PsaD and PsaC, PsaC and PsaE, and PsaE and PsaF. The novel cross-link between PsaE and PsaF was also observed following treatment with N-ethyl-1-3-[3-(dimethylamino)propyl]carbodiimide. These cross-linking results suggest a structural interaction between PsaE and PsaF and predict a transmembrane topology for PsaF.

Diepoxybutane Interstrand Cross-Links Induce DNA Bending

PubMed Central

Millard, Julie T.; McGowan, Erin E.; Bradley, Sharonda Q.

2011-01-01

The bifunctional alkylating agent 1,2,3,4-diepoxybutane (DEB) is thought to be a major contributor to the carcinogenicity of 1,3-butadiene, from which it is derived in vivo. DEB forms DNA interstrand cross-links primarily between distal deoxyguanosine residues at the duplex sequence 5’-GNC. In order for the short butanediol tether to span this distance, distortion of the DNA target has been postulated. We determined that the electrophoretic mobility of ligated DNA oligomers containing DEB cross-links was retarded in comparison with control, uncross-linked DNA. Our data are consistent with DNA bending of ~34° per lesion towards the major groove. PMID:21839139

Measurement and Analysis of in vitro Actin Polymerization

PubMed Central

Doolittle, Lynda K.; Rosen, Michael K.; Padrick, Shae B.

2014-01-01

Summary The polymerization of actin underlies force generation in numerous cellular processes. While actin polymerization can occur spontaneously, cells maintain control over this important process by preventing actin filament nucleation and then allowing stimulated polymerization and elongation by several regulated factors. Actin polymerization, regulated nucleation and controlled elongation activities can be reconstituted in vitro, and used to probe the signaling cascades cells use to control when and where actin polymerization occurs. Introducing a pyrene fluorophore allows detection of filament formation by an increase in pyrene fluorescence. This method has been used for many years and continues to be broadly used, owing to its simplicity and flexibility. Here we describe how to perform and analyze these in vitro actin polymerization assays, with an emphasis on extracting useful descriptive parameters from kinetic data. PMID:23868594

Fabrication of chemically cross-linked porous gelatin matrices.

PubMed

Bozzini, Sabrina; Petrini, Paola; Altomare, Lina; Tanzi, Maria Cristina

2009-01-01

The aim of this study was to chemically cross-link gelatin, by reacting its free amino groups with an aliphatic diisocyanate. To produce hydrogels with controllable properties, the number of reacting amino groups was carefully determined. Porosity was introduced into the gelatin-based hydrogels through the lyophilization process. Porous and non-porous matrices were characterized with respect to their chemical structure, morphology, water uptake and mechanical properties. The physical, chemical and mechanical properties of the porous matrices are related to the extent of their cross-linking, showing that they can be controlled by varying the reaction parameters. Water uptake values (24 hours) vary between 160% and 200% as the degree of cross-linking increases. The flexibility of the samples also decreases by changing the extent of cross-linking. Young's modulus shows values between 0.188 KPa, for the highest degree, and 0.142 KPa for the lowest degree. The matrices are potential candidates for use as tissue-engineering scaffolds by modulating their physical chemical properties according to the specific application.

Practical application of thermoreversibly Cross-linked rubber products

NASA Astrophysics Data System (ADS)

Polgar, L. M.; Picchioni, F.; de Ruiter, E.; van Duin, M.

2017-07-01

Currently, rubber products cannot simply be reprocessed after their product life, due to the irreversible cross-linking methods traditionally applied. The purpose of this work is to investigate how thermoreversible cross-linking of rubbers via Diels Alder chemistry can be used for the development of recyclable rubber products. Unfortunately, the applicability of the thermoreversible EPM-g-furan/BM system appears to be limited to room temperature applications, because of the rapid deterioration of the compression set at elevated temperatures compared to irreversibly cross-linked EPM. However, the use of EPM rubber modified with thiophene or cyclopentadiene moieties may extend the temperature application range and results in rubber products with acceptable properties. Finally, rubber products generally comprise fillers such as silica, carbon black or fibers. In this context, the reinforcing effect of short cut aramid fibers on the material properties of the newly developed thermoreversibly cross-linked EPM rubbers was also studied. The material properties of the resulting products were found to be comparable to those of a fiber reinforced, peroxide cured reference sample.

HcRed, a Genetically Encoded Fluorescent Binary Cross-Linking Agent for Cross-Linking of Mitochondrial ATP Synthase in Saccharomyces cerevisiae

PubMed Central

Gong, Lan; Ramm, Georg; Devenish, Rodney J.; Prescott, Mark

2012-01-01

Genetically encoded fluorescent cross-linking agents represent powerful tools useful both for visualising and modulating protein interactions in living cells. The far-red fluorescent protein HcRed, which is fluorescent only in a dimer form, can be used to promote the homo-dimerisation of target proteins, and thereby yield useful information about biological processes. We have in yeast cells expressed HcRed fused to a subunit of mitochondrial ATP synthase (mtATPase). This resulted in cross-linking of the large multi-subunit mtATPase complex within the inner-membrane of the mitochondrion. Fluorescence microscopy revealed aberrant mitochondrial morphology, and mtATPase complexes isolated from mitochondria were recovered as fluorescent dimers under conditions where complexes from control mitochondria were recovered as monomers. When viewed by electron microscopy normal cristae were absent from mitochondria in cells in which mATPase complexes were cross-linked. mtATPase dimers are believed to be the building blocks that are assembled into supramolecular mtATPase ribbons that promote the formation of mitochondrial cristae. We propose that HcRed cross-links mATPase complexes in the mitochondrial membrane hindering the normal assembly/disassembly of the supramolecular forms of mtATPase. PMID:22496895

On the Reproducibility of Label-Free Quantitative Cross-Linking/Mass Spectrometry

NASA Astrophysics Data System (ADS)

Müller, Fränze; Fischer, Lutz; Chen, Zhuo Angel; Auchynnikava, Tania; Rappsilber, Juri

2018-02-01

Quantitative cross-linking/mass spectrometry (QCLMS) is an emerging approach to study conformational changes of proteins and multi-subunit complexes. Distinguishing protein conformations requires reproducibly identifying and quantifying cross-linked peptides. Here we analyzed the variation between multiple cross-linking reactions using bis[sulfosuccinimidyl] suberate (BS3)-cross-linked human serum albumin (HSA) and evaluated how reproducible cross-linked peptides can be identified and quantified by LC-MS analysis. To make QCLMS accessible to a broader research community, we developed a workflow that integrates the established software tools MaxQuant for spectra preprocessing, Xi for cross-linked peptide identification, and finally Skyline for quantification (MS1 filtering). Out of the 221 unique residue pairs identified in our sample, 124 were subsequently quantified across 10 analyses with coefficient of variation (CV) values of 14% (injection replica) and 32% (reaction replica). Thus our results demonstrate that the reproducibility of QCLMS is in line with the reproducibility of general quantitative proteomics and we establish a robust workflow for MS1-based quantitation of cross-linked peptides.

Sensory role of actin in auxin-dependent responses of tobacco BY-2.

PubMed

Huang, Xiang; Maisch, Jan; Nick, Peter

2017-11-01

Polar auxin transport depends on the polar localization of auxin-efflux carriers. The cycling of these carriers between cell interior and plasma membrane depends on actin. The dynamic of actin not only affects auxin transport, but also changes the auxin-responsiveness. To study the potential link between auxin responsiveness and actin dynamics, we investigated developmental responses of the non-transformed BY-2 (Nicotiana tabacum L. cv Bright Yellow 2) cell line and the transgenic BY-2 strain GF11 (stably transformed BY-2 cells with a GFP-fimbrin actin-binding domain 2 construct). The developmental process was divided into three distinct stages: cell cycling, cell elongation and file disintegration. Several phenotypes were measured to monitor the cellular responses to different concentrations of exogenous natural auxin (Indole-3-acetic acid, IAA). We found that auxin stimulated and prolonged the mitotic activity, and delayed the exit from the proliferation phase. However, both responses were suppressed in the GF11 line. At the stationary phase of the cultivation cycle, auxin strongly accelerated the cell file disintegration. Interestingly, it was not suppressed but progressed to a more complete disintegration in the GF11 line. During the cultivation cycle, we also followed the organization of actin in the GF11 line and did not detect any significant difference in actin organization from untreated control or exogenous IAA treatment. Therefore, our findings indicate that the specific differences observed in the GF11 line must be linked with a function of actin that is not structural. It means that there is a sensory role of actin for auxin signaling. Copyright © 2017 Elsevier GmbH. All rights reserved.

Comparison of the properties of collagen-chitosan scaffolds after γ-ray irradiation and carbodiimide cross-linking.

PubMed

Chen, Zihao; Du, Tianming; Tang, Xiangyu; Liu, Changjun; Li, Ruixin; Xu, Cheng; Tian, Feng; Du, Zhenjie; Wu, Jimin

2016-07-01

The property of collagen-chitosan porous scaffold varies according to cross-linking density and scaffold composition. This study was designed to compare the properties of collagen-chitosan porous scaffolds cross-linked with γ-irradiation and carbodiimide (CAR) for the first time. Eleven sets of collagen-chitosan scaffolds containing different concentrations of chitosan at a 5% increasing gradient were fabricated. Fourier transform infrared spectroscopy was performed to confirm the success of cross-linking in the scaffolds. The scaffold morphology was evaluated under scanning electron microscope (SEM). SEM revealed that chitosan was an indispensable material for the fabrication of γ-ray irradiation scaffold. The microstructure of γ-ray irradiation scaffold was less stable than those of alternative scaffolds. Based upon swelling ratio, porosity factor, and collagenase degradation, γ-ray irradiation scaffold was less stable than CAR and 25% proportion of chitosan scaffolds. Mechanical property determines the orientation in γ-irradiation and CAR scaffold. In vitro degradation test indicated that γ-irradiation and CAR cross-linking can elevate the scaffold biocompatibility. Compared with γ-ray irradiation, CAR cross-linked scaffold containing 25% chitosan can more significantly enhance the bio-stability and biocompatibility of collagen-chitosan scaffolds. CAR cross-linked scaffold may be the best choice for future tissue engineering.

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

PubMed

Frölich, Sonja; Wallach, Michael

2015-06-01

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

Synthesis and enzymatic degradation of epichlorohydrin cross-linked pectins.

PubMed

Semdé, Rasmané; Moës, André J; Devleeschouwer, Michel J; Amighi, Karim

2003-02-01

The water solubility of pectin was successfully decreased by cross-linking with increasing amounts of epichlorohydrin in the reaction media. The initial molar ratios of epichlorohydrin/ galacturonic acid monomer in the reaction mixtures were 0, 0.37, 0.56, 0.74, 1.00, 1.47, and 2.44. The resulting epichlorohydrin cross-linked pectins were thus referred to as C-LP0, C-LP37, C-LP56, C-LP75, C-LP100, C-LP150, and C-LP250, respectively. Methoxylation degrees ranged from 60.5 +/- 0.9% to 68.0 +/- 0.6%, and the effective cross-linking degrees, determined by quantification of the hydroxyl anions consumed during the reaction, were 0, 17.8, 26.0, 38.3, 46.5, 53.5, and 58.7%. respectively. After incubating the different cross-linked pectins (0.5% w/v) in 25 mL of 0.05 M acetate-phosphate buffer (pH 4.5), containing 50 microL of Pectinex Ultra SP-L (pectinolytic enzymes), between 60 and 80% of the pectin osidic bounds were broken in less than 1 hr. Moreover, increasing the cross-linking degree only resulted in a weak slowing on the enzymatic degradation velocity.

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

Rebowski, Grzegorz; Namgoong, Suk; Boczkowska, Malgorzata

Actin filament nucleators initiate polymerization in cells in a regulated manner. A common architecture among these molecules consists of tandem WASP homology 2 domains (W domains) that recruit three to four actin subunits to form a polymerization nucleus. We describe a low-resolution crystal structure of an actin dimer assembled by tandem W domains, where the first W domain is cross-linked to Cys374 of the actin subunit bound to it, whereas the last W domain is followed by the C-terminal pointed end-capping helix of thymosin {beta}4. While the arrangement of actin subunits in the dimer resembles that of a long-pitch helixmore » of the actin filament, important differences are observed. These differences result from steric hindrance of the W domain with intersubunit contacts in the actin filament. We also determined the structure of the first W domain of Vibrio parahaemolyticus VopL cross-linked to actin Cys374 and show it to be nearly identical with non-cross-linked W-Actin structures. This result validates the use of cross-linking as a tool for the study of actin nucleation complexes, whose natural tendency to polymerize interferes with most structural methods. Combined with a biochemical analysis of nucleation, the structures may explain why nucleators based on tandem W domains with short inter-W linkers have relatively weak activity, cannot stay bound to filaments after nucleation, and are unlikely to influence filament elongation. The findings may also explain why nucleation-promoting factors of the Arp2/3 complex, which are related to tandem-W-domain nucleators, are ejected from branch junctions after nucleation. We finally show that the simple addition of the C-terminal pointed end-capping helix of thymosin {beta}4 to tandem W domains can change their activity from actin filament nucleation to monomer sequestration.« less

Lights, camera, actin.

PubMed

Rubenstein, Peter A; Wen, Kuo-Kuang

2005-10-01

Actin participates in many important biological processes. Currently, intensive investigation is being carried out in a number of laboratories concerning the function of actin in these processes and the molecular basis of its functions. We present a glimpse into four of these areas: actin-like proteins in bacterial cells, actin in the eukaryotic nucleus, the conformational plasticity of the actin filament, and finally, Arp2/3-dependent regulation of actin filament branching and creation of new filament barbed ends. IUBMB Life, 57: 683-687, 2005.

Towards the Structure Determination of a Modulated Protein Crystal: The Semicrystalline State of Profilin:Actin

NASA Technical Reports Server (NTRS)

Borgstahl, G.; Lovelace, J.; Snell, E. H.; Bellamy, H.

2003-01-01

microfilament system to be restructured in a controlled manner via polymerization, depolymerization, severing, cross-linking, and anchorage. The structure the semicrystalline state of profilin:actin will challenge and validate current models of muscle contraction and cell motility. The methodology and theory under development will be easily extendable to other systems.

Actin-binding proteins sensitively mediate F-actin bundle stiffness

NASA Astrophysics Data System (ADS)

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

2006-09-01

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

Highly cross-linked nanoporous polymers

DOEpatents

Steckle, Jr., Warren P.; Apen, Paul G.; Mitchell, Michael A.

1998-01-01

Condensation polymerization followed by a supercritical extraction step can be used to obtain highly cross-linked nanoporous polymers with high surface area, controlled pore sizes and rigid structural integrity. The invention polymers are useful for applications requiring separation membranes.

Highly cross-linked nanoporous polymers

DOEpatents

Steckle, Jr., Warren P.; Apen, Paul G.; Mitchell, Michael A.

1997-01-01

Condensation polymerization followed by a supercritical extraction step can be used to obtain highly cross-linked nanoporous polymers with high surface area, controlled pore sizes and rigid structural integrity. The invention polymers are useful for applications requiring separation membranes.

Formaldehyde cross-linking and structural proteomics: Bridging the gap.

PubMed

Srinivasa, Savita; Ding, Xuan; Kast, Juergen

2015-11-01

Proteins are dynamic entities constantly moving and altering their structures based on their functions and interactions inside and outside the cell. Formaldehyde cross-linking combined with mass spectrometry can accurately capture interactions of these rapidly changing biomolecules while maintaining their physiological surroundings. Even with its numerous established uses in biology and compatibility with mass spectrometry, formaldehyde has not yet been applied in structural proteomics. However, formaldehyde cross-linking is moving toward analyzing tertiary structure, which conventional cross-linkers have already accomplished. The purpose of this review is to describe the potential of formaldehyde cross-linking in structural proteomics by highlighting its applications, characteristics and current status in the field. Copyright © 2015 Elsevier Inc. All rights reserved.

Cross-Linked Nanotube Materials with Variable Stiffness Tethers

NASA Technical Reports Server (NTRS)

Frankland, Sarah-Jane V.; Odegard, Gregory M.; Herzog, Matthew N.; Gates, Thomas S.; Fay, Catherine C.

2004-01-01

The constitutive properties of a cross-linked single-walled carbon nanotube material are predicted with a multi-scale model. The material is modeled as a transversely isotropic solid using concepts from equivalent-continuum modeling. The elastic constants are determined using molecular dynamics simulation. Some parameters of the molecular force field are determined specifically for the cross-linker from ab initio calculations. A demonstration of how the cross-linked nanotubes may affect the properties of a nanotube/polyimide composite is included using a micromechanical analysis.

Highly cross-linked nanoporous polymers

DOEpatents

Steckle, W.P. Jr.; Apen, P.G.; Mitchell, M.A.

1998-01-20

Condensation polymerization followed by a supercritical extraction step can be used to obtain highly cross-linked nanoporous polymers with high surface area, controlled pore sizes and rigid structural integrity. The invention polymers are useful for applications requiring separation membranes. 1 fig.

Resistance of Actin to Cleavage during Apoptosis

NASA Astrophysics Data System (ADS)

Song, Qizhong; Wei, Tie; Lees-Miller, Susan; Alnemri, Emad; Watters, Dianne; Lavin, Martin F.

1997-01-01

A small number of cellular proteins present in the nucleus, cytosol, and membrane fraction are specifically cleaved by the interleukin-1β -converting enzyme (ICE)-like family of proteases during apoptosis. Previous results have demonstrated that one of these, the cytoskeletal protein actin, is degraded in rat PC12 pheochromocytoma cells upon serum withdrawal. Extracts from etoposide-treated U937 cells are also capable of cleaving actin. It was assumed that cleavage of actin represented a general phenomenon, and a mechanism coordinating proteolytic, endonucleolytic, and morphological aspects of apoptosis was proposed. We demonstrate here that actin is resistant to degradation in several different human cells induced to undergo apoptosis in response to a variety of stimuli, including Fas ligation, serum withdrawal, cytotoxic T-cell killing, and DNA damage. On the other hand, cell-free extracts from these cells and the ICE-like protease CPP32 were capable of cleaving actin in vitro. We conclude that while actin contains cleavage sites for ICE-like proteases, it is not degraded in vivo in human cells either because of lack of access of these proteases to actin or due to the presence of other factors that prevent degradation.

The wear of cross-linked polyethylene against itself.

PubMed

Joyce, T J; Ash, H E; Unsworth, A

1996-01-01

Cross-linked polyethylene (XLPE) may have an application as a material for an all-plastic surface replacement finger joint. It is inexpensive, biocompatible and can be injection-moulded into the complex shapes that are found on the ends of the finger bones. Further, the cross-linking of polyethylene has significantly improved its mechanical properties. Therefore, the opportunity exists for an all-XLPE joint, and so the wear characteristics of XLPE sliding against itself have been investigated. Wear tests were carried out on both reciprocating pin-on-plate machines and a finger function simulator. The reciprocating pin-on-plate machines had pins loaded at 10 N and 40 N. All pin-on-plate tests show wear factors from the plates very much greater than those of the pins. After 349 km of sliding, a mean wear factor of 0.46 x 10(-6) mm3/N m was found for the plates compared with 0.021 x 10(-6) mm3/N m for the pins. A fatigue mechanism may be causing this phenomenon of greater plate wear. Tests using the finger function simulator give an average wear rate of 0.22 x 10(-6) mm3/N m after 368 km. This sliding distance is equivalent to 12.5 years of use in vivo. The wear factors found were comparable with those of ultra-high molecular weight polyethylene (UHMWPE) against a metallic counterface and, therefore, as the loads across the finger joint are much less than those across the knee or the hip, it is probable that an all-XLPE finger joint will be viable from a wear point of view.

Managing actinic keratosis in primary care.

PubMed

Salmon, Nicola; Tidman, Michael J

2016-10-01

Actinic, or solar, keratosis is caused by chronic ultraviolet-induced damage to the epidermis. In the UK, 15-23% of individuals have actinic keratosis lesions. Risk factors include: advanced age; male gender; cumulative sun exposure or phototherapy; Fitzpatrick skin phototypes I-II; long-term immuno-suppression and genetic syndromes e.g. xeroderma pigmentosum and albinism. Actinic keratoses are regarded by some authorities as premalignant lesions that may transform into invasive squamous cell carcinoma (SCC) and by others as in situ SCC that may progress to an invasive stage. The risk of malignant change appears low; up to 0.5% per lesion per year. Up to 20-30% of lesions may spontaneously regress but in the absence of any reliable prognostic clinical indicators regarding malignant potential active treatment is considered appropriate. Actinic keratosis lesions may present as discrete hyperkeratotic papules, cutaneous horns, or more subtle flat lesions on sun-exposed areas of skin. The single most helpful diagnostic sign is an irregularly roughened surface texture: a sandpaper-like feel almost always indicates actinic damage. Dermatoscopy can be helpful in excluding signs of basal cell carcinoma when actinic keratosis is non-keratotic. It is always important to consider the possibility of SCC. The principal indication for referral to secondary care is the possibility of cutaneous malignancy. However, widespread and severe actinic damage in patients who are immunosuppressed is also a reason for referral.

Probing structures of large protein complexes using zero-length cross-linking.

PubMed

Rivera-Santiago, Roland F; Sriswasdi, Sira; Harper, Sandra L; Speicher, David W

2015-11-01

Structural mass spectrometry (MS) is a field with growing applicability for addressing complex biophysical questions regarding proteins and protein complexes. One of the major structural MS approaches involves the use of chemical cross-linking coupled with MS analysis (CX-MS) to identify proximal sites within macromolecules. Identified cross-linked sites can be used to probe novel protein-protein interactions or the derived distance constraints can be used to verify and refine molecular models. This review focuses on recent advances of "zero-length" cross-linking. Zero-length cross-linking reagents do not add any atoms to the cross-linked species due to the lack of a spacer arm. This provides a major advantage in the form of providing more precise distance constraints as the cross-linkable groups must be within salt bridge distances in order to react. However, identification of cross-linked peptides using these reagents presents unique challenges. We discuss recent efforts by our group to minimize these challenges by using multiple cycles of LC-MS/MS analysis and software specifically developed and optimized for identification of zero-length cross-linked peptides. Representative data utilizing our current protocol are presented and discussed. Copyright © 2015 Elsevier Inc. All rights reserved.

Study on the preparation process of cross-linked porous cassava starch

NASA Astrophysics Data System (ADS)

Yin, Xiulian; You, Qinghong; Wan, Miaomiao; Zhang, Xuejuan; Dai, Chunhua

2017-04-01

Using cassava starch as raw material, preparation process of porous cross-linked cassava starch was studied. Using TSTP as cross-linking agents, Orthogonal design was applied for the optimization of cross-linked porous starch preparation process. The results showed that the opitmal conditions of cross-linked porous cassava starch were as follows: reaction temperature 45°C, reaction time 20 h, 1% of the amount of the enzyme, the enzyme ratio of 1:5, pH 5.50, substrate concentration of 40%.

Actin polymerization‐dependent activation of Cas‐L promotes immunological synapse stability

PubMed Central

Santos, Luís C; Blair, David A; Kumari, Sudha; Cammer, Michael; Iskratsch, Thomas; Herbin, Olivier; Alexandropoulos, Konstantina

2016-01-01

The immunological synapse formed between a T‐cell and an antigen‐presenting cell is important for cell–cell communication during T‐cell‐mediated immune responses. Immunological synapse formation begins with stimulation of the T‐cell receptor (TCR). TCR microclusters are assembled and transported to the center of the immunological synapse in an actin polymerization‐dependent process. However, the physical link between TCR and actin remains elusive. Here we show that lymphocyte‐specific Crk‐associated substrate (Cas‐L), a member of a force sensing protein family, is required for transport of TCR microclusters and for establishing synapse stability. We found that Cas‐L is phosphorylated at TCR microclusters in an actin polymerization‐dependent fashion. Furthermore, Cas‐L participates in a positive feedback loop leading to amplification of Ca2+ signaling, inside–out integrin activation, and actomyosin contraction. We propose a new role for Cas‐L in T‐cell activation as a mechanical transducer linking TCR microclusters to the underlying actin network and coordinating multiple actin‐dependent structures in the immunological synapse. Our studies highlight the importance of mechanotransduction processes in T‐cell‐mediated immune responses. PMID:27359298

Modified gum arabic cross-linked gelatin scaffold for biomedical applications.

PubMed

Sarika, P R; Cinthya, Kuriakose; Jayakrishnan, A; Anilkumar, P R; James, Nirmala Rachel

2014-10-01

The present work deals with development of modified gum arabic cross-linked gelatin scaffold for cell culture. A new biocompatible scaffold was developed by cross-linking gelatin (Gel) with gum arabic, a polysaccharide. Gum arabic was subjected to periodate oxidation to obtain gum arabic aldehyde (GAA). GAA was reacted with gelatin under appropriate pH to prepare the cross-linked hydrogel. Cross-linking occurred due to Schiff's base reaction between aldehyde groups of oxidized gum arabic and amino groups of gelatin. The scaffold prepared from the hydrogel was characterized by swelling properties, degree of cross-linking, in vitro degradation and scanning electron microscopy (SEM). Cytocompatibility evaluation using L-929 and HepG2 cells confirmed non-cytotoxic and non-adherent nature of the scaffold. These properties are essential for generating multicellular spheroids and hence the scaffold is proposed to be a suitable candidate for spheroid cell culture. Copyright © 2014 Elsevier B.V. All rights reserved.

Enrichment of Cross-Linked Peptides Using Charge-Based Fractional Diagonal Chromatography (ChaFRADIC).

PubMed

Tinnefeld, Verena; Venne, A Saskia; Sickmann, Albert; Zahedi, René P

2017-02-03

Chemical cross-linking of proteins is an emerging field with huge potential for the structural investigation of proteins and protein complexes. Owing to the often relatively low yield of cross-linking products, their identification in complex samples benefits from enrichment procedures prior to mass spectrometry analysis. So far, this is mainly accomplished by using biotin moieties in specific cross-linkers or by applying strong cation exchange chromatography (SCX) for a relatively crude enrichment. We present a novel workflow to enrich cross-linked peptides by utilizing charge-based fractional diagonal chromatography (ChaFRADIC). On the basis of two-dimensional diagonal SCX separation, we could increase the number of identified cross-linked peptides for samples of different complexity: pure cross-linked BSA, cross-linked BSA spiked into a simple protein mixture, and cross-linked BSA spiked into a HeLa lysate. We also compared XL-ChaFRADIC with size exclusion chromatography-based enrichment of cross-linked peptides. The XL-ChaFRADIC approach is straightforward, reproducible, and independent of the cross-linking chemistry and cross-linker properties.

Enzymatically cross-linked injectable alginate-g-pyrrole hydrogels for neovascularization.

PubMed

Devolder, Ross; Antoniadou, Eleni; Kong, Hyunjoon

2013-11-28

Microparticles capable of releasing protein drugs are often incorporated into injectable hydrogels to minimize their displacement at an implantation site, reduce initial drug burst, and further control drug release rates over a broader range. However, there is still a need to develop methods for releasing drug molecules over extended periods of time, in order to sustain the bioactivity of drug molecules at an implantation site. In this study, we hypothesized that a hydrogel formed through the cross-linking of pyrrole units linked to a hydrophilic polymer would release protein drugs in a more sustained manner, because of an enhanced association between cross-linked pyrrole groups and the drug molecules. To examine this hypothesis, we prepared hydrogels of alginate substituted with pyrrole groups, alginate-g-pyrrole, through a horse-radish peroxidase (HRP)-activated cross-linking of the pyrrole groups. The hydrogels were encapsulated with poly(lactic-co-glycolic acid) (PLGA) microparticles loaded with vascular endothelial growth factor (VEGF). The resulting hydrogel system released VEGF in a more sustained manner than Ca(2+) alginate or Ca(2+) alginate-g-pyrrole gel systems. Finally, implantations of the VEGF-releasing HRP-activated alginate-g-pyrrole hydrogel system on chicken chorioallantoic membranes resulted in the formation of blood vessels in higher densities and with larger diameters, compared to other control conditions. Overall, the drug releasing system developed in this study will be broadly useful for regulating release rates of a wide array of protein drugs, and further enhance the quality of protein drug-based therapies. © 2013 Elsevier B.V. All rights reserved.

Citric-Acid-Derived Photo-cross-Linked Biodegradable Elastomers

PubMed Central

Gyawali, Dipendra; Tran, Richard T.; Guleserian, Kristine J.; Tang, Liping; Yang, Jian

2010-01-01

Citric-acid-derived thermally cross-linked biodegradable elastomers (CABEs) have recently received significant attention in various biomedical applications, including tissue-engineering orthopedic devices, bioimaging and implant coatings. However, citric-acid-derived photo-cross-linked biodegradable elastomers are rarely reported. Herein, we report a novel photo-cross-linked biodegradable elastomer, referred to as poly(octamethylene maleate citrate) (POMC), which preserves pendant hydroxyl and carboxylic functionalities after cross-linking for the potential conjugation of biologically active molecules. POMC is a low-molecular-mass pre-polymer with a molecular mass average between 701 and 1291 Da. POMC networks are soft and elastic with an initial modulus of 0.07 to 1.3 MPa and an elongation at break between 38 and 382%. FT-IR–ATR results confirmed the successful surface immobilization of type-I collagen onto POMC films, which enhanced in vitro cellular attachment and proliferation. Photo-polymerized POMC films implanted subcutaneously into Sprague–Dawley rats demonstrated minimal in vivo inflammatory responses. The development of POMC enriches the family of citric-acid-derived biodegradable elastomers and expands the available biodegradable polymers for versatile needs in biomedical applications. PMID:20557687

Glycosylated and nonglycosylated recombinant human granulocyte colony-stimulating factor differently modifies actin polymerization in neutrophils.

PubMed

Zucca, A; Brizzi, S; Riccioni, R; Azzarà, A; Ghimenti, M; Carulli, G

2006-01-01

Several neutrophil functions can be modified by rhG-CSF administration. Neutrophil morphology changes in the course of treatment with Filgrastim (nonglycosylated rhG-CSF), along with impairment of chemotaxis. Both morphology and chemotaxis are not affected by treatment with Lenograstim (glycosylated rhG-CSF). Thus, we evaluated actin polymerization in neutrophils induced by treatment with the two forms of rhG-CSF. In fact, actin polymerization is crucial for neutrophil motility. We evaluated twelve healthy subjects undergoing peripheral blood stem cells (PBSC) mobilization for allogeneic transplantation to HLA-identical siblings. Neutrophils were isolated by peripheral venous blood before and after administration of either Filgrastim (six PBSC donors) or Lenograstim (six PBSC donors). Actin polymerization was investigated by a flow cytometric assay, using FITC-phalloidin as a specific probe for F-actin, and two parameters were measured: spontaneous actin polymerization in resting neutrophils; fMLP-stimulated actin polymerization. Results were expressed as relative F-actin content. Fifteen blood donors were studied as a control group. Filgrastim administration induced an increased relative F-actin content in resting neutrophils; however, no further actin polymerization was observed after fMLP stimulation. Neutrophils from subjects treated with Lenograstim showed a normal behaviour in terms of both spontaneous and stimulated actin polymerization. Glycosylated and nonglycosylated rhG-CSF differently affect actin polymerization in newly generated neutrophils. Such effects may explain some previous findings concerning both morphology and chemotactic properties and may be due to different effects of the two forms of rhG-CSF on proteins involved in neutrophil motility regulation.

Cross-linked polymeric membranes for carbon dioxide separation

DOEpatents

Hong, Tao; Chatterjee, Sabornie; Mahurin, Shannon Mark; Long, Brian Keith; Jiang, De-en; Mays, Jimmy Wayne; Sokolov, Alexei P.; Saito, Tomonori

2018-01-23

A membrane useful in gas separation, the membrane comprising a cross-linked polysiloxane structure having a cross-link density of about 0.1.times.10.sup.-5 mol/cm.sup.3 to about 6.times.10.sup.-5 mol/cm.sup.3, where, in particular embodiments, the cross-linked polysiloxane structure has the following general structure: ##STR00001## wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, and R.sup.6 are independently selected from hydrocarbon groups having at least 1 and up to 6 carbon atoms; A.sup.1 and A.sup.2 are independently selected from cyclic hydrocarbon groups; L.sup.1 and L.sup.2 are linking groups or covalent bonds; n is an integer of at least 1; r and s are independently selected from integers of at least 1; and p is an integer of at least 10. The invention also includes methods for making and using the above-described membranes for gas separation.

Cross-Linked Enzyme Aggregates for Applications in Aqueous and Nonaqueous Media.

PubMed

Roy, Ipsita; Mukherjee, Joyeeta; Gupta, Munishwar N

2017-01-01

Extensive cross-linking of a precipitate of a protein by a cross-linking reagent (glutaraldehyde has been most commonly used) creates an insoluble enzyme preparation called cross-linked enzyme aggregates (CLEAs). CLEAs show high stability and performance in conventional aqueous as well as nonaqueous media. These are also stable at fairly high temperatures. CLEAs with more than one kind of enzyme activity can be prepared, and such CLEAs are called combi-CLEAs or multipurpose CLEAs. Extent of cross-linking often influences their morphology, stability, activity, and enantioselectivity.

A General Method for Targeted Quantitative Cross-Linking Mass Spectrometry.

PubMed

Chavez, Juan D; Eng, Jimmy K; Schweppe, Devin K; Cilia, Michelle; Rivera, Keith; Zhong, Xuefei; Wu, Xia; Allen, Terrence; Khurgel, Moshe; Kumar, Akhilesh; Lampropoulos, Athanasios; Larsson, Mårten; Maity, Shuvadeep; Morozov, Yaroslav; Pathmasiri, Wimal; Perez-Neut, Mathew; Pineyro-Ruiz, Coriness; Polina, Elizabeth; Post, Stephanie; Rider, Mark; Tokmina-Roszyk, Dorota; Tyson, Katherine; Vieira Parrine Sant'Ana, Debora; Bruce, James E

2016-01-01

Chemical cross-linking mass spectrometry (XL-MS) provides protein structural information by identifying covalently linked proximal amino acid residues on protein surfaces. The information gained by this technique is complementary to other structural biology methods such as x-ray crystallography, NMR and cryo-electron microscopy[1]. The extension of traditional quantitative proteomics methods with chemical cross-linking can provide information on the structural dynamics of protein structures and protein complexes. The identification and quantitation of cross-linked peptides remains challenging for the general community, requiring specialized expertise ultimately limiting more widespread adoption of the technique. We describe a general method for targeted quantitative mass spectrometric analysis of cross-linked peptide pairs. We report the adaptation of the widely used, open source software package Skyline, for the analysis of quantitative XL-MS data as a means for data analysis and sharing of methods. We demonstrate the utility and robustness of the method with a cross-laboratory study and present data that is supported by and validates previously published data on quantified cross-linked peptide pairs. This advance provides an easy to use resource so that any lab with access to a LC-MS system capable of performing targeted quantitative analysis can quickly and accurately measure dynamic changes in protein structure and protein interactions.

Nuclear Functions of Actin

PubMed Central

Visa, Neus; Percipalle, Piergiorgio

2010-01-01

Actin participates in several essential processes in the cell nucleus. Even though the presence of actin in the nucleus was proposed more than 30 years ago, nuclear processes that require actin have been only recently identified. Actin is part of chromatin remodeling complexes; it is associated with the transcription machineries; it becomes incorporated into newly synthesized ribonucleoproteins; and it influences long-range chromatin organization. As in the cytoplasm, nuclear actin works in conjunction with different types of actin-binding proteins that regulate actin function and bridge interactions between actin and other nuclear components. PMID:20452941

Molecular Model for HNBR with Tunable Cross-Link Density.

PubMed

Molinari, N; Khawaja, M; Sutton, A P; Mostofi, A A

2016-12-15

We introduce a chemically inspired, all-atom model of hydrogenated nitrile butadiene rubber (HNBR) and assess its performance by computing the mass density and glass-transition temperature as a function of cross-link density in the structure. Our HNBR structures are created by a procedure that mimics the real process used to produce HNBR, that is, saturation of the carbon-carbon double bonds in NBR, either by hydrogenation or by cross-linking. The atomic interactions are described by the all-atom "Optimized Potentials for Liquid Simulations" (OPLS-AA). In this paper, first, we assess the use of OPLS-AA in our models, especially using NBR bulk properties, and second, we evaluate the validity of the proposed model for HNBR by investigating mass density and glass transition as a function of the tunable cross-link density. Experimental densities are reproduced within 3% for both elastomers, and qualitatively correct trends in the glass-transition temperature as a function of monomer composition and cross-link density are obtained.

Elastic Coupling of Nascent apCAM Adhesions to Flowing Actin Networks

PubMed Central

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

2013-01-01

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

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

PubMed

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

2014-03-01

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

A Study into the Collision-induced Dissociation (CID) Behavior of Cross-Linked Peptides*

PubMed Central

Giese, Sven H.; Fischer, Lutz; Rappsilber, Juri

2016-01-01

Cross-linking/mass spectrometry resolves protein–protein interactions or protein folds by help of distance constraints. Cross-linkers with specific properties such as isotope-labeled or collision-induced dissociation (CID)-cleavable cross-linkers are in frequent use to simplify the identification of cross-linked peptides. Here, we analyzed the mass spectrometric behavior of 910 unique cross-linked peptides in high-resolution MS1 and MS2 from published data and validate the observation by a ninefold larger set from currently unpublished data to explore if detailed understanding of their fragmentation behavior would allow computational delivery of information that otherwise would be obtained via isotope labels or CID cleavage of cross-linkers. Isotope-labeled cross-linkers reveal cross-linked and linear fragments in fragmentation spectra. We show that fragment mass and charge alone provide this information, alleviating the need for isotope-labeling for this purpose. Isotope-labeled cross-linkers also indicate cross-linker-containing, albeit not specifically cross-linked, peptides in MS1. We observed that acquisition can be guided to better than twofold enrich cross-linked peptides with minimal losses based on peptide mass and charge alone. By help of CID-cleavable cross-linkers, individual spectra with only linear fragments can be recorded for each peptide in a cross-link. We show that cross-linked fragments of ordinary cross-linked peptides can be linearized computationally and that a simplified subspectrum can be extracted that is enriched in information on one of the two linked peptides. This allows identifying candidates for this peptide in a simplified database search as we propose in a search strategy here. We conclude that the specific behavior of cross-linked peptides in mass spectrometers can be exploited to relax the requirements on cross-linkers. PMID:26719564

Inverted bulk-heterojunction solar cell with cross-linked hole-blocking layer

PubMed Central

Udum, Yasemin; Denk, Patrick; Adam, Getachew; Apaydin, Dogukan H.; Nevosad, Andreas; Teichert, Christian; S. White, Matthew.; S. Sariciftci, Niyazi.; Scharber, Markus C.

2014-01-01

We have developed a hole-blocking layer for bulk-heterojunction solar cells based on cross-linked polyethylenimine (PEI). We tested five different ether-based cross-linkers and found that all of them give comparable solar cell efficiencies. The initial idea that a cross-linked layer is more solvent resistant compared to a pristine PEI layer could not be confirmed. With and without cross-linking, the PEI layer sticks very well to the surface of the indium–tin–oxide electrode and cannot be removed by solvents used to process PEI or common organic semiconductors. The cross-linked PEI hole-blocking layer functions for multiple donor–acceptor blends. We found that using cross-linkers improves the reproducibility of the device fabrication process. PMID:24817837

Selective Blockade of Cytoskeletal Actin Remodeling Reduces Experimental Choroidal Neovascularization

PubMed Central

Caballero, Sergio; Yang, Ru; Chaqour, Brahim

2011-01-01

Purpose. The efficacy of the peptide Ac-EEED on reducing cell adhesion and proliferation in vitro and choroidal neovascularization (CNV) in vivo was examined. Methods. The peptide chimera containing the Ac-EEED sequence was chemically linked to the N terminus of the XMTM delivery peptide from the Erns viral surface protein. Ac-EEED or scrambled control peptide (SCRAM) was added to cultures of vascular smooth muscle cells, pericytes, endothelial cells, and fibroblasts, and adhesion, growth, and matrix production was assessed. Ac-EEED or SCRAM was injected into the vitreous of mice undergoing laser rupture of Bruch's membrane to induce CNV and lesion volume, neovascularization and lesion fibrosis were assessed. Results. Ac-EEED–induced changes in the morphology of the actin cytoskeleton by inhibiting polymerization of G-actin and disrupting the formation of stress fibers. Pretreatment with Ac-EEED resulted in endothelial cells becoming less responsive to the mitogenic and pro-adhesive effects of VEGF. Ac-EEED treatment in fibroblasts reduced TGF-β–induced fibrosis as assessed by decreased levels of connective tissue growth factor, cysteine-rich 61, collagen I (COL1A2), and collagen III (COL3A1). CNV lesion size and fibrosis were reduced in a concentration-dependent manner by up to 60%. Conclusions. In vitro studies showed that Ac-EEED affects a broad range of mechanical properties associated with cytoskeletal actin to reduce growth factor effects. The utilization of Ac-EEED in vivo may offer a novel therapeutic strategy by both suppressed neovessel growth and curtailing fibrosis typically associated with the involutional stage of CNV. PMID:21178140

A Review of Collagen Cross-Linking in Cornea and Sclera

PubMed Central

Zhang, Xiao; Tao, Xiang-chen; Zhang, Jian; Li, Zhi-wei; Xu, Yan-yun; Wang, Yu-meng; Zhang, Chun-xiao; Mu, Guo-ying

2015-01-01

Riboflavin/UVA cross-linking is a technique introduced in the past decades for the treatment of keratoconus, keratectasia, and infectious keratitis. Its efficacy and safety have been investigated with clinical and laboratory studies since its first clinical application by Wollensak for the treatment of keratoconus. Although its complications are encountered during clinical practice, such as infection inducing risk, minimal invasion merits a further investigation on its future application in clinical practice. Recently, collagen cross-linking in sclera shows a promising prospect. In present study, we summarized the representative studies describing the clinical and laboratory application of collagen cross-linking published in past decades and provided our opinion on the positive and negative results of cross-linking in the treatment of ophthalmic disorders. PMID:25922758

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

PubMed

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

2011-04-20

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

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

PubMed Central

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

2012-01-01

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

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

PubMed

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

2018-04-12

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

Novel actin crosslinker superfamily member identified by a two step degenerate PCR procedure.

PubMed

Byers, T J; Beggs, A H; McNally, E M; Kunkel, L M

1995-07-24

Actin-crosslinking proteins link F-actin into the bundles and networks that constitute the cytoskeleton. Dystrophin, beta-spectrin, alpha-actinin, ABP-120, ABP-280, and fimbrin share homologous actin-binding domains and comprise an actin crosslinker superfamily. We have identified a novel member of this superfamily (ACF7) using a degenerate primer-mediated PCR strategy that was optimized to resolve less-abundant superfamily sequences. The ACF7 gene is on human chromosome 1 and hybridizes to high molecular weight bands on northern blots. Sequence comparisons argue that ACF7 does not fit into one of the existing families, but represents a new class within the superfamily.

Synthesis, Characterization, and Antibacterial Activity of Cross-Linked Chitosan-Glutaraldehyde

PubMed Central

Li, Bin; Shan, Chang-Lin; Zhou, Qing; Fang, Yuan; Wang, Yang-Li; Xu, Fei; Han, Li-Rong; Ibrahim, Muhammad; Guo, Long-Biao; Xie, Guan-Lin; Sun, Guo-Chang

2013-01-01

This present study deals with synthesis, characterization and antibacterial activity of cross-linked chitosan-glutaraldehyde. Results from this study indicated that cross-linked chitosan-glutaraldehyde markedly inhibited the growth of antibiotic-resistant Burkholderia cepacia complex regardless of bacterial species and incubation time while bacterial growth was unaffected by solid chitosan. Furthermore, high temperature treated cross-linked chitosan-glutaraldehyde showed strong antibacterial activity against the selected strain 0901 although the inhibitory effects varied with different temperatures. In addition, physical-chemical and structural characterization revealed that the cross-linking of chitosan with glutaraldehyde resulted in a rougher surface morphology, a characteristic Fourier transform infrared (FTIR) band at 1559 cm−1, a specific X-ray diffraction peak centered at 2θ = 15°, a lower contents of carbon, hydrogen and nitrogen, and a higher stability of glucose units compared to chitosan based on scanning electron microscopic observation, FTIR spectra, X-ray diffraction pattern, as well as elemental and thermo gravimetric analysis. Overall, this study indicated that cross-linked chitosan-glutaraldehyde is promising to be developed as a new antibacterial drug. PMID:23670533

Erbium laser resurfacing for actinic cheilitis.

PubMed

Cohen, Joel L

2013-11-01

Actinic cheilitis is a precancerous condition characterized by grayish-whitish area(s) of discoloration on the mucosal lip, often blunting the demarcation between mucosa and cutaneous lip. Actinic cheilitis is considered to be an early part of the spectrum of squamous cell carcinoma. Squamous cell carcinoma specifically of the lip has a high rate of recurrence and metastasis through the oral cavity leading to a poor overall survival. Risk factors for the development of actinic cheilitis include chronic solar irradiation, increasing age, male gender, light skin complexion, immunosuppression, and possibly tobacco and alcohol consumption. Treatment options include topical pharmacotherapy (eg, fluorouracil, imiquimod) or procedural interventions (eg, cryotherapy, electrosurgery, surgical vermillionectomy, laser resurfacing), each with their known advantages and disadvantages. There is little consensus as to which treatment options offer the most clinical utility given the paucity of comparative clinical data. In my practice, laser resurfacing has become an important tool for the treatment of actinic cheilitis owing to its ease of use and overall safety, tolerability, and cosmetic acceptability. Herein the use of erbium laser resurfacing is described for three actinic cheilitis presentations for which I find it particularly useful: clinically prominent actinic cheilitis, biopsy-proven actinic cheilitis, and treatment of the entire lip following complete tumor excision of squamous cell carcinoma. All patients were treated with a 2940-nm erbium laser (Sciton Profile Contour Tunable Resurfacing Laser [TRL], Sciton, Inc., Palo Alto, CA).

The Dynamic Actin Cytoskeleton in Smooth Muscle.

PubMed

Tang, Dale D

2018-01-01

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

Ice templated and cross-linked xylan/nanocrystalline cellulose hydrogels

Treesearch

Tobias Köhnke; Thomas Elder; Hans Theliander; Arthur J. Ragauskas

2014-01-01

Structured xylan-based hydrogels, reinforced with cellulose nanocrystals (CNCs), have successfully been prepared from water suspensions by cross-linking during freeze-casting. In order to induce cross-linking during the solidification/sublimation operation, xylan was first oxidized using sodium periodate to introduce dialdehydes. The oxidized xylan was then mixed with...

UV cross-linking of donor corneas confers resistance to keratolysis.

PubMed

Arafat, Samer N; Robert, Marie-Claude; Shukla, Anita N; Dohlman, Claes H; Chodosh, James; Ciolino, Joseph B

2014-09-01

The aim of this study was to develop a modified ex vivo corneal cross-linking method that increases stromal resistance to enzymatic degradation for use as a carrier for the Boston keratoprosthesis. Ex vivo cross-linking of human corneas was performed using Barron artificial anterior chambers. The corneas were deepithelialized, pretreated with riboflavin solution (0.1% riboflavin/20% dextran), and irradiated with ultraviolet A (UV-A) light (λ = 370 nm, irradiance = 3 mW/cm) for various durations. The combined effect of UV-A and gamma (γ) irradiation was also assessed using the commercially available γ-irradiated corneal donors. The corneas were then trephined and incubated at 37°C with 0.3% collagenase A solution. The time to dissolution of each cornea was compared across treatments. Deepithelialized corneas (no UV light, no riboflavin) dissolved in 5.8 ± 0.6 hours. Cross-linked corneas demonstrated increased resistance to dissolution, with a time to dissolution of 17.8 ± 2.6 hours (P < 0.0001). The corneal tissues' resistance to collagenase increased with longer UV-A exposure, reaching a plateau at 30 minutes. Cross-linking both the anterior and posterior corneas did not provide added resistance when compared with cross-linking the anterior corneas only (P > 0.05). γ-irradiated corneas dissolved as readily as deepithelialized controls regardless of whether they were further cross-linked (5.6 ± 1.2 hours) or not (6.1 ± 0.6 hours) (P = 0.43). Collagen cross-linking of the deepithelialized anterior corneal surface for 30 minutes conferred optimal resistance to in vitro keratolysis by collagenase A.

Morris Water Maze Training in Mice Elevates Hippocampal Levels of Transcription Factors Nuclear Factor (Erythroid-derived 2)-like 2 and Nuclear Factor Kappa B p65

PubMed Central

Snow, Wanda M.; Pahlavan, Payam S.; Djordjevic, Jelena; McAllister, Danielle; Platt, Eric E.; Alashmali, Shoug; Bernstein, Michael J.; Suh, Miyoung; Albensi, Benedict C.

2015-01-01

Research has identified several transcription factors that regulate activity-dependent plasticity and memory, with cAMP-response element binding protein (CREB) being the most well-studied. In neurons, CREB activation is influenced by the transcription factor nuclear factor kappa B (NF-κB), considered central to immunity but more recently implicated in memory. The transcription factor early growth response-2 (Egr-2), an NF-κB gene target, is also associated with learning and memory. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2), an antioxidant transcription factor linked to NF-κB in pathological conditions, has not been studied in normal memory. Given that numerous transcription factors implicated in activity-dependent plasticity demonstrate connections to NF-κB, this study simultaneously evaluated protein levels of NF-κB, CREB, Egr-2, Nrf2, and actin in hippocampi from young (1 month-old) weanling CD1 mice after training in the Morris water maze, a hippocampal-dependent spatial memory task. After a 6-day acquisition period, time to locate the hidden platform decreased in the Morris water maze. Mice spent more time in the target vs. non-target quadrants of the maze, suggestive of recall of the platform location. Western blot data revealed a decrease in NF-κB p50 protein after training relative to controls, whereas NF-κB p65, Nrf2 and actin increased. Nrf2 levels were correlated with platform crosses in nearly all tested animals. These data demonstrate that training in a spatial memory task results in alterations in and associations with particular transcription factors in the hippocampus, including upregulation of NF-κB p65 and Nrf2. Training-induced increases in actin protein levels caution against its use as a loading control in immunoblot studies examining activity-dependent plasticity, learning, and memory. PMID:26635523

The cell wall of Arabidopsis thaliana influences actin network dynamics.

PubMed

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

2017-07-20

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

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.

Collagen cross-linking of skin in patients with amyotrophic lateral sclerosis

NASA Technical Reports Server (NTRS)

Ono, S.; Yamauchi, M.

1992-01-01

Collagen cross-links of skin tissue (left upper arm) from 11 patients with amyotrophic lateral sclerosis (ALS) and 9 age-matched control subjects were quantified. It was found that patients with ALS had a significant reduction in the content of an age-related, stable cross-link, histidinohydroxylysinonorleucine, that was negatively correlated with the duration of illness. The contents of sodium borohydride-reducible labile cross-links, dehydro-hydroxylysinonorleucine and dehydro-histidinohydroxymerodesmosine, were significantly increased and were positively associated with the duration of illness (r = 0.703, p less than 0.05 and r = 0.684, p less than 0.05, respectively). The results clearly indicate that during the course of ALS, the cross-linking pathway of skin collagen runs counter to its normal aging, resulting in a "rejuvenation" phenomenon of skin collagen. Thus, cross-linking of skin collagen is affected in ALS.

Molecular cloning and characterization of human trabeculin-alpha, a giant protein defining a new family of actin-binding proteins.

PubMed

Sun, Y; Zhang, J; Kraeft, S K; Auclair, D; Chang, M S; Liu, Y; Sutherland, R; Salgia, R; Griffin, J D; Ferland, L H; Chen, L B

1999-11-19

We describe the molecular cloning and characterization of a novel giant human cytoplasmic protein, trabeculin-alpha (M(r) = 614,000). Analysis of the deduced amino acid sequence reveals homologies with several putative functional domains, including a pair of alpha-actinin-like actin binding domains; regions of homology to plakins at either end of the giant polypeptide; 29 copies of a spectrin-like motif in the central region of the protein; two potential Ca(2+)-binding EF-hand motifs; and a Ser-rich region containing a repeated GSRX motif. With similarities to both plakins and spectrins, trabeculin-alpha appears to have evolved as a hybrid of these two families of proteins. The functionality of the actin binding domains located near the N terminus was confirmed with an F-actin binding assay using glutathione S-transferase fusion proteins comprising amino acids 9-486 of the deduced peptide. Northern and Western blotting and immunofluorescence studies suggest that trabeculin is ubiquitously expressed and is distributed throughout the cytoplasm, though the protein was found to be greatly up-regulated upon differentiation of myoblasts into myotubes. Finally, the presence of cDNAs similar to, yet distinct from, trabeculin-alpha in both human and mouse suggests that trabeculins may form a new subfamily of giant actin-binding/cytoskeletal cross-linking proteins.

Increased actin polymerization reduces the inhibition of serum response factor activity by Yin Yang 1.

PubMed Central

Ellis, Peter D; Martin, Karen M; Rickman, Colin; Metcalfe, James C; Kemp, Paul R

2002-01-01

Recent evidence has implicated CC(A/T(richG))GG (CArG) boxes, binding sites for serum response factor (SRF), in the regulation of expression of a number of genes in response to changes in the actin cytoskeleton. In many cases, the activity of SRF at CArG boxes is modulated by transcription factors binding to overlapping (e.g. Yin Yang 1, YY1) or adjacent (e.g. ets) binding sites. However, the mechanisms by which SRF activity is regulated by the cytoskeleton have not been determined. To investigate these mechanisms, we screened for cells that did or did not increase the activity of a fragment of the promoter for a smooth-muscle (SM)-specific gene SM22alpha, in response to changes in actin cytoskeletal polymerization induced by LIM kinase. These experiments showed that vascular SM cells (VSMCs) and C2C12 cells increased the activity of promoters containing at least one of the SM22alpha CArG boxes (CArG near) in response to LIM kinase, whereas P19 cells did not. Bandshift assays using a probe to CArG near showed that P19 cells lacked detectable YY1 DNA binding to the CArG box in contrast with the other two cell types. Expression of YY1 in P19 cells inhibited SM22alpha promoter activity and conferred responsiveness to LIM kinase. Mutation of the CArG box to inhibit YY1 or SRF binding indicated that both factors were required for the LIM kinase response in VSMCs and C2C12 cells. The data indicate that changes in the actin cytoskeletal organization modify SRF activity at CArG boxes by modulating YY1-dependent inhibition. PMID:12023898

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

PubMed

Silveira, Paulo S P; Fredberg, Jeffrey J

2005-10-01

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

Actin isoform specificity is required for the maintenance of lactation

PubMed Central

Weymouth, Nate; Shi, Zengdun; Rockey, Don C.

2014-01-01

Smooth muscle α-actin (Acta2) is one of six highly conserved mammalian actin isoforms that appear to exhibit functional redundancy. Nonetheless, we have postulated a specific functional role for the smooth muscle specific isoform. Here, we show that Acta2 deficient mice have a remarkable mammary phenotype such that dams lacking Acta2 are unable to nurse their offspring effectively. The phenotype was rescued in cross fostering experiments with wild type mice, excluding a developmental defect in Acta2 null pups. The mechanism for the underlying phenotype is due to myoepithelial dysfunction postpartum resulting in precocious involution. Further, we demonstrate a specific defect in myoepithelial cell contractility in Acta2 null mammary glands, despite normal expression of cytoplasmic actins. We conclude that Acta2 specifically mediates myoepithelial cell contraction during lactation and that this actin isoform therefore exhibits functional specificity. PMID:22123032

Ara h 2 cross-linking catalyzed by MTGase decreases its allergenicity.

PubMed

Wu, Zhihua; Lian, Jun; Zhao, Ruifang; Li, Kun; Li, Xin; Yang, Anshu; Tong, Ping; Chen, Hongbing

2017-03-22

Peanuts, whose major allergen is Ara h 2, are included among the eight major food allergens. After reduction using dithiothreitol (DTT), cross-linking of Ara h 2 could be catalyzed by microbial transglutaminase (MTGase), a widely used enzyme in the food industry. In this study, Ara h 2 cross-linking was catalyzed by MTGase after it was reduced by DTT. Using mass spectrometry and PLINK software, five cross-linkers were identified, and five linear allergen epitopes were found to be involved in the reactions. The IgE binding capacity of cross-linked Ara h 2 was found to be significantly lower compared to that of native and reduced Ara h 2. After simulated gastric fluid (SGF) digestion, the digested products of the cross-linked Ara h 2, again, had a significantly lower IgE binding capacity compared to untreated and reduced Ara h 2. Furthermore, reduced and cross-linked Ara h 2 (RC-Ara h 2) induced lower sensitization in mice, indicating its lower allergenicity. Reduction and MTGase-catalyzed cross-linking are effective methods to decrease the allergenicity of Ara h 2. The reactions involved linear allergen epitopes destroying the material basis of the allergenicity, and this might develop a new direction for protein desensitization processes.

Linking Gestures: Cross-Cultural Variation during Instructional Analogies

ERIC Educational Resources Information Center

Richland, Lindsey Engle

2015-01-01

Deictic linking gestures, hand and arm motions that physically embody links being communicated between two or more objects in the shared communicative environment, are explored in a cross-cultural sample of mathematics instruction. Linking gestures are specifically examined here when they occur in the context of communicative analogies designed to…

Ac102 Participates in Nuclear Actin Polymerization by Modulating BV/ODV-C42 Ubiquitination during Autographa californica Multiple Nucleopolyhedrovirus Infection.

PubMed

Zhang, Yongli; Hu, Xue; Mu, Jingfang; Hu, Yangyang; Zhou, Yuan; Zhao, He; Wu, Chunchen; Pei, Rongjuan; Chen, Jizheng; Chen, Xinwen; Wang, Yun

2018-06-15

As a virus-encoded actin nucleation promoting factor (NPF), P78/83 induces actin polymerization to assist in Autographa californica multiple nucleopolyhedrovirus (AcMNPV) propagation. According to our previous study, although P78/83 actively undergoes ubiquitin-independent proteasomal degradation, AcMNPV encodes budded virus/occlusion derived virus (BV/ODV)-C42 (C42), which allows P78/83 to function as a stable NPF by inhibiting its degradation during viral infection. However, whether there are other viral proteins involved in regulating P78/83-induced actin polymerization has yet to be determined. In this study, we found that Ac102, an essential viral gene product previously reported to play a key role in mediating the nuclear accumulation of actin during AcMNPV infection, is a novel regulator of P78/83-induced actin polymerization. By characterizing an ac102 knockout bacmid, we demonstrated that Ac102 participates in regulating nuclear actin polymerization as well as the morphogenesis and distribution of capsid structures in the nucleus. These regulatory effects are heavily dependent on an interaction between Ac102 and C42. Further investigation revealed that Ac102 binds to C42 to suppress K48-linked ubiquitination of C42, which decreases C42 proteasomal degradation and consequently allows P78/83 to function as a stable NPF to induce actin polymerization. Thus, Ac102 and C42 form a regulatory cascade to control viral NPF activity, representing a sophisticated mechanism for AcMNPV to orchestrate actin polymerization in both a ubiquitin-dependent and ubiquitin-independent manner. IMPORTANCE Actin is one of the most functionally important proteins in eukaryotic cells. Morphologically, actin can be found in two forms: a monomeric form called globular actin (G-actin) and a polymeric form called filamentous actin (F-actin). G-actin can polymerize to form F-actin, and nucleation promoting factor (NPF) is the initiator of this process. Many viral pathogens harness the

Quantification of the Effect of Cross-shear on the Wear of Conventional and Highly Cross-linked UHMWPE

PubMed Central

Kang, Lu; Galvin, Alison L.; Brown, Thomas D.; Jin, Zhongmin; Fisher, John

2008-01-01

A computational model has been developed to quantify the degree of cross-shear of a polyethylene pin articulating against a metallic plate, based on the direct simulation of a multidirectional pin-on-plate wear machine. The principal molecular orientation (PMO) was determined for each polymer site. The frictional work in the direction perpendicular to the PMO was assumed to produce the greatest orientation softening (Wang et al., 1997). The cross-shear ratio (CS) was defined as the frictional work perpendicular to the PMO direction, divided by the total frictional work. Cross-shear on the pin contact surface was location-specific, and of continuously changing magnitude because the direction of frictional force continuously changed due to pin rotation. The polymer pin motion was varied from a purely linear track (CS=0) up to a maximum rotation of ±55° (CS=0.254). The relationship between wear factors (K) measured experimentally and theoretically predicted CS was defined using logarithmic functions for both conventional and highly cross-linked UHMWPE. Cross-shear increased the apparent wear factor for both polyethylenes by more than 5-fold compared to unidirectional wear. PMID:17936763

Cross-linked polyelectrolyte for direct methanol fuel cells applications based on a novel sulfonated cross-linker

NASA Astrophysics Data System (ADS)

Li, Mingyu; Zhang, Gang; Xu, Shuai; Zhao, Chengji; Han, Miaomiao; Zhang, Liyuan; Jiang, Hao; Liu, Zhongguo; Na, Hui

2014-06-01

A novel type of cross-linked proton exchange membrane of lower methanol permeation and high proton conductivity is prepared, based on a newly synthesized sulfonated cross-linker: carboxyl terminated benzimidazole trimer bearing sulfonic acid groups (s-BI). Compared to membranes cross-linked with non-sulfonated cross-linker (BI), SPEEK/s-BI-n membranes show higher IEC values and proton conductivities. Meanwhile, oxidative stability and mechanical property of SPEEK/s-BI-n membranes are obviously improved. Among SPEEK/s-BI-n membranes, SPEEK/s-BI-2 exhibits high proton conductivity, low swelling ratio (0.122 S cm-1 and 15.2% at 60 °C, respectively) and low methanol permeability coefficient. These results imply that the cross-linked membranes prepared with the newly sulfonated cross-linker are promising for the direct methanol fuel cells (DMFCs) application.

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

PubMed

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

2016-01-01

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

J-integral fracture toughness and tearing modulus measurement of radiation cross-linked UHMWPE.

PubMed

Gomoll, A; Wanich, T; Bellare, A

2002-11-01

Radiation and chemical cross-linking of medical grade ultrahigh molecular weight polyethylene (UHMWPE) has recently been utilized in an effort to improve wear performance of total joint replacement components. However, reductions in mechanical properties with cross-linking are cause for concern regarding the use of cross-linked UHMWPE for high-stress applications such as in total knee replacement prostheses. In this study, the fracture behavior of radiation cross-linked UHMWPE was compared to that of uncross-linked UHMWPE. The Rice and Sorensen model that utilizes mechanical parameters obtained from uniaxial tensile and compact tension tests was used to calculate the steady state J-integral fracture toughness, Jss, for radiation cross-linked UHMWPE. Jss decreased monotonically with increase in radiation dose. UHMWPE exhibited tough, ductile tearing behavior with stable crack growth when it was cross-linked using a gamma radiation dose of 0-50 kGy. However, in cross-linked UHMWPE irradiated to a dose of 100 and 200 kGy, unstable fracture occurred spontaneously upon attaining the initial crack driving force, J1c. This indicates that a high degree of cross-linking is less desirable for high-stress applications in orthopaedic implants. However, a substantial increase in J1c, even at a low degree of cross-linking, suggests that a low degree of cross-linking may be beneficial for resistance to delamination and catastrophic failure, both of which require an initiation step for the fracture to propagate in the material. This mechanical test should, however, be considered along with fatigue tests and joint simulator testing before determination of an appropriate amount of cross-linking for total joint replacement prostheses that experience high stresses.

Genome-Wide siRNA Screen Identifies Complementary Signaling Pathways Involved in Listeria Infection and Reveals Different Actin Nucleation Mechanisms during Listeria Cell Invasion and Actin Comet Tail Formation

PubMed Central

Kühbacher, Andreas; Emmenlauer, Mario; Rämo, Pauli; Kafai, Natasha; Dehio, Christoph

2015-01-01

ABSTRACT Listeria monocytogenes enters nonphagocytic cells by a receptor-mediated mechanism that is dependent on a clathrin-based molecular machinery and actin rearrangements. Bacterial intra- and intercellular movements are also actin dependent and rely on the actin nucleating Arp2/3 complex, which is activated by host-derived nucleation-promoting factors downstream of the cell receptor Met during entry and by the bacterial nucleation-promoting factor ActA during comet tail formation. By genome-wide small interfering RNA (siRNA) screening for host factors involved in bacterial infection, we identified diverse cellular signaling networks and protein complexes that support or limit these processes. In addition, we could precise previously described molecular pathways involved in Listeria invasion. In particular our results show that the requirements for actin nucleators during Listeria entry and actin comet tail formation are different. Knockdown of several actin nucleators, including SPIRE2, reduced bacterial invasion while not affecting the generation of comet tails. Most interestingly, we observed that in contrast to our expectations, not all of the seven subunits of the Arp2/3 complex are required for Listeria entry into cells or actin tail formation and that the subunit requirements for each of these processes differ, highlighting a previously unsuspected versatility in Arp2/3 complex composition and function. PMID:25991686

Actin turnover maintains actin filament homeostasis during cytokinetic ring contraction

PubMed Central

Palani, Saravanan; Sommese, Ruth; Kamnev, Anton; Hatano, Tomoyuki; Sivaramakrishnan, Sivaraj

2017-01-01

Cytokinesis in many eukaryotes involves a tension-generating actomyosin-based contractile ring. Many components of actomyosin rings turn over during contraction, although the significance of this turnover has remained enigmatic. Here, using Schizosaccharomyces japonicus, we investigate the role of turnover of actin and myosin II in its contraction. Actomyosin ring components self-organize into ∼1-µm-spaced clusters instead of undergoing full-ring contraction in the absence of continuous actin polymerization. This effect is reversed when actin filaments are stabilized. We tested the idea that the function of turnover is to ensure actin filament homeostasis in a synthetic system, in which we abolished turnover by fixing rings in cell ghosts with formaldehyde. We found that these rings contracted fully upon exogenous addition of a vertebrate myosin. We conclude that actin turnover is required to maintain actin filament homeostasis during ring contraction and that the requirement for turnover can be bypassed if homeostasis is achieved artificially. PMID:28655757

Characterization of the microtubule binding domain of microtubule actin crosslinking factor (MACF): identification of a novel group of microtubule associated proteins.

PubMed

Sun, D; Leung, C L; Liem, R K

2001-01-01

MACF (microtubule actin cross-linking factor) is a large, 608-kDa protein that can associate with both actin microfilaments and microtubules (MTs). Structurally, MACF can be divided into 3 domains: an N-terminal domain that contains both a calponin type actin-binding domain and a plakin domain; a rod domain that is composed of 23 dystrophin-like spectrin repeats; and a C-terminal domain that includes two EF-hand calcium-binding motifs, as well as a region that is homologous to two related proteins, GAR22 and Gas2. We have previously demonstrated that the C-terminal domain of MACF binds to MTs, although no homology was observed between this domain and other known microtubule-binding proteins. In this report, we describe the characterization of this microtubule-binding domain of MACF by transient transfection studies and in vitro binding assays. We found that the C-terminus of MACF contains at least two microtubule-binding regions, a GAR domain and a domain containing glycine-serine-arginine (GSR) repeats. In transfected cells, the GAR domain bound to and partially stabilized MTs to depolymerization by nocodazole. The GSR-containing domain caused MTs to form bundles that are still sensitive to nocodazole-induced depolymerization. When present together, these two domains acted in concert to bundle MTs and render them stable to nocodazole treatment. Recently, a study has shown that the N-terminal half of the plakin domain (called the M1 domain) of MACF also binds MTs. We therefore examined the microtubule binding ability of the M1 domain in the context of the entire plakin domain with and without the remaining N-terminal regions of two different MACF isoforms. Interestingly, in the presence of the surrounding sequences, the M1 domain did not bind MTs. In addition to MACF, cDNA sequences encoding the GAR and GSR-containing domains are also found in the partial human EST clone KIAA0728, which has high sequence homology to the 3' end of the MACF cDNA; hence, we refer to

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

PubMed Central

Stokasimov, Ema; Rubenstein, Peter A.

2009-01-01

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

Coordination of Actin- and Microtubule-Based Cytoskeletons Supports Transport of Spermatids and Residual Bodies/Phagosomes During Spermatogenesis in the Rat Testis

PubMed Central

Tang, Elizabeth I.; Lee, Will M.

2016-01-01

Germ cell transport across the seminiferous epithelium during spermatogenesis requires the intricate coordination of cell junctions, signaling proteins, and both actin- and microtubule (MT)-based cytoskeletons. Although the involvement of cytoskeletons in germ cell transport has been suggested, the precise mechanism(s) remains elusive. Based on growing evidence that actin and MT interactions underlie fundamental cellular processes, such as cell motility, it is unlikely that actin- and MT-based cytoskeletons work independently to regulate germ cell transport in the testis. Using rats treated with adjudin, a potential male contraceptive that disrupts spermatid adhesion and transport in the testis, as a study model, we show herein that actin- and MT-based cytoskeletons are both necessary for transport of spermatids and residual bodies/phagosomes across the seminiferous epithelium in adult rat testes. Analysis of intratubular expression of F-actin and tubulin revealed disruption of both actin and MT networks, concomitant with misdirected spermatids and phagosomes in rats treated with adjudin. Actin regulatory proteins, epidermal growth factor receptor pathway substrate 8 and actin-related protein 3, were mislocalized and down-regulated at the actin-rich anchoring junction between germ and Sertoli cells (apical ectoplasmic specialization) after adjudin treatment. Nonreceptor tyrosine kinase p-FAK-Tyr407, known to regulate F-actin nucleation via actin-related protein 3, was also mislocalized and down-regulated at the apical ectoplasmic specialization, corroborating the observation of actin cytoskeleton disruption. Additionally, spatiotemporal expression of MT regulatory protein end-binding protein 1, shown to be involved in MT-actin cross talk herein, was also disrupted after adjudin treatment. In summary, spermatid/phagosome transport across the epithelium during spermatogenesis requires the coordination between actin- and MT-based cytoskeletons. PMID:26894662

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

DOEpatents

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

2011-10-18

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

Energetics and kinetics of cooperative cofilin-actin filament interactions.

PubMed

Cao, Wenxiang; Goodarzi, Jim P; De La Cruz, Enrique M

2006-08-11

We have evaluated the thermodynamic parameters associated with cooperative cofilin binding to actin filaments, accounting for contributions of ion-linked equilibria, and determined the kinetic basis of cooperative cofilin binding. Ions weaken non-contiguous (isolated, non-cooperative) cofilin binding to an actin filament without affecting cooperative filament interactions. Non-contiguous cofilin binding is coupled to the dissociation of approximately 1.7 thermodynamically bound counterions. Counterion dissociation contributes approximately 40% of the total cofilin binding free energy (in the presence of 50 mM KCl). The non-contiguous and cooperative binding free energies are driven entirely by large, positive entropy changes, consistent with a cofilin-mediated increase in actin filament structural dynamics. The rate constant for cofilin binding to an isolated site on an actin filament is slow and likely to be limited by filament breathing. Cooperative cofilin binding arises from an approximately tenfold more rapid association rate constant and an approximately twofold slower dissociation rate constant. The more rapid association rate constant is presumably a consequence of cofilin-dependent changes in the average orientation of subdomain 2, subunit angular disorder and filament twist, which increase the accessibility of a neighboring cofilin-binding site on an actin filament. Cooperative association is more rapid than binding to an isolated site, but still slow for a second-order reaction, suggesting that cooperative binding is limited also by binding site accessibility. We suggest that the dissociation of actin-associated ions weakens intersubunit interactions in the actin filament lattice that enhance cofilin-binding site accessibility, favor cooperative binding and promote filament severing.

Mechanical Strength Improvements of Carbon Nanotube Threads through Epoxy Cross-Linking

PubMed Central

Yu, Qingyue; Alvarez, Noe T.; Miller, Peter; Malik, Rachit; Haase, Mark R.; Schulz, Mark; Shanov, Vesselin; Zhu, Xinbao

2016-01-01

Individual Carbon Nanotubes (CNTs) have a great mechanical strength that needs to be transferred into macroscopic fiber assemblies. One approach to improve the mechanical strength of the CNT assemblies is by creating covalent bonding among their individual CNT building blocks. Chemical cross-linking of multiwall CNTs (MWCNTs) within the fiber has significantly improved the strength of MWCNT thread. Results reported in this work show that the cross-linked thread had a tensile strength six times greater than the strength of its control counterpart, a pristine MWCNT thread (1192 MPa and 194 MPa, respectively). Additionally, electrical conductivity changes were observed, revealing 2123.40 S·cm−1 for cross-linked thread, and 3984.26 S·cm−1 for pristine CNT thread. Characterization suggests that the obtained high tensile strength is due to the cross-linking reaction of amine groups from ethylenediamine plasma-functionalized CNT with the epoxy groups of the cross-linking agent, 4,4-methylenebis(N,N-diglycidylaniline). PMID:28787868

Interaction between two adapter proteins, PAG and EBP50: a possible link between membrane rafts and actin cytoskeleton.

PubMed

Brdicková, N; Brdicka, T; Andera, L; Spicka, J; Angelisová, P; Milgram, S L; Horejsí, V

2001-10-26

Phosphoprotein associated with GEMs (PAG), also known as Csk-binding protein (Cbp), is a broadly expressed palmitoylated transmembrane adapter protein found in membrane rafts, also called GEMs (glycosphingolipid-enriched membrane microdomains). PAG is known to bind and activate the essential regulator of Src-family kinases, cytoplasmic protein tyrosine kinase Csk. In the present study we used the yeast 2-hybrid system to search for additional proteins which might bind to PAG. We have identified the abundant cytoplasmic adapter protein EBP50 (ezrin/radixin/moesin (ERM)-binding phosphoprotein of 50 kDa), also known as NHERF (Na(+)/H(+) exchanger regulatory factor), as a specific PAG-binding partner. The interaction involves the C-terminal sequence (TRL) of PAG and N-terminal PDZ domain(s) of EBP50. As EBP50 is known to interact via its C-terminal domain with the ERM-family proteins, which in turn bind to actin cytoskeleton, the PAG-EBP50 interaction may be important for connecting membrane rafts to the actin cytoskeleton.

Cross-Linked Micellar Spherical Nucleic Acids from Thermoresponsive Templates

PubMed Central

2017-01-01

A one-pot synthesis of micellar spherical nucleic acid (SNA) nanostructures using Pluronic F127 as a thermoresponsive template is reported. These novel constructs are synthesized in a chemically straightforward process that involves intercalation of the lipid tails of DNA amphiphiles (CpG motifs for TLR-9 stimulation) into the hydrophobic regions of Pluronic F127 micelles, followed by chemical cross-linking and subsequent removal of non-cross-linked structures. The dense nucleic acid shell of the resulting cross-linked micellar SNA enhances their stability in physiological media and facilitates their rapid cellular internalization, making them effective TLR-9 immunomodulatory agents. These constructs underscore the potential of SNAs in regulating immune response and address the relative lack of stability of noncovalent constructs. PMID:28207251

Microtubule actin crosslinking factor 1b: a novel plakin that localizes to the Golgi complex.

PubMed

Lin, Chung-Ming; Chen, Hui-Jye; Leung, Conrad L; Parry, David A D; Liem, Ronald K H

2005-08-15

MACF1 (microtubule actin crosslinking factor), also called ACF7 (actin crosslinking family 7) is a cytoskeletal linker protein that can associate with both actin filaments and microtubules. We have identified a novel alternatively spliced isoform of MACF1. We named this isoform MACF1b and renamed the original isoform MACF1a. MACF1b is identical to MACF1a, except that it has a region containing plakin (or plectin) repeats in the middle of the molecule. MACF1b is ubiquitously expressed in adult tissues with especially high levels in the lung. We studied the subcellular localization of MACF1b proteins in mammalian cell lines. In two lung cell lines, MACF1b was chiefly localized to the Golgi complex. Upon treatments that disrupt the Golgi complex, MACF1b redistributed into the cytosol, but remained co-localized with the dispersed Golgi ministacks. MACF1b proteins can be detected in the enriched Golgi fraction by western blotting. The domain of MACF1b that targets it to the Golgi was found at the N-terminal part of the region that contains the plakin repeats. Reducing the level of MACF1 proteins by small-interfering RNA resulted in the dispersal of the Golgi complex.

Recyclable cross-linked anion exchange membrane for alkaline fuel cell application

NASA Astrophysics Data System (ADS)

Hou, Jianqiu; Liu, Yazhi; Ge, Qianqian; Yang, Zhengjin; Wu, Liang; Xu, Tongwen

2018-01-01

Cross-linking can effectively solve the conductivity-swelling dilemma in anion exchange membranes (AEMs) but will generate solid wastes. To address this, we developed an AEM cross-linked via disulfide bonds, bearing quaternary ammonium groups, which can be easily recycled. The membrane (RC-QPPO) with IEC of 1.78 mmol g-1, when cross-linked, showed enhanced mechanical properties and good hydroxide conductivity (24.6 mS cm-1 at 30 °C). Even at higher IEC value (2.13 mmol g-1), it still has low water uptake, low swelling ratio and delivers a peak power density of 150 mW cm-2 at 65 °C. Exploiting the formation of disulfide bonds from -SH groups, the membrane can be readily cross-linked in alkaline condition and recycled by reversibly breaking disulfide bonds with dithiothreitol (DTT). The recycled membrane solution can be directly utilized to cast a brand-new AEM. By washing away the residual DTT with water and exposure to air, it can be cross-linked again and this process is repeatable. During the recycling and cross-linking processes, the membrane showed a slight IEC decrease of 5% due to functional group degradation. The strategy presented here is promising in enhancing AEM properties and reducing the impact of artificial polymers on the environment.

Yield and Failure Behavior Investigated for Cross-Linked Phenolic Resins Using Molecular Dynamics

NASA Technical Reports Server (NTRS)

Monk, Joshua D.; Lawson, John W.

2016-01-01

Molecular dynamics simulations were conducted to fundamentally evaluate the yield and failure behavior of cross-linked phenolic resins at temperatures below the glass transition. Yield stress was investigated at various temperatures, strain rates, and degrees of cross-linking. The onset of non-linear behavior in the cross-linked phenolic structures was caused by localized irreversible molecular rearrangements through the rotation of methylene linkers followed by the formation or annihilation of neighboring hydrogen bonds. The yield stress results, with respect to temperature and strain rate, could be fit by existing models used to describe yield behavior of amorphous glasses. The degree of cross-linking only indirectly influences the maximum yield stress through its influence on glass transition temperature (Tg), however there is a strong relationship between the degree of cross-linking and the failure mechanism. Low cross-linked samples were able to separate through void formation, whereas the highly cross-linked structures exhibited bond scission.

DNA-Protein Cross-Links: Formation, Structural Identities, and Biological Outcomes.

PubMed

Tretyakova, Natalia Y; Groehler, Arnold; Ji, Shaofei

2015-06-16

Noncovalent DNA-protein interactions are at the heart of normal cell function. In eukaryotic cells, genomic DNA is wrapped around histone octamers to allow for chromosomal packaging in the nucleus. Binding of regulatory protein factors to DNA directs replication, controls transcription, and mediates cellular responses to DNA damage. Because of their fundamental significance in all cellular processes involving DNA, dynamic DNA-protein interactions are required for cell survival, and their disruption is likely to have serious biological consequences. DNA-protein cross-links (DPCs) form when cellular proteins become covalently trapped on DNA strands upon exposure to various endogenous, environmental and chemotherapeutic agents. DPCs progressively accumulate in the brain and heart tissues as a result of endogenous exposure to reactive oxygen species and lipid peroxidation products, as well as normal cellular metabolism. A range of structurally diverse DPCs are found following treatment with chemotherapeutic drugs, transition metal ions, and metabolically activated carcinogens. Because of their considerable size and their helix-distorting nature, DPCs interfere with the progression of replication and transcription machineries and hence hamper the faithful expression of genetic information, potentially contributing to mutagenesis and carcinogenesis. Mass spectrometry-based studies have identified hundreds of proteins that can become cross-linked to nuclear DNA in the presence of reactive oxygen species, carcinogen metabolites, and antitumor drugs. While many of these proteins including histones, transcription factors, and repair proteins are known DNA binding partners, other gene products with no documented affinity for DNA also participate in DPC formation. Furthermore, multiple sites within DNA can be targeted for cross-linking including the N7 of guanine, the C-5 methyl group of thymine, and the exocyclic amino groups of guanine, cytosine, and adenine. This structural

DNA-Protein Cross-links: Formation, Structural Identities, and Biological Outcomes

PubMed Central

Tretyakova, Natalia Y.; Groehler, Arnold; Ji, Shaofei

2015-01-01

CONSPECTUS Non-covalent DNA-protein interactions are at the heart of normal cell function. In eukaryotic cells, genomic DNA is wrapped around histone octamers to allow for chromosomal packaging in the nucleus. Binding of regulatory protein factors to DNA directs replication, controls transcription, and mediates cellular responses to DNA damage. Because of their fundamental significance in all cellular processes involving DNA, dynamic DNA-protein interactions are required for cell survival, and their disruption is likely to have serious biological consequences. DNA-protein cross-links (DPCs) form when cellular proteins become covalently trapped on DNA strands upon exposure to various endogenous, environmental and chemotherapeutic agents. DPCs progressively accumulate in the brain and heart tissues as a result of endogenous exposure to reactive oxygen species and lipid peroxidation products, as well as normal cellular metabolism. A range of structurally diverse DPCs are found following treatment with chemotherapeutic drugs, transition metal ions, and metabolically activated carcinogens. Because of their considerable size and their helix-distorting nature, DPCs interfere with the progression of replication and transcription machineries and hence hamper the faithful expression of genetic information, potentially contributing to mutagenesis and carcinogenesis. Mass spectrometry-based studies have identified hundreds of proteins that can become cross-linked to nuclear DNA in the presence of reactive oxygen species, carcinogen metabolites, and antitumor drugs. While many of these proteins including histones, transcription factors, and repair proteins are known DNA binding partners, other gene products with no documented affinity for DNA also participate in DPC formation. Furthermore, multiple sites within DNA can be targeted for cross-linking including the N7 of guanine, the C-5 methyl group of thymine, and the exocyclic amino groups of guanine, cytosine, and adenine

The Bacterial Actin MamK

PubMed Central

Ozyamak, Ertan; Kollman, Justin; Agard, David A.; Komeili, Arash

2013-01-01

It is now recognized that actin-like proteins are widespread in bacteria and, in contrast to eukaryotic actins, are highly diverse in sequence and function. The bacterial actin, MamK, represents a clade, primarily found in magnetotactic bacteria, that is involved in the proper organization of subcellular organelles, termed magnetosomes. We have previously shown that MamK from Magnetospirillum magneticum AMB-1 (AMB-1) forms dynamic filaments in vivo. To gain further insights into the molecular mechanisms that underlie MamK dynamics and function, we have now studied the in vitro properties of MamK. We demonstrate that MamK is an ATPase that, in the presence of ATP, assembles rapidly into filaments that disassemble once ATP is depleted. The mutation of a conserved active site residue (E143A) abolishes ATPase activity of MamK but not its ability to form filaments. Filament disassembly depends on both ATPase activity and potassium levels, the latter of which results in the organization of MamK filaments into bundles. These data are consistent with observations indicating that accessory factors are required to promote filament disassembly and for spatial organization of filaments in vivo. We also used cryo-electron microscopy to obtain a high resolution structure of MamK filaments. MamK adopts a two-stranded helical filament architecture, but unlike eukaryotic actin and other actin-like filaments, subunits in MamK strands are unstaggered giving rise to a unique filament architecture. Beyond extending our knowledge of the properties and function of MamK in magnetotactic bacteria, this study emphasizes the functional and structural diversity of bacterial actins in general. PMID:23204522

Microscopy basics and the study of actin-actin-binding protein interactions.

PubMed

Thomasson, Maggie S; Macnaughtan, Megan A

2013-12-15

Actin is a multifunctional eukaryotic protein with a globular monomer form that polymerizes into a thin, linear microfilament in cells. Through interactions with various actin-binding proteins (ABPs), actin plays an active role in many cellular processes, such as cell motility and structure. Microscopy techniques are powerful tools for determining the role and mechanism of actin-ABP interactions in these processes. In this article, we describe the basic concepts of fluorescent speckle microscopy, total internal reflection fluorescence microscopy, atomic force microscopy, and cryoelectron microscopy and review recent studies that utilize these techniques to visualize the binding of actin with ABPs. Copyright © 2013 Elsevier Inc. All rights reserved.

Lysyl hydroxylase 2 induces a collagen cross-link switch in tumor stroma

PubMed Central

Chen, Yulong; Terajima, Masahiko; Yang, Yanan; Sun, Li; Ahn, Young-Ho; Pankova, Daniela; Puperi, Daniel S.; Watanabe, Takeshi; Kim, Min P.; Blackmon, Shanda H.; Rodriguez, Jaime; Liu, Hui; Behrens, Carmen; Wistuba, Ignacio I.; Minelli, Rosalba; Scott, Kenneth L.; Sanchez-Adams, Johannah; Guilak, Farshid; Pati, Debananda; Thilaganathan, Nishan; Burns, Alan R.; Creighton, Chad J.; Martinez, Elisabeth D.; Zal, Tomasz; Grande-Allen, K. Jane; Yamauchi, Mitsuo; Kurie, Jonathan M.

2015-01-01

Epithelial tumor metastasis is preceded by an accumulation of collagen cross-links that heighten stromal stiffness and stimulate the invasive properties of tumor cells. However, the biochemical nature of collagen cross-links in cancer is still unclear. Here, we postulated that epithelial tumorigenesis is accompanied by changes in the biochemical type of collagen cross-links. Utilizing resected human lung cancer tissues and a p21CIP1/WAF1-deficient, K-rasG12D-expressing murine metastatic lung cancer model, we showed that, relative to normal lung tissues, tumor stroma contains higher levels of hydroxylysine aldehyde–derived collagen cross-links (HLCCs) and lower levels of lysine aldehyde–derived cross-links (LCCs), which are the predominant types of collagen cross-links in skeletal tissues and soft tissues, respectively. Gain- and loss-of-function studies in tumor cells showed that lysyl hydroxylase 2 (LH2), which hydroxylates telopeptidyl lysine residues on collagen, shifted the tumor stroma toward a high-HLCC, low-LCC state, increased tumor stiffness, and enhanced tumor cell invasion and metastasis. Together, our data indicate that LH2 enhances the metastatic properties of tumor cells and functions as a regulatory switch that controls the relative abundance of biochemically distinct types of collagen cross-links in the tumor stroma. PMID:25664850

The Nf-actin gene is an important factor for food-cup formation and cytotoxicity of pathogenic Naegleria fowleri.

PubMed

Sohn, Hae-Jin; Kim, Jong-Hyun; Shin, Myeong-Heon; Song, Kyoung-Ju; Shin, Ho-Joon

2010-03-01

Naegleria fowleri destroys target cells by trogocytosis, a phagocytosis mechanism, and a process of piecemeal ingestion of target cells by food-cups. Phagocytosis is an actin-dependent process that involves polymerization of monomeric G-actin into filamentous F-actin. However, despite the numerous studies concerning phagocytosis, its role in the N. fowleri food-cup formation related with trogocytosis has been poorly reported. In this study, we cloned and characterized an Nf-actin gene to elucidate the role of Nf-actin gene in N. fowleri pathogenesis. The Nf-actin gene is composed of 1,128-bp and produced a 54.1-kDa recombinant protein (Nf-actin). The sequence identity was 82% with nonpathogenic Naegleria gruberi but has no sequence identity with other mammals or human actin gene. Anti-Nf-actin polyclonal antibody was produced in BALB/c mice immunized with recombinant Nf-actin. The Nf-actin was localized on the cytoplasm, pseudopodia, and especially, food-cup structure (amoebastome) in N. fowleri trophozoites using immunofluorescence assay. When N. fowleri co-cultured with Chinese hamster ovary cells, Nf-actin was observed to localize around on phagocytic food-cups. We also observed that N. fowleri treated with cytochalasin D as actin polymerization inhibitor or transfected with antisense oligomer of Nf-actin gene had shown the reduced ability of food-cup formation and in vitro cytotoxicity. Finally, it suggests that Nf-actin plays an important role in phagocytic activity of pathogenic N. fowleri.

Molecular architecture of the Spire-actin nucleus and its implication for actin filament assembly.

PubMed

Sitar, Tomasz; Gallinger, Julia; Ducka, Anna M; Ikonen, Teemu P; Wohlhoefler, Michael; Schmoller, Kurt M; Bausch, Andreas R; Joel, Peteranne; Trybus, Kathleen M; Noegel, Angelika A; Schleicher, Michael; Huber, Robert; Holak, Tad A

2011-12-06

The Spire protein is a multifunctional regulator of actin assembly. We studied the structures and properties of Spire-actin complexes by X-ray scattering, X-ray crystallography, total internal reflection fluorescence microscopy, and actin polymerization assays. We show that Spire-actin complexes in solution assume a unique, longitudinal-like shape, in which Wiskott-Aldrich syndrome protein homology 2 domains (WH2), in an extended configuration, line up actins along the long axis of the core of the Spire-actin particle. In the complex, the kinase noncatalytic C-lobe domain is positioned at the side of the first N-terminal Spire-actin module. In addition, we find that preformed, isolated Spire-actin complexes are very efficient nucleators of polymerization and afterward dissociate from the growing filament. However, under certain conditions, all Spire constructs--even a single WH2 repeat--sequester actin and disrupt existing filaments. This molecular and structural mechanism of actin polymerization by Spire should apply to other actin-binding proteins that contain WH2 domains in tandem.

Molecular architecture of the Spire–actin nucleus and its implication for actin filament assembly

PubMed Central

Sitar, Tomasz; Gallinger, Julia; Ducka, Anna M.; Ikonen, Teemu P.; Wohlhoefler, Michael; Schmoller, Kurt M.; Bausch, Andreas R.; Joel, Peteranne; Trybus, Kathleen M.; Noegel, Angelika A.; Schleicher, Michael; Huber, Robert; Holak, Tad A.

2011-01-01

The Spire protein is a multifunctional regulator of actin assembly. We studied the structures and properties of Spire–actin complexes by X-ray scattering, X-ray crystallography, total internal reflection fluorescence microscopy, and actin polymerization assays. We show that Spire–actin complexes in solution assume a unique, longitudinal-like shape, in which Wiskott–Aldrich syndrome protein homology 2 domains (WH2), in an extended configuration, line up actins along the long axis of the core of the Spire–actin particle. In the complex, the kinase noncatalytic C-lobe domain is positioned at the side of the first N-terminal Spire–actin module. In addition, we find that preformed, isolated Spire–actin complexes are very efficient nucleators of polymerization and afterward dissociate from the growing filament. However, under certain conditions, all Spire constructs—even a single WH2 repeat—sequester actin and disrupt existing filaments. This molecular and structural mechanism of actin polymerization by Spire should apply to other actin-binding proteins that contain WH2 domains in tandem. PMID:22106272

Macromolecular cross-linked enzyme aggregates (M-CLEAs) of α-amylase.

PubMed

Nadar, Shamraja S; Muley, Abhijeet B; Ladole, Mayur R; Joshi, Pranoti U

2016-03-01

Macromolecular cross-linked enzyme aggregates (M-CLEAs) of α-amylase were prepared by precipitation and subsequent cross-linking. The non-toxic, biodegradable, biocompatible, renewable polysaccharide based macromolecular cross-linkers viz. agar, chitosan, dextran, and gum arabic were used as a substitute for traditional glutaraldehyde to augment activity recovery toward macromolecular substrate. Macromolecular cross-linkers were prepared by periodate mediated controlled oxidation of polysaccharides. The effects of precipitating agent, concentration and different cross-linkers on activity recovery of α-amylase CLEAs were investigated. α-Amylase aggregated with ammonium sulphate and cross-linked by dextran showed 91% activity recovery, whereas glutaraldehyde CLEAs (G-CLEAs) exhibited 42% activity recovery. M-CLEAs exhibited higher thermal stability in correlation with α-amylase and G-CLEAs. Moreover, dextran and chitosan M-CLEAs showed same affinity for starch hydrolysis as of free α-amylase. The changes in secondary structures revealed the enhancements in structural and conformational rigidity attributed by cross-linkers. Finally, after five consecutive cycles dextran M-CLEAs retained 1.25 times higher initial activity than G-CLEAs. Copyright © 2015 Elsevier B.V. All rights reserved.

Role of actin depolymerizing factor cofilin in Aspergillus fumigatus oxidative stress response and pathogenesis.

PubMed

Jia, Xiaodong; Zhang, Xi; Hu, Yingsong; Hu, Mandong; Tian, Shuguang; Han, Xuelin; Sun, Yansong; Han, Li

2018-06-01

Aspergillus fumigatus is a major fungal pathogen that is responsible for approximately 90% of human aspergillosis. Cofilin is an actin depolymerizing factor that plays crucial roles in multiple cellular functions in many organisms. However, the functions of cofilin in A. fumigatus are still unknown. In this study, we constructed an A. fumigatus strain overexpressing cofilin (cofilin OE). The cofilin OE strain displayed a slightly different growth phenotype, significantly increased resistance against H 2 O 2 and diamide, and increased activation of the high osmolarity glycerol pathway compared to the wild-type strain (WT). The cofilin OE strain internalized more efficiently into lung epithelial A549 cells, and induced increased transcription of inflammatory factors (MCP-1, TNF-α and IL-8) compared to WT. Cofilin overexpression also resulted in increased polysaccharides including β-1, 3-glucan and chitin, and increased transcription of genes related to oxidative stress responses and polysaccharide synthesis in A. fumigatus. However, the cofilin OE strain exhibited similar virulence to the wild-type strain in murine and Galleria mellonella infection models. These results demonstrated for the first time that cofilin, a regulator of actin cytoskeleton dynamics, might play a critical role in the regulation of oxidative stress responses and cell wall polysaccharide synthesis in A. fumigatus.

Photouncaged Sequence-specific Interstrand DNA Cross-Linking with Photolabile 4-oxo-enal-modified Oligonucleotides

PubMed Central

Sun, Jingjing; Tang, Xinjing

2015-01-01

DNA cross-linking technology is an attractive tool for the detection, regulation, and manipulation of genes. In this study, a series of photolabile 4-oxo-enal-modified oligonucleotides functionalized with photosensitive ο-nitrobenzyl derivatives were rationally designed as a new kind of photocaged cross-linking agents. A comprehensive evaluation of cross-linking reactions for different nucleobases in complementary strands under different conditions suggested that the modified DNA oligonucleotides tended to form interstrand cross-linking to nucleobases with the potential of thymidine > guanosine » cytidine ~ adenosine. Different from previous literature reports that cytidine and adenosine were preferential cross-linked nucleobases with 4-oxo-enal moieties, our study represents the first example of DNA cross-linking for T and G selectivity using 4-oxo-enal moiety. The cross-linked adducts were identified and their cross-linking mechanism was also illustrated. This greatly expands the applications of 4-oxo-enal derivatives in the studies of DNA damage and RNA structure PMID:26020694

Photouncaged Sequence-specific Interstrand DNA Cross-Linking with Photolabile 4-oxo-enal-modified Oligonucleotides.

PubMed

Sun, Jingjing; Tang, Xinjing

2015-05-28

DNA cross-linking technology is an attractive tool for the detection, regulation, and manipulation of genes. In this study, a series of photolabile 4-oxo-enal-modified oligonucleotides functionalized with photosensitive ο-nitrobenzyl derivatives were rationally designed as a new kind of photocaged cross-linking agents. A comprehensive evaluation of cross-linking reactions for different nucleobases in complementary strands under different conditions suggested that the modified DNA oligonucleotides tended to form interstrand cross-linking to nucleobases with the potential of thymidine > guanosine » cytidine ~ adenosine. Different from previous literature reports that cytidine and adenosine were preferential cross-linked nucleobases with 4-oxo-enal moieties, our study represents the first example of DNA cross-linking for T and G selectivity using 4-oxo-enal moiety. The cross-linked adducts were identified and their cross-linking mechanism was also illustrated. This greatly expands the applications of 4-oxo-enal derivatives in the studies of DNA damage and RNA structure.

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

PubMed Central

Deshpande, Siddharth; Pfohl, Thomas

2015-01-01

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

Escherichia coli cytotoxic necrotizing factor 1: evidence for induction of actin assembly by constitutive activation of the p21 Rho GTPase.

PubMed Central

Fiorentini, C; Donelli, G; Matarrese, P; Fabbri, A; Paradisi, S; Boquet, P

1995-01-01

Cytotoxic necrotizing factor type 1 (CNF1) induces in HEp-2 cells an increase in F-actin structures, which was detectable by fluorescence-activated cell sorter analysis 24 h after addition of this factor to the culture medium. Increase in F-actin was correlated with the augmentation of both the cell volume and the total cell actin content. Actin assembly-disassembly is controlled by small GTP-binding proteins of the Rho family, which have been reported recently to be modified by CNF1 treatment. Clostridium difficile toxin B and Clostridium botulinum exoenzyme C3, both known to act on the Rho GTPase, were used as biological tools to study the effect of CNF1 on this protein. CNF1 incubated before, during, or after exposure to the chimeric toxin C3B (which is the product of a genetic fusion between the DNA coding for C3 and the one coding for the B fragment of diphtheria toxin) protected HEp-2 cells from the disruption of F-actin structures caused by inactivation of the Rho GTPase through its ADP-ribosylation. On the other hand, C. difficile toxin B cytopathic effect was not observed upon preincubation of cells with CNF1. Toxins acting through a Rho-independent mechanism, such as cytochalasin D and Clostridium spiroforme iota-like toxin, could not be modified in their cellular activities by CNF1 treatment. All of our results suggest that CNF1 modifies the Rho molecule, thus probably protecting this GTPase from further bacterial toxin modification. PMID:7558302

Escherichia coli cytotoxic necrotizing factor 1: evidence for induction of actin assembly by constitutive activation of the p21 Rho GTPase.

PubMed

Fiorentini, C; Donelli, G; Matarrese, P; Fabbri, A; Paradisi, S; Boquet, P

1995-10-01

Cytotoxic necrotizing factor type 1 (CNF1) induces in HEp-2 cells an increase in F-actin structures, which was detectable by fluorescence-activated cell sorter analysis 24 h after addition of this factor to the culture medium. Increase in F-actin was correlated with the augmentation of both the cell volume and the total cell actin content. Actin assembly-disassembly is controlled by small GTP-binding proteins of the Rho family, which have been reported recently to be modified by CNF1 treatment. Clostridium difficile toxin B and Clostridium botulinum exoenzyme C3, both known to act on the Rho GTPase, were used as biological tools to study the effect of CNF1 on this protein. CNF1 incubated before, during, or after exposure to the chimeric toxin C3B (which is the product of a genetic fusion between the DNA coding for C3 and the one coding for the B fragment of diphtheria toxin) protected HEp-2 cells from the disruption of F-actin structures caused by inactivation of the Rho GTPase through its ADP-ribosylation. On the other hand, C. difficile toxin B cytopathic effect was not observed upon preincubation of cells with CNF1. Toxins acting through a Rho-independent mechanism, such as cytochalasin D and Clostridium spiroforme iota-like toxin, could not be modified in their cellular activities by CNF1 treatment. All of our results suggest that CNF1 modifies the Rho molecule, thus probably protecting this GTPase from further bacterial toxin modification.

Identification of Actin-Binding Proteins from Maize Pollen

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

Staiger, C.J.

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

In vitro cross-linking of elastin peptides and molecular characterization of the resultant biomaterials.

PubMed

Heinz, Andrea; Ruttkies, Christoph K H; Jahreis, Günther; Schräder, Christoph U; Wichapong, Kanin; Sippl, Wolfgang; Keeley, Fred W; Neubert, Reinhard H H; Schmelzer, Christian E H

2013-04-01

Elastin is a vital protein and the major component of elastic fibers which provides resilience to many vertebrate tissues. Elastin's structure and function are influenced by extensive cross-linking, however, the cross-linking pattern is still unknown. Small peptides containing reactive allysine residues based on sequences of cross-linking domains of human elastin were incubated in vitro to form cross-links characteristic of mature elastin. The resultant insoluble polymeric biomaterials were studied by scanning electron microscopy. Both, the supernatants of the samples and the insoluble polymers, after digestion with pancreatic elastase or trypsin, were furthermore comprehensively characterized on the molecular level using MALDI-TOF/TOF mass spectrometry. MS(2) data was used to develop the software PolyLinX, which is able to sequence not only linear and bifunctionally cross-linked peptides, but for the first time also tri- and tetrafunctionally cross-linked species. Thus, it was possible to identify intra- and intermolecular cross-links including allysine aldols, dehydrolysinonorleucines and dehydromerodesmosines. The formation of the tetrafunctional cross-link desmosine or isodesmosine was unexpected, however, could be confirmed by tandem mass spectrometry and molecular dynamics simulations. The study demonstrated that it is possible to produce biopolymers containing polyfunctional cross-links characteristic of mature elastin from small elastin peptides. MALDI-TOF/TOF mass spectrometry and the newly developed software PolyLinX proved suitable for sequencing of native cross-links in proteolytic digests of elastin-like biomaterials. The study provides important insight into the formation of native elastin cross-links and represents a considerable step towards the characterization of the complex cross-linking pattern of mature elastin. Copyright © 2013 Elsevier B.V. All rights reserved.

Importance of Interaction between Integrin and Actin Cytoskeleton in Suspension Adaptation of CHO cells.

PubMed

Walther, Christa G; Whitfield, Robert; James, David C

2016-04-01

The biopharmaceutical production process relies upon mammalian cell technology where single cells proliferate in suspension in a chemically defined synthetic environment. This environment lacks exogenous growth factors, usually contributing to proliferation of fibroblastic cell types such as Chinese hamster ovary (CHO) cells. Use of CHO cells for production hence requires a lengthy 'adaptation' process to select clones capable of proliferation as single cells in suspension. The underlying molecular changes permitting proliferation in suspension are not known. Comparison of the non-suspension-adapted clone CHO-AD and a suspension-adapted propriety cell line CHO-SA by flow cytometric analysis revealed a highly variable bi-modal expression pattern for cell-to-cell contact proteins in contrast to the expression pattern seen for integrins. Those have a uni-modal expression on suspension and adherent cells. Integrins showed a conformation distinguished by regularly distributed clusters forming a sphere on the cell membrane of suspension-adapted cells. Actin cytoskeleton analysis revealed reorganisation from the typical fibrillar morphology found in adherent cells to an enforced spherical subcortical actin sheath in suspension cells. The uni-modal expression and specific clustering of integrins could be confirmed for CHO-S, another suspension cell line. Cytochalasin D treatment resulted in breakdown of the actin sheath and the sphere-like integrin conformation demonstrating the link between integrins and actin in suspension-adapted CHO cells. The data demonstrates the importance of signalling changes, leading to an integrin rearrangement on the cell surface, and the necessity of the reinforcement of the actin cytoskeleton for proliferation in suspension conditions.

Weak reversible cross links may decrease the strength of aligned fiber bundles.

PubMed

Nabavi, S Soran; Hartmann, Markus A

2016-02-21

Reversible cross-linking is an effective strategy to specifically tailor the mechanical properties of polymeric materials that can be found in a variety of biological as well as man-made materials. Using a simple model in this paper the influence of weak, reversible cross-links on the mechanical properties of aligned fiber bundles is investigated. Special emphasis in this analysis is put on the strength of the investigated structures. Using Monte Carlo methods two topologies of cross-links exceeding the strength of the covalent backbone are studied. Most surprisingly only two cross-links are sufficient to break the backbone of a multi chain system, resulting in a reduced strength of the material. The found effect crucially depends on the ratio of inter- to intra-chain cross-links and, thus, on the grafting density that determines this ratio.

General protein-protein cross-linking.

PubMed

Alegria-Schaffer, Alice

2014-01-01

This protocol describes a general protein-to-protein cross-linking procedure using the water-soluble amine-reactive homobifunctional BS(3) (bis[sulfosuccinimidyl] suberate); however, the protocol can be easily adapted using other cross-linkers of similar properties. BS(3) is composed of two sulfo-NHS ester groups and an 11.4 Å linker. Sulfo-NHS ester groups react with primary amines in slightly alkaline conditions (pH 7.2-8.5) and yield stable amide bonds. The reaction releases N-hydroxysuccinimide (see an application of NHS esters on Labeling a protein with fluorophores using NHS ester derivitization). © 2014 Elsevier Inc. All rights reserved.

Feedback Microtubule Control and Microtubule-Actin Cross-talk in Arabidopsis Revealed by Integrative Proteomic and Cell Biology Analysis of KATANIN 1 Mutants.

PubMed

Takáč, Tomáš; Šamajová, Olga; Pechan, Tibor; Luptovčiak, Ivan; Šamaj, Jozef

2017-09-01

Microtubule organization and dynamics are critical for key developmental processes such as cell division, elongation, and morphogenesis. Microtubule severing is an essential regulator of microtubules and is exclusively executed by KATANIN 1 in Arabidopsis In this study, we comparatively studied the proteome-wide effects in two KATANIN 1 mutants. Thus, shotgun proteomic analysis of roots and aerial parts of single nucleotide mutant fra2 and T-DNA insertion mutant ktn1-2 was carried out. We have detected 42 proteins differentially abundant in both fra2 and ktn1-2 KATANIN 1 dysfunction altered the abundance of proteins involved in development, metabolism, and stress responses. The differential regulation of tubulins and microtubule-destabilizing protein MDP25 implied a feedback microtubule control in KATANIN 1 mutants. Furthermore, deregulation of profilin 1, actin-depolymerizing factor 3, and actin 7 was observed. These findings were confirmed by immunoblotting analysis of actin and by microscopic observation of actin filaments using fluorescently labeled phalloidin. Results obtained by quantitative RT-PCR analysis revealed that changed protein abundances were not a consequence of altered expression levels of corresponding genes in the mutants. In conclusion, we show that abundances of several cytoskeletal proteins as well as organization of microtubules and the actin cytoskeleton are amended in accordance with defective microtubule severing. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Novel magnetic cross-linked lipase aggregates for improving the resolution of (R, S)-2-octanol.

PubMed

Liu, Ying; Guo, Chen; Liu, Chun-Zhao

2015-03-01

Novel magnetic cross-linked lipase aggregates were fabricated by immobilizing the cross-linked lipase aggregates onto magnetic particles with a high number of -NH2 terminal groups using p-benzoquinone as the cross-linking agent. At the optimal fabrication conditions, 100% of immobilization efficiency and 139% of activity recovery of the magnetic cross-linked lipase aggregates were achieved. The magnetic cross-linked lipase aggregates were able to efficiently resolve (R, S)-2-octanol, and retained 100% activity and 100% enantioselectivity after 10 cycles of reuse, whereas the cross-linked lipase aggregates only retained about 50% activity and 70% enantioselectivity due to insufficient cross-linking. These results provide a great potential for industrial applications of the magnetic cross-linked lipase aggregates. © 2014 Wiley Periodicals, Inc.

Cofilin Changes the Twist of F-Actin: Implications for Actin Filament Dynamics and Cellular Function

PubMed Central

McGough, Amy; Pope, Brian; Chiu, Wah; Weeds, Alan

1997-01-01

Cofilin is an actin depolymerizing protein found widely distributed in animals and plants. We have used electron cryomicroscopy and helical reconstruction to identify its binding site on actin filaments. Cofilin binds filamentous (F)-actin cooperatively by bridging two longitudinally associated actin subunits. The binding site is centered axially at subdomain 2 of the lower actin subunit and radially at the cleft between subdomains 1 and 3 of the upper actin subunit. Our work has revealed a totally unexpected (and unique) property of cofilin, namely, its ability to change filament twist. As a consequence of this change in twist, filaments decorated with cofilin have much shorter ‘actin crossovers' (∼75% of those normally observed in F-actin structures). Although their binding sites are distinct, cofilin and phalloidin do not bind simultaneously to F-actin. This is the first demonstration of a protein that excludes another actin-binding molecule by changing filament twist. Alteration of F-actin structure by cofilin/ADF appears to be a novel mechanism through which the actin cytoskeleton may be regulated or remodeled. PMID:9265645

Activated actin-depolymerizing factor/cofilin sequesters phosphorylated microtubule-associated protein during the assembly of alzheimer-like neuritic cytoskeletal striations.

PubMed

Whiteman, Ineka T; Gervasio, Othon L; Cullen, Karen M; Guillemin, Gilles J; Jeong, Erica V; Witting, Paul K; Antao, Shane T; Minamide, Laurie S; Bamburg, James R; Goldsbury, Claire

2009-10-14

In Alzheimer's disease (AD), rod-like cofilin aggregates (cofilin-actin rods) and thread-like inclusions containing phosphorylated microtubule-associated protein (pMAP) tau form in the brain (neuropil threads), and the extent of their presence correlates with cognitive decline and disease progression. The assembly mechanism of these respective pathological lesions and the relationship between them is poorly understood, yet vital to understanding the causes of sporadic AD. We demonstrate that, during mitochondrial inhibition, activated actin-depolymerizing factor (ADF)/cofilin assemble into rods along processes of cultured primary neurons that recruit pMAP/tau and mimic neuropil threads. Fluorescence resonance energy transfer analysis revealed colocalization of cofilin-GFP (green fluorescent protein) and pMAP in rods, suggesting their close proximity within a cytoskeletal inclusion complex. The relationship between pMAP and cofilin-actin rods was further investigated using actin-modifying drugs and small interfering RNA knockdown of ADF/cofilin in primary neurons. The results suggest that activation of ADF/cofilin and generation of cofilin-actin rods is required for the subsequent recruitment of pMAP into the inclusions. Additionally, we were able to induce the formation of pMAP-positive ADF/cofilin rods by exposing cells to exogenous amyloid-beta (Abeta) peptides. These results reveal a common pathway for pMAP and cofilin accumulation in neuronal processes. The requirement of activated ADF/cofilin for the sequestration of pMAP suggests that neuropil thread structures in the AD brain may be initiated by elevated cofilin activation and F-actin bundling that can be caused by oxidative stress, mitochondrial dysfunction, or Abeta peptides, all suspected initiators of synaptic loss and neurodegeneration in AD.

Torsional Rigidity of Single Actin Filaments and Actin-Actin Bond Breaking Force under Torsion Measured Directly by in vitro Micromanipulation

NASA Astrophysics Data System (ADS)

Tsuda, Yuri; Yasutake, Hironori; Ishijima, Akihiko; Yanagida, Toshio

1996-11-01

Knowledge of the elastic properties of actin filaments is crucial for considering its role in muscle contraction, cellular motile events, and formation of cell shape. The stiffness of actin filaments in the directions of stretching and bending has been determined. In this study, we have directly determined the torsional rigidity and breaking force of single actin filaments by measuring the rotational Brownian motion and tensile strength using optical tweezers and microneedles, respectively. Rotational angular fluctuations of filaments supplied the torsional rigidity as (8.0 ± 1.2) × 10-26 Nm2. This value is similar to that deduced from the longitudinal rigidity, assuming the actin filament to be a homogeneous rod. The breaking force of the actin-actin bond was measured while twisting a filament through various angles using microneedles. The breaking force decreased greatly under twist, e.g., from 600-320 pN when filaments were turned through 90 degrees, independent of the rotational direction. Our results indicate that an actin filament exhibits comparable flexibility in the rotational and longitudinal directions, but breaks more easily under torsional load.

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

PubMed Central

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

2015-01-01

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

Photoinduced DNA Interstrand Cross-Link Formation by Naphthalene Boronates via a Carbocation.

PubMed

Wang, Yibin; Lin, Zechao; Fan, Heli; Peng, Xiaohua

2016-07-18

Most photoinduced DNA cross-link formation by a bifunctional aryl derivative is through a bisquinone methide. DNA cross-linking via a bisarylcarbocation remains a less explored area. We designed and synthesized a series of naphthalene boronates that produce DNA interstrand cross-links via a carbocation upon UV irradiation. A free radical was generated from the naphthalene boronates with 350 nm irradiation and further converted to a carbocation by electron transfer. The activation mechanism was determined using the orthogonal traps, 2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPO) and methoxyamine that react with either the free radical or the carbocation but not both. This represents a novel example of photoinduced DNA cross-link formation via carbocations generated from a bisaryl derivative. This work provides information useful for the design of novel photoactivated DNA cross-linking agents. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Study of the effect of mixing approach on cross-linking efficiency of hyaluronic acid-based hydrogel cross-linked with 1,4-butanediol diglycidyl ether.

PubMed

Al-Sibani, Mohammed; Al-Harrasi, Ahmed; Neubert, Reinhard H H

2016-08-25

Regardless of various strategies reported for cross-linking hyaluronic acid (HA) with 1,4-butanediol diglycidyl ether (BDDE), seeking new strategies that enhance cross-linking efficiency with a low level of cross-linker is essential. In this work, we studied the influence of mixing approach on two cross-linked BDDE-HA hydrogels prepared by two different mixing approaches; the large-batch mixing approach in which the hydrogel quantities were all mixed as a single lump in one container (hydrogel 1), and the small-batches mixing approach in which the hydrogel quantities were divided into smaller batches, mixed separately at various HA/BDDE ratios then combined in one reaction mixture (hydrogel 2). The result showed that the cross-linking reaction was mixing process-dependent. Degradation tests proved that, in relation to hydrogel 1, hydrogel 2 was more stable, and exhibited a higher resistance towards hyaluronidase activity. The swelling ratio of hydrogel 1 was significantly higher than that of hydrogel 2 in distilled water; however, in phosphate buffer saline, both hydrogels showed no significant difference. SEM images demonstrated that hydrogel 2 composite showed a denser network structure and smaller pore-size than hydrogel 1. In comparison to native HA, the occurrence of chemical modification in the cross-linked hydrogels was confirmed by FTIR and NMR distinctive peaks. These peaks also provided evidence that hydrogel 2 exhibited a higher degree of modification than hydrogel 1. In conclusion, the small-batches mixing approach proved to be more effective than large-batch mixing in promoting HA-HA entanglement and increasing the probability of BDDE molecules for binding with HA chains. Copyright © 2016 Elsevier B.V. All rights reserved.

Induction of DNA-protein cross-links by platinum compounds.

PubMed

Woźniak, K; Walter, Z

2000-01-01

The differences between cis- and trans-diamminedichloroplatinum II (DDP) in forming DNA-protein cross-links in isolated human lymphocytes were investigated. Both cis- and trans-DDP can induce DNA-protein cross-links. We show that cis-DDP forms complexes between DNA and proteins faster than trans-DDP. This results from an increase in the quantity of DNA and platinum together with an increase in drug concentration. Under the same conditions trans-DDP causes a decrease in DNA-forming complexes with proteins. After a 12 h incubation of lymphocytes we observe a similar level of DNA in DNA-protein cross-links induced by DDP isomers, but more platinum appears in complexes induced by trans-DDP. The results obtained demonstrate that the antitumor drug - cis-DDP and the clinically ineffective trans-DDP induce links between DNA and proteins in a different manner. We suggest that the therapeutic activity of cis-DDP can in part arise from rapidly forming DNA-protein complexes which can destroy the most important cellular processes, such as replication and transcription.

The effect of toxins on inorganic phosphate release during actin polymerization.

PubMed

Vig, Andrea; Ohmacht, Róbert; Jámbor, Eva; Bugyi, Beáta; Nyitrai, Miklós; Hild, Gábor

2011-05-01

During the polymerization of actin, hydrolysis of bound ATP occurs in two consecutive steps: chemical cleavage of the high-energy nucleotide and slow release of the γ-phosphate. In this study the effect of phalloidin and jasplakinolide on the kinetics of P(i) release was monitored during the formation of actin filaments. An enzyme-linked assay based spectrophotometric technique was used to follow the liberation of inorganic phosphate. It was verified that jasplakinolide reduced the P(i) release in the same way as phalloidin. It was not possible to demonstrate long-range allosteric effects of the toxins by release of P(i) from F-actin. The products of ATP hydrolysis were released by denaturation of the actin filaments. HPLC analysis of the samples revealed that the ATP in the toxin-bound region was completely hydrolysed into ADP and P(i). The effect of both toxins can be sufficiently explained by local and mechanical blockade of P(i) dissociation.

[Complications and postoperative therapeutic strategies in cross-linking].

PubMed

Kohlhaas, M

2017-08-01

The reduced corneal mechanical stability in keratoconus and similar collagen diseases can lead to a progressive and irregular corneal shape and decrease of visual acuity. A progression of keratectatic diseases can be shown with corneal topography. Keratoconus can be treated by photo-oxidative cross-linking of the corneal collagen. In order to achieve a high absorption of irradiation energy in the cornea, riboflavin at a concentration of 0.1% and UVA light at a wavelength of 370 nm corresponding to the relative maximum absorption of riboflavin (vitamin B2) are used. Evidence for corneal cross-linking are the increase of biomechanical stiffness, the increased resistance against enzymatic degradation, a higher shrinkage temperature, a lower swelling rate and an increased diameter of collagen fibers. The currently available data demonstrate that the therapeutic cross-linking procedure is safe when respecting the important theoretical and clinical parameters and that a progression of the keratoconus can be avoided. In 80% of cases an average levelling of the curvature of approximately 2 dpt can be achieved, which leads not only to stabilization but also to an increase in visual acuity of approximately 1.2 lines. In a Cochrane review from 2015 publications about complications and results were reviewed. Complication rates ranged from 1-10% depending on the initial situation, comorbidities and stage of the keratoconus. The most important complications are early epithelial wound healing problems as well as extremely rare perforations. Corneal cross-linking is a well-established and safe procedure but is not free of complications.

Tissue-specific effects of aldose reductase inhibition on fluorescence and cross-linking of extracellular matrix in chronic galactosemia. Relationship to pentosidine cross-links.

PubMed

Richard, S; Tamas, C; Sell, D R; Monnier, V M

1991-08-01

Chronic experimental hyperglycemia mediated by galactose has been shown to induce browning and cross-linking of rat tail tendon collagen that could be duplicated in vitro by nonenzymatic galactosylation. To investigate the nature of these changes, Sprague-Dawley rats were placed on a 33% galactose diet without and with sorbinil for 6 and 12 mo. Collagen-linked fluorescence and pentosidine cross-links increased with age and galactosemia in tail tendons (P less than 0.001) and skin but were essentially unresponsive to aldose reductase inhibition (ARI). In contrast, tendon breaking time in urea, a likely parameter of cross-linking, was markedly improved (P less than 0.001) by ARI. Fluorescence that was inhibited by sorbinil treatment was increased in pepsin and proteinase K digest of aortic tissue from galactosemic rats (P less than 0.001), but impaired enzymatic digestibility was not observed. Systolic blood pressure as potential consequence of aortic stiffening was not increased in galactosemia. These data suggest that fluorescence in skin and tendon might be in part due to advanced glycosylation and pentosidine formation because these were not decreased by ARI. However, they also suggest that nonfluorescent cross-links may also be forming because, in contrast to fluorescence, tail tendon breaking time was partly corrected by ARI. Thus, it appears that extracellular matrix changes in chronic galactosemia are complex, being partly attributable to advanced glycosylation and partly to polyol-pathway activation.

Evaluation of hypoxia, angiogenesis, and lymphangiogenesis in actinic cheilitis.

PubMed

Barbosa, Natália G; Souza, Lélia B; Nonaka, Cassiano F W; Silveira, Ericka J D

2016-11-01

Actinic cheilitis is a potentially malignant condition caused mainly by chronic sun exposure. Here we aim to evaluate the role of hypoxia, angiogenesis, and lymphatic density in the clinical and morphological progression of a series of cases of actinic cheilitis. Immunohistochemistry was used to evaluate positivity to hypoxia-inducible factor (HIF)-1α, vascular endothelial growth factor (VEGF)-C, and D2-40 in 40 cases of actinic cheilitis of the lower lip. The cases studied exhibited variable degrees of positivity to the markers. The median number of lymphatic vessels was 3.2, 2.4, and 3.0 in lesions showing no epithelial dysplasia (NED) and with mild (MED) and moderate (MOED) epithelial dysplasia, respectively. The median VEGF-C positivity index was 82.44% (NED), 92.74% (MED), and 82.83% (MOED), and the median HIF-1α positivity index was 11.57% (NED), 5.26% (MED), and 13.55% (MOED). No significant differences in lymphatic density or median VEGF-C and HIF-1α positivity indices were observed between histological grades or clinical presentations of actinic cheilitis (P > 0.05). Although representing early events in lip carcinogenesis, the present results suggest that hypoxia, angiogenesis, and lymphangiogenesis do not influence the morphological or clinical progression of actinic cheilitis. © 2016 The International Society of Dermatology.

Application of a fast sorting algorithm to the assignment of mass spectrometric cross-linking data.

PubMed

Petrotchenko, Evgeniy V; Borchers, Christoph H

2014-09-01

Cross-linking combined with MS involves enzymatic digestion of cross-linked proteins and identifying cross-linked peptides. Assignment of cross-linked peptide masses requires a search of all possible binary combinations of peptides from the cross-linked proteins' sequences, which becomes impractical with increasing complexity of the protein system and/or if digestion enzyme specificity is relaxed. Here, we describe the application of a fast sorting algorithm to search large sequence databases for cross-linked peptide assignments based on mass. This same algorithm has been used previously for assigning disulfide-bridged peptides (Choi et al., ), but has not previously been applied to cross-linking studies. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Structure of the ribosome post-recycling complex probed by chemical cross-linking and mass spectrometry

PubMed Central

Kiosze-Becker, Kristin; Ori, Alessandro; Gerovac, Milan; Heuer, André; Nürenberg-Goloub, Elina; Rashid, Umar Jan; Becker, Thomas; Beckmann, Roland; Beck, Martin; Tampé, Robert

2016-01-01

Ribosome recycling orchestrated by the ATP binding cassette (ABC) protein ABCE1 can be considered as the final—or the first—step within the cyclic process of protein synthesis, connecting translation termination and mRNA surveillance with re-initiation. An ATP-dependent tweezer-like motion of the nucleotide-binding domains in ABCE1 transfers mechanical energy to the ribosome and tears the ribosome subunits apart. The post-recycling complex (PRC) then re-initiates mRNA translation. Here, we probed the so far unknown architecture of the 1-MDa PRC (40S/30S·ABCE1) by chemical cross-linking and mass spectrometry (XL-MS). Our study reveals ABCE1 bound to the translational factor-binding (GTPase) site with multiple cross-link contacts of the helix–loop–helix motif to the S24e ribosomal protein. Cross-linking of the FeS cluster domain to the ribosomal protein S12 substantiates an extreme lever-arm movement of the FeS cluster domain during ribosome recycling. We were thus able to reconstitute and structurally analyse a key complex in the translational cycle, resembling the link between translation initiation and ribosome recycling. PMID:27824037

Diverse mitotic functions of the cytoskeletal cross-linking protein Shortstop suggest a role in Dynein/Dynactin activity

PubMed Central

Dewey, Evan B.; Johnston, Christopher A.

2017-01-01

Proper assembly and orientation of the bipolar mitotic spindle is critical to the fidelity of cell division. Mitotic precision fundamentally contributes to cell fate specification, tissue development and homeostasis, and chromosome distribution within daughter cells. Defects in these events are thought to contribute to several human diseases. The underlying mechanisms that function in spindle morphogenesis and positioning remain incompletely defined, however. Here we describe diverse roles for the actin-microtubule cross-linker Shortstop (Shot) in mitotic spindle function in Drosophila. Shot localizes to mitotic spindle poles, and its knockdown results in an unfocused spindle pole morphology and a disruption of proper spindle orientation. Loss of Shot also leads to chromosome congression defects, cell cycle progression delay, and defective chromosome segregation during anaphase. These mitotic errors trigger apoptosis in Drosophila epithelial tissue, and blocking this apoptotic response results in a marked induction of the epithelial–mesenchymal transition marker MMP-1. The actin-binding domain of Shot directly interacts with Actin-related protein-1 (Arp-1), a key component of the Dynein/Dynactin complex. Knockdown of Arp-1 phenocopies Shot loss universally, whereas chemical disruption of F-actin does so selectively. Our work highlights novel roles for Shot in mitosis and suggests a mechanism involving Dynein/Dynactin activation. PMID:28747439

Transglutaminase catalyzed cross-linking of sodium caseinate improves oxidative stability of flaxseed oil emulsion.

PubMed

Ma, Hairan; Forssell, Pirkko; Kylli, Petri; Lampi, Anna-Maija; Buchert, Johanna; Boer, Harry; Partanen, Riitta

2012-06-20

Sodium caseinate was modified by transglutaminase catalyzed cross-linking reaction prior to the emulsification process in order to study the effect of cross-linking on the oxidative stability of protein stabilized emulsions. The extent of the cross-linking catalyzed by different dosages of transglutaminase was investigated by following the ammonia production during the reaction and using SDS-PAGE gel. O/W emulsions prepared with the cross-linked and non-cross-linked sodium caseinates were stored for 30 days under the same conditions. Peroxide value measurement, oxygen consumption measurement, and headspace gas chromatography analysis were used to study the oxidative stability of the emulsions. The emulsion made of the cross-linked sodium caseinate showed an improved oxidative stability with reduced formation of fatty acid hydroperoxides and volatiles and a longer period of low rate oxygen consumption. The improving effect of transglutaminase catalyzed cross-linking could be most likely attributed to the enhanced physical stability of the interfacial protein layer against competitive adsorption by oil oxidation products.

Controlled Cross-Linking with Glucose Oxidase for the Enhancement of Gelling Potential of Pork Myofibrillar Protein.

PubMed

Wang, Xu; Xiong, Youling L; Sato, Hiroaki; Kumazawa, Yoshiyuki

2016-12-21

Differential oxidative modifications of myofibrillar protein (MP) by hydroxyl radicals generated in an enzymatic system with glucose oxidase (GluOx) in the presence of glucose/FeSO 4 versus a Fenton system (H 2 O 2 /FeSO 4 ) were investigated. Pork MP was modified at 4 °C and pH 6.25 with hydroxyl radicals produced from 1 mg/mL glucose in the presence of 80, 160, or 320 μg/mL GluOx and 10 μM FeSO 4 . Total sulfhydryl content, solubility, cross-linking pattern, and gelation properties of MP were measured. H 2 O 2 production proceeded linearly with the concentration of GluOx and increased with reaction time. GluOx- and H 2 O 2 -dose-dependent protein polymerization, evidenced by faded myosin heavy chain and actin in SDS-PAGE as well as significant decreases in sulfhydryls, coincided with protein solubility loss. Firmer and more elastic MP gels were produced by GluOx than by the Fenton system at comparable H 2 O 2 levels due to an altered radical reaction pathway.

Live cell imaging of mitochondrial movement along actin cables in budding yeast.

PubMed

Fehrenbacher, Kammy L; Yang, Hyeong-Cheol; Gay, Anna Card; Huckaba, Thomas M; Pon, Liza A

2004-11-23

Mitochondrial inheritance is essential for cell division. In budding yeast, mitochondrial movement from mother to daughter requires (1) actin cables, F-actin bundles that undergo retrograde movement during elongation from buds into mother cells; (2) the mitochore, a mitochondrial protein complex implicated in linking mitochondria to actin cables; and (3) Arp2/3 complex-mediated force generation on mitochondria. We observed three new classes of mitochondrial motility: anterograde movement at velocities of 0.2-0.33 microm/s, retrograde movement at velocities of 0.26-0.51 microm/s, and no net anterograde or retrograde movement. In all cases, motile mitochondria were associated with actin cables undergoing retrograde flow at velocities of 0.18-0.62 microm/s. Destabilization of actin cables or mutations of the mitochore blocked all mitochondrial movements. In contrast, mutations in the Arp2/3 complex affected anterograde but not retrograde mitochondrial movements. Actin cables are required for movement of mitochondria, secretory vesicles, mRNA, and spindle alignment elements in yeast. We provide the first direct evidence that one of the proposed cargos use actin cables as tracks. In the case of mitochondrial inheritance, anterograde movement drives transfer of the organelle from mothers to buds, while retrograde movement contributes to retention of the organelle in mother cells. Interaction of mitochondria with actin cables is required for anterograde and retrograde movement. In contrast, force generation on mitochondria is required only for anterograde movement. Finally, we propose a novel mechanism in which actin cables serve as "conveyor belts" that drive retrograde organelle movement.

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

PubMed Central

Hong, Nan Hyung; Qi, Aidong

2015-01-01

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

Treatment of Actinic Purpura

PubMed Central

2017-01-01

Mature skin is prone to bruising, resulting in a condition known as actinic purpura, characterized by unsightly ecchymosis and purple patches. Similar to other skin conditions, the incidence of actinic purpura increases with advancing age and occurs with equal frequency among men and women. The unsightly appearance of actinic purpura may be a source of emotional distress among the elderly. A new product has been formulated specifically for the treatment of actinic purpura. This product contains retinol, α-hydroxy acids, arnica oil, ceramides, niacinamide, and phytonadione, which effectively treat actinic purpura by improving local circulation, thickening the skin, and repairing the skin barrier. The objective of this paper is to review the beneficial properties of these ingredients and their respective roles in the treatment of actinic purpura. PMID:28979656

Actin cytoskeleton modulates calcium signaling during maturation of starfish oocytes.

PubMed

Kyozuka, Keiichiro; Chun, Jong T; Puppo, Agostina; Gragnaniello, Gianni; Garante, Ezio; Santella, Luigia

2008-08-15

Before successful fertilization can occur, oocytes must undergo meiotic maturation. In starfish, this can be achieved in vitro by applying 1-methyladenine (1-MA). The immediate response to 1-MA is the fast Ca2+ release in the cell cortex. Here, we show that this Ca2+ wave always initiates in the vegetal hemisphere and propagates through the cortex, which is the space immediately under the plasma membrane. We have observed that alteration of the cortical actin cytoskeleton by latrunculin-A and jasplakinolide can potently affect the Ca2+ waves triggered by 1-MA. This indicates that the cortical actin cytoskeleton modulates Ca2+ release during meiotic maturation. The Ca2+ wave was inhibited by the classical antagonists of the InsP(3)-linked Ca2+ signaling pathway, U73122 and heparin. To our surprise, however, these two inhibitors induced remarkable actin hyper-polymerization in the cell cortex, suggesting that their inhibitory effect on Ca2+ release may be attributed to the perturbation of the cortical actin cytoskeleton. In post-meiotic eggs, U73122 and jasplakinolide blocked the elevation of the vitelline layer by uncaged InsP(3), despite the massive release of Ca2+, implying that exocytosis of the cortical granules requires not only a Ca2+ rise, but also regulation of the cortical actin cytoskeleton. Our results suggest that the cortical actin cytoskeleton of starfish oocytes plays critical roles both in generating Ca2+ signals and in regulating cortical granule exocytosis.

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

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.

The actin cytoskeleton may control the polar distribution of an auxin transport protein.

PubMed

Muday, G K; Hu, S; Brady, S R

2000-06-01

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.

T Lymphocyte Migration: An Action Movie Starring the Actin and Associated Actors.

PubMed

Dupré, Loïc; Houmadi, Raïssa; Tang, Catherine; Rey-Barroso, Javier

2015-01-01

The actin cytoskeleton is composed of a dynamic filament meshwork that builds the architecture of the cell to sustain its fundamental properties. This physical structure is characterized by a continuous remodeling, which allows cells to accomplish complex motility steps such as directed migration, crossing of biological barriers, and interaction with other cells. T lymphocytes excel in these motility steps to ensure their immune surveillance duties. In particular, actin cytoskeleton remodeling is a key to facilitate the journey of T lymphocytes through distinct tissue environments and to tune their stop and go behavior during the scanning of antigen-presenting cells. The molecular mechanisms controlling actin cytoskeleton remodeling during T lymphocyte motility have been only partially unraveled, since the function of many actin regulators has not yet been assessed in these cells. Our review aims to integrate the current knowledge into a comprehensive picture of how the actin cytoskeleton drives T lymphocyte migration. We will present the molecular actors that control actin cytoskeleton remodeling, as well as their role in the different T lymphocyte motile steps. We will also highlight which challenges remain to be addressed experimentally and which approaches appear promising to tackle them.

Cross-Linked Amylose Bio-Plastic: A Transgenic-Based Compostable Plastic Alternative

PubMed Central

Sagnelli, Domenico; Kemmer, Gerdi Christine; Holse, Mette; Hebelstrup, Kim H.; Bao, Jinsong; Stelte, Wolfgang; Bjerre, Anne-Belinda; Blennow, Andreas

2017-01-01

Bio-plastics and bio-materials are composed of natural or biomass derived polymers, offering solutions to solve immediate environmental issues. Polysaccharide-based bio-plastics represent important alternatives to conventional plastic because of their intrinsic biodegradable nature. Amylose-only (AO), an engineered barley starch with 99% amylose, was tested to produce cross-linked all-natural bioplastic using normal barley starch as a control. Glycerol was used as plasticizer and citrate cross-linking was used to improve the mechanical properties of cross-linked AO starch extrudates. Extrusion converted the control starch from A-type to Vh- and B-type crystals, showing a complete melting of the starch crystals in the raw starch granules. The cross-linked AO and control starch specimens displayed an additional wide-angle diffraction reflection. Phospholipids complexed with Vh-type single helices constituted an integrated part of the AO starch specimens. Gas permeability tests of selected starch-based prototypes demonstrated properties comparable to that of commercial Mater-Bi© plastic. The cross-linked AO prototypes had composting characteristics not different from the control, indicating that the modified starch behaves the same as normal starch. The data shows the feasibility of producing all-natural bioplastic using designer starch as raw material. PMID:28973963

Cross-Linked Amylose Bio-Plastic: A Transgenic-Based Compostable Plastic Alternative.

PubMed

Sagnelli, Domenico; Hooshmand, Kourosh; Kemmer, Gerdi Christine; Kirkensgaard, Jacob J K; Mortensen, Kell; Giosafatto, Concetta Valeria L; Holse, Mette; Hebelstrup, Kim H; Bao, Jinsong; Stelte, Wolfgang; Bjerre, Anne-Belinda; Blennow, Andreas

2017-09-30

Bio-plastics and bio-materials are composed of natural or biomass derived polymers, offering solutions to solve immediate environmental issues. Polysaccharide-based bio-plastics represent important alternatives to conventional plastic because of their intrinsic biodegradable nature. Amylose-only (AO), an engineered barley starch with 99% amylose, was tested to produce cross-linked all-natural bioplastic using normal barley starch as a control. Glycerol was used as plasticizer and citrate cross-linking was used to improve the mechanical properties of cross-linked AO starch extrudates. Extrusion converted the control starch from A-type to Vh- and B-type crystals, showing a complete melting of the starch crystals in the raw starch granules. The cross-linked AO and control starch specimens displayed an additional wide-angle diffraction reflection. Phospholipids complexed with Vh-type single helices constituted an integrated part of the AO starch specimens. Gas permeability tests of selected starch-based prototypes demonstrated properties comparable to that of commercial Mater-Bi © plastic. The cross-linked AO prototypes had composting characteristics not different from the control, indicating that the modified starch behaves the same as normal starch. The data shows the feasibility of producing all-natural bioplastic using designer starch as raw material.

Effect of Rubber Polarity on Cluster Formation in Rubbers Cross-Linked with Diels–Alder Chemistry

PubMed Central

2017-01-01

Diels–Alder chemistry has been used for the thermoreversible cross-linking of furan-functionalized ethylene/propylene (EPM) and ethylene/vinyl acetate (EVM) rubbers. Both furan-functionalized elastomers were successfully cross-linked with bismaleimide to yield products with a similar cross-link density. NMR relaxometry and SAXS measurements both show that the apolar EPM-g-furan precursor contains phase-separated polar clusters and that cross-linking with polar bismaleimide occurs in these clusters. The heterogeneously cross-linked network of EPM-g-furan contrasts with the homogeneous network in the polar EVM-g-furan. The heterogeneous character of the cross-links in EPM-g-furan results in a relatively high Young’s modulus, whereas the more uniform cross-linking in EVM-g-furan results in a higher tensile strength and elongation at break. PMID:29213149

Actin Cytoskeleton Manipulation by Effector Proteins Secreted by Diarrheagenic Escherichia coli Pathotypes

PubMed Central

Navarro-Garcia, Fernando; Serapio-Palacios, Antonio; Ugalde-Silva, Paul; Tapia-Pastrana, Gabriela; Chavez-Dueñas, Lucia

2013-01-01

The actin cytoskeleton is a dynamic structure necessary for cell and tissue organization, including the maintenance of epithelial barriers. Disruption of the epithelial barrier coincides with alterations of the actin cytoskeleton in several disease states. These disruptions primarily affect the paracellular space, which is normally regulated by tight junctions. Thereby, the actin cytoskeleton is a common and recurring target of bacterial virulence factors. In order to manipulate the actin cytoskeleton, bacteria secrete and inject toxins and effectors to hijack the host cell machinery, which interferes with host-cell pathways and with a number of actin binding proteins. An interesting model to study actin manipulation by bacterial effectors is Escherichia coli since due to its genome plasticity it has acquired diverse genetic mobile elements, which allow having different E. coli varieties in one bacterial species. These E. coli pathotypes, including intracellular and extracellular bacteria, interact with epithelial cells, and their interactions depend on a specific combination of virulence factors. In this paper we focus on E. coli effectors that mimic host cell proteins to manipulate the actin cytoskeleton. The study of bacterial effector-cytoskeleton interaction will contribute not only to the comprehension of the molecular causes of infectious diseases but also to increase our knowledge of cell biology. PMID:23509714

Identification of Pyridinoline Trivalent Collagen Cross-Links by Raman Microspectroscopy.

PubMed

Gamsjaeger, Sonja; Robins, Simon P; Tatakis, Dimitris N; Klaushofer, Klaus; Paschalis, Eleftherios P

2017-06-01

Intermolecular cross-linking of bone collagen is intimately related to the way collagen molecules are arranged in a fibril, imparts certain mechanical properties to the fibril, and may be involved in the initiation of mineralization. Raman microspectroscopy allows the analysis of minimally processed bone blocks and provides simultaneous information on both the mineral and organic matrix (mainly type I collagen) components, with a spatial resolution of ~1 μm. The aim of the present study was to validate Raman spectroscopic parameters describing one of the major mineralizing type I trivalent cross-links, namely pyridinoline (PYD). To achieve this, a series of collagen cross-linked peptides with known PYD content (as determined by HPLC analysis), human bone, porcine skin, predentin and dentin animal model tissues were analyzed by Raman microspectroscopy. The results of the present study confirm that it is feasible to monitor PYD trivalent collagen cross-links by Raman spectroscopic analysis in mineralized tissues, exclusively through a Raman band ~1660 wavenumbers. This allows determination of the relative PYD content in undecalcified bone tissues with a spatial resolution of ~1 μm, thus enabling correlations with histologic and histomorphometric parameters.

Removing Cross-Linked Telopeptides Enhances the Production of Low-Molecular-Weight Collagen Peptides from Spent Hens.

PubMed

Hong, Hui; Chaplot, Shreyak; Chalamaiah, Meram; Roy, Bimol C; Bruce, Heather L; Wu, Jianping

2017-08-30

The low-molecular-weight (LMW) peptides derived from collagen have shown a potential for various nutritional and pharmaceutical applications. However, production of LMW peptides from vertebrate collagen remains a challenge. Herein, we report a new method to produce LMW collagen peptides using pepsin pretreatment that removed cross-linked telopeptides in collagen molecules. After the pretreatment, the proportion of LMW collagen peptides (<1.4 kDa) that were obtained from pepsin-soluble collagen increased to 32.59% compared to heat-soluble collagen peptides (16.10%). Fourier transform infrared spectroscopy results indicated that telopeptide cleavage retained the triple-helical conformation of collagen. Liquid chromatography-tandem mass spectrometry analysis suggested that Gly-X-Y (X is often proline, while Y is either hydroxyproline or hydroxylysine) repeats were not the main factors that hindered the enzymatic hydrolysis of collagen molecules. However, cross-link quantification demonstrated that trivalent cross-links that included pyridinolines and pyrroles were the primary obstacles to producing small peptides from collagen of spent hens. This study demonstrated for the first time that removing cross-linked telopeptides could enhance the production of LMW peptides from spent hen collagen, which is also of interest to manufacturers who produce LMW collagen peptides from other vertebrate animals, such as bovids and porcids.

Radiation cross-linking in ultra-high molecular weight polyethylene for orthopaedic applications

NASA Astrophysics Data System (ADS)

Oral, Ebru; Muratoglu, Orhun K.

2007-12-01

The motivation for radiation cross-linking of ultra-high molecular weight polyethylene (UHMWPE) is to increase its wear resistance to be used as bearing surfaces for total joint arthroplasty. However, radiation also leaves behind long-lived residual free radicals in this polymer, the reactions of which can detrimentally affect mechanical properties. In this review, we focus on the radiation cross-linking and oxidative stability of first and second generation highly cross-linked UHMWPEs developed in our laboratory.

Alteration of mineral crystallinity and collagen cross-linking of bones in osteopetrotic toothless (tl/tl) rats and their improvement after treatment with colony stimulating factor-1

NASA Technical Reports Server (NTRS)

Wojtowicz, A.; Dziedzic-Goclawska, A.; Kaminski, A.; Stachowicz, W.; Wojtowicz, K.; Marks, S. C. Jr; Yamauchi, M.

1997-01-01

A common feature of various types of mammalian osteopetroses is a marked increase in bone mass accompanied by spontaneous bone fractures. The toothless (tl/tl) rat osteopetrotic mutation is characterized by drastically reduced bone resorption due to a profound deficiency of osteoclasts and their precursors. An altered bone morphology has also been observed. The mutants cannot be cured by bone marrow transplantation, but skeletal defects are greatly reduced after treatment with colony stimulating factor 1 (CSF-1). The objectives of this study were to characterize mineral and collagen matrices in cancellous and compact bone isolated from long bones of 6-week-old normal littermates, tl/tl osteopetrotic mutants and mutants (tl/tl) treated with CSF-1. There were no differences in bone mineral content, but a significant decrease in the crystallinity of mineral evaluated by the method based on electron paramagnetic resonance spectrometry was observed in all bones of tl/tl mutants as compared to that of controls. Within the collagen matrix, slight decreases in the labile cross-links, but significant increases in the content of the stable cross-links, pyridinoline, and deoxypyridinoline, were observed in both cancellous and compact bone of osteopetrotic mutants. In tl/tl mutants treated with human recombinant CSF-1, the normalization of the crystallinity of bone mineral as well as collagen cross-links was found. Our results indicate that remodeling of bone matrix in tl/tl mutants is highly suppressed, but that after treatment with CSF-1, this activity recovers significantly. Taken together, these data provide further support for the hypothesis that CSF-1 is an essential factor for normal osteoclast differentiation and bone remodelling.

Interrelation of electret properties of polyethylene foam from the method of cross-linking

NASA Astrophysics Data System (ADS)

Gilmanov, I. R.; Galikhanov, M. F.; Gilmanova, A. R.

2017-09-01

The electret properties of chemically cross-linked polyethylene foam and physically cross-linked polyethylene foam have been studied. It has been shown that chemically cross-linked polyethylene foam has higher surface potential, effective surface charge density and electric field strength compared to physically bonded polyethylene foam. This is due to the presence of molecules and fragments of dicumyl peroxide, which can play the role of traps for injection charge carriers, a greater degree of cross-linking and with the oxidation of polyethylene, which occurs during irradiation during physical cross-linking. When the foam is deformed, its electret properties are reduced, and when the volume is relaxed, they are restored. This is due to the partial mutual compensation of homo- and heterocharge during compression and the return of the structure of the gas-filled polymer to its former position when the load is removed.

Filopodia-like Actin Cables Position Nuclei in Association with Perinuclear Actin in Drosophila Nurse Cells

PubMed Central

Huelsmann, Sven; Ylänne, Jari; Brown, Nicholas H.

2013-01-01

Summary Controlling the position of the nucleus is vital for a number of cellular processes from yeast to humans. In Drosophila nurse cells, nuclear positioning is crucial during dumping, when nurse cells contract and expel their contents into the oocyte. We provide evidence that in nurse cells, continuous filopodia-like actin cables, growing from the plasma membrane and extending to the nucleus, achieve nuclear positioning. These actin cables move nuclei away from ring canals. When nurse cells contract, actin cables associate laterally with the nuclei, in some cases inducing nuclear turning so that actin cables become partially wound around the nuclei. Our data suggest that a perinuclear actin meshwork connects actin cables to nuclei via actin-crosslinking proteins such as the filamin Cheerio. We provide a revised model for how actin structures position nuclei in nurse cells, employing evolutionary conserved machinery. PMID:24091012

Multiple roles for the actin cytoskeleton during regulated exocytosis

PubMed Central

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

2014-01-01

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

[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

Nucleus-associated actin in Amoeba proteus.

PubMed

Berdieva, Mariia; Bogolyubov, Dmitry; Podlipaeva, Yuliya; Goodkov, Andrew

2016-10-01

The presence, spatial distribution and forms of intranuclear and nucleus-associated cytoplasmic actin were studied in Amoeba proteus with immunocytochemical approaches. Labeling with different anti-actin antibodies and staining with TRITC-phalloidin and fluorescent deoxyribonuclease I were used. We showed that actin is abundant within the nucleus as well as in the cytoplasm of A. proteus cells. According to DNase I experiments, the predominant form of intranuclear actin is G-actin which is associated with chromatin strands. Besides, unpolymerized actin was shown to participate in organization of a prominent actin layer adjacent to the outer surface of nuclear envelope. No significant amount of F-actin was found in the nucleus. At the same time, the amoeba nucleus is enclosed in a basket-like structure formed by circumnuclear actin filaments and bundles connected with global cytoplasmic actin cytoskeleton. A supposed architectural function of actin filaments was studied by treatment with actin-depolymerizing agent latrunculin A. It disassembled the circumnuclear actin system, but did not affect the intranuclear chromatin structure. The results obtained for amoeba cells support the modern concept that actin is involved in fundamental nuclear processes that have evolved in the cells of multicellular organisms. Copyright © 2016 Elsevier GmbH. All rights reserved.

Physicochemical properties of collagen solutions cross-linked by glutaraldehyde.

PubMed

Tian, Zhenhua; Li, Conghu; Duan, Lian; Li, Guoying

2014-06-01

The physicochemical properties of collagen solutions (5 mg/ml) cross-linked by various amounts of glutaraldehyde (GTA) [GTA/collagen (w/w) = 0-0.5] under acidic condition (pH 4.00) were examined. Based on the results of the determination of residual amino group content, sodium dodecyl sulphate-polyacrylamide gel electrophoresis, dynamic rheological measurements, differential scanning calorimetry and atomic force microscopy (AFM), it was proved that the collagen solutions possessed strikingly different physicochemical properties depending on the amount of GTA. At low GTA amounts [GTA/collagen (w/w) ≤ 0.1], the residual amino group contents of the cross-linked collagens decreased largely from 100% to 32.76%, accompanied by an increase in the molecular weight. Additionally, increases of the fiber diameter and the values of G', G″ and η* were measured, while the thermal denaturation temperature (Td) did not change visibly and the fluidity of collagen samples was still retained with increasing the GTA amount. When the ratio of GTA to collagen exceeded 0.1, although the residual amino group content only decreased by ~8.2%, the cross-linked collagen solution [GTA/collagen (w/w) = 0.3] displayed a clear loss of flow and a sudden rise (~2.0 °C) of the Td value compared to the uncross-linked collagen solution, probably illustrating that the collagen solution was converted into a gel with mature network structure-containing nuclei observed in AFM image. It was conjectured that the physicochemical properties of the collagen solutions might be in connection with the cross-linking between collagen molecules from the same aggregate or different aggregates.

Cell Adhesions: Actin-Based Modules that Mediate Cell-Extracellular Matrix and Cell-Cell Interactions

PubMed Central

Bachir, Alexia; Horwitz, Alan Rick; Nelson, W. James; Bianchini, Julie M.

2018-01-01

Cell adhesions link cells to the extracellular matrix (ECM) and to each other, and depend on interactions with the actin cytoskeleton. Both cell-ECM and cell-cell adhesion sites contain discrete, yet overlapping functional modules. These modules establish physical association with the actin cytoskeleton, locally modulate actin organization and dynamics, and trigger intracellular signaling pathways. Interplay between these modules generates distinct actin architectures that underlie different stages, types, and functions of cell-ECM and cell-cell adhesions. Actomyosin contractility is required to generate mature, stable adhesions, as well as sense and translate the mechanical properties of the cellular environment to changes in cell organization and behavior. In this chapter we discuss the organization and function of different adhesion modules and how they interact with the actin cytoskeleton. We highlight the molecular mechanisms of mechanotransduction in adhesions, and how adhesion molecules mediate crosstalk between cell-ECM and cell-cell adhesion sites. PMID:28679638

Cross-linking reveals laminin coiled-coil architecture

PubMed Central

Armony, Gad; Jacob, Etai; Moran, Toot; Levin, Yishai; Mehlman, Tevie; Levy, Yaakov; Fass, Deborah

2016-01-01

Laminin, an ∼800-kDa heterotrimeric protein, is a major functional component of the extracellular matrix, contributing to tissue development and maintenance. The unique architecture of laminin is not currently amenable to determination at high resolution, as its flexible and narrow segments complicate both crystallization and single-particle reconstruction by electron microscopy. Therefore, we used cross-linking and MS, evaluated using computational methods, to address key questions regarding laminin quaternary structure. This approach was particularly well suited to the ∼750-Å coiled coil that mediates trimer assembly, and our results support revision of the subunit order typically presented in laminin schematics. Furthermore, information on the subunit register in the coiled coil and cross-links to downstream domains provide insights into the self-assembly required for interaction with other extracellular matrix and cell surface proteins. PMID:27815530

Anti-actin IgA antibodies in severe coeliac disease

PubMed Central

Granito, A; Muratori, P; Cassani, F; Pappas, G; Muratori, L; Agostinelli, D; Veronesi, L; Bortolotti, R; Petrolini, N; Bianchi, F B; Volta, U

2004-01-01

Anti-actin IgA antibodies have been found in sera of coeliacs. Our aim was to define the prevalence and clinical significance of anti-actin IgA in coeliacs before and after gluten withdrawal. One hundred and two biopsy-proven coeliacs, 95 disease controls and 50 blood donors were studied. Anti-actin IgA were evaluated by different methods: (a) antimicrofilament positivity on HEp-2 cells and on cultured fibroblasts by immunofluorescence; (b) anti-actin positivity by enzyme-linked immuosorbent assay (ELISA); and (c) presence of the tubular/glomerular pattern of anti-smooth muscle antibodies on rat kidney sections by immunofluorescence. Antimicrofilament IgA were present in 27% of coeliacs and in none of the controls. Antimicrofilament antibodies were found in 25 of 54 (46%) coeliacs with severe villous atrophy and in three of 48 (6%) with mild damage (P < 0·0001). In the 20 patients tested, antimicrofilaments IgA disappeared after gluten withdrawal in accordance with histological recovery. Our study shows a significant correlation between antimicrofilament IgA and the severity of intestinal damage in untreated coeliacs. The disappearance of antimicrofilament IgA after gluten withdrawal predicts the normalization of intestinal mucosa and could be considered a useful tool in the follow-up of severe coeliac disease. PMID:15270857

Assessment of UVA-Riboflavin Corneal Cross-Linking Using Small Amplitude Oscillatory Shear Measurements.

PubMed

Aslanides, Ioannis M; Dessi, Claudia; Georgoudis, Panagiotis; Charalambidis, Georgios; Vlassopoulos, Dimitris; Coutsolelos, Athanassios G; Kymionis, George; Mukherjee, Achyut; Kitsopoulos, Theofanis N

2016-04-01

The effect of ultraviolet (UV)-riboflavin cross-linking (CXL) has been measured primarily using the strip extensometry technique. We propose a simple and reliable methodology for the assessment of CXL treatment by using an established rheologic protocol based on small amplitude oscillatory shear (SAOS) measurements. It provides information on the average cross-link density and the elastic modulus of treated cornea samples. Three fresh postmortem porcine corneas were used to study the feasibility of the technique, one serving as control and two receiving corneal collagen cross-linking treatment. Subsequently, five pairs of fresh postmortem porcine corneas received corneal collagen cross-linking treatment with riboflavin and UVA-irradiation (370 nm; irradiance of 3 mW/cm2) for 30 minutes (Dresden protocol); the contralateral porcine corneas were used as control samples. After the treatment, the linear viscoelastic moduli of the corneal samples were measured using SAOS measurements and the average cross-linking densities extracted. For all cases investigated, the dynamic moduli of the cross-linked corneas were higher compared to those of the corresponding control samples. The increase of the elastic modulus of the treated samples was between 122% and 1750%. The difference was statistically significant for all tested samples (P = 0.018, 2-tailed t-test). We report a simple and accurate methodology for quantifying the effects of cross-linking on porcine corneas treated with the Dresden protocol by means of SAOS measurements in the linear regime. The measured dynamic moduli, elastic and viscous modulus, represent the energy storage and energy dissipation, respectively. Hence, they provide a means to assess the changing physical properties of the cross-linked collagen networks after CXL treatment.

Triple shape memory effects of cross-linked polyethylene/polypropylene blends with cocontinuous architecture.

PubMed

Zhao, Jun; Chen, Min; Wang, Xiaoyan; Zhao, Xiaodong; Wang, Zhenwen; Dang, Zhi-Min; Ma, Lan; Hu, Guo-Hua; Chen, Fenghua

2013-06-26

In this paper, the triple shape memory effects (SMEs) observed in chemically cross-linked polyethylene (PE)/polypropylene (PP) blends with cocontinuous architecture are systematically investigated. The cocontinuous window of typical immiscible PE/PP blends is the volume fraction of PE (v(PE)) of ca. 30-70 vol %. This architecture can be stabilized by chemical cross-linking. Different initiators, 2,5-dimethyl-2,5-di(tert-butylperoxy)-hexane (DHBP), dicumylperoxide (DCP) coupled with divinylbenzene (DVB) (DCP-DVB), and their mixture (DHBP/DCP-DVB), are used for the cross-linking. According to the differential scanning calorimetry (DSC) measurements and gel fraction calculations, DHBP produces the best cross-linking and DCP-DVB the worst, and the mixture, DHBP/DCP-DVB, is in between. The chemical cross-linking causes lower melting temperature (Tm) and smaller melting enthalpy (ΔHm). The prepared triple shape memory polymers (SMPs) by cocontinuous immiscible PE/PP blends with v(PE) of 50 vol % show pronounced triple SMEs in the dynamic mechanical thermal analysis (DMTA) and visual observation. This new strategy of chemically cross-linked immiscible blends with cocontinuous architecture can be used to design and prepare new SMPs with triple SMEs.

On-Demand Dissolution of Chemically Cross-Linked Hydrogels.

PubMed

Konieczynska, Marlena D; Grinstaff, Mark W

2017-02-21

The formation and subsequent on-demand dissolution of chemically cross-linked hydrogels is of keen interest to chemists, engineers, and clinicians. In this Account, we summarize our recent advances in the area of dissolvable chemically cross-linked hydrogels and provide a comparative discussion of other recent hydrogel systems. Using biocompatible macromonomers, we developed a library of cross-linked dendritic hydrogels that possess favorable properties, including biocompatibility, tissue adhesion, and swelling. Additionally, these hydrogels possess the unique ability to dissolve on-demand via application of a biocompatible aqueous solution. Each of the three hydrogel systems described employs a thiol-thioester exchange reaction as the mechanism of dissolution. These new materials successfully decrease bleeding in in vivo models of hepatic and aortic hemorrhage and dissolve on-demand, providing easy removal. In addition, we evaluated these hydrogels as dressings for second-degree burn wounds and performed proof-of-concept in vivo studies. These hydrogel wound dressings provide a means of repeatedly changing the dressing in a minimally invasive and atraumatic manner while also serving as a protective barrier against bacterial infection. Finally, we highlight the seminal work of other researchers in the field of dissolvable chemically cross-linked hydrogels using thiol-disulfide exchange, retro-Michael-type, and retro-Diels-Alder reactions. These chemistries provide a versatile synthetic toolbox to dissolve hydrogels in a controlled manner on time scales from minutes to weeks. Continued investigation of these dissolution approaches as well as the development of new chemical reactions will open doors to other avenues of on-demand dissolution and expand the application space for these materials. In summary, the management and closure of wounds after traumatic injury or surgical intervention are of significant clinical importance. Stimuli-responsive hydrogels that

Dependence of nanomechanical modification of polymers on plasma-induced cross-linking

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

Tajima, S.; Komvopoulos, K.

2007-01-01

The nanomechanical properties of low-density polyethylene (LDPE) modified by inductively coupled, radio-frequency Ar plasma were investigated by surface force microscopy. The polymer surface was modified under plasma conditions of different ion energy fluences and radiation intensities obtained by varying the sample distance from the plasma power source. Nanoindentation results of the surface stiffness versus maximum penetration depth did not reveal discernible differences between untreated and plasma-treated LDPE, presumably due to the small thickness of the modified surface layer that resulted in a substrate effect. On the contrary, nanoscratching experiments demonstrated a significant increase in the surface shear resistance of plasma-modifiedmore » LDPE due to chain cross-linking. These experiments revealed an enhancement of cross-linking with increasing ion energy fluence and radiation intensity, and a tip size effect on the friction force and dominant friction mechanisms (adhesion, plowing, and microcutting). In addition, LDPE samples with a LiF crystal shield were exposed to identical plasma conditions to determine the role of vacuum ultraviolet (VUV) and ultraviolet (UV) radiation in the cross-linking process. The cross-linked layer of plasma-treated LDPE exhibited much higher shear strength than that of VUV/UV-treated LDPE. Plasma-induced surface modification of the nanomechanical properties of LDPE is interpreted in the context of molecular models of the untreated and cross-linked polymer surfaces derived from experimental findings.« less

Actin retrograde flow actively aligns and orients ligand-engaged integrins in focal adhesions

PubMed Central

Swaminathan, Vinay; Kalappurakkal, Joseph Mathew; Moore, Travis I.; Koga, Nobuyasu; Baker, David A.; Oldenbourg, Rudolf; Tani, Tomomi; Springer, Timothy A.; Waterman, Clare M.

2017-01-01

Integrins are transmembrane receptors that, upon activation, bind extracellular ligands and link them to the actin filament (F-actin) cytoskeleton to mediate cell adhesion and migration. Cytoskeletal forces in migrating cells generated by polymerization- or contractility-driven “retrograde flow” of F-actin from the cell leading edge have been hypothesized to mediate integrin activation for ligand binding. This predicts that these forces should align and orient activated, ligand-bound integrins at the leading edge. Here, polarization-sensitive fluorescence microscopy of GFP-αVβ3 integrins in fibroblasts shows that integrins are coaligned in a specific orientation within focal adhesions (FAs) in a manner dependent on binding immobilized ligand and a talin-mediated linkage to the F-actin cytoskeleton. These findings, together with Rosetta modeling, suggest that integrins in FA are coaligned and may be highly tilted by cytoskeletal forces. Thus, the F-actin cytoskeleton sculpts an anisotropic molecular scaffold in FAs, and this feature may underlie the ability of migrating cells to sense directional extracellular cues. PMID:29073038

Actin polymerization drives polar growth in Arabidopsis root hair cells.

PubMed

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

2014-01-01

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

Fiber optic immunosensor for cross-linked fibrin concentration

NASA Astrophysics Data System (ADS)

Moskowitz, Samuel E.

2000-08-01

Working with calcium ions in the blood, platelets produce thromboplastin which transforms prothrombin into thrombin. Removing peptides, thrombin changes fibrinogen into fibrin. Cross-linked insoluble fibrin polymers are solubilized by enzyme plasmin found in blood plasma. Resulting D-dimers are elevated in patients with intravascular coagulation, deep venous thrombosis, pulmonary embolism, myocardial infarction, multiple trauma, cancer, impaired renal and liver functions, and sepsis. Consisting principally of a NIR 780 nm GaAlAs laser diode and a 800 nm avalanche photodiode (APD), the fiber-optic immunosensor can determined D-dimer concentration to levels <0.1 ng/ml. A capture monoclonal antibody to the antigen soluble cross-linked fibrin is employed. Immobilized at the tip of an optical fiber by avidin-biotin, the captured antigen is detected by a second antibody which is labeled with NN 382 fluorescent dye. An evanescent wave traveling on an excitation optical fiber excites the antibody-antigen fluorophore complex. Concentration of cross-linked fibrin is directly proportional to the APD measured intensity of fluorescence. NIR fluorescence has advantages of low background interference, short fluorescence lifetime, and large difference between excitation and emission peaks. Competitive ELISA test for D-dimer concentration requires trained personnel performing a time consuming operation.

Role of special cross-links in structure formation of bacterial DNA polymer

NASA Astrophysics Data System (ADS)

Agarwal, Tejal; Manjunath, G. P.; Habib, Farhat; Lakshmi Vaddavalli, Pavana; Chatterji, Apratim

2018-01-01

Using data from contact maps of the DNA-polymer of Escherichia coli (E. Coli) (at kilobase pair resolution) as an input to our model, we introduce cross-links between monomers in a bead-spring model of a ring polymer at very specific points along the chain. Via suitable Monte Carlo simulations, we show that the presence of these cross-links leads to a particular organization of the chain at large (micron) length scales of the DNA. We also investigate the structure of a ring polymer with an equal number of cross-links at random positions along the chain. We find that though the polymer does get organized at the large length scales, the nature of the organization is quite different from the organization observed with cross-links at specific biologically determined positions. We used the contact map of E. Coli bacteria which has around 4.6 million base pairs in a single circular chromosome. In our coarse-grained flexible ring polymer model, we used 4642 monomer beads and observed that around 80 cross-links are enough to induce the large-scale organization of the molecule accounting for statistical fluctuations caused by thermal energy. The length of a DNA chain even of a simple bacterial cell such as E. Coli is much longer than typical proteins, hence we avoided methods used to tackle protein folding problems. We define new suitable quantities to identify the large scale structure of a polymer chain with a few cross-links.

WHAMM Directs the Arp2/3 Complex to the ER for Autophagosome Biogenesis through an Actin Comet Tail Mechanism.

PubMed

Kast, David J; Zajac, Allison L; Holzbaur, Erika L F; Ostap, E Michael; Dominguez, Roberto

2015-06-29

Nucleation-promoting factors (NPFs) control the spatio-temporal activity of Arp2/3 complex in cells]. Thus, WASP and the WAVE complex direct the formation of branched actin networks at the leading edge during cell motility and endo/exocytosis, whereas the WASH complex is involved in endosomal transport. Less understood are WHAMM and JMY, two NPFs with similar domain architecture. JMY is found in the nucleus and the cytosol and is involved in transcriptional regulation, cell motility, and trans-Golgi transport. WHAMM was reported to bind microtubules and to be involved in ER to cis-Golgi transport. Here, we show that WHAMM directs the activity of Arp2/3 complex for autophagosome biogenesis through an actin-comet tail motility mechanism. Macroautophagy--the process by which cytosolic material is engulfed into autophagosomes for degradation and/or recycling--was recently shown to involve actin, but the mechanism is unknown. We found that WHAMM forms puncta that colocalize and comigrate with the autophagy markers LC3, DFCP1, and p62 through a WHAMM-dependent actin-comet tail mechanism. Under starvation, WHAMM and actin are observed at the interface between neighboring autophagosomes, whose number and size increase with WHAMM expression. Interfering with actin polymerization, inhibiting Arp2/3 complex, knocking down WHAMM, or blocking its interaction with Arp2/3 complex through mutagenesis all inhibit comet tail formation and reduce the size and number of autophagosomes. Finally, JMY shows similar localization to WHAMM and could be involved in similar processes. These results reveal a link between Arp2/3-complex-dependent actin assembly and autophagy. Copyright © 2015 Elsevier Ltd. All rights reserved.

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

PubMed

Kopecká, Marie; Kawamoto, Susumu; Yamaguchi, Masashi

2013-04-01

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

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

NASA Astrophysics Data System (ADS)

Marshall Mccall, Patrick

-equilibrium turnover on the steady-state properties of collections of filaments remained unclear. Here, I reconstituted tunable, non-equilibrium actin turnover dynamics in entangled solutions of actin filaments as a model of the actin cortex of living cells. We found that this non-equilibrium turnover decouples solution mechanics from microstructure, enabling structurally indistinguishable materials to behave effectively as either viscous fluids or elastic gels. Additionally, we employed computer simulations to identify the dynamical regime in which actin turnover controls the effective viscosity of 2D cross-linked actin networks in the presence of motors. Additionally, I examine in this thesis the localization and self-assembly of actin filaments in condensed liquid phases called polyelectrolyte coacervates as a model membrane-less organelle. We find that concentration of actin through spontaneous partitioning preferentially to the coacervate phase accelerates the assembly of filaments. These filaments then localize to the coacervate-bulk interface, generating particles with visco-elastic shells surrounding liquid cores. In this case, the properties of the condensed phase enable regulation of actin assembly dynamics.

Influence of cross-linked arabinoxylans on the postprandial blood glucose response in rats.

PubMed

Vogel, Barbara; Gallaher, Daniel D; Bunzel, Mirko

2012-04-18

Viscous dietary fibers are well established to reduce the blood glucose response to a meal. In this study, arabinoxylans, the most abundant dietary fiber in most cereals, were extracted under alkaline conditions and cross-linked by using laccase. Cross-linking of the arabinoxylans led to gel formation and increased in vitro viscosity almost 100-fold after drying and rehydration. To determine the ability of these cross-linked arabinoxylans to blunt the postprandial blood glucose curve of a meal, arabinoxylans, either native or cross-linked, and either prehydrated or not, were fed to rats as part of a meal, and blood glucose was monitored at intervals after the meal. Cellulose, a nonviscous fiber, served as a control. Cross-linked, but not native, arabinoxylans significantly reduced the area under the blood glucose time curve 5-9% relative to cellulose, indicating that they remained viscous within the gastrointestinal tract, and thus likely provide the health benefits found with other viscous fibers.

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

PubMed Central

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

2015-01-01

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

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

PubMed

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

2015-10-01

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

Competition between dewetting and cross-linking in poly(N-vinylpyrrolidone)/polystyrene bilayer films.

PubMed

Telford, Andrew M; Thickett, Stuart C; James, Michael; Neto, Chiara

2011-12-06

We investigated the dewetting of metastable poly(N-vinylpyrrolidone) (PNVP) thin films (45 nm) on top of polystyrene (PS) thin films (58 nm) as a function of annealing temperature and molecular weight of PS (96 and 6850 kg/mol). We focused on the competition between dewetting, occurring as a result of unfavorable intermolecular interactions at the PNVP/PS interface, and spontaneous cross-linking of PNVP, occurring during thermal annealing, as we recently reported (Telford, A. M.; James, M.; Meagher, L.; Neto, C. ACS Appl. Mater. Interfaces 2010, 2, 2399-2408). Using optical microscopy, we studied how the dewetting morphology and dynamics at different temperatures depended on the relative viscosity of the top PNVP film, which increased with cross-linking time, and of the bottom PS film. In the PNVP/PS96K system, cross-linking dominated over dewetting at temperatures below 180 °C, reducing drastically nucleated hole density and their maximum size, while above 180 °C the two processes reversed, with complete dewetting occurring at 200 °C. On the other hand, the PNVP/PS6850K system never achieved advanced dewetting stages as the dewetting was slower than cross-linking in the investigated temperature range. In both systems, dewetting of the PNVP films could be avoided altogether by thermally annealing the bilayers at temperatures where cross-linking dominated. The cross-linking was characterized quantitatively using neutron reflectometry, which indicated shrinkage and densification of the PNVP film, and qualitatively through selective removal of the bottom PS film. A simple model accounting for progressive cross-linking during the dewetting process predicted well the observed hole growth profiles and produced estimates of the PNVP cross-linking rate coefficients and of the activation energy of the process, in good agreement with literature values for similar systems. © 2011 American Chemical Society

Calreticulin attenuated microwave radiation-induced human microvascular endothelial cell injury through promoting actin acetylation and polymerization.

PubMed

Xu, Feifei; Wang, You; Tao, Tianqi; Song, Dandan; Liu, Xiuhua

2017-01-01

Recent work reveals that actin acetylation modification has been linked to different normal and disease processes and the effects associated with metabolic and environmental stressors. Herein, we highlight the effects of calreticulin on actin acetylation and cell injury induced by microwave radiation in human microvascular endothelial cell (HMEC). HMEC injury was induced by high-power microwave of different power density (10, 30, 60, 100 mW/cm 2 , for 6 min) with or without exogenous recombinant calreticulin. The cell injury was assessed by lactate dehydrogenase (LDH) activity and Cell Counting Kit-8 in culture medium, migration ability, intercellular junction, and cytoskeleton staining in HMEC. Western blotting analysis was used to detected calreticulin expression in cytosol and nucleus and acetylation of globular actin (G-actin). We found that HMEC injury was induced by microwave radiation in a dose-dependent manner. Pretreatment HMEC with calreticulin suppressed microwave radiation-induced LDH leakage and increased cell viability and improved microwave radiation-induced decrease in migration, intercellular junction, and cytoskeleton. Meanwhile, pretreatment HMEC with exogenous calreticulin upregulated the histone acetyltransferase activity and the acetylation level of G-actin and increased the fibrous actin (F-actin)/G-actin ratio. We conclude that exogenous calreticulin protects HMEC against microwave radiation-induced injury through promoting actin acetylation and polymerization.

Durability and mechanical properties of silane cross-linked wood thermoplastic composites

Treesearch

Magnus Bengtsson; Nicole M. Stark; Kristiina Oksman

2007-01-01

In this study, silane cross-linked wood–polyethylene composite profiles were manufactured by reactive extrusion. These composites were evaluated regarding their durability and mechanical properties in comparison with two non-cross-linked wood– polyethylene composites. An addition of only 2% w/w of silane solution during manufacturing was enough to achieve almost 60%...

Cross-Linked Nanohybrid Polymer Electrolytes With POSS Cross-Linker for Solid-State Lithium Ion Batteries.

PubMed

Zhang, Jinfang; Li, Xiaofeng; Li, Ying; Wang, Huiqi; Ma, Cheng; Wang, Yanzhong; Hu, Shengliang; Wei, Weifeng

2018-01-01

A new class of freestanding cross-linked hybrid polymer electrolytes (HPEs) with POSS as the cross-linker was prepared by a one-step free radical polymerization reaction. Octavinyl octasilsesquioxane (OV-POSS) with eight functional corner groups was used to provide cross-linking sites for the connection of polymer segments and the required mechanical strength to separate the cathode and anode. The unique cross-linked structure offers additional free volume for the motion of EO chains and provides fast and continuously interconnected ion-conducting channels along the nanoparticles/polymer matrix interface. The HPE exhibits the highest ionic conductivity of 1.39 × 10 -3 S cm -1 , as well as excellent interfacial compatibility with the Li electrode at 80°C. In particular, LiFePO 4 /Li cells based on the HPE deliver good rate capability and long-term cycling performance with an initial discharge capacity of 152.1 mAh g -1 and a capacity retention ratio of 88% after 150 cycles with a current density of 0.5 C at 80°C, demonstrating great potential application in high-performance LIBs at elevated temperatures.

Cross-linked Nanohybrid Polymer Electrolytes with POSS Cross-linker for Solid-state Lithium Ion Batteries

NASA Astrophysics Data System (ADS)

Zhang, Jinfang; Li, Xiaofeng; Li, Ying; Wang, Huiqi; Ma, Cheng; Wang, Yanzhong; Hu, Shengliang; Wei, Weifeng

2018-05-01