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1
Liu, Yi, Keyvan Mollaeian, and Juan Ren. "An Image Recognition-Based Approach to Actin Cytoskeleton Quantification." Electronics 7, no. 12 (December 17, 2018): 443. http://dx.doi.org/10.3390/electronics7120443.
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Quantification of the actin cytoskeleton is of prime importance to unveil the cellular force sensing and transduction mechanism. Although fluorescence imaging provides a convenient tool for observing the morphology of the actin cytoskeleton, due to the lack of approaches to accurate actin cytoskeleton quantification, the dynamics of mechanotransduction is still poorly understood. Currently, the existing image-based actin cytoskeleton analysis tools are either incapable of quantifying both the orientation and the quantity of the actin cytoskeleton simultaneously or the quantified results are subject to analysis artifacts. In this study, we propose an image recognition-based actin cytoskeleton quantification (IRAQ) approach, which quantifies both the actin cytoskeleton orientation and quantity by using edge, line, and brightness detection algorithms. The actin cytoskeleton is quantified through three parameters: the partial actin-cytoskeletal deviation (PAD), the total actin-cytoskeletal deviation (TAD), and the average actin-cytoskeletal intensity (AAI). First, Canny and Sobel edge detectors are applied to skeletonize the actin cytoskeleton images, then PAD and TAD are quantified using the line directions detected by Hough transform, and AAI is calculated through the summational brightness over the detected cell area. To verify the quantification accuracy, the proposed IRAQ was applied to six artificially-generated actin cytoskeleton mesh work models. The average error for both the quantified PAD and TAD was less than 1.22 ∘ . Then, IRAQ was implemented to quantify the actin cytoskeleton of NIH/3T3 cells treated with an F-actin inhibitor (latrunculin B). The quantification results suggest that the local and total actin-cytoskeletal organization became more disordered with the increase of latrunculin B dosage, and the quantity of the actin cytoskeleton showed a monotonically decreasing relation with latrunculin B dosage.
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Breuer, David, Alexander Ivakov, Arun Sampathkumar, Florian Hollandt, Staffan Persson, and Zoran Nikoloski. "Quantitative analyses of the plant cytoskeleton reveal underlying organizational principles." Journal of The Royal Society Interface 11, no. 97 (August 6, 2014): 20140362. http://dx.doi.org/10.1098/rsif.2014.0362.
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The actin and microtubule (MT) cytoskeletons are vital structures for cell growth and development across all species. While individual molecular mechanisms underpinning actin and MT dynamics have been intensively studied, principles that govern the cytoskeleton organization remain largely unexplored. Here, we captured biologically relevant characteristics of the plant cytoskeleton through a network-driven imaging-based approach allowing us to quantitatively assess dynamic features of the cytoskeleton. By introducing suitable null models, we demonstrate that the plant cytoskeletal networks exhibit properties required for efficient transport, namely, short average path lengths and high robustness. We further show that these advantageous features are maintained during temporal cytoskeletal rearrangements. Interestingly, man-made transportation networks exhibit similar properties, suggesting general laws of network organization supporting diverse transport processes. The proposed network-driven analysis can be readily used to identify organizational principles of cytoskeletons in other organisms.
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Jack, R. M., R. M. Ezzell, J. Hartwig, and D. T. Fearon. "Differential interaction of the C3b/C4b receptor and MHC class I with the cytoskeleton of human neutrophils." Journal of Immunology 137, no. 12 (December 15, 1986): 3996–4003. http://dx.doi.org/10.4049/jimmunol.137.12.3996.
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Abstract As measured by fluorescence microscopy and radioligand binding, C3b/C4b receptors (CR1) became attached to the detergent-insoluble cytoskeleton of human neutrophils when receptors were cross-linked by affinity-purified polyclonal F(ab')2 anti-CR1, dimeric C3b, or Fab monoclonal anti-CR1 followed by F(ab')2 goat anti-mouse F(ab')2. CR1 on neutrophils bearing monovalent anti-CR1 was not attached to the cytoskeleton. In contrast, cross-linked CR1 on erythrocytes and cross-linked MHC Class I on neutrophils were not cytoskeleton associated. A possible role for filamentous actin (F-actin) in the binding of cross-linked CR1 to neutrophil cytoskeleton was suggested by three observations. When neutrophils were differentially extracted with either Low Salt-detergent buffer or High Salt-detergent buffer, stained with FITC-phalloidin, and examined by fluorescent flow cytometry, the residual cytoskeletons generated with the former buffer were shown to contain polymerized F-actin, whereas cytoskeletons generated with the latter buffer were found to be depleted of F-actin. In parallel experiments, High Salt-detergent buffer was also found to release cross-linked CR1 from neutrophils. Second, depolymerization of F-actin by DNAse I released half of the cytoskeletal-associated cross-linked CR1. Third, immunoadsorbed neutrophil CR1, but not MHC Class I or erythrocyte CR1, specifically bound soluble 125I-actin. In addition, Fc receptor and CR3, other phagocytic membrane proteins of neutrophils, specifically bound 125I-actin. These data demonstrate that CR1 cross-linked on neutrophils becomes associated with detergent-insoluble cytoskeleton and that this interaction is mediated either directly or indirectly by actin.
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Vaduva, Gabriela, Nancy C. Martin, and Anita K. Hopper. "Actin-binding Verprolin Is a Polarity Development Protein Required for the Morphogenesis and Function of the Yeast Actin Cytoskeleton." Journal of Cell Biology 139, no. 7 (December 29, 1997): 1821–33. http://dx.doi.org/10.1083/jcb.139.7.1821.
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Yeast verprolin, encoded by VRP1, is implicated in cell growth, cytoskeletal organization, endocytosis and mitochondrial protein distribution and function. We show that verprolin is also required for bipolar bud-site selection. Previously we reported that additional actin suppresses the temperature-dependent growth defect caused by a mutation in VRP1. Here we show that additional actin suppresses all known defects caused by vrp1-1 and conclude that the defects relate to an abnormal cytoskeleton. Using the two-hybrid system, we show that verprolin binds actin. An actin-binding domain maps to the LKKAET hexapeptide located in the first 70 amino acids. A similar hexapeptide in other acting-binding proteins was previously shown to be necessary for actin-binding activity. The entire 70– amino acid motif is conserved in novel higher eukaryotic proteins that we predict to be actin-binding, and also in the actin-binding proteins, WASP and N-WASP. Verprolin-GFP in live cells has a cell cycle-dependent distribution similar to the actin cortical cytoskeleton. In fixed cells hemagglutinin-tagged Vrp1p often co-localizes with actin in cortical patches. However, disassembly of the actin cytoskeleton using Latrunculin-A does not alter verprolin's location, indicating that verprolin establishes and maintains its location independent of the actin cytoskeleton. Verprolin is a new member of the actin-binding protein family that serves as a polarity development protein, perhaps by anchoring actin. We speculate that the effects of verprolin upon the actin cytoskeleton might influence mitochondrial protein sorting/function via mRNA distribution.
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Bezanilla, Magdalena, Amy S. Gladfelter, David R. Kovar, and Wei-Lih Lee. "Cytoskeletal dynamics: A view from the membrane." Journal of Cell Biology 209, no. 3 (May 11, 2015): 329–37. http://dx.doi.org/10.1083/jcb.201502062.
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Many aspects of cytoskeletal assembly and dynamics can be recapitulated in vitro; yet, how the cytoskeleton integrates signals in vivo across cellular membranes is far less understood. Recent work has demonstrated that the membrane alone, or through membrane-associated proteins, can effect dynamic changes to the cytoskeleton, thereby impacting cell physiology. Having identified mechanistic links between membranes and the actin, microtubule, and septin cytoskeletons, these studies highlight the membrane’s central role in coordinating these cytoskeletal systems to carry out essential processes, such as endocytosis, spindle positioning, and cellular compartmentalization.
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SAUMET, Anne, Nando de JESUS, Chantal LEGRAND, and Véronique DUBERNARD. "Association of thrombospondin-1 with the actin cytoskeleton of human thrombin-activated platelets through an αIIbβ3- or CD36-independent mechanism." Biochemical Journal 363, no. 3 (April 24, 2002): 473–82. http://dx.doi.org/10.1042/bj3630473.
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Thrombospondin-1 (TSP-1) is an adhesive glycoprotein which, when secreted from α-granules of activated platelets, can bind to the cell surface and participate in platelet aggregate formation. In this study, we show that thrombin activation leads to the rapid and specific association of a large amount of secreted α-granular TSP-1 with the actin cytoskeleton. This cytoskeletal association of TSP-1 was correlated with platelet secretion, but not aggregation, and was inhibited by cytochalasin D, an inhibitor of actin polymerization. Association of TSP-1 with the actin cytoskeleton was mediated by membrane receptors, as shown by using MAII, a TSP-1-specific monoclonal antibody that inhibited both TSP-1 surface binding to activated platelets and cytoskeletal association. TSP-1 and its potential membrane receptors, e.g. αIIbβ3 integrin, CD36 and CD47, concomitantly associated with the actin cytoskeleton. However, studies on platelets from a patient with type I Glanzmann's thrombasthenia lacking αIIbβ3 and another with barely detectable CD36 showed normal TSP-1 surface expression and association with the actin cytoskeleton. Likewise, no involvement of CD47 in TSP-1 association with the actin cytoskeleton could be inferred from experiments with control platelets using the function-blocking anti-CD47 antibody B6H12. Finally, assembly of signalling complexes, as observed through translocation of tyrosine-phosphorylated proteins and kinases to the actin cytoskeleton, was found to occur in concert with cytoskeletal association of TSP-1, in control platelets as well as in thrombasthenic and CD36-deficient platelets. Our results imply a role for the actin cytoskeleton in the membrane-surface expression process of TSP-1 molecules and suggest a possible coupling of TSP-1 receptors to signalling events occurring independently of αIIbβ3 or CD36. These results provide new insights into the link between surface-bound TSP-1 and the contractile actin microfilament system which may promote platelet aggregate cohesion.
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Ballestrem, C., B. Wehrle-Haller, and B. A. Imhof. "Actin dynamics in living mammalian cells." Journal of Cell Science 111, no. 12 (June 15, 1998): 1649–58. http://dx.doi.org/10.1242/jcs.111.12.1649.
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The actin cytoskeleton maintains the cellular architecture and mediates cell movements. To explore actin cytoskeletal dynamics, the enhanced green fluorescent protein (EGFP) was fused to human β-actin. The fusion protein was incorporated into actin fibers which became depolymerized upon cytochalasin B treatment. This functional EGFP-actin construct enabled observation of the actin cytoskeleton in living cells by time lapse fluorescence microscopy. Stable expression of the construct was obtained in mammalian cell lines of different tissue origins. In stationary cells, actin rich, ring-like structured ‘actin clouds’ were observed in addition to stress fibers. These ruffle-like structures were found to be involved in the reorganization of the actin cytoskeleton. In migratory cells, EGFP-actin was found in the advancing lamellipodium. Immobile actin spots developed in the lamellipodium and thin actin fibers formed parallel to the leading edge. Thus EGFP-actin expressed in living cells unveiled structures involved in the dynamics of the actin cytoskeleton.
8
Holly, Stephen P., and Kendall J. Blumer. "Pak-Family Kinases Regulate Cell and Actin Polarization Throughout the Cell Cycle of Saccharomyces cerevisiae." Journal of Cell Biology 147, no. 4 (November 15, 1999): 845–56. http://dx.doi.org/10.1083/jcb.147.4.845.
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During the cell cycle of the yeast Saccharomyces cerevisiae, the actin cytoskeleton and cell surface growth are polarized, mediating bud emergence, bud growth, and cytokinesis. We have determined whether p21-activated kinase (PAK)-family kinases regulate cell and actin polarization at one or several points during the yeast cell cycle. Inactivation of the PAK homologues Ste20 and Cla4 at various points in the cell cycle resulted in loss of cell and actin cytoskeletal polarity, but not in depolymerization of F-actin. Loss of PAK function in G1 depolarized the cortical actin cytoskeleton and blocked bud emergence, but allowed isotropic growth and led to defects in septin assembly, indicating that PAKs are effectors of the Rho–guanosine triphosphatase Cdc42. PAK inactivation in S/G2 resulted in depolarized growth of the mother and bud and a loss of actin polarity. Loss of PAK function in mitosis caused a defect in cytokinesis and a failure to polarize the cortical actin cytoskeleton to the mother-bud neck. Cla4–green fluorescent protein localized to sites where the cortical actin cytoskeleton and cell surface growth are polarized, independently of an intact actin cytoskeleton. Thus, PAK family kinases are primary regulators of cell and actin cytoskeletal polarity throughout most or all of the yeast cell cycle. PAK-family kinases in higher organisms may have similar functions.
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Uray, Karen, Evelin Major, and Beata Lontay. "MicroRNA Regulatory Pathways in the Control of the Actin–Myosin Cytoskeleton." Cells 9, no. 7 (July 9, 2020): 1649. http://dx.doi.org/10.3390/cells9071649.
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MicroRNAs (miRNAs) are key modulators of post-transcriptional gene regulation in a plethora of processes, including actin–myosin cytoskeleton dynamics. Recent evidence points to the widespread effects of miRNAs on actin–myosin cytoskeleton dynamics, either directly on the expression of actin and myosin genes or indirectly on the diverse signaling cascades modulating cytoskeletal arrangement. Furthermore, studies from various human models indicate that miRNAs contribute to the development of various human disorders. The potentially huge impact of miRNA-based mechanisms on cytoskeletal elements is just starting to be recognized. In this review, we summarize recent knowledge about the importance of microRNA modulation of the actin–myosin cytoskeleton affecting physiological processes, including cardiovascular function, hematopoiesis, podocyte physiology, and osteogenesis.
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Vindin, Howard, Leanne Bischof, Peter Gunning, and Justine Stehn. "Validation of an Algorithm to Quantify Changes in Actin Cytoskeletal Organization." Journal of Biomolecular Screening 19, no. 3 (September 9, 2013): 354–68. http://dx.doi.org/10.1177/1087057113503494.
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The actin cytoskeleton plays an important role in most, if not all, processes necessary for cell survival. Given the fundamental role that the actin cytoskeleton plays in the progression of cancer, it is an ideal target for chemotherapy. Although it is possible to image the actin cytoskeleton in a high-throughput manner, there is currently no validated method to quantify changes in the cytoskeleton in the same capacity, which makes research into its organization and the development of anticytoskeletal drugs difficult. We have validated the use of a linear feature detection algorithm, allowing us to measure changes in actin filament organization. Its ability to quantify changes associated with cytoskeletal disruption will make it a valuable tool in the development of compounds that target the cytoskeleton in cancer. Our results show that this algorithm can quantify cytoskeletal changes in a cell-based system after addition of both well-established and novel anticytoskeletal agents using either fluorescence microscopy or a high-content imaging approach. This novel method gives us the potential to screen compounds in a high-throughput manner for cancer and other diseases in which the cytoskeleton plays a key role.
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Jones, Steven L., and Tatyana M. Svitkina. "Axon Initial Segment Cytoskeleton: Architecture, Development, and Role in Neuron Polarity." Neural Plasticity 2016 (2016): 1–19. http://dx.doi.org/10.1155/2016/6808293.
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The axon initial segment (AIS) is a specialized structure in neurons that resides in between axonal and somatodendritic domains. The localization of the AIS in neurons is ideal for its two major functions: it serves as the site of action potential firing and helps to maintain neuron polarity. It has become increasingly clear that the AIS cytoskeleton is fundamental to AIS functions. In this review, we discuss current understanding of the AIS cytoskeleton with particular interest in its unique architecture and role in maintenance of neuron polarity. The AIS cytoskeleton is divided into two parts, submembrane and cytoplasmic, based on localization, function, and molecular composition. Recent studies using electron and subdiffraction fluorescence microscopy indicate that submembrane cytoskeletal components (ankyrin G,βIV-spectrin, and actin filaments) form a sophisticated network in the AIS that is conceptually similar to the polygonal/triangular network of erythrocytes, with some important differences. Components of the AIS cytoplasmic cytoskeleton (microtubules, actin filaments, and neurofilaments) reside deeper within the AIS shaft and display structural features distinct from other neuronal domains. We discuss how the AIS submembrane and cytoplasmic cytoskeletons contribute to different aspects of AIS polarity function and highlight recent advances in understanding their AIS cytoskeletal assembly and stability.
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Breuer, David, Jacqueline Nowak, Alexander Ivakov, Marc Somssich, Staffan Persson, and Zoran Nikoloski. "System-wide organization of actin cytoskeleton determines organelle transport in hypocotyl plant cells." Proceedings of the National Academy of Sciences 114, no. 28 (June 27, 2017): E5741—E5749. http://dx.doi.org/10.1073/pnas.1706711114.
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The actin cytoskeleton is an essential intracellular filamentous structure that underpins cellular transport and cytoplasmic streaming in plant cells. However, the system-level properties of actin-based cellular trafficking remain tenuous, largely due to the inability to quantify key features of the actin cytoskeleton. Here, we developed an automated image-based, network-driven framework to accurately segment and quantify actin cytoskeletal structures and Golgi transport. We show that the actin cytoskeleton in both growing and elongated hypocotyl cells has structural properties facilitating efficient transport. Our findings suggest that the erratic movement of Golgi is a stable cellular phenomenon that might optimize distribution efficiency of cell material. Moreover, we demonstrate that Golgi transport in hypocotyl cells can be accurately predicted from the actin network topology alone. Thus, our framework provides quantitative evidence for system-wide coordination of cellular transport in plant cells and can be readily applied to investigate cytoskeletal organization and transport in other organisms.
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Liu, Lingling, Qing Luo, Jinghui Sun, and Guanbin Song. "Cytoskeletal control of nuclear morphology and stiffness are required for OPN-induced bone-marrow-derived mesenchymal stem cell migration." Biochemistry and Cell Biology 97, no. 4 (August 2019): 463–70. http://dx.doi.org/10.1139/bcb-2018-0263.
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During cell migration, the movement of the nucleus must be coordinated with the cytoskeletal dynamics that influence the efficiency of cell migration. Our previous study demonstrated that osteopontin (OPN) significantly promotes the migration of bone-marrow-derived mesenchymal stem cells (BMSCs). However, the mechanism that regulates nuclear mechanics of the cytoskeleton during OPN-promoted BMSC migration remains unclear. In this study, we investigated how the actin cytoskeleton influences nuclear mechanics in BMSCs. We assessed the morphology and mechanics of the nuclei in the OPN-treated BMSCs subjected to disruption or polymerization of the actin cytoskeleton. We found that disruption of actin organization by cytochalasin D (Cyto D) resulted in a decrease in the nuclear projected area and nuclear stiffness. Stabilizing the actin assembly with jasplakinolide (JASP) resulted in an increase in the nuclear projected area and nuclear stiffness. SUN1 (Sad-1/UNC-84 1) is a component of the LINC (linker of nucleoskeleton and cytoskeleton) complex involved in the connections between the nucleus and the cytoskeleton. We found that SUN1 depletion by RNAi decreased the nuclear stiffness and OPN-promoted BMSC migration. Thus, the F-actin cytoskeleton plays an important role in determining the morphology and mechanical properties of the nucleus. We suggest that the cytoskeletal–nuclear interconnectivity through SUN1 proteins plays an important role in OPN-promoted BMSC migration.
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Akram, Zain, Ishtiaq Ahmed, Heike Mack, Ramandeep Kaur, Richard C. Silva, Beatriz A. Castilho, Sylvie Friant, Evelyn Sattlegger, and Alan L. Munn. "Yeast as a Model to Understand Actin-Mediated Cellular Functions in Mammals—Illustrated with Four Actin Cytoskeleton Proteins." Cells 9, no. 3 (March 10, 2020): 672. http://dx.doi.org/10.3390/cells9030672.
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The budding yeast Saccharomyces cerevisiae has an actin cytoskeleton that comprises a set of protein components analogous to those found in the actin cytoskeletons of higher eukaryotes. Furthermore, the actin cytoskeletons of S. cerevisiae and of higher eukaryotes have some similar physiological roles. The genetic tractability of budding yeast and the availability of a stable haploid cell type facilitates the application of molecular genetic approaches to assign functions to the various actin cytoskeleton components. This has provided information that is in general complementary to that provided by studies of the equivalent proteins of higher eukaryotes and hence has enabled a more complete view of the role of these proteins. Several human functional homologues of yeast actin effectors are implicated in diseases. A better understanding of the molecular mechanisms underpinning the functions of these proteins is critical to develop improved therapeutic strategies. In this article we chose as examples four evolutionarily conserved proteins that associate with the actin cytoskeleton: (1) yeast Hof1p/mammalian PSTPIP1, (2) yeast Rvs167p/mammalian BIN1, (3) yeast eEF1A/eEF1A1 and eEF1A2 and (4) yeast Yih1p/mammalian IMPACT. We compare the knowledge on the functions of these actin cytoskeleton-associated proteins that has arisen from studies of their homologues in yeast with information that has been obtained from in vivo studies using live animals or in vitro studies using cultured animal cell lines.
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Aunis, D., and M. F. Bader. "The cytoskeleton as a barrier to exocytosis in secretory cells." Journal of Experimental Biology 139, no. 1 (September 1, 1988): 253–66. http://dx.doi.org/10.1242/jeb.139.1.253.
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Chromaffin cells of the adrenal medulla synthesize, store and secrete catecholamines. These cells contain numerous electron-dense secretory granules which discharge their contents into the extracellular space by exocytosis. The subplasmalemmal area of the chromaffin cell is characterized by the presence of a highly organized cytoskeletal network. F-Actin seems to be exclusively localized in this area and together with specific actin-binding proteins forms a dense viscoelastic gel; fodrin, vinculin and caldesmon, three actin cross-linking proteins, and gelsolin, an actin-severing protein, are found in this subplasmalemmal region. Since fodrin-, caldesmon- and alpha-actinin-binding sites exist on secretory granule membranes, actin filaments can also link secretory granules. Chromaffin granules can be entrapped in this subplasmalemmal lattice and thus the cytoskeleton acts as a barrier preventing exocytosis. When cells are stimulated, molecular rearrangements of the subplasmalemmal cytoskeleton take place: F-actin depolymerizes and fodrin reorganizes into patches. In addition, introduction of monospecific antifodrin immunoglobulins into digitonin-permeabilized cells blocks exocytosis, demonstrating the crucial role of this actin-binding protein. In bacterial toxin-permeabilized chromaffin cells, experiments using actin-perturbing agents such as cytochalasin D and DNAase I suggest that exocytosis is in part controlled by the cytoskeleton. The intracellular signal governing the cytoskeletal reorganization (associated with exocytosis) is calcium. Calcium inhibits some and activates other actin-binding proteins and consequently causes dissolution of the subplasmalemmal cytoskeleton. This dissolution of cytoskeletal filaments should result in granule detachment and permit granules free access to exocytotic sites on the plasma membrane.
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Liu, Yi-Jun, Ting Zhang, Daxiao Cheng, Junhua Yang, Sicong Chen, Xingyue Wang, Xia Li, et al. "Late endosomes promote microglia migration via cytosolic translocation of immature protease cathD." Science Advances 6, no. 50 (December 2020): eaba5783. http://dx.doi.org/10.1126/sciadv.aba5783.
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Organelle transport requires dynamic cytoskeleton remodeling, but whether cytoskeletal dynamics are, in turn, regulated by organelles remains elusive. Here, we demonstrate that late endosomes, a type of prelysosomal organelles, facilitate actin-cytoskeleton remodeling via cytosolic translocation of immature protease cathepsin D (cathD) during microglia migration. After cytosolic translocation, late endosome–derived cathD juxtaposes actin filaments at the leading edge of lamellipodia. Suppressing cathD expression or blocking its cytosolic translocation impairs the maintenance but not the initiation of lamellipodial extension. Moreover, immature cathD balances the activity of the actin-severing protein cofilin to maintain globular-actin (G-actin) monomer pool for local actin recycling. Our study identifies cathD as a key lysosomal molecule that unconventionally contributes to actin cytoskeleton remodeling via cytosolic translocation during adenosine triphosphate–evoked microglia migration.
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Durand-Smet, Pauline, Tamsin A. Spelman, Elliot M. Meyerowitz, and Henrik Jönsson. "Cytoskeletal organization in isolated plant cells under geometry control." Proceedings of the National Academy of Sciences 117, no. 29 (July 8, 2020): 17399–408. http://dx.doi.org/10.1073/pnas.2003184117.
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The cytoskeleton plays a key role in establishing robust cell shape. In animals, it is well established that cell shape can also influence cytoskeletal organization. Cytoskeletal proteins are well conserved between animal and plant kingdoms; nevertheless, because plant cells exhibit major structural differences to animal cells, the question arises whether the plant cytoskeleton also responds to geometrical cues. Recent numerical simulations predicted that a geometry-based rule is sufficient to explain the microtubule (MT) organization observed in cells. Due to their high flexural rigidity and persistence length of the order of a few millimeters, MTs are rigid over cellular dimensions and are thus expected to align along their long axis if constrained in specific geometries. This hypothesis remains to be testedin cellulo. Here, we explore the relative contribution of geometry to the final organization of actin and MT cytoskeletons in single plant cells ofArabidopsis thaliana. We show that the cytoskeleton aligns with the long axis of the cells. We find that actin organization relies on MTs but not the opposite. We develop a model of self-organizing MTs in three dimensions, which predicts the importance of MT severing, which we confirm experimentally. This work is a first step toward assessing quantitatively how cellular geometry contributes to the control of cytoskeletal organization in living plant cells.
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Harris, Andrew R., Pamela Jreij, and Daniel A. Fletcher. "Mechanotransduction by the Actin Cytoskeleton: Converting Mechanical Stimuli into Biochemical Signals." Annual Review of Biophysics 47, no. 1 (May 20, 2018): 617–31. http://dx.doi.org/10.1146/annurev-biophys-070816-033547.
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Force transmission through the actin cytoskeleton plays a central role in cell movements, shape change, and internal organization. Dynamic reorganization of actin filaments by an array of specialized binding proteins creates biochemically and architecturally distinct structures, many of which are finely tuned to exert or resist mechanical loads. The molecular complexity of the actin cytoskeleton continues to be revealed by detailed biochemical assays, and the architectural diversity and dynamics of actin structures are being uncovered by advances in super-resolution fluorescence microscopy and electron microscopy. However, our understanding of how mechanical forces feed back on cytoskeletal architecture and actin-binding protein organization is comparatively limited. In this review, we discuss recent work investigating how mechanical forces applied to cytoskeletal proteins are transduced into biochemical signals. We explore multiple mechanisms for mechanical signal transduction, including the mechanosensitive behavior of actin-binding proteins, the effect of mechanical force on actin filament dynamics, and the influence of mechanical forces on the structure of single actin filaments. The emerging picture is one in which the actin cytoskeleton is defined not only by the set of proteins that constitute a network but also by the constant interplay of mechanical forces and biochemistry.
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Benoit, Béatrice, Anita Baillet, and Christian Poüs. "Cytoskeleton and Associated Proteins: Pleiotropic JNK Substrates and Regulators." International Journal of Molecular Sciences 22, no. 16 (August 4, 2021): 8375. http://dx.doi.org/10.3390/ijms22168375.
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This review extensively reports data from the literature concerning the complex relationships between the stress-induced c-Jun N-terminal kinases (JNKs) and the four main cytoskeleton elements, which are actin filaments, microtubules, intermediate filaments, and septins. To a lesser extent, we also focused on the two membrane-associated cytoskeletons spectrin and ESCRT-III. We gather the mechanisms controlling cytoskeleton-associated JNK activation and the known cytoskeleton-related substrates directly phosphorylated by JNK. We also point out specific locations of the JNK upstream regulators at cytoskeletal components. We finally compile available techniques and tools that could allow a better characterization of the interplay between the different types of cytoskeleton filaments upon JNK-mediated stress and during development. This overview may bring new important information for applied medical research.
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Swaminathan, Vinay, Joseph Mathew Kalappurakkal, Shalin B. Mehta, Pontus Nordenfelt, Travis I. Moore, Nobuyasu Koga, David A. Baker, et al. "Actin retrograde flow actively aligns and orients ligand-engaged integrins in focal adhesions." Proceedings of the National Academy of Sciences 114, no. 40 (October 3, 2017): 10648–53. http://dx.doi.org/10.1073/pnas.1701136114.
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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.
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Nurko, S., K. Sogabe, J. A. Davis, N. F. Roeser, M. Defrain, A. Chien, D. Hinshaw, et al. "Contribution of actin cytoskeletal alterations to ATP depletion and calcium-induced proximal tubule cell injury." American Journal of Physiology-Renal Physiology 270, no. 1 (January 1, 1996): F39—F52. http://dx.doi.org/10.1152/ajprenal.1996.270.1.f39.
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The actin cytoskeleton of rabbit proximal tubules was assessed by deoxyribonuclease (DNase) binding, sedimentability of detergent-insoluble actin, laser-scanning confocal microscopy, and ultrastructure during exposure to hypoxia, antimycin, or antimycin plus ionomycin. One-third of total actin was DNase reactive in control cells prior to deliberate depolymerization, and a similar proportion was unsedimentable from detergent lysates during 2.5 h at 100,000 g. Tubules injured by hypoxia or antimycin alone, without glycine, showed Ca(2+)-dependent pathology of the cytoskeleton, consisting of increases in DNase-reactive actin, redistribution of pelletable actin, and loss of microvilli concurrent with lethal membrane damage. In contrast, tubules similarly depleted of ATP and incubated with glycine showed no significant changes of DNase-reactive actin or actin sedimentability for up to 60 min, but, nevertheless, developed substantial loss of basal membrane-associated actin within 15 min and disruption of actin cores and clubbing of microvilli at durations > 30 min. These structural changes that occurred in the presence of glycine were not prevented by limiting Ca2+ availability or pH 6.9. Very rapid and extensive cytoskeletal disruption followed antimycin-plus-ionomycin treatment. In this setting, glycine and pH 6.9 decreased lethal membrane damage but did not ameliorate pathology in the cytoskeleton or microvilli; limiting Ca2+ availability partially protected the cytoskeleton but did not prevent lethal membrane damage. The data suggest that both ATP depletion-dependent but Ca(2+)-independent, as well as Ca(2+)-mediated, processes can disrupt the actin cytoskeleton during acute proximal tubule cell injury; that both types of change occur, despite protection afforded by glycine and reduced pH against lethal membrane damage; and that Ca(2+)-independent processes primarily account for prelethal actin cytoskeletal alterations during simple ATP depletion of proximal tubule cells.
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Li, S., V. C. Duance, and E. J. Blain. "F-actin cytoskeletal organization in intervertebral disc health and disease." Biochemical Society Transactions 35, no. 4 (July 20, 2007): 683–85. http://dx.doi.org/10.1042/bst0350683.
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The cytoskeleton, which in most cell types, including the intervertebral disc described here, comprises microfilaments, microtubules and intermediate filaments, plays important functions in many fundamental cellular events, including cell division, motility, protein trafficking and secretion. The cytoskeleton is also critical for communication; for example, alterations to the architecture of the F-actin (filamentous actin) cytoskeletal networks can affect communication between the cells and the extracellular matrix, potentially compromising tissue homoeostasis. Although there are limited studies to date, this paper aims to review current knowledge on F-actin cytoskeletal element organization in intervertebral disc cells, how F-actin differs with pathology and its implications for mechanotransduction.
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Wang, Jia, Spencer Freeman, Jeff Lee, and Michael Gold. "Antigen-induced reorientation of the microtubule organizing centre to the B cell immune synapse requires Rap activation and actin dynamics (IRM8P.704)." Journal of Immunology 192, no. 1_Supplement (May 1, 2014): 127.5. http://dx.doi.org/10.4049/jimmunol.192.supp.127.5.
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Abstract B cell activation can be initiated by antigen presenting cells (APC) that display intact antigen (Ag) on their surface. Reorientation of the B cell microtubule-organizing centre (MTOC) towards the APC facilitates Ag internalization at the immune synapse (IS), which is critical for B cells to present Ag to T cells and elicit T cell help. We showed previously that BCR-induced activation of the Rap GTPase, a key regulator of cell polarity and actin dynamics, is required for IS formation. We now show that BCR-induced Rap activation is critical for MTOC polarization towards anti-Ig-coated beads and towards APCs. Activated Rap increases actin dynamics and promotes cytoskeletal reorganization by activating the actin-severing protein cofilin. We found that both disrupting the actin cytoskeleton with latrunculin A and blocking cofilin function prevented MTOC polarization, suggesting that Rap controls MTOC reorientation via its effects on actin dynamics. To assess how actin dynamics regulates the microtubule (MT) network, we tested the role of the actin-MT crosslinking protein IQGAP1 in BCR-induced MTOC polarization. We found that both Rap and cofilin controlled the accumulation of IQGAP1 at the site of Ag contact, and that depleting IQGAP1 prevented MTOC polarization. Thus Rap regulates the polarity of the MT network via its effects on the actin cytoskeleton and this may involve proteins such as IQGAP1 that link the actin and MT cytoskeletons.
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Templeton, Douglas M., and Ying Liu. "Effects of cadmium on the actin cytoskeleton in renal mesangial cells." Canadian Journal of Physiology and Pharmacology 91, no. 1 (January 2013): 1–7. http://dx.doi.org/10.1139/cjpp-2012-0229.
Full textAbstract:
We provide an overview of our studies on cadmium and the actin cytoskeleton in mesangial cells, from earlier work on the effects of Cd2+ on actin polymerization in vivo and in vitro, to a role of disruption or stabilization of the cytoskeleton in apoptosis and apoptosis-like death. More recent studies implicate cadmium-dependent association of gelsolin and the Ca2+/calmodulin-dependent protein kinase II (CaMK-II) with actin filaments in cytoskeletal effects. We also present previously unpublished data concerning cadmium and the disruption of focal adhesions. The work encompasses studies on rat, mouse, and human mesangial cells. The major conclusions are that Cd2+ acts independently of direct effects on cellular Ca2+ levels to nevertheless activate Ca2+-dependent proteins that shift the actin polymerization–depolymerization in favour of depolymerization. Cadmium-dependent translocation of CaMK-IIδ, gelsolin, and a 50 kDa gelsolin cleavage fragment to the filamentous (F-)actin cytoskeleton appear to be involved. An intact filamentous actin cytoskeleton is required to initiate apoptotic and apoptotic-like death, but F-actin depolymerization is an eventual result.
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Kalfa, Theodosia A., Suvarnamala Pushkaran, Narla Mohandas, John H. Hartwig, Velia M. Fowler, James F. Johnson, Clinton H. Joiner, David A. Williams, and Yi Zheng. "Rac GTPases regulate the morphology and deformability of the erythrocyte cytoskeleton." Blood 108, no. 12 (December 1, 2006): 3637–45. http://dx.doi.org/10.1182/blood-2006-03-005942.
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Abstract Actin oligomers are a significant structural component of the erythrocyte cytoskeleton. Rac1 and Rac2 GTPases regulate actin structures and have multiple overlapping as well as distinct roles in hematopoietic cells; therefore, we studied their role in red blood cells (RBCs). Conditional gene targeting with a loxP-flanked Rac1 gene allowed Crerecombinase–induced deletion of Rac1 on a Rac2 null genetic background. The Rac1–/–;Rac2–/– mice developed microcytic anemia with a hemoglobin drop of about 20% and significant anisocytosis and poikilocytosis. Reticulocytes increased more than 2-fold. Rac1–/–;Rac2–/– RBCs stained with rhodamine-phalloidin demonstrated F-actin meshwork gaps and aggregates under confocal microscopy. Transmission electron microscopy of the cytoskeleton demonstrated junctional aggregates and pronounced irregularity of the hexagonal spectrin scaffold. Ektacytometry confirmed that these cytoskeletal changes in Rac1–/–;Rac2–/– erythrocytes were associated with significantly decreased cellular deformability. The composition of the cytoskeletal proteins was altered with an increased actin-to-spectrin ratio and increased phosphorylation (Ser724) of adducin, an F-actin capping protein. Actin and phosphorylated adducin of Rac1–/–;Rac2–/– erythrocytes were more easily extractable by Triton X-100, indicating weaker association to the cytoskeleton. Thus, deficiency of Rac1 and Rac2 GTPases in mice alters actin assembly in RBCs and causes microcytic anemia with reticulocytosis, implicating Rac GTPases as dynamic regulators of the erythrocyte cytoskeleton organization.
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Payrastre, B., P. M. van Bergen en Henegouwen, M. Breton, J. C. den Hartigh, M. Plantavid, A. J. Verkleij, and J. Boonstra. "Phosphoinositide kinase, diacylglycerol kinase, and phospholipase C activities associated to the cytoskeleton: effect of epidermal growth factor." Journal of Cell Biology 115, no. 1 (October 1, 1991): 121–28. http://dx.doi.org/10.1083/jcb.115.1.121.
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In this paper we demonstrate that cytoskeletons isolated from A431 cells have associated with them high activities of several kinases involved in inositol lipid metabolism, such as phosphatidylinositol kinase, phosphatidylinositol phosphate kinase, and diacylglycerol kinase. In addition also phospholipase C activity was detected on isolated cytoskeletons. Controlled extraction of the cytoskeletons followed by in vitro polymerization of actin demonstrated an association of the kinases to the actin filament system consisting of actin and a number of actin-binding proteins. The cytoskeleton-associated lipid kinase activities were significantly increased upon treatment of intact cells with EGF. These data suggest that the association of the phosphoinositide kinases, diacylglycerol kinase, phospholipase C, and also the EGF receptor to the cytoskeleton may play a role in the efficient signal transduction induced by EGF, by providing a matrix for the various components involved in signal transduction.
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Chinthalapudi, Krishna, Erumbi Rangarajan, Dipak Patil, and Tina Izard. "Lipid-directed cytoskeletal protein oligomerization at sites of cell adhesion." Acta Crystallographica Section A Foundations and Advances 70, a1 (August 5, 2014): C1833. http://dx.doi.org/10.1107/s2053273314081674.
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Vertebrate cell growth, division, migration, morphogenesis, and development, rely on the dynamic interactions of cells with components the extracellular matrix (ECM) via cell surface complexes. These focal adhesions (FAs) are comprised of integrin receptors, associated signaling molecules, and talin, which is required for "inside-out" signaling that stabilizes contacts of integrin receptors with the ECM by linking FAs to the actin cytoskeleton by binding to vinculin. The highly dynamic interactions with the actin cytoskeleton are also essential for the formation of membrane protrusions (lamellopodia and filopodia). Second messengers are found at the plasma cell membrane and include signaling lipids such as phosphoinositides, which play essential roles in signal transduction pathways and in directing the oligomerization of cytoskeletal proteins that function as essential links of FAs to the actin cytoskeleton. Notably, the most abundant phosphoinositide, phosphatidyl (4,5) bisphosphate (PIP2), directly binds to key cytoskeletal proteins, where it triggers homotypic and heterotypic interactions that amplify binding to the actin network. Binding of the inositol head group and the hydrophobic acyl chain pose difficulties in generating protein/PIP2 complex crystals and here we present the only second non-membrane protein structure of such a complex. Our crystal structure and biochemical approaches define the roles of PIP2 in controlling the oligomerization of cytoskeletal proteins and their binding to adhesion receptors and to the actin cytoskeleton. Importantly, we also determined the contribution of PIP2-directed oligomerization of cytoskeletal proteins to the formation and stabilization of adhesion complexes. These studies provide important new insights into how dynamic interactions of cytoskeletal proteins with the lipid membrane, adhesion complexes, and the actin network direct the mechanical behaviors of cells.
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REDONDO, Pedro C., Ana I. LAJAS, Ginés M. SALIDO, Antonio GONZALEZ, Juan A. ROSADO, and José A. PARIENTE. "Evidence for secretion-like coupling involving pp60src in the activation and maintenance of store-mediated Ca2+ entry in mouse pancreatic acinar cells." Biochemical Journal 370, no. 1 (February 15, 2003): 255–63. http://dx.doi.org/10.1042/bj20021505.
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Store-mediated Ca2+ entry (SMCE) is one of the main pathways for Ca2+ influx in non-excitable cells. Recent studies favour a secretion-like coupling mechanism to explain SMCE, where Ca2+ entry is mediated by an interaction of the endoplasmic reticulum (ER) with the plasma membrane (PM) and is modulated by the actin cytoskeleton. To explore this possibility further we have now investigated the role of the actin cytoskeleton in the activation and maintenance of SMCE in pancreatic acinar cells, a more specialized secretory cell type which might be an ideal cellular model to investigate further the properties of the secretion-like coupling model. In these cells, the cytoskeletal disrupters cytochalasin D and latrunculin A inhibited both the activation and maintenance of SMCE. In addition, stabilization of a cortical actin barrier by jasplakinolide prevented the activation, but not the maintenance, of SMCE, suggesting that, as for secretion, the actin cytoskeleton plays a double role in SMCE as a negative modulator of the interaction between the ER and PM, but is also required for this mechanism, since the cytoskeleton disrupters impaired Ca2+ entry. Finally, depletion of the intracellular Ca2+ stores induces cytoskeletal association and activation of pp60src, which is independent on Ca2+ entry. pp60src activation requires the integrity of the actin cytoskeleton and participates in the initial phase of the activation of SMCE in pancreatic acinar cells.
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Sechi, A. S., and J. Wehland. "The actin cytoskeleton and plasma membrane connection: PtdIns(4,5)P(2) influences cytoskeletal protein activity at the plasma membrane." Journal of Cell Science 113, no. 21 (November 1, 2000): 3685–95. http://dx.doi.org/10.1242/jcs.113.21.3685.
Full textAbstract:
The co-ordination of rearrangements of the actin cytoskeleton depends on its tight connection to the plasma membrane. Phosphatidylinositol 4,5-bisphosphate is thought to transmit signals originating at the plasma membrane to the underlying actin cytoskeleton. This lipid binds to, and influences the activity of, several actin-associated proteins in vitro that regulate the architecture of the actin cytoskeleton. Signalling intermediates in this process include focal adhesion molecules such as vinculin and members of two families of proteins, ERM and WASP. These proteins interact with phosphatidylinositol 4,5-bisphosphate and appear to be regulated by interplay between small GTPases and phosphatidylinositol 4,5-bisphosphate metabolism, and thus link the plasma membrane with cytoskeletal remodelling.
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Vafiadaki, Elizabeth, Demetrios A. Arvanitis, Aristides G. Eliopoulos, Evangelia G. Kranias, and Despina Sanoudou. "The Cardioprotective PKA-Mediated Hsp20 Phosphorylation Modulates Protein Associations Regulating Cytoskeletal Dynamics." International Journal of Molecular Sciences 21, no. 24 (December 16, 2020): 9572. http://dx.doi.org/10.3390/ijms21249572.
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The cytoskeleton has a primary role in cardiomyocyte function, including the response to mechanical stimuli and injury. The small heat shock protein 20 (Hsp20) conveys protective effects in cardiac muscle that are linked to serine-16 (Ser16) Hsp20 phosphorylation by stress-induced PKA, but the link between Hsp20 and the cytoskeleton remains poorly understood. Herein, we demonstrate a physical and functional interaction of Hsp20 with the cytoskeletal protein 14-3-3. We show that, upon phosphorylation at Ser16, Hsp20 translocates from the cytosol to the cytoskeleton where it binds to 14-3-3. This leads to dissociation of 14-3-3 from the F-actin depolymerization regulator cofilin-2 (CFL2) and enhanced F-actin depolymerization. Importantly, we demonstrate that the P20L Hsp20 mutation associated with dilated cardiomyopathy exhibits reduced physical interaction with 14-3-3 due to diminished Ser16 phosphorylation, with subsequent failure to translocate to the cytoskeleton and inability to disassemble the 14-3-3/CFL2 complex. The topological sequestration of Hsp20 P20L ultimately results in impaired regulation of F-actin dynamics, an effect implicated in loss of cytoskeletal integrity and amelioration of the cardioprotective functions of Hsp20. These findings underscore the significance of Hsp20 phosphorylation in the regulation of actin cytoskeleton dynamics, with important implications in cardiac muscle physiology and pathophysiology.
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Grandy, Carolin, Fabian Port, Jonas Pfeil, and Kay-Eberhard Gottschalk. "Influence of ROCK Pathway Manipulation on the Actin Cytoskeleton Height." Cells 11, no. 3 (January 26, 2022): 430. http://dx.doi.org/10.3390/cells11030430.
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The actin cytoskeleton with its dynamic properties serves as the driving force for the movement and division of cells and gives the cell shape and structure. Disorders in the actin cytoskeleton occur in many diseases. Deeper understanding of its regulation is essential in order to better understand these biochemical processes. In our study, we use metal-induced energy transfer (MIET) as a tool to quantitatively examine the rarely considered third dimension of the actin cytoskeleton with nanometer accuracy. In particular, we investigate the influence of different drugs acting on the ROCK pathway on the three-dimensional actin organization. We find that cells treated with inhibitors have a lower actin height to the substrate while treatment with a stimulator for the ROCK pathway increases the actin height to the substrate, while the height of the membrane remains unchanged. This reveals the precise tuning of adhesion and cytoskeleton tension, which leads to a rich three-dimensional structural behaviour of the actin cytoskeleton. This finetuning is differentially affected by either inhibition or stimulation. The high axial resolution shows the importance of the precise finetuning of the actin cytoskeleton and the disturbed regulation of the ROCK pathway has a significant impact on the actin behavior in the z dimension.
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Higuchi-Sanabria, Ryo, Joseph W. Paul, Jenni Durieux, Camila Benitez, Phillip A. Frankino, Sarah U. Tronnes, Gilberto Garcia, Joseph R. Daniele, Samira Monshietehadi, and Andrew Dillin. "Spatial regulation of the actin cytoskeleton by HSF-1 during aging." Molecular Biology of the Cell 29, no. 21 (October 15, 2018): 2522–27. http://dx.doi.org/10.1091/mbc.e18-06-0362.
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There are many studies suggesting an age-associated decline in the actin cytoskeleton, and this has been adopted as common knowledge in the field of aging biology. However, a direct identification of this phenomenon in aging multicellular organisms has not been performed. Here, we express LifeAct::mRuby in a tissue-specific manner to interrogate cytoskeletal organization as a function of age. We show for the first time in Caenorhabditis elegans that the organization and morphology of the actin cytoskeleton deteriorate at advanced age in the muscles, intestine, and hypodermis. Moreover, hsf-1 is essential for regulating cytoskeletal integrity during aging, so that knockdown of hsf-1 results in premature aging of actin and its overexpression protects actin cytoskeletal integrity in the muscles, the intestine, and the hypodermis. Finally, hsf-1 overexpression in neurons alone is sufficient to protect cytoskeletal integrity in nonneuronal cells.
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Fox, Joan E. B. "The Platelet Cytoskeleton." Thrombosis and Haemostasis 70, no. 06 (1993): 0884–93. http://dx.doi.org/10.1055/s-0038-1649694.
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SummaryThe platelet cytoskeleton contains two actin filament-based components. One is the cytoplasmic actin filaments which fill the cytoplasm and mediate contractile events. The other is the membrane skeleton, which coats the plasma membrane and regulates properties of the membrane such as its contours and stability. In the unstimulated platelet, only 30-40% of the actin is polymerized into filaments; the rest is thought to be prevented from polymerizing by the association of thymosin β4 with monomeric actin and by the association of gelsolin with the barbed ends of pre-existing actin filaments. When platelets are activated, there is a rapid increase in actin polymerization; new filaments fill the extending filopodia and form a network at the periphery of the platelet. As a result of activation, myosin binds to cytoplasmic actin filaments, causing them to move towards the center of the platelet. As platelets aggregate, additional cytoskeletal reorganizations occur: GP Ilb-IIIa associates with adhesive ligand in a platelet aggregate; this results in the association of GP Ilb-IIIa, membrane skeleton proteins, and signaling molecules with cytoplasmic actin. Future studies should help to elucidate the significance of the cytoskeleton in regulating signal transduction events in platelets.
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Lehtonen, Sanna, Fang Zhao, and Eero Lehtonen. "CD2-associated protein directly interacts with the actin cytoskeleton." American Journal of Physiology-Renal Physiology 283, no. 4 (October 1, 2002): F734—F743. http://dx.doi.org/10.1152/ajprenal.00312.2001.
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CD2-associated protein (CD2AP) is an adapter protein associating with several membrane proteins, including nephrin, mutated in congenital nephrotic syndrome of the Finnish type, and polycystin-2, mutated in type 2 autosomal dominant polycystic kidney disease. Both proteins have critical roles in the maintenance of the integrity of the nephrons. Previous studies have suggested a role for CD2AP in the regulation of the organization of the actin cytoskeleton, but it has not been known whether the postulated association between CD2AP and actin is direct or mediated by other proteins. In this study, we address this question by using various cellular and biochemical approaches. We show that CD2AP and F-actin partially colocalize in cultured cells and that disruption of the actin cytoskeleton results in disorganization of endogenous CD2AP. Using cytoskeletal fractionation by differential centrifugation, we demonstrate that a proportion of CD2AP associates with the actin cytoskeleton. Furthermore, using pure actin and purified CD2AP fusion proteins in an F-actin coprecipitation assay, we show that CD2AP directly associates with filamentous actin and that this interaction is mediated by means of the COOH terminus of CD2AP. The present results suggest that CD2AP is involved in the regulation of the actin cytoskeleton and indicate that CD2AP may act as a direct adapter between the actin cytoskeleton and cell membrane proteins, such as nephrin and polycystin-2. Alterations in these interactions could explain some of the pathophysiological changes in congenital nephrotic syndrome and polycystic kidney disease.
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Molitoris, B. A. "Putting the actin cytoskeleton into perspective: pathophysiology of ischemic alterations." American Journal of Physiology-Renal Physiology 272, no. 4 (April 1, 1997): F430—F433. http://dx.doi.org/10.1152/ajprenal.1997.272.4.f430.
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The actin cytoskeleton plays an ever-increasingly understood role in mediating a myriad of processes necessary for cellular structure and function. New and exciting information regarding the dynamic aspects of the actin cytoskeleton and its intracellular regulation are unfolding at a rapid rate. Actin cytoskeletal-surface membrane interactions mediating such diverse cellular events as cell polarity, endocytosis, exocytosis, cell division, cellular migration, cell adhesion, signal transduction, and ion channel activity are part of an ever-growing list of cellular processes dependent on precise actin polarization and regulation of assembly and disassembly. The purpose of this review is to highlight recent advances in the understanding of actin cytoskeleton-mediated cellular processes, to provide a framework that interrelates the complex protein-protein interactions necessary for localization, regulation, and mediation of these essential cellular functions, and to outline the role of actin effector proteins in the pathophysiology of ischemic cell injury.
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Wang, Jingyi, Na Lian, Yue Zhang, Yi Man, Lulu Chen, Haobo Yang, Jinxing Lin, and Yanping Jing. "The Cytoskeleton in Plant Immunity: Dynamics, Regulation, and Function." International Journal of Molecular Sciences 23, no. 24 (December 8, 2022): 15553. http://dx.doi.org/10.3390/ijms232415553.
Full textAbstract:
The plant cytoskeleton, consisting of actin filaments and microtubules, is a highly dynamic filamentous framework involved in plant growth, development, and stress responses. Recently, research has demonstrated that the plant cytoskeleton undergoes rapid remodeling upon sensing pathogen attacks, coordinating the formation of microdomain immune complexes, the dynamic and turnover of pattern-recognizing receptors (PRRs), the movement and aggregation of organelles, and the transportation of defense compounds, thus serving as an important platform for responding to pathogen infections. Meanwhile, pathogens produce effectors targeting the cytoskeleton to achieve pathogenicity. Recent findings have uncovered several cytoskeleton-associated proteins mediating cytoskeletal remodeling and defense signaling. Furthermore, the reorganization of the actin cytoskeleton is revealed to further feedback-regulate reactive oxygen species (ROS) production and trigger salicylic acid (SA) signaling, suggesting an extremely complex role of the cytoskeleton in plant immunity. Here, we describe recent advances in understanding the host cytoskeleton dynamics upon sensing pathogens and summarize the effectors that target the cytoskeleton. We highlight advances in the regulation of cytoskeletal remodeling associated with the defense response and assess the important function of the rearrangement of the cytoskeleton in the immune response. Finally, we propose suggestions for future research in this area.
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Edmonds, B. T., J. Wyckoff, Y. G. Yeung, Y. Wang, E. R. Stanley, J. Jones, J. Segall, and J. Condeelis. "Elongation factor-1 alpha is an overexpressed actin binding protein in metastatic rat mammary adenocarcinoma." Journal of Cell Science 109, no. 11 (November 1, 1996): 2705–14. http://dx.doi.org/10.1242/jcs.109.11.2705.
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Overexpression of elongation factor-1 alpha (EF1 alpha) mRNA has been correlated with increased metastatic potential in mammary adenocarcinoma; however, this relationship was not explored at the level of protein expression. As EF1 alpha has been shown in other cell types to be a component of the actin cytoskeleton, a likely effector in metastasis, the actin binding activity of EF1 alpha from metastatic and nonmetastatic rat breast tumors and cell lines was investigated. We have shown that EF1 alpha protein is overexpressed in metastatic compared to nonmetastatic cells and whole tumors. Similarly to other EF1 alpha s, both types of tumor EF1 alpha bind to F-actin, but EF1 alpha from metastatic cells has a reduced affinity for actin. In addition, there is a high correlation between the intracellular distribution of filamentous actin and EF1 alpha in those cytoskeletal structures thought to be important for supporting the cellular motility required for metastasis. Following stimulation with EGF, there is a parallel increase in the amount of F-actin and EF1 alpha associated with the cytoskeleton. The response to EGF can be blocked with cytochalasin D indicating that the binding of EF1 alpha to the cytoskeleton is mediated by F-actin. We propose that a weakened association of EF1 alpha with actin may be related to the metastatic process via an altered organization of the actin cytoskeleton and the differential translation of mRNAs associated with the cytoskeleton.
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Flaumenhaft, Robert, James R. Dilks, Nataliya Rozenvayn, and Kamil Woronowicz. "The Platelet Actin Cytoskeleton Associates Directly with Syntaxin-4 and Participates in α-Granule Secretion." Blood 112, no. 11 (November 16, 2008): 1839. http://dx.doi.org/10.1182/blood.v112.11.1839.1839.
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Abstract Following platelet activation, platelets undergo a dramatic shape change directed by the actin cytoskeleton and accompanied by secretion of granule contents. Secretion of platelet granules requires soluble NSF Attachment Protein Receptors (SNAREs) that mediate fusion events required for granule release. While the actin cytoskeleton is thought to influence platelet granule secretion, the mechanism of this influence is not known. We sought to determine whether actin controls α-granule release by interacting with platelet SNAREs. We found that disruption of the actin cytoskeleton by latrunculin A prevented pseudopodia formation and inhibited thrombin-induced α-granule secretion by 90±4% without significantly affecting activation-induced platelet aggregation. Latrunculin A inhibited α-granule secretion induced by either calcium ionophore A23187 or the phorbol ester PMA, indicating that latrunculin A blocked a relatively distal component of the secretory signaling pathway. To study distal mechanisms of α-granule release, we developed a cell-free secretory system using an α-granule-enriched membrane preparation isolated from a metrizamide gradient. When incubated with either platelet cytosol alone or ATP alone, P-selectin expression was not observed in this system. However, upon incubation of the membrane preparation with both platelet cytosol and ATP, a 7.1±2.5-fold increase in P-selectin expression was observed, indicating α-granule secretion. Release of β-thromboglobulin, a soluble α-granule component, was also observed following incubation of the membrane preparation with ATP and cytosol. α-Granule secretion in the cell-free secretory system required the distal secretory machinery as evidenced by the fact that anti-syntaxin-4 antibody inhibited P-selectin expression by 61±19%. To determine whether the α-granule preparation supported actin polymerization, FITC-phalloidin was used to monitor F-actin formation by flow cytometry. Incubation of the α-granule-enriched preparation with platelet cytosol plus ATP resulted in a 3.4±0.3-fold increase in the binding of FITC-phalloidin. Inhibition of actin polymerization using cytochalasins prevented both FITC-phalloidin binding and P-selectin expression in the cell-free platelet system. Incubation of the α-granule-enriched preparation with purified platelet actin and ATP resulted in actin polymerization and α-granule release. These results showed that actin polymerization was required for α-granule release in the cell-free secretory system and that purified platelet actin could substitute for platelet cytosol to facilitate granule release. To determine whether platelet SNAREs associate with the actin cytoskeleton, we isolated the Triton X-100 insoluble actin cytoskeleton from platelets. VAMP-8 and syntaxin-2 associated only with the actin cytoskeletons of activated platelets. Syntaxin-4 and SNAP-23 associated with actin cytoskeletons isolated from either resting or activated platelets. Association of all SNAREs was inhibited by cytochalasin B, indicating that SNAREs precipitated secondary to association with the actin cytoskeleton rather than with lipid microdomains or protein aggregates. Since syntaxin-4 and SNAP-23 bound the actin cytoskeleton in both the resting and activated platelets, we evaluated whether they bound purified polymerized platelet actin directly. Recombinant syntaxin-4 bound directly to polymerized platelet actin to approximately the same extent as purified α-actinin, a known actin-binding protein. In contrast, recombinant SNAP-23 failed to bind significantly to polymerized platelet actin. Since SNAP-23 binds syntaxin-4 in resting platelets, its association with the resting actin cytoskeleton is likely mediated via its interaction with syntaxin-4, which binds actin directly. Based on these data, we propose a model whereby activation-induced actin polymerization facilitates SNARE complex formation and membrane fusion through the direct association of actin with syntaxin-4.
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Chen, Yuejun, Feifei Wang, Hui Long, Ying Chen, Ziyan Wu, and Lan Ma. "GRK5 promotes F-actin bundling and targets bundles to membrane structures to control neuronal morphogenesis." Journal of Cell Biology 194, no. 6 (September 19, 2011): 905–20. http://dx.doi.org/10.1083/jcb.201104114.
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Neuronal morphogenesis requires extensive membrane remodeling and cytoskeleton dynamics. In this paper, we show that GRK5, a G protein–coupled receptor kinase, is critically involved in neurite outgrowth, dendrite branching, and spine morphogenesis through promotion of filopodial protrusion. Interestingly, GRK5 is not acting as a kinase but rather provides a key link between the plasma membrane and the actin cytoskeleton. GRK5 promoted filamentous actin (F-actin) bundling at the membranes of dynamic neuronal structures by interacting with both F-actin and phosphatidylinositol-4,5-bisphosphate. Moreover, separate domains of GRK5 mediated the coupling of actin cytoskeleton dynamics and membrane remodeling and were required for its effects on neuronal morphogenesis. Accordingly, GRK5 knockout mice exhibited immature spine morphology and deficient learning and memory. Our findings identify GRK5 as a critical mediator of dendritic development and suggest that coordinated actin cytoskeleton and membrane remodeling mediated by bifunctional actin-bundling and membrane-targeting molecules, such as GRK5, is crucial for proper neuronal morphogenesis and the establishment of functional neuronal circuitry.
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Kamińska, Joanna, Beata Gajewska, Anita K. Hopper, and Teresa ˙Zołądek. "Rsp5p, a New Link between the Actin Cytoskeleton and Endocytosis in the Yeast Saccharomyces cerevisiae." Molecular and Cellular Biology 22, no. 20 (October 15, 2002): 6946–48. http://dx.doi.org/10.1128/mcb.22.20.6946-6958.2002.
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ABSTRACT Rsp5p is an ubiquitin-protein ligase of Saccharomyces cerevisiae that has been implicated in numerous processes including transcription, mitochondrial inheritance, and endocytosis. Rsp5p functions at multiple steps of endocytosis, including ubiquitination of substrates and other undefined steps. We propose that one of the roles of Rsp5p in endocytosis involves maintenance and remodeling of the actin cytoskeleton. We report the following. (i) There are genetic interactions between rsp5 and several mutant genes encoding actin cytoskeletal proteins. rsp5 arp2, rsp5 end3, and rsp5 sla2 double mutants all show synthetic growth defects. Overexpressed wild-type RSP5 or mutant rsp5 genes with lesions of some WW domains suppress growth defects of arp2 and end3 cells. The defects in endocytosis, actin cytoskeleton, and morphology of arp2 are also suppressed. (ii) Rsp5p and Sla2p colocalize in abnormal F-actin-containing clumps in arp2 and pan1 mutants. Immunoprecipitation experiments confirmed that Rsp5p and Act1p colocalize in pan1 mutants. (iii) Rsp5p and Sla2p coimmunoprecipitate and partially colocalize to punctate structures in wild-type cells. These studies provide the first evidence for an interaction of an actin cytoskeleton protein with Rsp5p. (iv) rsp5-w1 mutants are resistant to latrunculin A, a drug that sequesters actin monomers and depolymerizes actin filaments, consistent with the fact that Rsp5p is involved in actin cytoskeleton dynamics.
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Vlahovich, Nicole, Anthony J. Kee, Chris Van der Poel, Emma Kettle, Delia Hernandez-Deviez, Christine Lucas, Gordon S. Lynch, Robert G. Parton, Peter W. Gunning, and Edna C. Hardeman. "Cytoskeletal Tropomyosin Tm5NM1 Is Required for Normal Excitation–Contraction Coupling in Skeletal Muscle." Molecular Biology of the Cell 20, no. 1 (January 2009): 400–409. http://dx.doi.org/10.1091/mbc.e08-06-0616.
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The functional diversity of the actin microfilaments relies in part on the actin binding protein tropomyosin (Tm). The muscle-specific Tms regulate actin-myosin interactions and hence contraction. However, there is less known about the roles of the numerous cytoskeletal isoforms. We have shown previously that a cytoskeletal Tm, Tm5NM1, defines a Z-line adjacent cytoskeleton in skeletal muscle. Recently, we identified a second cytoskeletal Tm in this region, Tm4. Here we show that Tm4 and Tm5NM1 define separate actin filaments; the former associated with the terminal sarcoplasmic reticulum (SR) and other tubulovesicular structures. In skeletal muscles of Tm5NM1 knockout (KO) mice, Tm4 localization was unchanged, demonstrating the specificity of the membrane association. Tm5NM1 KO muscles exhibit potentiation of T-system depolarization and decreased force rundown with repeated T-tubule depolarizations consistent with altered T-tubule function. These results indicate that a Tm5NM1-defined actin cytoskeleton is required for the normal excitation–contraction coupling in skeletal muscle.
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Resch, Guenter P., Kenneth N. Goldie, Angelika Krebs, Andreas Hoenger, and J. Victor Small. "Visualisation of the actin cytoskeleton by cryo-electron microscopy." Journal of Cell Science 115, no. 9 (May 1, 2002): 1877–82. http://dx.doi.org/10.1242/jcs.115.9.1877.
Full textAbstract:
An understanding of the mechanisms driving cell motility requires clarification of the structural organisation of actin filament arrays in the regions of cell protrusion termed lamellipodia. Currently, there is a lack of consensus on lamellipodia organisation stemming from the application of alternative procedures for ultrastructural visualisation of cytoskeleton networks. In this study, we show that cryo-electron microscopy of extracted cytoskeletons embedded in a thin layer of vitreous ice can reveal the organisation of cytoskeletal elements at high resolution. Since this method involves no dehydration, drying and contrasting steps that can potentially introduce subtle distortions of filament order and interactions, its application opens the way to resolving the controversial details of lamellipodia architecture.
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Gonzalez-Quevedo, Rosa, Marina Shoffer, Lily Horng, and Anthony E. Oro. "Receptor tyrosine phosphatase–dependent cytoskeletal remodeling by the hedgehog-responsive gene MIM/BEG4." Journal of Cell Biology 168, no. 3 (January 31, 2005): 453–63. http://dx.doi.org/10.1083/jcb.200409078.
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During development, dynamic remodeling of the actin cytoskeleton allows the precise placement and morphology of tissues. Morphogens such as Sonic hedgehog (Shh) and local cues such as receptor protein tyrosine phosphatases (RPTPs) mediate this process, but how they regulate the cytoskeleton is poorly understood. We previously identified Basal cell carcinoma–enriched gene 4 (BEG4)/Missing in Metastasis (MIM), a Shh-inducible, Wiskott-Aldrich homology 2 domain–containing protein that potentiates Gli transcription (Callahan, C.A., T. Ofstad, L. Horng, J.K. Wang, H.H. Zhen, P.A. Coulombe, and A.E. Oro. 2004. Genes Dev. 18:2724–2729). Here, we show that endogenous MIM is induced in a patched1-dependent manner and regulates the actin cytoskeleton. MIM functions by bundling F-actin, a process that requires self-association but is independent of G-actin binding. Cytoskeletal remodeling requires an activation domain distinct from sequences required for bundling in vitro. This domain associates with RPTPδ and, in turn, enhances RPTPδ membrane localization. MIM-dependent cytoskeletal changes can be inhibited using a soluble RPTPδ-D2 domain. Our data suggest that the hedgehog-responsive gene MIM cooperates with RPTP to induce cytoskeletal changes.
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Sameshima, Masazumi, Yoshiro Kishi, Masako Osumi, Dana Mahadeo, and David A. Cotter. "Novel Actin Cytoskeleton. Actin Tubules." Cell Structure and Function 25, no. 5 (2000): 291–95. http://dx.doi.org/10.1247/csf.25.291.
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Liu, Yi, Keyvan Mollaeian, Muhammad Huzaifah Shamim, and Juan Ren. "Effect of F-actin and Microtubules on Cellular Mechanical Behavior Studied Using Atomic Force Microscope and an Image Recognition-Based Cytoskeleton Quantification Approach." International Journal of Molecular Sciences 21, no. 2 (January 8, 2020): 392. http://dx.doi.org/10.3390/ijms21020392.
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Cytoskeleton morphology plays a key role in regulating cell mechanics. Particularly, cellular mechanical properties are directly regulated by the highly cross-linked and dynamic cytoskeletal structure of F-actin and microtubules presented in the cytoplasm. Although great efforts have been devoted to investigating the qualitative relation between the cellular cytoskeleton state and cell mechanical properties, comprehensive quantification results of how the states of F-actin and microtubules affect mechanical behavior are still lacking. In this study, the effect of both F-actin and microtubules morphology on cellular mechanical properties was quantified using atomic force microscope indentation experiments together with the proposed image recognition-based cytoskeleton quantification approach. Young’s modulus and diffusion coefficient of NIH/3T3 cells with different cytoskeleton states were quantified at different length scales. It was found that the living NIH/3T3 cells sense and adapt to the F-actin and microtubules states: both the cellular elasticity and poroelasticity are closely correlated to the depolymerization degree of F-actin and microtubules at all measured indentation depths. Moreover, the significance of the quantitative effects of F-actin and microtubules in affecting cellular mechanical behavior is depth-dependent.
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Chia, C. P., I. Khrebtukova, J. McCluskey, and W. F. Wade. "MHC class II molecules that lack cytoplasmic domains are associated with the cytoskeleton." Journal of Immunology 153, no. 8 (October 15, 1994): 3398–407. http://dx.doi.org/10.4049/jimmunol.153.8.3398.
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Abstract MHC class II molecules, composed of alpha- and beta-chain heterodimers, are required for Ag presentation. The carboxyl-terminal domains of class II molecules are believed to mediate the location of class II in the plasma membrane and are important for signal transduction and Ag presentation. These domains contain typical transmembrane sequences, and cytoplasmic sequences of 12 or 18 amino acids for the alpha- and beta-chains, respectively. We examined these domains to determine whether they linked class II molecules to the actin-based cytoskeleton. Our analyses of class II-cytoskeleton interactions, such as a colocalization with actin filaments during capping, association with the detergent-insoluble cytoskeleton, and direct binding of filamentous actin, revealed that both the cytoplasmic and transmembrane domains contributed to class II interactions with the cytoskeleton. Detergent-extracted and immunoprecipitated full-length class II molecules had quantitatively stronger interactions with the cytoskeleton than did molecules with deleted cytoplasmic domains. A secondary Ab, which was used to cross-link primary Ab bound to class II, up-regulated the class II-cytoskeletal associations. This association was efficiently inhibited by dihydrocytochalasin B, but only partially disrupted by chlorpromazine. The mechanism of interaction with actin filaments after ligation of class II occurred without a measurable increase in filamentous actin levels. This suggested that enhanced class II-cytoskeleton associations involved a rearrangement of existing actin filaments, possibly through the multiple kinases that are activated after class II transmembrane signaling.
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Sousa, Vítor L., Serena Bellani, Maila Giannandrea, Malikmohamed Yousuf, Flavia Valtorta, Jacopo Meldolesi, and Evelina Chieregatti. "α-Synuclein and Its A30P Mutant Affect Actin Cytoskeletal Structure and Dynamics." Molecular Biology of the Cell 20, no. 16 (August 15, 2009): 3725–39. http://dx.doi.org/10.1091/mbc.e08-03-0302.
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The function of α-synuclein, a soluble protein abundant in the brain and concentrated at presynaptic terminals, is still undefined. Yet, α-synuclein overexpression and the expression of its A30P mutant are associated with familial Parkinson's disease. Working in cell-free conditions, in two cell lines as well as in primary neurons we demonstrate that α-synuclein and its A30P mutant have different effects on actin polymerization. Wild-type α-synuclein binds actin, slows down its polymerization and accelerates its depolymerization, probably by monomer sequestration; A30P mutant α-synuclein increases the rate of actin polymerization and disrupts the cytoskeleton during reassembly of actin filaments. Consequently, in cells expressing mutant α-synuclein, cytoskeleton-dependent processes, such as cell migration, are inhibited, while exo- and endocytic traffic is altered. In hippocampal neurons from mice carrying a deletion of the α-synuclein gene, electroporation of wild-type α-synuclein increases actin instability during remodeling, with growth of lamellipodia-like structures and apparent cell enlargement, whereas A30P α-synuclein induces discrete actin-rich foci during cytoskeleton reassembly. In conclusion, α-synuclein appears to play a major role in actin cytoskeletal dynamics and various aspects of microfilament function. Actin cytoskeletal disruption induced by the A30P mutant might alter various cellular processes and thereby play a role in the pathogenesis of neurodegeneration.
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López-Contreras, L., V. I. Hernández-Ramírez, A. E. Lagunes-Guillén, Sarita Montaño, B. Chávez-Munguía, B. Sánchez-Ramírez, and P. Talamás-Rohana. "Exploring the Possible Role of Lysine Acetylation onEntamoeba histolyticaVirulence: A Focus on the Dynamics of the Actin Cytoskeleton." BioMed Research International 2013 (2013): 1–9. http://dx.doi.org/10.1155/2013/757392.
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Cytoskeleton remodeling can be regulated, among other mechanisms, by lysine acetylation. The role of acetylation on cytoskeletal and other proteins ofEntamoeba histolyticahas been poorly studied. Dynamic rearrangements of the actin cytoskeleton are crucial for amebic motility and capping formation, processes that may be effective means of evading the host immune response. Here we report the possible effect of acetylation on the actin cytoskeleton dynamics andin vivovirulence ofE. histolytica. Using western blot, immunoprecipitation, microscopy assays, andin silicoanalysis, we show results that strongly suggest that the increase in Aspirin-induced cytoplasm proteins acetylation reduced cell movement and capping formation, likely as a consequence of alterations in the structuration of the actin cytoskeleton. Additionally, intrahepatic inoculation of Aspirin-treated trophozoites in hamsters resulted in severe impairment of the amebic virulence. Taken together, these results suggest an important role for lysine acetylation in amebic invasiveness and virulence.
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Vu, Vivian, Phuong Bui, Megumi Eguchi, Aimin Xu, and Gary Sweeney. "Globular adiponectin induces LKB1/AMPK-dependent glucose uptake via actin cytoskeleton remodeling." Journal of Molecular Endocrinology 51, no. 1 (May 24, 2013): 155–65. http://dx.doi.org/10.1530/jme-13-0059.
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Previous studies have shown that many metabolic actions of adiponectin are mediated via the activation of AMP kinase and that adiponectin stimulates GLUT4 translocation and glucose uptake in the muscle. In this study, we demonstrate that adiponectin stimulates actin cytoskeleton remodeling, with increased phosphorylation of cofilin, and that blocking of cytoskeletal remodeling with cytochalasin D prevents adiponectin-stimulated AMPK phosphorylation in L6 myoblasts. LKB1 is an upstream kinase of AMPK, and we observed the colocalization of LKB1 with filamentous actin in response to adiponectin. Adiponectin-stimulated translocation of LKB1 from a nuclear to a cytoplasmic location to activate AMPK was also dependent on actin cytoskeleton remodeling. Cytoskeletal remodeling visualized by rhodamine–phalloidin immunofluorescence indicated that adiponectin-stimulated reorganization resulted in the formation membrane ruffles, which were also clearly visible by scanning electron microscopy in L6-GLUT4myc myoblasts. The stimulation of glucose uptake, but not of GLUT4-myc translocation to the cell surface, by adiponectin was also dependent on actin cytoskeleton remodeling. These results suggest that actin remodeling induced by adiponectin is essential for mediating LKB1/AMPK signaling and glucose uptake in skeletal muscle cells.
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Wender, Nomy, Eduardo Villalobo, and David Mirelman. "EhLimA, a Novel LIM Protein, Localizes to the Plasma Membrane in Entamoeba histolytica." Eukaryotic Cell 6, no. 9 (July 13, 2007): 1646–55. http://dx.doi.org/10.1128/ec.00177-07.
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ABSTRACT The parasitic protozoan Entamoeba histolytica relies on a very dynamic cytoskeleton in order to invade and survive in host tissues. Identification of cytoskeletal elements is key to understanding these processes. Here we present the characterization of EhLimA, the first LIM protein of E. histolytica. EhLimA consists of a single LIM domain at its N terminus and exhibits the highest degree of homology with DdLimE from Dictyostelium discoideum. Immunofluorescence localization of EhLimA using anti-EhLimA antibodies revealed that EhLimA is highly concentrated at the plasma membrane of cells. Silencing or overexpression of the EhLimA gene did not have a significant effect on the growth or morphology of the parasite. EhLimA associates with the cytoskeleton as demonstrated by the enrichment of the protein in cytoskeleton fractions as well as in pull-down assays that revealed that cytoskeleton association involves interaction with actin. EhLimA binding to actin was shown to be dependent on the N-terminal LIM domain of EhLimA, as removal of even half of the LIM domain resulted in almost complete inhibition of the binding to actin. We also found that a portion of EhLimA floats to the lower-density regions of a sucrose gradient together with portions of the Gal-lectin light subunit and actin. Treatment of cells with the cholesterol-sequestering agent digitonin resulted in increased solubility of EhLimA. These results indicate that in addition to cytoskeletal association, EhLimA may also associate with lipid rafts in the parasite plasma membrane and suggest that EhLimA may be part of the molecular system connecting the actin cytoskeleton to membrane rafts.