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Donna M Peters - One of the best experts on this subject based on the ideXlab platform.
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dexamethasone associated cross linked Actin Network formation in human trabecular meshwork cells involves β3 integrin signaling
Investigative Ophthalmology & Visual Science, 2011Co-Authors: Mark S Filla, Marie K Schwinn, Amanda Nosie, Ross Clark, Donna M PetersAbstract:Actin-mediated processes play an important role in regulating aqueous humor outflow through the trabecular meshwork (TM).1 In human and bovine TM cells, steroid treatment leads to increased formation of a novel Actin structure called a cross-linked Actin Network (CLAN).2,3 This structure has been suggested to participate in the pathogenesis of steroid-induced glaucoma (SIG)2,4,5 and possibly primary open angle glaucoma.6 The exact role of CLANs in these diseases, however, has not been defined. CLANs were originally observed in spreading cells and were described as Actin geodesic domes.7 Although they have been found within the TM of healthy eyes, they appear to be more abundant in glaucomatous meshworks.8,9 More recently, CLANs have been observed in human and bovine lamina cribrosa cells that were either steroid treated or glaucomatous.10 CLANs are composed of interconnected arrays of three to five Actin filaments extending outward from a central vertex. They may be precursors to Actin stress fibers7 that regulate contractility in cells. Tropomyosin, α-Actinin, and filamin were found along the Actin filaments in CLANs, whereas the transmembrane heparan sulfate proteoglycan syndecan-4, phosphatidylinositol 4,5-bisphosphate (PIP2), and α-Actinin appear to form a molecular complex, or vertisome, at the vertices of the Actin filaments.7,11–13 CLANs appear to be attached to the apical cell surface,11,12,14,15 presumably by syndecan-4. CLANs are often found within lamellipodia as cells spread, but they can also be found specifically over the nucleus or throughout the cytoplasm.2,7,15 Treatment with glucocorticoids such as dexamethasone (DEX) increases CLAN formation in confluent monolayers of TM cells2,5,16 and in the TM of cultured eyes.17 CLAN formation can also be induced by activating a cooperative β1/β3 integrin signaling pathway that uses, in part, the G-protein–coupled receptor CD47 as a coreceptor coupled to the Rac1 GTPase and the guanine nucleotide exchange factor Trio.13,18 Integrins belong to a ubiquitously expressed family of transmembrane heterodimeric glycoproteins composed of an α and a β subunit. The extracellular domain binds a number of extracellular matrix (ECM) proteins through the conserved sequence, arg-gly-asp, whereas their cytoplasmic tails interact with a variety of tyrosine kinases, adaptor proteins, and Actin-binding proteins.19,20 As a result, integrins form an important physical link between the extracellular environment and the Actin cytoskeleton and may provide a mechanism for sensing changes in external forces in the microenvironment of the TM. In this study, we compared the roles of DEX and αvβ3 integrins in CLAN formation. These studies show that CLANs induced by DEX treatment are structurally similar to those formed by β3 integrins and that CLAN formation involves the same signaling pathway activated by αvβ3 integrins. We also show that DEX treatment increases the level of expression and activation of β3 integrin and that overexpression of αvβ3 integrin can increase CLAN formation. These studies suggest that glucocorticoids may trigger CLAN formation by enhancing β3 integrin signaling in HTM cells through the upregulation of β3 integrins or the activation of an inside-out signaling mechanism that triggers αvβ3 signaling. Elucidating the signaling pathways that direct the formation and disassembly of CLANs in the TM may further our understanding of SIG and other glaucomas.
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regulation of cross linked Actin Network clan formation in human trabecular meshwork htm cells by convergence of distinct β1 and β3 integrin pathways
Investigative Ophthalmology & Visual Science, 2009Co-Authors: Mark S Filla, Marie K Schwinn, Nader Sheibani, Paul L Kaufman, Donna M PetersAbstract:PURPOSE. To determine the β1/β3 integrin-mediated pathways that regulate cross-linked Actin Network (CLAN) formation in human trabecular meshwork (HTM) cells. CLANs form in glaucomatous and steroid-treated TM cells, which may contribute to reducing outflow facility through the TM. METHODS. Expression of CD47 (an αvβ3 integrin coreceptor/ thrombospondin-1 receptor) and integrins avβ3 and β1 was assessed by FACS. CLANs were induced by plating cells on fibronectin (a β1 integrin ligand) in the absence or presence of the β3 integrin-activating mAb AP-5 and were identified by phalloidin labeling. The role of Src kinases, PI-3 kinase (PI-3K), Rac1, and CD47 was determined by incubating cells with the inhibitors PP2 and EPA (Src kinases), LY294002 (PI-3K), or NSC23766 (Rac1). Tiam1 and Trio siRNAs and dominant-negative Tiaml were used to determine which Rac1-specific guanine nucleotide exchange factor was involved. The role of CD47 was determined using the thrombospondin-1-derived agonist peptide 4N1K and the CD47 function blocking antibody B6H12.2. RESULTS. HTM cells expressed CD47 and integrins αvβ3 and β1. β3 Integrin or CD47 activation significantly increased CLAN formation over β1 integrin-induced levels, whereas anti-CD47 mAb B6H12.2 inhibited this increase. PP2, NSC23766, and Trio siRNA decreased β3-induced CLAN formation by 72%, 45%, and 67%, respectively, whereas LY294002 and dominant negative Tiam1 had no effect. LY294002 decreased β1 integrin-mediated CLAN formation by 42%, and PP2 completely blocked it. CONCLUSIONS. Distinct β1 and αvβ3 integrin signaling pathways converge to enhance CLAN formation. β1-Mediated CLAN formation was PI-3K dependent, whereas β3-mediated CLAN formation was CD47 and Rac1/Trio dependent and might have been regulated by thrombospondin-1. Both integrin pathways were Src dependent.
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regulation of cross linked Actin Network clan formation in human trabecular meshwork htm cells by convergence of distinct β1 and β3 integrin pathways
Investigative Ophthalmology & Visual Science, 2009Co-Authors: Mark S Filla, Marie K Schwinn, Nader Sheibani, Paul L Kaufman, Donna M PetersAbstract:Purpose To determine the beta1/beta3 integrin-mediated pathways that regulate cross-linked Actin Network (CLAN) formation in human trabecular meshwork (HTM) cells. CLANs form in glaucomatous and steroid-treated TM cells, which may contribute to reducing outflow facility through the TM. Methods Expression of CD47 (an alphavbeta3 integrin coreceptor/thrombospondin-1 receptor) and integrins alphavbeta3 and beta1 was assessed by FACS. CLANs were induced by plating cells on fibronectin (a beta1 integrin ligand) in the absence or presence of the beta3 integrin-activating mAb AP-5 and were identified by phalloidin labeling. The role of Src kinases, PI-3 kinase (PI-3K), Rac1, and CD47 was determined by incubating cells with the inhibitors PP2 and EPA (Src kinases), LY294002 (PI-3K), or NSC23766 (Rac1). Tiam1 and Trio siRNAs and dominant-negative Tiam1 were used to determine which Rac1-specific guanine nucleotide exchange factor was involved. The role of CD47 was determined using the thrombospondin-1-derived agonist peptide 4N1K and the CD47 function blocking antibody B6H12.2. Results HTM cells expressed CD47 and integrins alphavbeta3 and beta1. beta3 Integrin or CD47 activation significantly increased CLAN formation over beta1 integrin-induced levels, whereas anti-CD47 mAb B6H12.2 inhibited this increase. PP2, NSC23766, and Trio siRNA decreased beta3-induced CLAN formation by 72%, 45%, and 67%, respectively, whereas LY294002 and dominant negative Tiam1 had no effect. LY294002 decreased beta1 integrin-mediated CLAN formation by 42%, and PP2 completely blocked it. Conclusions Distinct beta1 and alphavbeta3 integrin signaling pathways converge to enhance CLAN formation. beta1-Mediated CLAN formation was PI-3K dependent, whereas beta3-mediated CLAN formation was CD47 and Rac1/Trio dependent and might have been regulated by thrombospondin-1. Both integrin pathways were Src dependent.
Mark S Filla - One of the best experts on this subject based on the ideXlab platform.
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dexamethasone associated cross linked Actin Network formation in human trabecular meshwork cells involves β3 integrin signaling
Investigative Ophthalmology & Visual Science, 2011Co-Authors: Mark S Filla, Marie K Schwinn, Amanda Nosie, Ross Clark, Donna M PetersAbstract:Actin-mediated processes play an important role in regulating aqueous humor outflow through the trabecular meshwork (TM).1 In human and bovine TM cells, steroid treatment leads to increased formation of a novel Actin structure called a cross-linked Actin Network (CLAN).2,3 This structure has been suggested to participate in the pathogenesis of steroid-induced glaucoma (SIG)2,4,5 and possibly primary open angle glaucoma.6 The exact role of CLANs in these diseases, however, has not been defined. CLANs were originally observed in spreading cells and were described as Actin geodesic domes.7 Although they have been found within the TM of healthy eyes, they appear to be more abundant in glaucomatous meshworks.8,9 More recently, CLANs have been observed in human and bovine lamina cribrosa cells that were either steroid treated or glaucomatous.10 CLANs are composed of interconnected arrays of three to five Actin filaments extending outward from a central vertex. They may be precursors to Actin stress fibers7 that regulate contractility in cells. Tropomyosin, α-Actinin, and filamin were found along the Actin filaments in CLANs, whereas the transmembrane heparan sulfate proteoglycan syndecan-4, phosphatidylinositol 4,5-bisphosphate (PIP2), and α-Actinin appear to form a molecular complex, or vertisome, at the vertices of the Actin filaments.7,11–13 CLANs appear to be attached to the apical cell surface,11,12,14,15 presumably by syndecan-4. CLANs are often found within lamellipodia as cells spread, but they can also be found specifically over the nucleus or throughout the cytoplasm.2,7,15 Treatment with glucocorticoids such as dexamethasone (DEX) increases CLAN formation in confluent monolayers of TM cells2,5,16 and in the TM of cultured eyes.17 CLAN formation can also be induced by activating a cooperative β1/β3 integrin signaling pathway that uses, in part, the G-protein–coupled receptor CD47 as a coreceptor coupled to the Rac1 GTPase and the guanine nucleotide exchange factor Trio.13,18 Integrins belong to a ubiquitously expressed family of transmembrane heterodimeric glycoproteins composed of an α and a β subunit. The extracellular domain binds a number of extracellular matrix (ECM) proteins through the conserved sequence, arg-gly-asp, whereas their cytoplasmic tails interact with a variety of tyrosine kinases, adaptor proteins, and Actin-binding proteins.19,20 As a result, integrins form an important physical link between the extracellular environment and the Actin cytoskeleton and may provide a mechanism for sensing changes in external forces in the microenvironment of the TM. In this study, we compared the roles of DEX and αvβ3 integrins in CLAN formation. These studies show that CLANs induced by DEX treatment are structurally similar to those formed by β3 integrins and that CLAN formation involves the same signaling pathway activated by αvβ3 integrins. We also show that DEX treatment increases the level of expression and activation of β3 integrin and that overexpression of αvβ3 integrin can increase CLAN formation. These studies suggest that glucocorticoids may trigger CLAN formation by enhancing β3 integrin signaling in HTM cells through the upregulation of β3 integrins or the activation of an inside-out signaling mechanism that triggers αvβ3 signaling. Elucidating the signaling pathways that direct the formation and disassembly of CLANs in the TM may further our understanding of SIG and other glaucomas.
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regulation of cross linked Actin Network clan formation in human trabecular meshwork htm cells by convergence of distinct β1 and β3 integrin pathways
Investigative Ophthalmology & Visual Science, 2009Co-Authors: Mark S Filla, Marie K Schwinn, Nader Sheibani, Paul L Kaufman, Donna M PetersAbstract:PURPOSE. To determine the β1/β3 integrin-mediated pathways that regulate cross-linked Actin Network (CLAN) formation in human trabecular meshwork (HTM) cells. CLANs form in glaucomatous and steroid-treated TM cells, which may contribute to reducing outflow facility through the TM. METHODS. Expression of CD47 (an αvβ3 integrin coreceptor/ thrombospondin-1 receptor) and integrins avβ3 and β1 was assessed by FACS. CLANs were induced by plating cells on fibronectin (a β1 integrin ligand) in the absence or presence of the β3 integrin-activating mAb AP-5 and were identified by phalloidin labeling. The role of Src kinases, PI-3 kinase (PI-3K), Rac1, and CD47 was determined by incubating cells with the inhibitors PP2 and EPA (Src kinases), LY294002 (PI-3K), or NSC23766 (Rac1). Tiam1 and Trio siRNAs and dominant-negative Tiaml were used to determine which Rac1-specific guanine nucleotide exchange factor was involved. The role of CD47 was determined using the thrombospondin-1-derived agonist peptide 4N1K and the CD47 function blocking antibody B6H12.2. RESULTS. HTM cells expressed CD47 and integrins αvβ3 and β1. β3 Integrin or CD47 activation significantly increased CLAN formation over β1 integrin-induced levels, whereas anti-CD47 mAb B6H12.2 inhibited this increase. PP2, NSC23766, and Trio siRNA decreased β3-induced CLAN formation by 72%, 45%, and 67%, respectively, whereas LY294002 and dominant negative Tiam1 had no effect. LY294002 decreased β1 integrin-mediated CLAN formation by 42%, and PP2 completely blocked it. CONCLUSIONS. Distinct β1 and αvβ3 integrin signaling pathways converge to enhance CLAN formation. β1-Mediated CLAN formation was PI-3K dependent, whereas β3-mediated CLAN formation was CD47 and Rac1/Trio dependent and might have been regulated by thrombospondin-1. Both integrin pathways were Src dependent.
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regulation of cross linked Actin Network clan formation in human trabecular meshwork htm cells by convergence of distinct β1 and β3 integrin pathways
Investigative Ophthalmology & Visual Science, 2009Co-Authors: Mark S Filla, Marie K Schwinn, Nader Sheibani, Paul L Kaufman, Donna M PetersAbstract:Purpose To determine the beta1/beta3 integrin-mediated pathways that regulate cross-linked Actin Network (CLAN) formation in human trabecular meshwork (HTM) cells. CLANs form in glaucomatous and steroid-treated TM cells, which may contribute to reducing outflow facility through the TM. Methods Expression of CD47 (an alphavbeta3 integrin coreceptor/thrombospondin-1 receptor) and integrins alphavbeta3 and beta1 was assessed by FACS. CLANs were induced by plating cells on fibronectin (a beta1 integrin ligand) in the absence or presence of the beta3 integrin-activating mAb AP-5 and were identified by phalloidin labeling. The role of Src kinases, PI-3 kinase (PI-3K), Rac1, and CD47 was determined by incubating cells with the inhibitors PP2 and EPA (Src kinases), LY294002 (PI-3K), or NSC23766 (Rac1). Tiam1 and Trio siRNAs and dominant-negative Tiam1 were used to determine which Rac1-specific guanine nucleotide exchange factor was involved. The role of CD47 was determined using the thrombospondin-1-derived agonist peptide 4N1K and the CD47 function blocking antibody B6H12.2. Results HTM cells expressed CD47 and integrins alphavbeta3 and beta1. beta3 Integrin or CD47 activation significantly increased CLAN formation over beta1 integrin-induced levels, whereas anti-CD47 mAb B6H12.2 inhibited this increase. PP2, NSC23766, and Trio siRNA decreased beta3-induced CLAN formation by 72%, 45%, and 67%, respectively, whereas LY294002 and dominant negative Tiam1 had no effect. LY294002 decreased beta1 integrin-mediated CLAN formation by 42%, and PP2 completely blocked it. Conclusions Distinct beta1 and alphavbeta3 integrin signaling pathways converge to enhance CLAN formation. beta1-Mediated CLAN formation was PI-3K dependent, whereas beta3-mediated CLAN formation was CD47 and Rac1/Trio dependent and might have been regulated by thrombospondin-1. Both integrin pathways were Src dependent.
Patricia T Yam - One of the best experts on this subject based on the ideXlab platform.
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myosin ii contributes to cell scale Actin Network treadmilling through Network disassembly
Nature, 2010Co-Authors: Cyrus A Wilso, Mark A Tsuchida, Greg M Alle, Eri L Arnha, Kathry T Applegate, Patricia T Yam, Kinnere Kere, Gaudenz DanuseAbstract:In animals, most cells when on the move migrate using a crawling motion, in which the front of the cell is propelled forward by the force provided by polymerization of Actin filaments. Cell biologists have generally assumed that the rear of the crawling cell is then pushed forward by a contractile force generated by non-muscle myosin II. Observations of fish keratocytes in motion now show that no actual contraction is required for rear retraction. Rather, the myosin II has a direct role in facilitating Actin Network treadmilling via Actin disassembly. Eukaryotic cells crawl through a process in which the front of the cell is propelled forwards by the force provided by polymerization of Actin filaments. These must be disassembled at the rear of the cell to allow sustained motility. It is now shown that non-muscle myosin II protein is needed for the disassembly of Actin Networks at the rear of crawling cells. Crawling locomotion of eukaryotic cells is achieved by a process dependent on the Actin cytoskeleton1: protrusion of the leading edge requires assembly of a Network of Actin filaments2, which must be disassembled at the cell rear for sustained motility. Although ADF/cofilin proteins have been shown to contribute to Actin disassembly3, it is not clear how activity of these locally Acting proteins could be coordinated over the distance scale of the whole cell. Here we show that non-muscle myosin II has a direct role in Actin Network disassembly in crawling cells. In fish keratocytes undergoing motility, myosin II is concentrated in regions at the rear with high rates of Network disassembly. Activation of myosin II by ATP in detergent-extracted cytoskeletons results in rear-localized disassembly of the Actin Network. Inhibition of myosin II activity and stabilization of Actin filaments synergistically impede cell motility, suggesting the existence of two disassembly pathways, one of which requires myosin II activity. Our results establish the importance of myosin II as an enzyme for Actin Network disassembly; we propose that gradual formation and reorganization of an actomyosin Network provides an intrinsic destruction timer, enabling long-range coordination of Actin Network treadmilling in motile cells.
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myosin ii contributes to cell scale Actin Network treadmilling through Network disassembly
Nature, 2010Co-Authors: Cyrus A Wilson, Mark A Tsuchida, Kathry T Applegate, Patricia T Yam, Greg M Allen, Erin L Barnhart, Kinneret Keren, Gaudenz DanuserAbstract:Crawling locomotion of eukaryotic cells is achieved by a process dependent on the Actin cytoskeleton: protrusion of the leading edge requires assembly of a Network of Actin filaments, which must be disassembled at the cell rear for sustained motility. Although ADF/cofilin proteins have been shown to contribute to Actin disassembly, it is not clear how activity of these locally Acting proteins could be coordinated over the distance scale of the whole cell. Here we show that non-muscle myosin II has a direct role in Actin Network disassembly in crawling cells. In fish keratocytes undergoing motility, myosin II is concentrated in regions at the rear with high rates of Network disassembly. Activation of myosin II by ATP in detergent-extracted cytoskeletons results in rear-localized disassembly of the Actin Network. Inhibition of myosin II activity and stabilization of Actin filaments synergistically impede cell motility, suggesting the existence of two disassembly pathways, one of which requires myosin II activity. Our results establish the importance of myosin II as an enzyme for Actin Network disassembly; we propose that gradual formation and reorganization of an actomyosin Network provides an intrinsic destruction timer, enabling long-range coordination of Actin Network treadmilling in motile cells.
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Actin myosin Network reorganization breaks symmetry at the cell rear to spontaneously initiate polarized cell motility
Journal of Cell Biology, 2007Co-Authors: Patricia T Yam, Cyrus A Wilson, Erin L Barnhart, Gaudenz Danuser, Benedict Hebert, Natalie A Dye, Paul W Wiseman, Julie A. TheriotAbstract:We have analyzed the spontaneous symmetry breaking and initiation of Actin-based motility in keratocytes (fish epithelial cells). In stationary keratocytes, the Actin Network flow was inwards and radially symmetric. Immediately before motility initiation, the Actin Network flow increased at the prospective cell rear and reoriented in the perinuclear region, aligning with the prospective axis of movement. Changes in Actin Network flow at the cell front were detectable only after cell polarization. Inhibition of myosin II or Rho kinase disrupted Actin Network organization and flow in the perinuclear region and decreased the motility initiation frequency, whereas increasing myosin II activity with calyculin A increased the motility initiation frequency. Local stimulation of myosin activity in stationary cells by the local application of calyculin A induced directed motility initiation away from the site of stimulation. Together, these results indicate that large-scale Actin–myosin Network reorganization and contractility at the cell rear initiate spontaneous symmetry breaking and polarized motility of keratocytes.
Marie K Schwinn - One of the best experts on this subject based on the ideXlab platform.
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dexamethasone associated cross linked Actin Network formation in human trabecular meshwork cells involves β3 integrin signaling
Investigative Ophthalmology & Visual Science, 2011Co-Authors: Mark S Filla, Marie K Schwinn, Amanda Nosie, Ross Clark, Donna M PetersAbstract:Actin-mediated processes play an important role in regulating aqueous humor outflow through the trabecular meshwork (TM).1 In human and bovine TM cells, steroid treatment leads to increased formation of a novel Actin structure called a cross-linked Actin Network (CLAN).2,3 This structure has been suggested to participate in the pathogenesis of steroid-induced glaucoma (SIG)2,4,5 and possibly primary open angle glaucoma.6 The exact role of CLANs in these diseases, however, has not been defined. CLANs were originally observed in spreading cells and were described as Actin geodesic domes.7 Although they have been found within the TM of healthy eyes, they appear to be more abundant in glaucomatous meshworks.8,9 More recently, CLANs have been observed in human and bovine lamina cribrosa cells that were either steroid treated or glaucomatous.10 CLANs are composed of interconnected arrays of three to five Actin filaments extending outward from a central vertex. They may be precursors to Actin stress fibers7 that regulate contractility in cells. Tropomyosin, α-Actinin, and filamin were found along the Actin filaments in CLANs, whereas the transmembrane heparan sulfate proteoglycan syndecan-4, phosphatidylinositol 4,5-bisphosphate (PIP2), and α-Actinin appear to form a molecular complex, or vertisome, at the vertices of the Actin filaments.7,11–13 CLANs appear to be attached to the apical cell surface,11,12,14,15 presumably by syndecan-4. CLANs are often found within lamellipodia as cells spread, but they can also be found specifically over the nucleus or throughout the cytoplasm.2,7,15 Treatment with glucocorticoids such as dexamethasone (DEX) increases CLAN formation in confluent monolayers of TM cells2,5,16 and in the TM of cultured eyes.17 CLAN formation can also be induced by activating a cooperative β1/β3 integrin signaling pathway that uses, in part, the G-protein–coupled receptor CD47 as a coreceptor coupled to the Rac1 GTPase and the guanine nucleotide exchange factor Trio.13,18 Integrins belong to a ubiquitously expressed family of transmembrane heterodimeric glycoproteins composed of an α and a β subunit. The extracellular domain binds a number of extracellular matrix (ECM) proteins through the conserved sequence, arg-gly-asp, whereas their cytoplasmic tails interact with a variety of tyrosine kinases, adaptor proteins, and Actin-binding proteins.19,20 As a result, integrins form an important physical link between the extracellular environment and the Actin cytoskeleton and may provide a mechanism for sensing changes in external forces in the microenvironment of the TM. In this study, we compared the roles of DEX and αvβ3 integrins in CLAN formation. These studies show that CLANs induced by DEX treatment are structurally similar to those formed by β3 integrins and that CLAN formation involves the same signaling pathway activated by αvβ3 integrins. We also show that DEX treatment increases the level of expression and activation of β3 integrin and that overexpression of αvβ3 integrin can increase CLAN formation. These studies suggest that glucocorticoids may trigger CLAN formation by enhancing β3 integrin signaling in HTM cells through the upregulation of β3 integrins or the activation of an inside-out signaling mechanism that triggers αvβ3 signaling. Elucidating the signaling pathways that direct the formation and disassembly of CLANs in the TM may further our understanding of SIG and other glaucomas.
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regulation of cross linked Actin Network clan formation in human trabecular meshwork htm cells by convergence of distinct β1 and β3 integrin pathways
Investigative Ophthalmology & Visual Science, 2009Co-Authors: Mark S Filla, Marie K Schwinn, Nader Sheibani, Paul L Kaufman, Donna M PetersAbstract:PURPOSE. To determine the β1/β3 integrin-mediated pathways that regulate cross-linked Actin Network (CLAN) formation in human trabecular meshwork (HTM) cells. CLANs form in glaucomatous and steroid-treated TM cells, which may contribute to reducing outflow facility through the TM. METHODS. Expression of CD47 (an αvβ3 integrin coreceptor/ thrombospondin-1 receptor) and integrins avβ3 and β1 was assessed by FACS. CLANs were induced by plating cells on fibronectin (a β1 integrin ligand) in the absence or presence of the β3 integrin-activating mAb AP-5 and were identified by phalloidin labeling. The role of Src kinases, PI-3 kinase (PI-3K), Rac1, and CD47 was determined by incubating cells with the inhibitors PP2 and EPA (Src kinases), LY294002 (PI-3K), or NSC23766 (Rac1). Tiam1 and Trio siRNAs and dominant-negative Tiaml were used to determine which Rac1-specific guanine nucleotide exchange factor was involved. The role of CD47 was determined using the thrombospondin-1-derived agonist peptide 4N1K and the CD47 function blocking antibody B6H12.2. RESULTS. HTM cells expressed CD47 and integrins αvβ3 and β1. β3 Integrin or CD47 activation significantly increased CLAN formation over β1 integrin-induced levels, whereas anti-CD47 mAb B6H12.2 inhibited this increase. PP2, NSC23766, and Trio siRNA decreased β3-induced CLAN formation by 72%, 45%, and 67%, respectively, whereas LY294002 and dominant negative Tiam1 had no effect. LY294002 decreased β1 integrin-mediated CLAN formation by 42%, and PP2 completely blocked it. CONCLUSIONS. Distinct β1 and αvβ3 integrin signaling pathways converge to enhance CLAN formation. β1-Mediated CLAN formation was PI-3K dependent, whereas β3-mediated CLAN formation was CD47 and Rac1/Trio dependent and might have been regulated by thrombospondin-1. Both integrin pathways were Src dependent.
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regulation of cross linked Actin Network clan formation in human trabecular meshwork htm cells by convergence of distinct β1 and β3 integrin pathways
Investigative Ophthalmology & Visual Science, 2009Co-Authors: Mark S Filla, Marie K Schwinn, Nader Sheibani, Paul L Kaufman, Donna M PetersAbstract:Purpose To determine the beta1/beta3 integrin-mediated pathways that regulate cross-linked Actin Network (CLAN) formation in human trabecular meshwork (HTM) cells. CLANs form in glaucomatous and steroid-treated TM cells, which may contribute to reducing outflow facility through the TM. Methods Expression of CD47 (an alphavbeta3 integrin coreceptor/thrombospondin-1 receptor) and integrins alphavbeta3 and beta1 was assessed by FACS. CLANs were induced by plating cells on fibronectin (a beta1 integrin ligand) in the absence or presence of the beta3 integrin-activating mAb AP-5 and were identified by phalloidin labeling. The role of Src kinases, PI-3 kinase (PI-3K), Rac1, and CD47 was determined by incubating cells with the inhibitors PP2 and EPA (Src kinases), LY294002 (PI-3K), or NSC23766 (Rac1). Tiam1 and Trio siRNAs and dominant-negative Tiam1 were used to determine which Rac1-specific guanine nucleotide exchange factor was involved. The role of CD47 was determined using the thrombospondin-1-derived agonist peptide 4N1K and the CD47 function blocking antibody B6H12.2. Results HTM cells expressed CD47 and integrins alphavbeta3 and beta1. beta3 Integrin or CD47 activation significantly increased CLAN formation over beta1 integrin-induced levels, whereas anti-CD47 mAb B6H12.2 inhibited this increase. PP2, NSC23766, and Trio siRNA decreased beta3-induced CLAN formation by 72%, 45%, and 67%, respectively, whereas LY294002 and dominant negative Tiam1 had no effect. LY294002 decreased beta1 integrin-mediated CLAN formation by 42%, and PP2 completely blocked it. Conclusions Distinct beta1 and alphavbeta3 integrin signaling pathways converge to enhance CLAN formation. beta1-Mediated CLAN formation was PI-3K dependent, whereas beta3-mediated CLAN formation was CD47 and Rac1/Trio dependent and might have been regulated by thrombospondin-1. Both integrin pathways were Src dependent.
Julie A. Theriot - One of the best experts on this subject based on the ideXlab platform.
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leading edge stability in motile cells is an emergent property of branched Actin Network growth
bioRxiv, 2020Co-Authors: Rikki M Garner, Julie A. TheriotAbstract:Abstract Animal cell migration is predominantly driven by the coordinated, yet stochastic, polymerization of thousands of nanometer-scale Actin filaments across micron-scale cell leading edges. It remains unclear how such inherently noisy processes generate robust cellular behavior. We employed high-speed, high-resolution imaging of migrating neutrophil-like HL-60 cells to explore the fine-scale dynamic shape fluctuations that emerge and relax throughout the process of leading edge maintenance. We then developed a minimal stochastic model of the leading edge that is able to reproduce this stable relaxation behavior. Remarkably, we find that lamellipodial stability naturally emerges from the interplay between branched Actin Network growth and leading edge shape – with no additional feedback required – based on a synergy between membrane-proximal branching and lateral spreading of filaments. These results thus demonstrate a novel biological noise-suppression mechanism based entirely on system geometry. Furthermore, our model suggests that the Arp2/3-mediated ∼70-80o branching angle optimally smooths lamellipodial shape, addressing its long-mysterious conservation from protists to mammals. One sentence summary An experimental and computational investigation of fluctuation dynamics at the leading edge of motile cells demonstrates that the specific angular geometry of Arp2/3-mediated Actin Network branch formation lies at the core of a successful biological noise-suppression strategy.
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material properties of Actin Networks from motile fish keratocytes under large deformations
Biophysical Journal, 2012Co-Authors: Mark A Tsuchida, Julie A. TheriotAbstract:In Actin-based crawling motility, cells continuously build, reorganize, and disassemble an Actin Network in a process driven jointly by biochemical reactions and mechanical work. A thorough understanding of how forces produced by Actin and myosin contribute to whole-cell movement will thus require detailed knowledge of the material properties of the cytoskeletal Network at the relevant spatial and temporal scales (micrometer-scale deformations over tens of seconds to minutes). Measurements of mechanical properties have largely been limited to microscopic strains, whole-cell bulk measurements, or reconstituted gels that do not fully capture the cellular cytoskeletal organization.We have therefore sought to characterize the deformation of Actin Networks driven from motile cells under large (up to several hundred percent) applied strains. Using detergent-extracted cytoskeletons from fish epithelial keratocytes, we have applied arbitrary strains to the Actin Network between the cell body at the rear of the cell and the native adhesions in the lamellipodial (front) region of the cell, using a glass needle. Deformations through the cell body propagated through a significant portion of the lamellipodium, in some cases reaching the leading edge, indicating good mechanical linkage between the cell body and the lamellipodium. The lamellipodial Actin Network is surprisingly flexible and exhibits strain hardening. At rates of deformation comparable to the speeds of live, crawling cells, the Network exhibited significantly elastic behavior, suggesting that elastic forces might contribute to the myosin-driven reorganization of the Actin Network in the rear of the cell. These results will help refine current physical models for crawling cell motility.
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Actin myosin Network reorganization breaks symmetry at the cell rear to spontaneously initiate polarized cell motility
Journal of Cell Biology, 2007Co-Authors: Patricia T Yam, Cyrus A Wilson, Erin L Barnhart, Gaudenz Danuser, Benedict Hebert, Natalie A Dye, Paul W Wiseman, Julie A. TheriotAbstract:We have analyzed the spontaneous symmetry breaking and initiation of Actin-based motility in keratocytes (fish epithelial cells). In stationary keratocytes, the Actin Network flow was inwards and radially symmetric. Immediately before motility initiation, the Actin Network flow increased at the prospective cell rear and reoriented in the perinuclear region, aligning with the prospective axis of movement. Changes in Actin Network flow at the cell front were detectable only after cell polarization. Inhibition of myosin II or Rho kinase disrupted Actin Network organization and flow in the perinuclear region and decreased the motility initiation frequency, whereas increasing myosin II activity with calyculin A increased the motility initiation frequency. Local stimulation of myosin activity in stationary cells by the local application of calyculin A induced directed motility initiation away from the site of stimulation. Together, these results indicate that large-scale Actin–myosin Network reorganization and contractility at the cell rear initiate spontaneous symmetry breaking and polarized motility of keratocytes.
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Loading history determines the velocity of Actin-Network growth.
Nature cell biology, 2005Co-Authors: Sapun H. Parekh, Ovijit Chaudhuri, Julie A. Theriot, Daniel A. FletcherAbstract:Directional polymerization of Actin filaments in branched Networks is one of the most powerful force-generating systems in eukaryotic cells. Growth of densely cross-linked Actin Networks drives cell crawling, intracellular transport of vesicles and organelles, and movement of intracellular pathogens such as Listeria monocytogenes. Using a modified atomic force microscope (AFM), we obtained force-velocity (Fv) measurements of growing Actin Networks in vitro until Network elongation ceased at the stall force. We found that the growth velocity of a branched Actin Network against increasing forces is load-independent over a wide range of forces before a convex decline to stall. Surprisingly, when force was decreased on a growing Network, the velocity increased to a value greater than the previous velocity, such that two or more stable growth velocities can exist at a single load. These results demonstrate that a single Fv relationship does not capture the complete behaviour of this system, unlike other molecular motors in cells, because the growth velocity depends on loading history rather than solely on the instantaneous load.
