Journal articles on the topic 'Talin inhibitor'
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1
BASS, Mark D., Bipin PATEL, Igor G. BARSUKOV, Ian J. FILLINGHAM, Robert MASON, Beverley J. SMITH, Clive R. BAGSHAW, and David R. CRITCHLEY. "Further characterization of the interaction between the cytoskeletal proteins talin and vinculin." Biochemical Journal 362, no. 3 (March 8, 2002): 761–68. http://dx.doi.org/10.1042/bj3620761.
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The cytoskeletal protein talin, which is thought to couple integrins to F-actin, contains three binding sites (VBS1—VBS3) for vinculin, a protein implicated in the negative regulation of cell motility and whose activity is modulated by an intramolecular interaction between the vinculin head (Vh) and vinculin tail (Vt) domains. In the present study we show that recombinant talin polypeptides containing the three VBSs (VBS1, residues 498–636; VBS2, residues 727–965; and VBS3, residues 1943–2157) each bind tightly to the same or overlapping sites within vinculin1–258. A short synthetic talin VBS3 peptide (residues 1944–1969) was sufficient to inhibit binding of a 125I-labelled talin VBS3 polypeptide to vinculin1–258, and NMR spectroscopy confirmed that this peptide forms a 1:1 complex in slow exchange with vinculin1–258. Binding of the 125I-labelled VBS3 polypeptide was markedly temperature dependent, but was not inhibited by 1M salt or 10% (v/v) 2-methyl-2-propanol. Attempts to further define the talin-binding site within vinculin1–258 using a gel-blot assay were unsuccessful, but near maximal talin-binding activity was retained by a construct spanning vinculin residues 1–131 in a yeast two-hybrid assay. Interestingly, the talin VBS3 polypeptide was a potent inhibitor of the Vh—Vt interaction, and the VBS3 synthetic peptide was able to expose the actin-binding site in intact vinculin, which is otherwise masked by the Vh—Vt interaction. The results suggest that under certain conditions, talin may be an effective activator of vinculin.
2
Dai, Baiyun, Peng Wu, Feng Xue, Renchi Yang, Ziqiang Yu, Kesheng Dai, Changgeng Ruan, Gang Liu, Peter J. Newman, and Cunji Gao. "Integrin-αIIbβ3-mediated outside-in signalling activates a negative feedback pathway to suppress platelet activation." Thrombosis and Haemostasis 116, no. 11 (September 2016): 918–30. http://dx.doi.org/10.1160/th16-02-0096.
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SummaryIntegrin-αIIbβ3-mediated outside-in signalling is widely accepted as an amplifier of platelet activation; accumulating evidence suggests that outside-in signalling can, under certain conditions, also function as an inhibitor of platelet activation. The role of integrin-αIIbβ3-mediated outside-in signalling in platelet activation is disputable. We employed flow cytometry, aggregometry, immunoprecipitation, and immunoblotting to investigate the role of integrin-αIIbβ3-mediated outside-in signalling in platelet activation. Integrin αIIbβ3 inhibition enhances agonist-induced platelet ATP secretion. Human platelets lacking expression of αIIbβ3 exhibited more platelet ATP secretion than their wild-type counterparts. Moreover, integrin-αIIbβ3-mediated outside-in signals activate SHIP-1, which in turn mediates p-Akt dep-hosphorylation, leading to inactivation of PI3K/Akt signalling. Furthermore, 3AC (SHIP-1 inhibitor) inhibits platelet disaggregation, and promotes platelet ATP secretion. Upon ADP stimulation, Talin is recruited to αIIbβ3, and it is dissociated from αIIbβ3 when platelets disaggregate. In addition, treatment with RUC2, an inhibitor of αIIbβ3, which blocks αIIbβ3-mediated outside-in signalling, can markedly prevent the dissociation of talin from integrin. SHIP1 Inhibitor 3AC inhibits the dissociation of talin from integrin-β3. These results suggest that integrin-αIIbβ3-mediated outside-in signalling can serve as a brake to restrict unnecessary platelet activation by activated SHIP-1, which mediated the disassociation of talin from β3, leading to integrin inactivation and blocking of PI3K/Akt signalling to restrict platelet ATP secretion.
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Koh, Timothy J., and James G. Tidball. "Nitric oxide inhibits calpain-mediated proteolysis of talin in skeletal muscle cells." American Journal of Physiology-Cell Physiology 279, no. 3 (September 1, 2000): C806—C812. http://dx.doi.org/10.1152/ajpcell.2000.279.3.c806.
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We tested the hypothesis that nitric oxide can inhibit cytoskeletal breakdown in skeletal muscle cells by inhibiting calpain cleavage of talin. The nitric oxide donor sodium nitroprusside prevented many of the effects of calcium ionophore on C2C12muscle cells, including preventing talin proteolysis and release into the cytosol and reducing loss of vinculin, cell detachment, and loss of cellular protein. These results indicate that nitric oxide inhibition of calpain protected the cells from ionophore-induced proteolysis. Calpain inhibitor I and a cell-permeable calpastatin peptide also protected the cells from proteolysis, confirming that ionophore-induced proteolysis was primarily calpain mediated. The activity of m-calpain in a casein zymogram was inhibited by sodium nitroprusside, and this inhibition was reversed by dithiothreitol. Previous incubation with the active site-targeted calpain inhibitor I prevented most of the sodium nitroprusside-induced inhibition of m-calpain activity. These data suggest that nitric oxide inhibited m-calpain activity via S-nitrosylation of the active site cysteine. The results of this study indicate that nitric oxide produced endogenously by skeletal muscle and other cell types has the potential to inhibit m-calpain activity and cytoskeletal proteolysis.
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Zhang, Jingying Sarah, William E. Kraus, and George A. Truskey. "Stretch-induced nitric oxide modulates mechanical properties of skeletal muscle cells." American Journal of Physiology-Cell Physiology 287, no. 2 (August 2004): C292—C299. http://dx.doi.org/10.1152/ajpcell.00018.2004.
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In this study, we examined the hypothesis that stretch-induced (nitric oxide) NO modulates the mechanical properties of skeletal muscles by increasing accumulation of protein levels of talin and vinculin and by inhibiting calpain-induced proteolysis, thereby stabilizing the focal contacts and the cytoskeleton. Differentiating C2C12 myotubes were subjected to a single 10% step stretch for 0–4 days. The apparent elastic modulus of the cells, Eapp, was subsequently determined by atomic force microscopy. Static stretch led to significant increases ( P < 0.01) in Eapp beginning at 2 days. These increases were correlated with increases in NO activity and neuronal NO synthase (nNOS) protein expression. Expression of talin was upregulated throughout, whereas expression of vinculin was significantly increased only on days 3 and 4. Addition of the NO donor l-arginine onto stretched cells further enhanced Eapp, NOS activity, and nNOS expression, whereas the presence of the NO inhibitor Nω-nitro-l-arginine methyl ester (l-NAME) reversed the effects of mechanical stimulation and of l-arginine. Overall, viscous dissipation, as determined by the value of hysteresis, was not significantly altered. For assessment of the role of vinculin and talin stability, cells treated with l-NAME showed a significant decrease in Eapp, whereas addition of a calpain inhibitor abolished the effect. Thus our results show that NO inhibition of calpain-initiated cleavage of cytoskeleton proteins was correlated with the changes in Eapp. Together, our data suggest that NO modulates the mechanical behavior of skeletal muscle cells through the combined action of increased talin and vinculin levels and a decrease in calpain-mediated talin proteolysis.
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Shen, Bo, Xiaojuan Zhao, Kelly A. O'Brien, Aleksandra Stojanovic-Terpo, Michael Keegan Delaney, Kyungho Kim, Jaehyung Cho, Stephen C. T. Lam, and Xiaoping Du. "A Mechanism For Switch Of Integrin Signaling Direction and a New Anti-Thrombotic Strategy Through Selective Outside-In Signaling Inhibition." Blood 122, no. 21 (November 15, 2013): 2295. http://dx.doi.org/10.1182/blood.v122.21.2295.2295.
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Abstract Antagonists of platelet integrin alphaIIbbeta3 are potent anti-thrombotics due to critical roles of integrins in thrombosis. However, integrins are also important in hemostasis, and thus integrin antagonists have potentially life-threatening bleeding side effect. It would be ideal if we can develop integrin antagonists without bleeding side effect. Integrins transmit signals bidirectionally. Intracellular signals activate integrin alphaIIbbeta3, leading to talin-dependent integrin ligation, which is required for platelet adhesion and initial hemostatic thrombus formation. Integrin ligation in turn mediates Galpha13/Src-dependent outside-in signaling that stabilizes and amplify thrombi, which is crucial for occlusive arterial thrombosis. Here we show that talin and Galpha13 bind to mutually exclusive but distinct sites in integrin beta3, and their bindings are dynamically regulated during integrin signaling. The first talin binding wave mediates inside-out signaling and also “ligand-induced integrin activation”, but is not required for early phase outside-in signaling. Integrin ligation induces talin dissociation and Galpha13 binding, which selectively mediates early phase outside-in signaling. The second talin binding wave is associated with late phase outside-in signaling and clot retraction. Based on these findings, we have designed a selective inhibitor of Galpha13-integrin interaction, which specifically abolished outside-in signaling without affecting inside-out signaling and integrin ligation. Strikingly, this inhibitor potently inhibits occlusive thrombosis in vivo, but has no effect on tail bleeding time, in contrast to the current integrin antagonists. Thus, we have discovered a mechanism for switching the direction of integrin signaling and a new anti-thrombotic that does not cause bleeding. Disclosures: No elevant conflicts of interest to declare.
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Yuan, Weiping, Tina M. Leisner, Andrew W. McFadden, Zhengyan Wang, Mark K. Larson, Shantres Clark, Christel Boudignon-Proudhon, Stephen C. T. Lam, and Leslie V. Parise. "CIB1 is an endogenous inhibitor of agonist-induced integrin αIIbβ3 activation." Journal of Cell Biology 172, no. 2 (January 16, 2006): 169–75. http://dx.doi.org/10.1083/jcb.200505131.
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In response to agonist stimulation, the αIIbβ3 integrin on platelets is converted to an active conformation that binds fibrinogen and mediates platelet aggregation. This process contributes to both normal hemostasis and thrombosis. Activation of αIIbβ3 is believed to occur in part via engagement of the β3 cytoplasmic tail with talin; however, the role of the αIIb tail and its potential binding partners in regulating αIIbβ3 activation is less clear. We report that calcium and integrin binding protein 1 (CIB1), which interacts directly with the αIIb tail, is an endogenous inhibitor of αIIbβ3 activation; overexpression of CIB1 in megakaryocytes blocks agonist-induced αIIbβ3 activation, whereas reduction of endogenous CIB1 via RNA interference enhances activation. CIB1 appears to inhibit integrin activation by competing with talin for binding to αIIbβ3, thus providing a model for tightly controlled regulation of αIIbβ3 activation.
7
Mason, Christopher, Stephen Lynch, James Benjamin, Dani Ashak, Jamunabai M. Prakash, Andrew Moore, Pamela Bagsiyao, et al. "Intracellular matrix metalloproteinase-2 (MMP-2) regulates human platelet activation via hydrolysis of talin." Thrombosis and Haemostasis 111, no. 01 (2014): 140–53. http://dx.doi.org/10.1160/th13-03-0248.
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SummaryMatrix metalloproteinase (MMP) activity is generally associated with normal or pathological extracellular processes such as tissue remodeling in growth and development or in tumor metastasis and angiogenesis. Platelets contain at least three MMPs, 1, 2 and 9 that have been reported to stimulate or inhibit agonist-induced platelet aggregation via extracellular signals. The non-selective Zn+2 chelating MMP inhibitor, 1,10-phenanthroline, and the serine protease inhibitor, AEBSF, were found to inhibit all tested agonist-induced platelet aggregation reactions. In vitro analysis demonstrated that 1,10-phenanthroline completely inhibited MMP-1,2,and 9 but had little to no effect on calpain activity while the converse was true with AEBSF. We now demonstrate that MMP-2 functions intracellularly to regulate agonistinduced platelet aggregations via the hydrolytic activation of talin, the presumed final activating factor of glycoprotein (GP)IIb/IIIa integrin (the inside-out signal). Once activated GPIIb/IIIa binds the dimeric fibrinogen molecule required for platelet aggregation. The active intracellular MMP-2 molecule is complexed with JAK 2/STAT 3, as demonstrated by the fact that all three proteins are co-immunoprecipitated with either anti-JAK 2, or anti-STAT 3 antibodies and by immunofluorescence studies. The MMP-2 platelet activation pathway can be synergistically inhibited with the non-selective MMP inhibitor, 1,10-phenanthroline, plus a JAK 2 inhibitor. This activation pathway is distinct from the previously reported calpain-talin activating pathway. The identification of a new central pathway for platelet aggregation presents new potential targets for drug regulation and furthers our understanding of the complexity of platelet activation mechanisms.
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Lewis, J. M., D. A. Cheresh, and M. A. Schwartz. "Protein kinase C regulates alpha v beta 5-dependent cytoskeletal associations and focal adhesion kinase phosphorylation." Journal of Cell Biology 134, no. 5 (September 1, 1996): 1323–32. http://dx.doi.org/10.1083/jcb.134.5.1323.
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Integrins alpha v beta 3 and alpha v beta 5 both mediate cell adhesion to vitronectin yet trigger different postligand binding events. Integrin alpha v beta 3 is able to induce cell spreading, migration, angiogenesis, and tumor metastasis without additional stimulators, whereas alpha v beta 5 requires exogenous activation of protein kinase C (PKC) to mediate these processes. To investigate this difference, the ability of beta 3 or beta 5 to induce colocalization of intracellular proteins was assessed by immunofluorescence in hamster CS-1 melanoma cells. We found that alpha v beta 5 induced colocalization of talin, alpha-actinin, tensin, and actin very weakly relative to alpha v beta 3. alpha v beta 5 was able to efficiently induce colocalization of focal adhesion kinase (FAK); however, it was unable to increase phosphorylation of FAK on tyrosine. Activation of PKC by adding phorbol ester to alpha v beta 5-expressing cells induced spreading, increased colocalization of alpha-actinin, tensin, vinculin, p130cas and actin, and triggered tyrosine phosphorylation of FAK. Unexpectedly, talin colocalization remained low even after activation of PKC. Treatment of cells with the PKC inhibitor calphostin C inhibited spreading and the colocalization of talin, alpha-actinin, tensin, and actin for both alpha v beta 3 and alpha v beta 5. We conclude that PKC regulates localization of cytoskeletal proteins and phosphorylation of FAK induced by alpha v beta 5. Our results also show that FAK can be localized independent of its phosphorylation and that cells can spread and induce localization of other focal adhesion proteins in the absence of detectable talin.
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Feng, Shuju, Xin Lu, and Michael H. Kroll. "A Contractile Cytoskeletal Tether Modulates Signaling between Glycoprotein Ibα and αIIbβ3." Blood 104, no. 11 (November 16, 2004): 1554. http://dx.doi.org/10.1182/blood.v104.11.1554.1554.
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Abstract In resting platelets, the cytoplasmic domains of glycoprotein (Gp) Ibα and β3 integrin link the GpIb-IX-V and αIIbβ3 complex with adapter proteins, cytoskeletal elements and lipid and tyrosine kinases. Biochemical and pharmacological analyses of intact platelets and genetic analyses of recombinant GpIb-IX-V and αIIbβ3 in CHO cells point towards the hypothesis that a series of cytoskeletal proteins form a functional tether connecting the cytoplasmic domains of GpIbα and β3 integrin. To test the hypothesis that dynamic contractility of this tether modulates VWF-induced signaling between GpIb-IX-V and αIIbβ3, we examined how pathological shear stress affects connections between tethering elements and how altering the contractile function of the tether affects VWF-induced platelet adherence and aggregation. We observed in resting platelets that there is no co-immunoprecipitation of GpIbα and αIIbβ3. The large dimeric actin-binding protein filamin A co-immunoprecipitates with both GpIbα and β3 integrin, but its association with αIIbβ3 is inhibited by DNaseI, indicating that it binds indirectly to αIIbβ3 through cytoskeletal connections. These connections were investigated in resting platelets by examining a series of immunoprecipitates (IP) for co-precipitating proteins using immunoblotting (IB). We observed the following IP/IB pairs [(+) designates that the co-precipitation is DNaseI-sensitive]: β3/talin (+); talin/α-actinin (+); talin/vinculin (+); and talin/filamin A (−). We also observed that myosin heavy chain (MHC) and the tyrosine kinase Syk co-immunoprecipitate with αIIbβ3 in resting platelets. When washed platelets in buffer containing calcium (1 mM) and VWF (5 μg/ml) were sheared at 120 dynes/cm2 for 2 minutes, the cytoskeletal linkage separates: there was enhanced filamin A, actin and α-actinin binding to GpIbα, enhanced vinculin binding to α-actinin, enhanced talin binding to αIIbβ3, and both myosin and the tyrosine kinase Syk disassociated from the β3 tail. Inhibiting myosin contractility with the myosin light chain kinase (MLCK) inhibitor ML-9 (1 mM) inhibited the shear-induced association between talin and β3, as well as platelet aggregation in response to 120 dynes/cm2 in the cone plate viscometer and platelet-dependent thrombosis from whole blood onto type I collagen in a parallel plate flow chamber with a shear rate of 500 sec−1 (shear stress of ~ 20 dynes/cm2). The data presented support a model of shear-induced platelet aggregation in which a series of cytoskeletal proteins serve as a mechanotransducing scaffolding linking the cytoplasmic domains of GpIbα and β3 integrin. When GpIb-IX-V engages ligand under shearing forces, the force is transduced from GpIbα to filamin to actin/α-actinin to vinculin to talin to β3, and contractility driven by the activation of αIIbβ3-associated myosin enhances talin binding to β3, thereby effecting a conformation change that creates a ligand-receptive αIIbβ3. Such interactions may be regulated by cytosolic ionized calcium (which effects MLCK activation) and tyrosine kinases (αIIbβ3-associated Syk disassociates from β3 following shear). These results provide evidence that the structural scaffolding connecting GpIb-IX-V with αIIbβ3 localizes signaling elements to functionally important compartments that modulate αIIbβ3 activation and shear-induced platelet aggregation.
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Litvinov, Rustem I., Karen Pei Yi Fong, Oleg V. Kim, Kathleen I. Molnar, Paul C. Billings, Alex Sternisha, James A. Wells, John W. Weisel, William F. DeGrado, and Joel S. Bennett. "Active Calpain Promotes Fibrin Clot Contraction By Strengthening the Coupling of Fibrin-Bound αIIbβ3 to the Platelet Cytoskeleton." Blood 132, Supplement 1 (November 29, 2018): 1128. http://dx.doi.org/10.1182/blood-2018-99-113063.
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Abstract Platelet-driven blood clot contraction reduces and compacts clot volume, promoting hemostasis while restoring blood flow past otherwise obstructive thrombi. Clot contraction is driven by traction forces in the range of tens of pN per platelet that are generated by platelet non-muscle myosin IIa and actin on αIIbβ3 bound to extracellular fibrin fibers. In an analysis of the kinetics of thrombin-induced clot formation and contraction in platelet-rich plasma, we found that clot contraction has two discernable phases: an exponential initiation phase and a second exponential contraction phase. It is noteworthy that Azam et al (Mol Cell Biol21:2213-20, 2001) observed that deleting the µ-calpain gene in mice impairs platelet-mediated clot contraction. In this context, we found that inhibiting calpain activity with ALLM (N-Acetyl-Leu-Leu-Methional), a cell-permeable calpain inhibitor, significantly prolonged the duration of the initiation phase and decreased the kinetic rate constants for both the initiation and contraction phases of thrombin-stimulated platelet-mediated clot contraction, but without affecting the overall extent of contraction. The calpain inhibitor ALLN (N-acetyl-Leu-Leu-Norleu) had the same effect. Thus, these studies imply that activation of platelet calpain facilitates the transmission of traction forces from the platelet cytoskeleton to the αIIbβ3 bound to fibrin fibers. μ-Calpain is calcium-dependent cytosolic neutral cysteine protease that is activated 30-60 seconds after the onset of platelet aggregation stimulated by platelet agonists such as thrombin. To identify the calpain-cleaved protein or proteins involved in clot contraction, we incubated washed human platelets with the thrombin activation peptide TRAP in the presence or absence of calpain inhibitors and identified calpain-cleaved proteins using subtiligase-mediated biotin-labeling of nascent N-termini followed by mass spectrometry. We identified 32 proteins in the platelet cytosol that undergo calpain-mediated cleavage after TRAP stimulation. Many of these are cytoskeletal proteins, most prominently talin which connects integrins to the cytoskeleton and vinculin which is required for myosin-contractility-dependent effects on traction force and adhesion strength. Accordingly, we focused our attention on these two proteins. Talin, a 250 kD protein, is composed of a 45 kD N-terminal head domain attached via a flexible linker to a 200 kD C-terminal rod domain. The rod domain consists of 62 amphipathic α-helices organized into a series of four- and five-helix bundles. Vinculin is a 116 kD protein composed of 5 domains and has an autoinhibited structure in which domains 1-3 bind to domain 5. We detected 8 calpain-mediated cleavages in talin, 2 previously identified cleavage sites in unstructured regions and 6 others in α-helical regions known to interact with other proteins. Four of the latter are located within the first 12 talin helices, a region that contains four vinculin binding sites (VBSs). Likewise, the remaining two cleavage sites are in proximity to a VBS. A VBS is a vinculin-binding α-helix that is buried in a helical bundle due to extensive hydrophobic interactions with other amphipathic helices. Because VBS are packed into the interior of a helical bundle, a structural rearrangement is required to initiate vinculin binding. Using magnetic tweezers and atomic force microscopy, del Rio et al demonstrated that force-induced stretching of single talin rod molecules activates VBSs (Science323:638-41, 2009)), implying that stretching causes the conformational change required to expose buried VBSs. Thus, based on the location of the calpain-mediated cleavages in talin, we hypothesize that by cleaving talin in proximity to a VBS, calpain facilitates vinculin to binding talin, thereby strengthening the coupling of fibrin-bound αIIbβ3 to the platelet cytoskeleton to promote fibrin clot contraction. Disclosures No relevant conflicts of interest to declare.
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Verheijden, Kim A. T., Ramon Sonneveld, Marinka Bakker-van Bebber, Jack F. M. Wetzels, Johan van der Vlag, and Tom Nijenhuis. "The Calcium-Dependent Protease Calpain-1 Links TRPC6 Activity to Podocyte Injury." Journal of the American Society of Nephrology 29, no. 8 (June 28, 2018): 2099–109. http://dx.doi.org/10.1681/asn.2016111248.
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BackgroundThe hallmark of podocytopathies, such as FSGS, is podocyte injury resulting in proteinuria. Transient receptor potential channel C6 (TRPC6) is a calcium-conducting ion channel expressed at the slit diaphragm. TRPC6 gain-of-function mutations and glomerular TRPC6 overexpression are associated with proteinuria. However, the pathways linking TRPC6 to podocyte injury, which is characterized by loss of the slit diaphragm protein nephrin, activation of several intracellular pathways (including calcineurin-NFAT signaling), and cytoskeletal rearrangement, remain elusive.MethodsWe tested whether the calcium-dependent protease calpain-1 mediates TRPC6-dependent podocyte injury in human and experimental FSGS and cultured podocytes.ResultsCompared with kidneys of healthy controls, kidneys of patients with FSGS had increased TRPC6 expression, increased calpain and calcineurin activity, and reduced expression of the calpain target Talin-1, which links the actin cytoskeleton to integrins and is critical for podocyte cytoskeletal stability. In a rat model of human FSGS, increased glomerular and urinary calpain activity associated with reduced Talin-1 abundance, enhanced calcineurin activity, and increased proteinuria. Treatment with the calpain inhibitor calpeptin prevented these effects. In cultured podocytes, pharmacologic stimulation of TRPC6-dependent calcium influx increased calpain-1 and calcineurin activity and reduced Talin-1 expression, and knockdown of TRPC6 or calpain-1 prevented these effects.ConclusionsWe elucidated a novel mechanism that links TRPC6 activity to calpain-1 activation and through Talin-1 loss and possibly, calcineurin activation, the podocyte injury characterizing FSGS. Therefore, calpain-1 and/or TRPC6 inhibition could be future therapeutic options to treat patients with FSGS or other podocytopathies.
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Eddy, R. J., L. M. Pierini, F. Matsumura, and F. R. Maxfield. "Ca2+-dependent myosin II activation is required for uropod retraction during neutrophil migration." Journal of Cell Science 113, no. 7 (April 1, 2000): 1287–98. http://dx.doi.org/10.1242/jcs.113.7.1287.
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Buffering of intracellular Ca2+ transients in human neutrophils leads to reduced motility due to defective uropod detachment on fibronectin and vitronectin-coated surfaces. Since one potential target of a rise in [Ca2+]i is the activation of myosin II, we characterized the role of myosin II during motility. Treatment of neutrophils with a myosin inhibitor (2,3-butanedione monoxime), or myosin light chain kinase inhibitors (ML-7, ML-9, or KT5926) resulted in impaired uropod retraction and a dose-dependent decrease in chemokinesis following stimulation with N-formyl-Met-Leu-Phe (fMLP). Treatment with ML-9 resulted in a redistribution of F-actin and talin to the non-retracted uropods, mimicking the redistribution observed during [Ca2+]i buffering. Impairment of uropod retraction and redistribution of F-actin and talin by myosin II inhibition was only observed on adhesive substrates such as fibronectin and not on poorly adhesive substrates such as human serum-coated glass. At higher concentrations of ML-9, cell polarization was inhibited and pseudopod extension occurred radially. Using an antibody specific for serine 19-phosphorylated regulatory light chain of myosin II, regions of activated myosin II were found at the leading edge as well as the uropod in motile fMLP-stimulated cells. [Ca2+]i depletion caused a 50% decrease in the level of serine 19-phosphorylated myosin II suggesting that activation of myosin II by intracellular Ca2+ transients may be an essential step in establishing a polarized pseudopod and providing the force required for uropod retraction during PMN motility on adhesive surfaces.
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Yan, Rong, Zhicheng Wang, Weilin Zhang, Suping Li, Changgeng Ruan, and Kesheng Dai. "Calpain Regulates ADAM17-Mediated Platelet Glycoprotein Ibalpha Ectodomain Shedding." Blood 114, no. 22 (November 20, 2009): 2996. http://dx.doi.org/10.1182/blood.v114.22.2996.2996.
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Abstract Abstract 2996 Poster Board II-974 Introduction: The interaction of platelet glycoprotein (GP) Ibalpha with von Willebrand factor (VWF) exposed at the injured vessel wall initiates platelet adhesion and thrombus formation. Thus GPIbalpha ectodomain shedding has important implications for thrombosis and hemostasis. A disintegrin and metalloproteinase 17 (ADAM17) was identified recently to play an essential role in agonist induced GPIbalpha shedding. Calpain, the calcium-dependent neutral protease, has been thought to play a key role in GPIbalpha shedding, whereas it still remains unclear the role of calpain in ADAM17-mediated GPIbalpha shedding. Methods: Washed platelets isolated from vein blood of healthy donors were pre-incubated with various reagents. GPIbalpha fragments were detected by Western blot with anti- GPIbalpha C- or N-terminal antibodies. GPIbalpha surface expression was determined by flow cytometry with anti-GPIbalpha N-terminal antibody. Results: Western blot and flow cytometry results showed that cell permeable calpain inhibitors, MDL 28170, calpain inhibit I and calpain inhibitor II, significantly reduced calmodulin inhibitor W7- and calcium ionophore A23187-induced GPIbalpha shedding. Dibucaine induced GPIbalpha shedding was potently inhibited by calpain inhibitors. W7 and A23187 induced talin cleavage was abolished by calpain inhibitors. Calpain inhibitors had no obvious effect on N-ethylmaleimide-induced GPIbalpha shedding. In order to investigate the role of protein kinase C (PKC) in calpain-mediated GPIbalpha shedding, calpain inhibitors or PKC inhibitor (bisindolylmaleimide I hydrochloride, BIM) were incubated with platelets respectively, and then platelets were further incubated with PKC activator phorbol-ester (PMA) or dibucaine. PMA-induced or dibucaine-mediated GPIbalpha shedding did not inhibit by calpain inhibitors or PKC inhibitor respectively, indicating that PKC is not involved in calpain-mediated GPIbalpha shedding. In addition, two CHO cell lines expressing wild-type GPIb-IX and GPIb-IX mutant with truncated GPIbalpha lacking filamin A binding site were used to test the role of filamin A in calpain-regulated GPIbalpha shedding. Dibucaine-induced GPIbalpha shedding was inhibited by MDL28170 in both of the cell lines. Conclusion: The data indicate that calpain plays key roles in ADAM17-mediated GPIbalpha ectodomain shedding and the calpain-regualted GPIbalpha shedding is independent of PKC activity or filamin A cleavage. Disclosures: No relevant conflicts of interest to declare.
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Yao, X., A. Thibodeau, and J. G. Forte. "Ezrin-calpain I interactions in gastric parietal cells." American Journal of Physiology-Cell Physiology 265, no. 1 (July 1, 1993): C36—C46. http://dx.doi.org/10.1152/ajpcell.1993.265.1.c36.
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Gastric ezrin, a membrane-cytoskeletal linker with sequence homology to talin and erythrocyte band 4.1, has been associated with the remodeling of parietal cell apical membrane that occurs with adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase stimulation. Here we examine the interrelationship between parietal cell ezrin and Ca(2+)-dependent protease activity. Addition of Ca2+ to sonicated gastric gland preparations rendered a relatively selective proteolysis of the 80-kDa ezrin, accompanied by the appearance of a 55-kDa breakdown product. Ca(2+)-dependent proteolysis of ezrin was blocked by E64, a cysteine protease inhibitor, or calpastatin, indicating calpain as the responsible protease. Degradation of ezrin in intact gastric glands was achieved by varying extracellular [Ca2+] and [ionomycin]. Ezrin degradation in situ was rapid and relatively selective, although Ca(2+)-dependent degradation of some spectrin-like bands was also observed. The effect of activated calpain I on parietal cell function was assessed by probing the secretory response to histamine stimulation using [14C]aminopyrine uptake, along with parallel measurements of calpain activity, over a wide range of ionomycin. Activation of calpain, as evidenced by loss of parietal cell ezrin, was correlated with decreased AP uptake by stimulated gastric glands, supporting a role for ezrin in the oxyntic secretory process. The calpain-ezrin interaction established here, and the similarities of calpain with talin and erythrocyte band 4.1, suggest a common feature to this family of ezrin/band 4.1/talin proteins that have been implicated in membrane-cytoskeletal association.
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Nguyen, Bao, M. Eloísa Carbajal, and María L. Vitale. "Intracellular Mechanisms Involved in Dopamine-Induced Actin Cytoskeleton Organization and Maintenance of a Round Phenotype in Cultured Rat Lactotrope Cells*." Endocrinology 140, no. 8 (August 1, 1999): 3467–77. http://dx.doi.org/10.1210/endo.140.8.6905.
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Abstract The participation of the actin cytoskeleton in the control of PRL secretion by dopamine (DA) is not yet fully understood. Recently, we demonstrated that DA induces cortical actin assembly and stabilization in anterior pituitary PRL-secreting cells (lactotropes) that can be linked to DA-induced inhibition of PRL secretion. Here we show that DA prevents cell flattening and the formation of cytoplasmic actin cables in cultured rat lactotropes. The effects of DA were reversible, mediated by D2 receptors, exclusive to lactotropes, and independent of other anterior pituitary cells present in the cultures. Because cAMP and Ca2+ mediate DA-induced inhibition of PRL secretion and synthesis, we investigated whether morphological responses to DA were dependent on these second messengers. Either inhibition of protein kinase A activity with the specific inhibitor KT5720 or blockade of Ca2+ channels with nifedipine inhibited cell flattening and induced cytoplasmic actin filament breakdown. Nifedipine was as effective as DA, but KT5720 was less effective than DA. Increased intracellular cAMP levels provoked cell flattening, which was blocked by nifedipine and KT5720, but not by DA. The results suggest that Ca2+-dependent pathways control cell shape in most lactotropes; however, in a subpopulation of lactotropes, cAMP-dependent pathways may also contribute to DA morphological responses. Next, we studied the participation of the Rho family of guanosine triphosphatases, which is known to regulate the dynamics of actin filaments. Inactivation of Rho by C3 exoenzyme induced cytoplasmic actin cable disassembly and lactotrope rounding up. No additive effects were observed among Rho-, cAMP-, and Ca2+-dependent pathways. However, C3-induced morphological responses were blocked by increased cAMP levels, suggesting that Rho-dependent steps are upstream cAMP-dependent steps. DA-induced actin cytoskeleton reorganization in lactotropes may involve modifications in the expression and localization of actin-binding proteins. DA increased expression of the actin anchoring proteins talin and α-actinin, but not of vinculin. DA enhanced association of talin to cell membranes. Increased talin-membrane interaction may be implicated in DA-induced maintenance of a round phenotype in lactotrope cells.
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Supinski, Gerald S., Lin Wang, Xiao-Hong Song, Jennifer S. Moylan, and Leigh Ann Callahan. "Muscle-specific calpastatin overexpression prevents diaphragm weakness in cecal ligation puncture-induced sepsis." Journal of Applied Physiology 117, no. 8 (October 15, 2014): 921–29. http://dx.doi.org/10.1152/japplphysiol.00975.2013.
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Recent work indicates that infections are a major contributor to diaphragm weakness in patients who are critically ill and mechanically ventilated, and that diaphragm weakness is a risk factor for death and prolonged mechanical ventilation. Infections activate muscle calpain, but many believe this is an epiphenomenon and that other proteolytic processes are responsible for infection-induced muscle weakness. We tested the hypothesis that muscle-specific overexpression of calpastatin (CalpOX; an endogenous calpain inhibitor) would attenuate diaphragm dysfunction in cecal ligation puncture (CLP)-induced sepsis. We studied 1) wild-type (WT) sham-operated mice, 2) WT CLP-operated mice, 3) CalpOX sham-operated mice, and 4) CalpOX CLP-operated mice ( n = 9–10/group). Twenty-four hours after surgery, we assessed the diaphragm force-frequency relationship, diaphragm mass, and total protein content and diaphragm levels of talin and myosin heavy chain (MHC). CLP markedly reduced diaphragm-specific force generation (force/cross-sectional area), which was prevented by calpastatin overexpression (force averaged 21.4 ± 0.5, 6.9 ± 0.8, 22.4 ± 1.0, and 18.3 ± 1.3 N/cm2, respectively, for WT sham, WT CLP, CalpOX sham, and CalpOX CLP groups, P < 0.001). Diaphragm mass and total protein content were similar in all groups. CLP induced talin cleavage and reduced MHC levels; CalpOX prevented these alterations. CLP-induced sepsis rapidly reduces diaphragm-specific force generation and is associated with cleavage and/or depletion of key muscle proteins (talin, MHC), effects prevented by muscle-specific calpastatin overexpression. These data indicate that calpain activation is a major cause of diaphragm weakness in response to CLP-induced sepsis.
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Lynch, Christopher D., Andre M. Lazar, Thomas Iskratsch, Xian Zhang, and Michael P. Sheetz. "Endoplasmic spreading requires coalescence of vimentin intermediate filaments at force-bearing adhesions." Molecular Biology of the Cell 24, no. 1 (January 2013): 21–30. http://dx.doi.org/10.1091/mbc.e12-05-0377.
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For cells to develop long-range forces and carry materials to the periphery, the microtubule and organelle-rich region at the center of the cell—the endoplasm—needs to extend to near the cell edge. Depletion of the actin cross-linking protein filamin A (FlnA) causes a collapse of the endoplasm into a sphere around the nucleus of fibroblasts and disruption of matrix adhesions, indicating that FlnA is involved in endoplasmic spreading and adhesion growth. Here, we report that treatment with the calpain inhibitor N-[N-(N-acetyl-l-leucyl)-l-leucyl]-l-norleucine (ALLN) restores endoplasmic spreading as well as focal adhesion (FA) growth on fibronectin-coated surfaces in a Fln-depleted background. Addback of calpain-uncleavable talin, not full-length talin, achieves a similar effect in Fln-depleted cells and indicates a crucial role for talin in endoplasmic spreading. Because FA maturation involves the vimentin intermediate filament (vIF) network, we also examined the role of vIFs in endoplasmic spreading. Wild-type cells expressing a vimentin variant incapable of polymerization exhibit deficient endoplasmic spreading as well as defects in FA growth. ALLN treatment restores FA growth despite the lack of vIFs but does not restore endoplasmic spreading, implying that vIFs are essential for endoplasm spreading. Consistent with that hypothesis, vIFs are always displaced from adhesions when the endoplasm does not spread. In Fln-depleted cells, vIFs extend beyond adhesions, nearly to the cell edge. Finally, inhibiting myosin II–mediated contraction blocks endoplasmic spreading and adhesion growth. Thus we propose a model in which myosin II–mediated forces and coalescence of vIFs at mature FAs are required for endoplasmic spreading.
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Petrich, Brian G., Patrizia Marchese, Zaverio M. Ruggeri, Saskia Spiess, Rachel A. M. Weichert, Feng Ye, Ralph Tiedt, et al. "Talin is required for integrin-mediated platelet function in hemostasis and thrombosis." Journal of Experimental Medicine 204, no. 13 (December 17, 2007): 3103–11. http://dx.doi.org/10.1084/jem.20071800.
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Integrins are critical for hemostasis and thrombosis because they mediate both platelet adhesion and aggregation. Talin is an integrin-binding cytoplasmic adaptor that is a central organizer of focal adhesions, and loss of talin phenocopies integrin deletion in Drosophila. Here, we have examined the role of talin in mammalian integrin function in vivo by selectively disrupting the talin1 gene in mouse platelet precursor megakaryocytes. Talin null megakaryocytes produced circulating platelets that exhibited normal morphology yet manifested profoundly impaired hemostatic function. Specifically, platelet-specific deletion of talin1 led to spontaneous hemorrhage and pathological bleeding. Ex vivo and in vitro studies revealed that loss of talin1 resulted in dramatically impaired integrin αIIbβ3-mediated platelet aggregation and β1 integrin–mediated platelet adhesion. Furthermore, loss of talin1 strongly inhibited the activation of platelet β1 and β3 integrins in response to platelet agonists. These data establish that platelet talin plays a crucial role in hemostasis and provide the first proof that talin is required for the activation and function of mammalian α2β1 and αIIbβ3 integrins in vivo.
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Wang, Yang-Kao, Hsi-Hui Lin, and Ming-Jer Tang. "Collagen gel overlay induces two phases of apoptosis in MDCK cells." American Journal of Physiology-Cell Physiology 280, no. 6 (June 1, 2001): C1440—C1448. http://dx.doi.org/10.1152/ajpcell.2001.280.6.c1440.
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We previously demonstrated that collagen gel overlay induced cell remodeling to form lumen and apoptosis in Madin-Darby canine kidney cells. In the present study, we established that collagen gel overlay-induced apoptosis was initiated at areas exclusive of cell remodeling within 24 h (first phase) and extended into areas of cell remodeling within 48 h (second phase). Collagen gel overlay-induced apoptosis was accompanied by selective proteolysis of focal adhesion kinase (FAK), talin, p130cas, and c- src. Upon collagen gel overlay, FAK was initially degraded into a 90-kDa product during the first phase and subsequently into a 80-kDa product during the second phase. Collagen gel overlay-induced apoptosis of focal adhesion complex proteins and apoptosis of the first phase could be blocked only by a protease inhibitor cocktail. In addition, we found that both DEVD-fmk and ZVAD-fmk inhibited secondary proteolysis of FAK, but only ZVAD-fmk blocked collagen gel overlay-induced apoptosis of the second phase. Finally, collagen gel overlay-induced apoptosis and proteolysis of focal adhesion complex proteins were completely inhibited by the combination of protease inhibitor cocktail and ZVAD-fmk. Taken together, collagen gel overlay induces two phases of apoptosis; the first phase is dependent on proteolysis of focal adhesion complex proteins, and the second phase on activation of caspases.
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Miyazaki, Takuro, Kazuo Honda, and Hisayuki Ohata. "Requirement of Ca2+ influx- and phosphatidylinositol 3-kinase-mediated m-calpain activity for shear stress-induced endothelial cell polarity." American Journal of Physiology-Cell Physiology 293, no. 4 (October 2007): C1216—C1225. http://dx.doi.org/10.1152/ajpcell.00083.2007.
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Proteolytic activity in sheared human umbilical vein endothelial cells (HUVECs) was measured using a fluorogenic substrate and laser scanning confocal microscopy to clarify the key role of an intracellular Ca2+-sensitive protease, calpain, in these cells in response to shear stress. Within physiological shear range, activity in the cells was enhanced in shear-dependent fashion. Short interfering RNA-induced silencing of m-calpain, but not of μ-calpain, suppressed the activity. Either removal of extracellular Ca2+ or application of an intracellular Ca2+ chelator (BAPTA/AM) or nonselective cation channel blocker (Gd3+) reduced proteolytic activity. Furthermore, activity was suppressed by phosphatidylinositol bisphosphate (PIP2) chelator (neomycin) or phosphatidylinositol 3-kinase (PI3K) inhibitor (LY294002); in contrast, activity, which was partially inhibited by ERK kinase inhibitor (U0126, PD98059), was unaffected by PLC inhibitor (U73122). Moreover, Akt phosphorylation downstream of PI3K, which was elicited by shear, was attenuated by neomycin but not by calpain inhibitor (calpeptin). Following assessment of shear stress-induced focal adhesion (FA) and cytoskeletal dynamics using interference reflection/green fluorescence protein-actin microscopy, we found that either calpain or PI3K inhibition impaired shear stress-induced polarization of FAs via stabilization of FA structures. Additionally, HUVEC alignment and cytoskeletal remodeling, which was accompanied by calpain-mediated cleavage of vinculin and talin, were also elicited by prolonged application of shear and impaired by m-calpain knockdown. Thus, these results revealed that physiological shear stress elicits Ca2+ influx-sensitive activation of m-calpain in HUVECs. This activity is facilitated primarily through the PI3K pathway; furthermore, it is essential for subsequent FA reorganization and cell alignment under shear conditions.
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Dubois, Christophe, Beat Steiner, Nelly Kieffer, and Sylvie Meyer Reigner. "Thrombin binding to GPIbα induces platelet aggregation and fibrin clot retraction supported by resting αIIbβ3 interaction with polymerized fibrin." Thrombosis and Haemostasis 89, no. 05 (2003): 853–65. http://dx.doi.org/10.1055/s-0037-1613473.
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SummaryWe have investigated the mechanisms leading to platelet aggregation following thrombin interaction with the glycoprotein (GP) Ib-IX-V complex. We show that platelets desensitized for the two thrombin receptors, PAR-1 and PAR-4, are still able to aggregate in response to thrombin and that this aggregation can be inhibited by a monoclonal antibody (VM16d) that blocks thrombin binding to GPIbα, or by pretreatment of platelets with Mocarhagin, a protease that specifically cleaves GPIbα. The thrombin/GPIbα-initiated signaling cascade induces platelet shape change through activation of the Rho kinase p160ROCK, independent of calcium mobilization, transient MEK-1 phosphorylation as well as the cleavage of talin through a calcium-independent mechanism. This signaling cascade does not induce the exposure of high affinity αIIbβ3 integrin receptors, nor does it lead to µ-calpain cleavage of filamin or the integrin cytoplasmic tail. In contrast, we provide evidence that binding of thrombin to GPIbα induces fibrin binding to resting αIIbβ3 leading to fibrin-dependent platelet aggregation and clot retraction, that can be selectively inhibited by αIIbβ3 antagonists such as RGDS, the dodecapeptide or lamifiban, as well as by the fibrin polymerization inhibitor GPRP-amide.
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Glogauer, M., P. Arora, G. Yao, I. Sokholov, J. Ferrier, and C. A. McCulloch. "Calcium ions and tyrosine phosphorylation interact coordinately with actin to regulate cytoprotective responses to stretching." Journal of Cell Science 110, no. 1 (January 1, 1997): 11–21. http://dx.doi.org/10.1242/jcs.110.1.11.
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The actin-dependent sensory and response elements of stromal cells that are involved in mechanical signal transduction are poorly understood. To study mechanotransduction we have described previously a collagen-magnetic bead model in which application of well-defined forces to integrins induces an immediate (< 1 second) calcium influx. In this report we used the model to determine the role of calcium ions and tyrosine-phosphorylation in the regulation of force-mediated actin assembly and the resulting change in membrane rigidity. Collagen-beads were bound to cells through the focal adhesion-associated proteins talin, vinculin, alpha 2-integrin and beta-actin, indicating that force application was mediated through cytoskeletal elements. When force (2 N/m2) was applied to collagen beads, confocal microscopy showed a marked vertical extension of the cell which was counteracted by an actin-mediated retraction. Immunoblotting showed that force application induced F-actin accumulation at the bead-membrane complex but vinculin, talin and alpha 2-integrin remained unchanged. Atomic force microscopy showed that membrane rigidity increased 6-fold in the vicinity of beads which had been exposed to force. Force also induced tyrosine phosphorylation of several cytoplasmic proteins including paxillin. The force-induced actin accumulation was blocked in cells loaded with BAPTA/AM or in cells preincubated with genistein, an inhibitor of tyrosine phosphorylation. Repeated force application progressively inhibited the amplitude of force-induced calcium ion flux. As force-induced actin reorganization was dependent on calcium and tyrosine phosphorylation, and as progressive increases of filamentous actin in the submembrane cortex were correlated with increased membrane rigidity and dampened calcium influx, we suggest that cortical actin regulates stretch-activated cation permeable channel activity and provides a desensitization mechanism for cells exposed to repeated long-term mechanical stimuli. The actin response may be cytoprotective since it counteracts the initial force-mediated membrane extension and potentially strengthens cytoskeletal integrity at force-transfer points.
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Fu, Ying, Guang Wu, and Zhenbiao Yang. "ROP Gtpase–Dependent Dynamics of Tip-Localized F-Actin Controls Tip Growth in Pollen Tubes." Journal of Cell Biology 152, no. 5 (March 5, 2001): 1019–32. http://dx.doi.org/10.1083/jcb.152.5.1019.
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Tip-growing pollen tubes provide a useful model system to study polar growth. Although roles for tip-focused calcium gradient and tip-localized Rho-family GTPase in pollen tube growth is established, the existence and function of tip-localized F-actin have been controversial. Using the green fluorescent protein–tagged actin-binding domain of mouse talin, we found a dynamic form of tip-localized F-actin in tobacco pollen tubes, termed short actin bundles (SABs). The dynamics of SABs during polar growth in pollen tubes is regulated by Rop1At, a Rop GTPase belonging to the Rho family. When overexpressed, Rop1At transformed SAB into a network of fine filaments and induced a transverse actin band behind the tip, leading to depolarized growth. These changes were due to ectopic Rop1At localization to the apical region of the plasma membrane and were suppressed by guanine dissociation inhibitor overexpression, which removed ectopically localized Rop1At. Rop GTPase–activating protein (RopGAP1) overexpression, or Latrunculin B treatments, also recovered normal actin organization and tip growth in Rop1At-overexpressing tubes. Moreover, overexpression of RopGAP1 alone disrupted SABs and inhibited growth. Finally, SAB oscillates and appears at the tip before growth. Together, these results indicate that the dynamics of tip actin are essential for tip growth and provide the first direct evidence to link Rho GTPase to actin organization in controlling cell polarity and polar growth in plants.
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Ruchalski, Kathleen, Haiping Mao, Satish K. Singh, Yihan Wang, Dick D. Mosser, Fanghong Li, John H. Schwartz, and Steven C. Borkan. "HSP72 inhibits apoptosis-inducing factor release in ATP-depleted renal epithelial cells." American Journal of Physiology-Cell Physiology 285, no. 6 (December 2003): C1483—C1493. http://dx.doi.org/10.1152/ajpcell.00049.2003.
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Inhibition of the mitochondrial release and nuclear translocation of apoptosis-inducing factor (AIF) by heat stress protein (HSP)72 may ameliorate apoptosis in renal epithelial cells exposed to a metabolic inhibitor. To evaluate this hypothesis, cells were transiently exposed to 5 mM sodium cyanide in the absence of medium glucose, a maneuver known to induce apoptosis. ATP depletion for 1-2 h resulted in the progressive accumulation of mitochondrial AIF in the cytosol of samples obtained by selectively permeabilizing the plasma membrane with digitonin. During recovery from ATP depletion, time-dependent nuclear AIF accumulation (but not cytochrome c, an F0F1 ATP synthase subunit, or talin) was observed in isolated nuclei. Nuclear AIF accumulation was associated with peripheral chromatin condensation and DNA degradation. Prior heat stress (HS) significantly reduced AIF leakage into the cytosol, decreased nuclear accumulation of AIF, and inhibited DNA degradation. HS also increased the interaction between AIF and HSP72 detected by immunoprecipitation. In ATP depleted cells, selective overexpression of human HSP72 reduced the leakage of mitochondrial AIF in a dose-dependent manner ( r = 0.997). This study suggests that mitochondrial membrane injury and subsequent AIF release contribute to nuclear injury and apoptosis in ATP-depleted renal cells. HSP72, an antiapoptotic protein, inhibits cell injury in part by preventing mitochondrial AIF release and perhaps by decreasing its nuclear accumulation.
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Manso, Ana Maria, Seok-Min Kang, Sergey V. Plotnikov, Ingo Thievessen, Jaewon Oh, Hilary E. Beggs, and Robert S. Ross. "Cardiac fibroblasts require focal adhesion kinase for normal proliferation and migration." American Journal of Physiology-Heart and Circulatory Physiology 296, no. 3 (March 2009): H627—H638. http://dx.doi.org/10.1152/ajpheart.00444.2008.
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Migration and proliferation of cardiac fibroblasts (CFs) play an important role in the myocardial remodeling process. While many factors have been identified that regulate CF growth and migration, less is known about the signaling mechanisms involved in these processes. Here, we utilized Cre-LoxP technology to obtain focal adhesion kinase (FAK)-deficient adult mouse CFs and studied how FAK functioned in modulating cell adhesion, proliferation, and migration of these cells. Treatment of FAKflox/flox CFs with Ad/Cre virus caused over 70% reduction of FAK protein levels within a cell population. FAK-deficient CFs showed no changes in focal adhesions, cell morphology, or protein expression levels of vinculin, talin, or paxillin; proline-rich tyrosine kinase 2 (Pyk2) expression and activity were increased. Knockdown of FAK protein in CFs increased PDGF-BB-induced proliferation, while it reduced PDGF-BB-induced migration. Adhesion to fibronectin was not altered. To distinguish between the function of FAK and Pyk2, FAK function was inhibited via adenoviral-mediated overexpression of the natural FAK inhibitor FAK-related nonkinase (FRNK). Ad/FRNK had no effect on Pyk2 expression, inhibited the PDGF-BB-induced migration, but did not change the PDGF-BB-induced proliferation. FAK deficiency had only modest effects on increasing PDGF-BB activation of p38 and JNK MAPKs, with no alteration in the ERK response vs. control cells. These results demonstrate that FAK is required for the PDGF-BB-induced migratory response of adult mouse CFs and suggest that FAK could play an essential role in the wound-healing response that occurs in numerous cardiac pathologies.
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Eddy, Robert J., Lynda M. Pierini, and Frederick R. Maxfield. "Microtubule Asymmetry during Neutrophil Polarization and Migration." Molecular Biology of the Cell 13, no. 12 (December 2002): 4470–83. http://dx.doi.org/10.1091/mbc.e02-04-0241.
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The development of cell polarity in response to chemoattractant stimulation in human polymorphonuclear neutrophils (PMNs) is characterized by the rapid conversion from round to polarized morphology with a leading lamellipod at the front and a uropod at the rear. During PMN polarization, the microtubule (MT) array undergoes a dramatic reorientation toward the uropod that is maintained during motility and does not require large-scale MT disassembly or cell adhesion to the substratum. MTs are excluded from the leading lamella during polarization and motility, but treatment with a myosin light chain kinase inhibitor (ML-7) or the actin-disrupting drug cytochalasin D causes an expansion of the MT array and penetration of MTs into the lamellipod. Depolymerization of the MT array before stimulation caused 10% of the cells to lose their polarity by extending two opposing lateral lamellipodia. These multipolar cells showed altered localization of a leading lamella-specific marker, talin, and a uropod-specific marker, CD44. In summary, these results indicate that F-actin– and myosin II-dependent forces lead to the development and maintenance of MT asymmetry that may act to reinforce cell polarity during PMN migration.
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Hodivala-Dilke, Kairbaan M., C. Michael DiPersio, Jordan A. Kreidberg, and Richard O. Hynes. "Novel Roles for α3β1 Integrin as a Regulator of Cytoskeletal Assembly and as a Trans-dominant Inhibitor of Integrin Receptor Function in Mouse Keratinocytes." Journal of Cell Biology 142, no. 5 (September 7, 1998): 1357–69. http://dx.doi.org/10.1083/jcb.142.5.1357.
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Previously we found that α3β1 integrin–deficient neonatal mice develop micro-blisters at the epidermal–dermal junction. These micro-blisters were associated with poor basement membrane organization. In the present study we have investigated the effect of α3β1-deficiency on other keratinocyte integrins, actin-associated proteins and F-actin organization. We show that the absence of α3β1 results in an increase in stress fiber formation in keratinocytes grown in culture and at the basal face of the basal keratinocytes of α3-null epidermis. Moreover, we see a higher concentration of actin-associated proteins such as vinculin, talin, and α-actinin at focal contact sites in the α3-deficient keratinocytes. These changes in focal contact composition were not due to a change in steady-state levels of these proteins, but rather to reorganization due to α3β1 deficiency. Apart from the loss of α3β1 there is no change in expression of the other integrins expressed by the α3-null keratinocytes. However, in functional assays, α3β1 deficiency allows an increase in fibronectin and collagen type IV receptor activities. Thus, our findings provide evidence for a role of α3β1 in regulating stress fiber formation and as a trans-dominant inhibitor of the functions of the other integrins in mouse keratinocytes. These results have potential implications for the regulation of keratinocyte adhesion and migration during wound healing.
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Shimada, Masako, Matthew J. Mahon, Peter A. Greer, and Gino V. Segre. "The Receptor for Parathyroid Hormone and Parathyroid Hormone-Related Peptide Is Hydrolyzed and Its Signaling Properties Are Altered by Directly Binding the Calpain Small Subunit." Endocrinology 146, no. 5 (May 1, 2005): 2336–44. http://dx.doi.org/10.1210/en.2004-1637.
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Abstract We show calcium-dependent, direct binding between the N-terminal portion of the PTH/PTHrP receptor (PTH1R) C-terminal intracellular tail and the calpain small subunit. Binding requires, but may not be limited to, amino acids W474, S475, and W477. The wild-type, full-length rat (r) PTH1R, but not rPTH1R with W474A/W477A substitutions, copurifies with the endogenous calpain small subunit in HEK293 cells. Calpain hydrolyzes ΔNt-rPTH1R, a receptor with a 156-amino acid N-terminal deletion, in a calcium-dependent manner in vitro and in intact cells. Most importantly, PTH stimulation increases the cleavage of ΔNt-rPTH1R and rPTH1R-yellow fluorescent protein in HEK293 cells, and of talin in HEK293 cells expressing rPTH1R-yellow fluorescent protein and in ROS17/2.8 osteoblast-like cells that express rPTH1R endogenously. The absence of calpain in Capn4-null embryonic fibroblasts and the lowered calpain activity in MC3T3-E1 osteoblastic cells due to stable expression of the calpain inhibitor, calpastatin, reduce PTH-stimulated cAMP accumulation. The calpain small subunit is the second protein, in addition to the sodium-hydrogen exchanger regulatory factor, and the first enzyme that binds the PTH1R; PTH1R bound to both of these proteins results in altered PTH signaling.
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McClung, J. M., A. R. Judge, E. E. Talbert, and S. K. Powers. "Calpain-1 is required for hydrogen peroxide-induced myotube atrophy." American Journal of Physiology-Cell Physiology 296, no. 2 (February 2009): C363—C371. http://dx.doi.org/10.1152/ajpcell.00497.2008.
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Recent reports suggest numerous roles for cysteine proteases in the progression of skeletal muscle atrophy due to disuse or disease. Nonetheless, a specific requirement for these proteases in the progression of skeletal muscle atrophy has not been demonstrated. Therefore, this investigation determined whether calpains or caspase-3 is required for oxidant-induced C2C12 myotube atrophy. We demonstrate that exposure to hydrogen peroxide (25 μM H2O2) induces myotube oxidative damage and atrophy, with no evidence of cell death. Twenty-four hours of exposure to H2O2 significantly reduced both myotube diameter and the abundance of numerous proteins, including myosin (−81%), α-actinin (−40%), desmin (−79%), talin (−37%), and troponin I (−80%). Myotube atrophy was also characterized by increased cleavage of the cysteine protease substrate αII-spectrin following 4 h and 24 h of H2O2 treatment. This degradation was blocked by administration of the protease inhibitor leupeptin (10 μM). Using small interfering RNA transfection of mature myotubes against the specific proteases calpain-1, calpain-2, and caspase-3, we demonstrated that calpain-1 is required for H2O2-induced myotube atrophy. Collectively, our data provide the first evidence for an absolute requirement for calpain-1 in the development of skeletal muscle myotube atrophy in response to oxidant-induced cellular stress.
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Crowley, E., and A. F. Horwitz. "Tyrosine phosphorylation and cytoskeletal tension regulate the release of fibroblast adhesions." Journal of Cell Biology 131, no. 2 (October 15, 1995): 525–37. http://dx.doi.org/10.1083/jcb.131.2.525.
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We have investigated the mechanisms by which fibroblasts release their adhesions to the extracellular matrix substrata using a permeabilized cell system in which the adhesions remain relatively stable. A large number of different molecules were assayed for their effect on focal adhesion stability using immunofluorescence with antibodies against different focal adhesion constituents. ATP uniquely stimulates a rapid breakdown of focal adhesions, and at high ATP concentrations (> 5 mM), many cells are released from the dish. The remaining cells appear contracted with talin, alpha-actinin, and vinculin localized diffusely throughout the cell. Integrin containing tracks of variable intensity outline the regions where cells had resided before they detached from the substratum. At lower ATP concentrations (0.5-5 mM) the cells remain spread; however the focal adhesion components, including integrin, show an array of phenotypes ranging from diffusely localized throughout the cell to a localization in small, thin focal adhesions. Okadaic acid, a serine, threonine phosphatase inhibitor, enhances the contracted phenotype, even at low concentrations (0.5 mM) of ATP. The localization of focal adhesion components is different in okadaic acid-treated cells. In highly contracted cells, integrin is present in tracks where the cells resided before the contraction; however focal adhesions are no longer apparent. Talin, vinculin, and alpha-actinin localize in trabecular networks toward the periphery of the cell. Interestingly, phosphotyrosine staining as well as nascent, intracellular integrin precedes the recruitment of focal adhesion constituents into the trabecular network. The ATP-stimulated focal adhesion breakdown appears to operate through two mechanisms. First, ATP stimulates the tyrosine phosphorylation of several cytoskeletally associated proteins. These tyrosine phosphorylations correlated well with focal adhesion breakdown. Furthermore, addition of a recombinant, constitutively active tyrosine phosphatase inhibits both the tyrosine phosphorylations and the breakdown of the focal adhesions. None of the major tyrosine phosphoproteins are FAK, integrin, tensin, paxillin, or other phosphoproteins implicated in focal adhesion assembly. The second mechanism is cell contraction. High ATP concentrations, or lower ATP concentrations in the presence of okadaic acid induce cell contraction. Inhibiting the contraction by addition of a heptapeptide IRICRKG, which blocks the actin-myosin interaction, also inhibits focal adhesion breakdown. Neither the peptide nor the phosphatase inhibits focal adhesion breakdown under all conditions suggesting that both tension and tyrosine phosphorylations mediate the release of adhesions.
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Bloch, Daria, Meirav Lavy, Yael Efrat, Idan Efroni, Keren Bracha-Drori, Mohamad Abu-Abied, Einat Sadot, and Shaul Yalovsky. "Ectopic Expression of an Activated RAC inArabidopsisDisrupts Membrane Cycling." Molecular Biology of the Cell 16, no. 4 (April 2005): 1913–27. http://dx.doi.org/10.1091/mbc.e04-07-0562.
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Rho GTPases regulate the actin cytoskeleton, exocytosis, endocytosis, and other signaling cascades. Rhos are subdivided into four subfamilies designated Rho, Racs, Cdc42, and a plant-specific group designated RACs/Rops. This research demonstrates that ectopic expression of a constitutive active Arabidopsis RAC, AtRAC10, disrupts actin cytoskeleton organization and membrane cycling. We created transgenic plants expressing either wild-type or constitutive active AtRAC10 fused to the green fluorescent protein. The activated AtRAC10 induced deformation of root hairs and leaf epidermal cells and was primarily localized in Triton X-100–insoluble fractions of the plasma membrane. Actin cytoskeleton reorganization was revealed by creating double transgenic plants expressing activated AtRAC10 and the actin marker YFP-Talin. Plants were further analyzed by membrane staining with N-[3-triethylammoniumpropyl]-4-[p-diethylaminophenylhexatrienyl] pyridinium dibromide (FM4-64) under different treatments, including the protein trafficking inhibitor brefeldin A or the actin-depolymeryzing agents latrunculin-B (Lat-B) and cytochalasin-D (CD). After drug treatments, activated AtRAC10 did not accumulate in brefeldin A compartments, but rather reduced their number and colocalized with FM4-64–labeled membranes in large intracellular vesicles. Furthermore, endocytosis was compromised in root hairs of activated AtRAC10 transgenic plants. FM4-64 was endocytosed in nontransgenic root hairs treated with the actin-stabilizing drug jasplakinolide. These findings suggest complex regulation of membrane cycling by plant RACs.
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Anderson, S. I., N. A. Hotchin, and G. B. Nash. "Role of the cytoskeleton in rapid activation of CD11b/CD18 function and its subsequent downregulation in neutrophils." Journal of Cell Science 113, no. 15 (August 1, 2000): 2737–45. http://dx.doi.org/10.1242/jcs.113.15.2737.
Full textAbstract:
When rolling adherent neutrophils are stimulated, they rapidly immobilize through activation of integrin CD11b/CD18, and then modulate attachment through this integrin to allow migration. We investigated links between cytoskeletal rearrangement and changes in function of integrin CD11b/CD18 in neutrophils stimulated with formyl peptide (fMLP). Neutrophils treated with the actin-polymerizing agent jasplakinolide became rolling adherent on monolayers of activated platelets, but could not use CD11b/CD18 to become immobilised when fMLP was perfused over them. If treated with jasplakinolide after fMLP, the cells stopped migrating but could not detach when fMLP was removed. Jasplakinolide did not inhibit changes in intracellular Ca(2+) seen after fMLP treatment, or inhibit neutrophil immobilisation induced by externally added Mn(2+). Thus cytoskeletal rearrangement was directly implicated in upregulation and, later, downregulation of CD11b/CD18 binding. Inhibition of RhoA with C3-transferase caused a dose-dependent reduction of initial rolling adhesion of neutrophils, and reduced the rate of migration after stimulation; however, neither the conversion of rolling to stationary adhesion, nor the ability of neutrophils to detach on removal of the stimulus, were inhibited. Thus, Rho may regulate actin polymerisation and motility in neutrophils, but did not appear to control integrin-mediated adhesion itself. Integrin binding may be promoted by disruption of links to the cytoskeleton, effected through depolymerisation of actin or cleavage of linking protein talin by calpain. Disruption of actin filaments with cytochalasin D did not, however, cause integrin-mediated immobilisation of rolling neutrophils. Although the calpain inhibitor calpeptin did inhibit the adhesion response to fMLP, this was only at doses where actin polymerisation was also ablated. We suggest that the cytoskeleton actively regulates binding conformation of CD11b/CD18 as well as its mobility in the membrane.
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Lindsay, Cory, Seema Bhatlekar, Raul Teruel-Montoya, Lin Ma, Martha Sola-Visner, Joseph E. Italiano, Leonard C. Edelstein, and Paul F. Bray. "Functionally Distinct Cell Populations Characterize Cord Blood Derived Megakaryocytes." Blood 126, no. 23 (December 3, 2015): 4754. http://dx.doi.org/10.1182/blood.v126.23.4754.4754.
Full textAbstract:
Abstract A fundamental understanding of platelet biology requires functional assessment of megakaryocyte-platelet expressed genes and transcripts. The need for gene functional studies has recently increased with unbiased genome wide associations with platelet phenotypes. Animal models with an over-expressed or knocked-out gene have been particularly valuable systems, although species differences may preclude optimal evaluation. Assessing protein function in human platelets can be accomplished with pharmacologic inhibitors, although inhibitor availability and specificity can plague this approach. Studies with primary human megakaryocytes (MKs) are greatly challenged by inaccessibility, low yields and contamination with other cells. Cultured human megakaryocyte-like cell lines (e.g., Meg-01, HEL and others) have proven valuable, but these cells poorly recapitulate platelet physiology. Increasingly, investigators are using cultured human MKs to study gene function. CD34+ hematopoietic stem cells from human cord blood are routinely used to generate megakaryocytes in culture. This system has advantages of being human, relatively easy to obtain, cultured from primary cells and able to be genetically manipulated. Although cord blood-derived megakaryocytes (CBDMs) have proven useful for studying aspects of megakaryocyte/platelet biology, there is little information about the heterogeneous cell populations observed in CBDMs. In the course of our studies with CBDMs we observed multiple distinct cell populations by flow cytometry. Similar populations were also observed in the megakaryocytic Meg-01 cell line. The largest cells (called P1) were the most abundant (consisting of nearly 100% of cells) at day 3 in culture. P2 cells, which are smaller and more granular than P1, appeared at day 6 and by day 13 were ~50% of the total. P3 appeared at day 6 and are the smallest, with size and granularity roughly similar to platelets; by day 13 these were ~30% of the total. P1 but not P2 nor P3 became CD61/CD41/CD42 positive and CD34 negative over 13 days in culture. Ninety-seven percent and 93% of P2 and P3 cells, respectively, were phosphatidyl serine (PS) positive, whereas 93% of P1 cells were PS negative. Electron microscopy revealed many typical features of bone marrow MKs in the PS negative (P1) cells, including large size, polyploid nucleus, mitochondria and immature granules. However, the demarcation membrane system was poorly developed. Virtually all of the PS positive (P2) cells were apoptotic, lacked granules and had no discernable nuclei. Purification of P1 and P2 populations followed by re-culturing revealed that P1 gives rise to both the P2 and P3 populations, whereas P2 gave rise to no other population. CD34+ cells cultured with the pan-caspase inhibitor Z-VAD-FMK developed a much smaller proportion of P2 cells with a corresponding increase in P1 cells. Stimulation of these 3 populations with collagen related peptide, thrombin, protease activated receptor 1-activating peptide (PAR1-AP) and PAR4-AP showed strong integrin activation in P1 cells, but not in P2 nor P3 cells. To assess which population was susceptible to genetic manipulation, day 3 cultures were infected with a lentiviral construct containing a shRNA to talin. Talin knockdown prevented agonist-induced integrin activation in P1 cells and as expected, had no effect on the P2 or P3 population. In summary, these data indicate that only a portion of CBDMs are functional and only the P1 population should be used to assess MK gene function. Over time, the majority of CBDMs become apoptotic and the smaller P3, platelet-like particles have minimal response to agonist compared to peripheral blood platelets. These results may partially explain the difficulties in generating functional platelets in vitro and are consistent with the need to restrain apoptosis for successful megakaryocytopoiesis. Disclosures No relevant conflicts of interest to declare.
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Schubert, Peter, Jonathan N. Thon, Geraldine M. Walsh, Katherine Serrano, Edwin D. Moore, Juergen Kast, and Dana V. Devine. "A Novel Marker for Blood Platelet Storage Lesion: Newly Synthesized Rap1 in Integrin Signaling." Blood 110, no. 11 (November 16, 2007): 4020. http://dx.doi.org/10.1182/blood.v110.11.4020.4020.
Full textAbstract:
Abstract Platelet transfusion is a standard live-saving medical procedure for patients with platelet-deficient diseases like leukemia. Platelets have a limited shelf-life of 5–7 days for transfusion purposes. This is in part due to a storage-related deterioration typified by altered morphology features and platelet metabolism as well as increased platelet activation. While the manifestations of the platelet storage lesion (PSL) are well known, the precise biochemical pathways involved in the initiation or exacerbation of this process have yet to be identified. Recently, we analyzed protein changes in the platelet proteome at day 1 and day 7 of storage by using a comprehensive proteomic approach. Out of 503 proteins, twelve were identified as significantly and consistently changing in relative concentration across all proteomic probes, including glycoprotein (GP) IIb/IIIa, Rap1 and talin, which showed an increase in their concentration paralleled with an increase in surface expression of GPIIb/IIIa. Synthesis of Rap1 in stored platelets could be demonstrated after incubation with the translational inhibitor rapamycin at a final concentration of 10 nM for twelve hours and subsequent activation with thrombin. Flow cytometry revealed that storage lead to a moderate level of platelet activation (10.5 ± 1.4% at day1 and 28.8 ± 1.3% at day 7 of storage) compared to ADP-treated controls (73.7 ± 1.2%) monitored by CD62P surface expression, in a rapamycin-independent manner. Microscopic analyses revealed similar re-localization patterns for GPIIIa, Rap1 and talin comparing changes during platelet storage and agonist induced activation. A significant correlation (p=0.007) between Rap1 activation and CD62P surface expression was seen. This observation is in strong agreement with a model for platelet GP IIb/IIIa activation [Han et al., Current Biology, 16, 1796–1806, 2007] also suggesting a calcium-dependent initiation of signal transduction. To analyze this hypothesis in more detail, treatment of platelets sampled at days 1, 4 and 7 of storage with 1 mM EDTA for 2 hours resulted in decreased Rap1 activation, in reduced surface expression of CD41 (GPIIb) and CD61 (GPIIIa) as well as in a lower level of platelet activation compared to untreated controls, respectively. This study unravels one aspect of the PSL, showing involvement of integrin signaling and identifying Rap1 as a novel marker for PSL. Therefore, this pathway offers potential targets for intervention which might lead to a reduction in platelet activation during storage, and may enable platelets to be stored for longer periods of time. From a transfusion point of view, however, extending the shelf-life of platelet units will ultimately need to be balanced with maintaining the quality of transfused platelets, their functionality, and efficacy in the patient.
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Scheffold, Annika, Billy Michael Chelliah Jebaraj, Eugen Tausch, Anella Yahiaoui, Anna Dolnik, Tamara Jacqueline Blaette, Lars Bullinger, et al. "In Vivo modeling of Resistance to PI3Kδ Inhibitor Treatment Using EµTCL1-Tg Tumor Transfer Model." Blood 128, no. 22 (December 2, 2016): 190. http://dx.doi.org/10.1182/blood.v128.22.190.190.
Full textAbstract:
Abstract Inhibitors of B-cell receptor (BCR) signaling have proven effective in the treatment of chronic lymphocytic leukemia (CLL). An important downstream mediator of BCR signaling is phosphoinositide 3-kinase delta (PI3Kd), which through activation of AKT, controls cell survival, growth and proliferation. Since it is expressed predominantly in cells of hematopoietic origin, PI3Kd is a promising target in CLL, and the novel PI3Kd inhibitor idelalisib is effective in treating relapsed/refractory CLL. However, a subset of patients relapse under therapy, and the mechanisms leading to resistance are not understood. To generate and study resistance mechanisms in vivo, a tool compound GS-649443 with favorable murine pharmacokinetic properties in vivo as compared to Idelalisib was used. GS-649443 is a highly specific and potent small molecule inhibitor of the PI3K isoform d, with an IC50 of 0.3nM and 86/420/4120 fold selectivity over PI3K isoforms g/b/a respectively. We performed an in vivo serial transfer and treatment scheme to generate specific resistance against GS-649443 using the transferable murine CLL clone TCL1-192(Chen SS et al., 2013). 5 million TCL1-192 cells were transferred by intravenous injection into the tail vein of CB17.SCID mice, followed by treatment with vehicle (N=12) or 5mg/kg GS-649443 (N=12) twice daily by oral gavage, starting from day 10. Analysis of the animals (N=6 vs. 6) at the death of the vehicle treated mice (11 days) showed a drastic reduction in spleen weight (median 0.650g vs. 0.345g, P=0.005), liver weight (median2.025g vs. 1.145g, P=0.0022) and WBC count (median220.4 cells/nl vs. 1.7 cells/nl, P=0.0078) as compared to vehicle treated mice. Also, GS-649443 treatment almost doubled the survival of the mice in comparison to vehicle treatment (Fig. 1, P=0.0011). To generate resistance against GS-649443 in vivo, TCL1-192 cells were kept under selection pressure using serial transfers and subsequent treatment. After three rounds of transfer and treatment, the mice treated with GS-649443 failed to respond and showed a survival identical to vehicle controls (Fig.1). Mutations acquired over time during the serial transfers in TCL1-192 cells from resistant mice were identified using whole exome sequencing. 64 treatment specific mutations were identified with a tumor variant allele frequency of ≥10%. Intriguingly, no single recurrent mutation that would likely contribute to drug resistance was identified. This is similar to the observation in patients refractory to idelalisib (abstract Ghia et al., submitted to ASH 2016). However, a subset of the genes such as Grb2, Mylk, Srfbp1, Ptk2, Cd44 and Prkd1 harboring mutations could be functionally grouped into integrin and extracellular matrix signaling. Consistent with this, all the resistant tumors showed a significant upregulation of genes involved in the integrin receptor complex such as talin 1 (P=0.004), talin 2 (P=0.004) and vinculin (P=0.009). RNAseq analysis for detecting changes in gene expression identified an upregulation of Igf1r expression in the resistant samples compared to vehicle treated samples (P<0.0001), which could be confirmed at the protein level using western blotting. The upregulation of Igf1r may be attributed to downregulation of WT1 in all the resistant samples (P=0.0061). WT1 is a transcriptional repressor of Igf1r, whose expression is mediated by PI3K/AKT. Treatment with GS-649443 in general led to an increase in Igf1r levels in all tumor samples compared to vehicle treatment but the increase was much more robust in the resistant TCL1-192 samples. Targeting Igf1r using linsitinib as a single agent failed to show a response (P=0.6538) since Igf1r was found to revert to normal levels in the absence of GS-649443. In contrast,the combination treatment with linsitinib and GS-649443 significantly improved survival of mice with resistant TCL1-192 as well as mice with sensitive TCL1-192 (P=0.0074 vs. P=0.0023). The synergy between the receptor tyrosine kinases such as IGF1R and integrin mediated signaling is established in different cancers and a similar mechanism may play a role in compensating for the inhibition of BCR mediated PI3K/AKT signaling. In conclusion, resistance to a tool PI3Kd inhibitor is not mediated by unique recurrent mutations but by alterations in survival signaling with mutations playing a probable cooperative role. Treatment scheme for generation of resistance in vivo. Treatment scheme for generation of resistance in vivo. Disclosures Tausch: Celgene: Other: Travel support; Amgen: Other: Travel support; Gilead: Other: Travel support, Speakers Bureau. Yahiaoui:Gilead Sciences: Employment. Munugalavadla:Gilead Sciences: Employment, Equity Ownership. Tannheimer:Gilead Sciences: Employment. Stilgenbauer:Pharmacyclics: Consultancy, Honoraria, Other: Travel grants , Research Funding; GSK: Consultancy, Honoraria, Other: Travel grants , Research Funding; Novartis: Consultancy, Honoraria, Other: Travel grants , Research Funding; Amgen: Consultancy, Honoraria, Other: Travel grants, Research Funding; Sanofi: Consultancy, Honoraria, Other: Travel grants , Research Funding; Boehringer Ingelheim: Consultancy, Honoraria, Other: Travel grants , Research Funding; Gilead: Consultancy, Honoraria, Other: Travel grants , Research Funding; Janssen: Consultancy, Honoraria, Other: Travel grants , Research Funding; Hoffmann-La Roche: Consultancy, Honoraria, Other: Travel grants , Research Funding; AbbVie: Consultancy, Honoraria, Other: Travel grants, Research Funding; Mundipharma: Consultancy, Honoraria, Other: Travel grants , Research Funding; Genzyme: Consultancy, Honoraria, Other: Travel grants , Research Funding; Genentech: Consultancy, Honoraria, Other: Travel grants , Research Funding; Celgene: Consultancy, Honoraria, Other: Travel grants , Research Funding.
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Brohard-Bohn, Brigitte, Sabine Pain, Christilla Bachelot-Loza, Jacques Auger, and Francine Rendu. "Thiosulfinates Inhibit Platelet Aggregation and Microparticle Shedding at a Calpain-dependent Step." Thrombosis and Haemostasis 86, no. 11 (2001): 1284–91. http://dx.doi.org/10.1055/s-0037-1616063.
Full textAbstract:
SummaryThiosulfinates (TSs) are sulfur compounds generated through the processing of different Allium species with antiplatelet property. To further define this platelet inhibitory effect we studied diallyl-TS (Al2TS), dipropyl-TS (Pr2TS), and dimethyl-TS (Me2TS) on platelet responses. The three TSs inhibited dose-dependent platelet aggregation, with IC50 values of 15 ± 2, 19 ± 2, and 9 ± 1 μM for Al2TS, Pr2TS and Me2TS, respectively. TSs had no effect on the expression of a platelet procoagulant surface, measured by flow cytometry as the binding of annexin V-FITC. They inhibited the microparticle shedding and clot retraction. Since the microparticle shedding is a calpain-activation dependent step, we assessed calpain activation by analysis of autoproteolysis in shorter active forms and by talin proteolysis in the presence of TSs. Calpain activation was inhibited by TSs independently of fibrinogen binding. Thus, TSs represent a new category of platelet inhibitors, acting on cal-pain-induced events.
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Márquez, María Gabriela, María del Carmen Fernández-Tome, Nicolás Octavio Favale, Lucila Gisele Pescio, and Norma Beatriz Sterin-Speziale. "Bradykinin modulates focal adhesions and induces stress fiber remodeling in renal papillary collecting duct cells." American Journal of Physiology-Renal Physiology 294, no. 3 (March 2008): F603—F613. http://dx.doi.org/10.1152/ajprenal.00234.2007.
Full textAbstract:
Focal adhesions (FAs) are specialized regions of cell attachment to the extracellular matrix. Previous works have suggested that bradykinin (BK) can modulate cell-matrix interaction. In the present study, we used a physiological cellular model to evaluate the potential role of BK in modulating FAs and stress fibers. We performed a quantitative morphometric analysis of FAs in primary cultured rat renal papillary collecting duct cells, which included size, axial ratio (shape), and average length. After 1, 5, or 10 min of incubation with BK, cultured cells were immunostained and analyzed by confocal microscopy. Although the shape of FAs was not altered, BK induced a decrease in the number of vinculin-stained FAs per cell, and a decrease in both their size and their average length, but not in talin-containing FAs, thus suggesting that BK could be inducing a restructuring of FAs. BK also induced a remodeling of the actin filament assemblies rather than their dissipation. Since we have previously demonstrated that BK stimulates activation of PLCβ in rat renal papillae, we attempted to determine whether BK can modulate FA restructuring by this mechanism, by pretreating cultured cells with the PLCβ inhibitor U73122. The present study, performed under physiological conditions with cells that were not genetically manipulated, provides new experimental evidence supporting the notion that the intrarenal hormone BK modulates FAs and actin cytoskeleton organization through a mechanism that involves the activation of PLCβ. We propose this finding as a novel mechanism for BK modulation of tubular collecting duct function.
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Sedwick, Caitlin E., Margaret M. Morgan, Lismaida Jusino, Judy L. Cannon, Jim Miller, and Janis K. Burkhardt. "TCR, LFA-1, and CD28 Play Unique and Complementary Roles in Signaling T Cell Cytoskeletal Reorganization." Journal of Immunology 162, no. 3 (February 1, 1999): 1367–75. http://dx.doi.org/10.4049/jimmunol.162.3.1367.
Full textAbstract:
Abstract T cells interacting with APCs undergo rearrangement of surface receptors and cytoskeletal elements to face the zone of contact with the APC. This polarization process is thought to affect T cell signaling by organizing a specialized domain on the T cell surface and to direct T cell effector function toward the appropriate APC. We have investigated the contribution of TCR, CD28, and LFA-1 signaling to T cell cytoskeletal polarization by assaying the response of an Ag-specific Th1 clone toward a panel of transfected APCs expressing MHC class II alone or in combination with ICAM-1 or B7-1. We show that polarization of talin, an actin-binding protein, occurs in response to integrin engagement. In contrast, reorientation of the T cell microtubule-organizing center (MTOC) is dependent on and directed toward the site of TCR signaling, regardless of whether integrins or costimulatory molecules are engaged. MTOC reorientation in response to peptide-MHC complexes is sensitive to the phosphatidylinositol 3-kinase inhibitor wortmannin. CD28 coengagement overcomes this sensitivity, as does activation via Ab cross-linking of the TCR or via covalent peptide-MHC complexes, suggesting that phosphatidylinositol 3-kinase is not required per se but rather plays a role in signal amplification. Engagement of TCR in trans with LFA-1 results in separation of MTOC reorientation and cortical cytoskeletal polarization events, indicating that the two processes are not directly mechanistically linked. These studies show that T cells mobilize individual cytoskeletal components in response to distinct and specific cell surface interactions.
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Finlay, B. B., S. Ruschkowski, and S. Dedhar. "Cytoskeletal rearrangements accompanying salmonella entry into epithelial cells." Journal of Cell Science 99, no. 2 (June 1, 1991): 283–96. http://dx.doi.org/10.1242/jcs.99.2.283.
Full textAbstract:
Salmonella bacteria can enter (invade) eukaryotic cells, and exist as intracellular parasites. Confocal, light immunofluorescence and electron microscopy were used to examine various cytoskeletal components of cultured Madin Darby canine kidney (MDCK) and HeLa epithelial cells after infection with Salmonella typhimurium. These bacteria entered and remained within membrane-bound vacuoles and were surrounded by large (5–10 microns) dense structures composed of various cytoskeletal components. These structures consisted of extensive aggregations of polymerized actin, alpha-actinin and tropomyosin above and beside the invading bacterium in both epithelial cell lines. These structures were evident soon after bacterial addition (maximal at 20 min for HeLa cells, 60 min for MDCK cells), and disappeared later in the infection as the cytoskeletal components returned to a more normal distribution after bacterial internalization. Surprisingly, tubulin also aggregated above internalized Salmonella although bacterial entry or penetration through polarized monolayers was not disrupted by the microtubule-inhibiting agent nocadazole (this treatment actually enhanced tubulin accumulation around these organisms). There were little if any rearrangements in intermediate filaments composed of keratin or vimentin. Large amounts of talin also accumulated above and around invading Salmonella, but there was only a minor accumulation of vinculin around a few organisms. Pretreatment of epithelial cells with the microfilament inhibitor cytochalasin D blocked bacterial internalization but did not prevent accumulation of polymerized actin and alpha-actinin directly beneath uninternalized bacteria, yet prevented accumulation of the other cytoskeletal components. These results suggest that Salmonella bind to the surface and trigger a signal in epithelial cells that causes marked rearrangements in various cytoskeletal components, including recruitment of actin filaments and alpha-actinin, which then generates the force necessary for bacterial uptake.
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Rodríguez-Fernández, Lucía, Iván Ferrer-Vicens, Concha García, Sara S. Oltra, Rosa Zaragozá, Juan R. Viña, and Elena R. García-Trevijano. "Isoform-specific function of calpains in cell adhesion disruption: studies in postlactational mammary gland and breast cancer." Biochemical Journal 473, no. 18 (September 12, 2016): 2893–909. http://dx.doi.org/10.1042/bcj20160198.
Full textAbstract:
Cleavage of adhesion proteins is the first step for physiological clearance of undesired cells during postlactational regression of the mammary gland, but also for cell migration in pathological states such as breast cancer. The intracellular Ca2+-dependent proteases, calpains (CAPNs), are known to cleave adhesion proteins. The isoform-specific function of CAPN1 and CAPN2 was explored and compared in two models of cell adhesion disruption: mice mammary gland during weaning-induced involution and breast cancer cell lines according to tumor subtype classification. In both models, E-cadherin, β-catenin, p-120, and talin-1 were cleaved as assessed by western blot analysis. Both CAPNs were able to cleave adhesion proteins from lactating mammary gland in vitro. Nevertheless, CAPN2 was the only isoform found to co-localize with E-cadherin in cell junctions at the peak of lactation. CAPN2/E-cadherin in vivo interaction, analyzed by proximity ligation assay, was dramatically increased during involution. Calpain inhibitor administration prevented the cytosolic accumulation of truncated E-cadherin cleaved by CAPN2. Conversely, in breast cancer cells, CAPN2 was restricted to the nuclear compartment. The isoform-specific expression of CAPNs and CAPN activity was dependent on the breast cancer subtype. However, CAPN1 and CAPN2 knockdown cells showed that cleavage of adhesion proteins and cell migration was mediated by CAPN1, independently of the breast cancer cell line used. Data presented here suggest that the subcellular distribution of CAPN1 and CAPN2 is a major issue in target-substrate recognition; therefore, it determines the isoform-specific role of CAPNs during disruption of cell adhesion in either a physiological or a pathological context.
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Kawashima, Ikko, Zhilin Liu, Lisa K. Mullany, Toshihiro Mihara, Joanne S. Richards, and Masayuki Shimada. "EGF-Like Factors Induce Expansion of the Cumulus Cell-Oocyte Complexes by Activating Calpain-Mediated Cell Movement." Endocrinology 153, no. 8 (June 6, 2012): 3949–59. http://dx.doi.org/10.1210/en.2012-1059.
Full textAbstract:
Cumulus cell-oocyte complex (COC) expansion is obligatory for LH-induced ovulation and is initiated by LH induction of the epidermal growth factor (EGF)-like factors that mediate the synthesis of the hyaluronan-rich matrix and hyaluronan-stabilizing factors. COC expansion also involves the movement of cumulus cells within the matrix by mechanisms that have not been characterized. We document herein that two proteases, calpain 2 and to a lesser extent calpain 1, are expressed in cumulus cells and that the proteolytic activity of these enzymes is rapidly and significantly increased in COC isolated from human chorionic gonadotropin-induced ovulatory follicles in vivo. Stimulation of calpain activity was associated with proteolytic degradation of paxillin and talin (two components of focal adhesion complexes), cell detachment, and the formation of cell surface bleb-like protrusions. Injection of a calpain inhibitor in vivo reduced 1) human chorionic gonadotropin-stimulated calpain enzyme activity, 2) cell detachment, 3) membrane protrusion formation, and 4) COC expansion by mechanisms that did not alter Has2 expression. During EGF-like factor induction of COC expansion in culture, calpain activity was increased by ERK1/2 and intracellular Ca2+ signaling pathways. Inhibition of calpain activity in cultured COC blocked cumulus cell detachment, protrusion formation, and the vigorous movement of cumulus cells. As a consequence, COC expansion was impaired. Collectively, these results show that two highly coordinated processes control COC expansion. One process involves the synthesis of the hyaluronan matrix, and the other mediates cumulus cell detachment and movement. The latter are controlled by calpain activation downstream of the EGF receptor activation of the Ca2+ pathway and ERK1/2 pathways.
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Gong, Haixia, and Xiaoping Du. "Ga13 and Talin Coordinate integrin Inside-out and Outside-In Signaling by “time-sharing” of b3 cytoplasmic Domain." Blood 116, no. 21 (November 19, 2010): 162. http://dx.doi.org/10.1182/blood.v116.21.162.162.
Full textAbstract:
Abstract Abstract 162 The bidirectional signaling of integrins mediates cell adhesion, spreading, retraction and migration. The binding of talin and kindlins to the cytoplasmic domain of integrin b3 subunit transmits inside-out signals to induce integrin activation. Ligand-induced outside-in signaling requires the binding of a G protein subunit, Ga13, and a tyrosine kinase, c-Src, to the b3 cytoplasmic domain. It is unclear how the short cytoplasmic domain of b3 accommodates these molecules and allows coordinated bidirectional signaling. Here we show that Ga13 and talin are mutually exclusive in binding to b3 both in vivo and in vitro. Increasing expression level of talin head or full-length talin in CHO123 cell decreases Ga13-b3 association. Ga13 also competes with talin head for GST-b3 binding in purified binding system. More importantly, talin is associated with b3 only in inside-out signaling during platelet aggregation. Following integrin ligation, however, Ga13 binds to b3, replacing talin. The Ga13 binding site located between K729-T741 within the talin binding region. However, Ga13 binding and signaling require a distinct ExE733 motif (EEE in b3) conserved in most integrin b subunits that is not required for talin binding but flanked by talin binding sequences on both sides. Interference of Ga13 binding to integrin b3 cytoplasmic domain by myristorylated b3 peptide (Myr-EEERA735) or by point-mutating the EEE motif to AAA selectively inhibits outside-in signaling, thus inhibited cell spreading on fibrinogen, accelerated RhoA activation and inhibited c-Src activity. But they have no effect on talin-dependent inside-out signaling judged by fibrinogen binding assay. In conclusion, our data suggest that the timed share of binding sites in b3 between Ga13 and talin coordinates bidirectional integrin signaling. Disclosures: No relevant conflicts of interest to declare.
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Lawson, Christine, Ssang-Taek Lim, Sean Uryu, Xiao Lei Chen, David A. Calderwood, and David D. Schlaepfer. "FAK promotes recruitment of talin to nascent adhesions to control cell motility." Journal of Cell Biology 196, no. 2 (January 23, 2012): 223–32. http://dx.doi.org/10.1083/jcb.201108078.
Full textAbstract:
Cell migration is a dynamic process that involves the continuous formation, maturation, and turnover of matrix–cell adhesion sites. New (nascent) adhesions form at the protruding cell edge in a tension-independent manner and are comprised of integrin receptors, signaling, and cytoskeletal-associated proteins. Integrins recruit focal adhesion kinase (FAK) and the cytoskeletal protein talin to nascent adhesions. Canonical models support a role for talin in mediating FAK localization and activation at adhesions. Here, alternatively, we show that FAK promotes talin recruitment to nascent adhesions occurring independently of talin binding to β1 integrins. The direct binding site for talin on FAK was identified, and a point mutation in FAK (E1015A) prevented talin association and talin localization to nascent adhesions but did not alter integrin-mediated FAK recruitment and activation at adhesions. Moreover, FAK E1015A inhibited cell motility and proteolytic talin cleavage needed for efficient adhesion dynamics. These results support an alternative linkage for FAK–talin interactions within nascent adhesions essential for the control of cell migration.
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Barry, S. T., and D. R. Critchley. "The RhoA-dependent assembly of focal adhesions in Swiss 3T3 cells is associated with increased tyrosine phosphorylation and the recruitment of both pp125FAK and protein kinase C-delta to focal adhesions." Journal of Cell Science 107, no. 7 (July 1, 1994): 2033–45. http://dx.doi.org/10.1242/jcs.107.7.2033.
Full textAbstract:
Mouse Swiss 3T3 fibroblasts cultured in serum-free medium lose their actin stress fibres and vinculin-containing focal adhesions, a process that can be reversed by the addition of serum, lysophosphatidic acid (LPA) or bombesin, and is mediated by rhoA (A. J. Ridley and A. Hall (1992) Cell 70, 389–399). We have shown that the addition of serum to these cells induces the recruitment of the cytoskeletal proteins talin, vinculin and paxillin, and the protein kinases pp125FAK and PKC-delta, to newly formed focal adhesions, and that alpha-actinin is distributed along the actin stress fibres associated with these structures. The newly formed focal adhesions stained heavily with an antibody to phosphotyrosine. A similar response was elicited by 100 ng/ml LPA. The effect of serum was rapid, with focal staining for paxillin largely restricted to cell margins seen within 2 minutes of serum addition, and preceding the assembly of actin filaments. Phosphotyrosine staining differed in that it was predominantly punctate and was widely distributed throughout the cell. By 5 minutes, the paxillin and phosphotyrosine staining was concentrated at the ends of actin filaments largely at the cell margins. The structures stained ranged from circular to oval, but by 10 minutes they more closely resembled the elongated focal adhesions found in cultured fibroblasts. Within 10 minutes, the addition of serum or LPA induced a marked increase in the levels of pp125FAK and paxillin immune-precipitated by an anti-phosphotyrosine antibody. The results suggest that both pp125FAK and paxillin undergo changes in tyrosine phosphorylation upon activation of rhoA, and that these changes are associated with the assembly of focal adhesions and actin stress fibres. The observation that formation of focal adhesions can be induced by the tyrosine phosphatase inhibitor vanadyl hydroperoxide is consistent with the direct involvement of tyrosine phosphorylation in the assembly process. The localisation of PKC-delta to newly formed focal adhesions suggests that serine/threonine phosphorylation may also be important in this regard.
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Podolnikova, Nataly P., Benjamin Bowen, Valeryi K. Lishko, Andriy V. Podolnikov, and Tatiana Ugarova. "Control of Platelet Adhesion by Rigidity Sensing at the Surface of Fibrin Clot." Blood 110, no. 11 (November 16, 2007): 3906. http://dx.doi.org/10.1182/blood.v110.11.3906.3906.
Full textAbstract:
Abstract Thrombus formation at sites of vascular injury must occur quickly to reduce blood loss, but is carefully controlled to limit vessel occlusion. Arrest of bleeding is mediated by adhesion and aggregation of platelets and the formation of the fibrin clot. While the interactions responsible for platelet adhesion and thrombus growth have been extensively researched, the mechanisms that limit platelet adhesion are not clear. We have previously demonstrated that plasma fibrinogen is a potent inhibitor of integrin-mediated leukocyte adhesion to fibrin clots and surface-bound fibrinogen, and have provided evidence that fibrinogen reduces cell adhesion by binding to the surface of fibrin rather than blocking leukocyte integrins. Accordingly, cells that engage fibrinogen molecules loosely bound to fibrin (soft substrate) are not able to consolidate their grip on the surface; subsequently, cells detach. Conversely, cells that adhere to the naked fibrin clot (rigid substrate) adhere firmly. Since fibrin and immobilized fibrinogen support platelet adhesion, we examined the effect of soluble fibrinogen on integrin αIIbβ3-mediated adhesion. We show that the anti-adhesive fibrinogen layer formed on the surface of fibrin inhibits platelet adhesion. We also demonstrate that fibrinogen immobilized on plastic at high densities (>20 μg/ml) supports weak platelet adhesion whereas at low concentrations (∼2 μg/ml) it is highly adhesive. An investigation of the mechanism underlying differential platelet adhesion indicates that platelet adhesion to rigid substrates (low-density fibrinogen and naked fibrin gel) induces much stronger phosphorylation of FAK and Syk kinases than that to soft substrates (high-density fibrinogen and fibrin exposed to soluble fibrinogen). Furthermore, the rigid, but not the soft substrates induce recruitment of signaling molecules talin and skelemin to αIIbβ3-containing focal adhesions. Consistent with their limited ability to induce sufficient signaling, soft substrates do not support platelet spreading. These data suggest that circulating fibrinogen prevents stable platelet adhesion by modifying the mechanical properties of the fibrin clot’s surface which results in reduced force generation and insufficient signaling.
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CHEN, Huaiyang, Akiko ISHII, Wai-Keung WONG, Lan Bo CHEN, and Su Hao LO. "Molecular characterization of human tensin." Biochemical Journal 351, no. 2 (October 10, 2000): 403–11. http://dx.doi.org/10.1042/bj3510403.
Full textAbstract:
Tensin is a focal-adhesion molecule that binds to actin filaments and interacts with phosphotyrosine-containing proteins. To analyse tensin's function in mammals, we have cloned tensin cDNAs from human and cow. The isolated approx. 7.7-kb human cDNA contains an open reading frame encoding 1735 amino acid residues. The amino acid sequence of human tensin shares 60% identity with chicken tensin, and contains all the structural features described previously in chicken tensin. This includes the actin-binding domains, the Src homology domain 2, and the region similar to a tumour suppressor, PTEN. Two major differences between human and chicken tensin are (i) the lack of the first 54 residues present in chicken tensin, and (ii) the addition of 34- and 38-residue inserts in human and bovine tensin. In addition, our interspecies sequencing data have uncovered the presence of a glutamine/CAG repeat that appears to have expanded in the course of evolution. Northern-blot analysis reveals a 10-kb message in most of the human tissues examined. An additional 9-kb message is detected in heart and skeletal muscles. The molecular mass predicted from the human cDNA is 185kDa, although both endogenous and recombinant human tensin migrate as 220-kDa proteins on SDS/PAGE. The discrepancy is due to the unusually low electrophoretic mobility of the central region of the tensin polypeptide (residues 306–981). A survey of human prostate and breast cancer cell lines by Western-blot analysis shows a lack of tensin expression in most cancer cell lines, whereas these lines express considerable amounts of focal-adhesion molecules such as talin and focal-adhesion kinase. Finally, tensin is rapidly cleaved by a focal-adhesion protease, calpain II. Incubation of cells with a calpain inhibitor, MDL, prevented tensin cleavage and induced morphological change in these cells, suggesting that cleavage of tensin and other focal-adhesion constituents by calpain disrupts maintenance of normal cell shape.
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Woods, A., and J. R. Couchman. "Protein kinase C involvement in focal adhesion formation." Journal of Cell Science 101, no. 2 (February 1, 1992): 277–90. http://dx.doi.org/10.1242/jcs.101.2.277.
Full textAbstract:
Matrix molecules such as fibronectin can promote cell attachment, spreading and focal adhesion formation. Although some interactions of fibronectin with cell surface receptors have now been identified, the consequent activation of intracellular messenger systems by cell/matrix interactions have still to be elucidated. We show here that the kinase inhibitors H7 and HA1004 reduce focal adhesion and stress fiber formation in response to fibronectin in a dose-dependent manner, and that activators of protein kinase C can promote their formation under conditions where they do not normally form. Fibroblasts spread within 1h on substrata composed of fibronectin and formed focal adhesions by 3h, as monitored by interference reflection microscopy (IRM) and by labeling for talin, vinculin and integrin beta 1 subunits. In addition, stress fibers were visible. When cells were allowed to spread for 1h and then treated with kinase inhibitors H7 and HA1004 for 2h, IRM indicated a reduction in focal adhesion formation at concentrations where protein kinase C (PKC) should be inhibited. In contrast, focal adhesions formed normally at concentrations of these inhibitors where cyclic AMP- or cyclic GMP-dependent kinases should be inactivated. Inhibition of PKC, but not that of cyclic AMP- or cyclic GMP-dependent kinases, also prevented the formation of stress fibers and induced a dispersal of talin and vinculin, but not integrin beta 1 subunits, from small condensations present at 1h. Consistent with the reduction in focal adhesion formation when PKC was inhibited, activation of PKC by 30 minutes of treatment with phorbol esters induced focal adhesion formation in cells spread for 3h on substrata composed of the cell-binding (RGD-containing) fragment of fibronectin, while untreated cells or those treated with inactive phorbol esters did not form these structures.
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Jones, P., P. Jackson, G. J. Price, B. Patel, V. Ohanion, A. L. Lear, and D. R. Critchley. "Identification of a talin binding site in the cytoskeletal protein vinculin." Journal of Cell Biology 109, no. 6 (December 1, 1989): 2917–27. http://dx.doi.org/10.1083/jcb.109.6.2917.
Full textAbstract:
Binding of the cytoskeletal protein vinculin to talin is one of a number of interactions involved in linking F-actin to cell-matrix junctions. To identify the talin binding domain in vinculin, we expressed the NH2-terminal region of the molecule encoded by two closely similar, but distinct vinculin cDNAs, using an in vitro transcription translation system. The 5' Eco RI-Bam HI fragment of a partial 2.89-kb vinculin cDNA encodes a 45-kD polypeptide containing the first 398 amino acids of the molecule. The equivalent restriction enzyme fragment of a second vinculin cDNA (cVin5) lacks nucleotides 746-867, and encodes a 41-kD polypeptide missing amino acids 167-207. The radiolabeled 45-kD vinculin polypeptide bound to microtiter wells coated with talin, but not BSA, and binding was inhibited by unlabeled vinculin. In contrast, the 41-kD vinculin polypeptide was devoid of talin binding activity. The role of residues 167-207 in talin binding was further analyzed by making a series of deletions spanning this region, each deletion of seven amino acids contiguous with the next. Loss of residues 167-173, 174-180, 181-187, 188-194, or 195-201 resulted in a marked reduction in talin binding activity, although loss of residues 202-208 had much less effect. When the 45-kD vinculin polypeptide was expressed in Cos cells, it localized to cell matrix junctions, whereas the 41-kD polypeptide, lacking residues 167-207, was unable to do so. Interestingly, some deletion mutants with reduced ability to bind talin in vitro, were still able to localize to cell matrix junctions.
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Park, Da Som, Se Eun Kim, Deog-Bon Koo, and Man-Jong Kang. "Histone deacetylases inhibitor and RAD51 recombinase increase transcription activator-like effector nucleases-mediated homologous recombination on the bovine β-casein gene locus." Asian-Australasian Journal of Animal Sciences 33, no. 6 (June 1, 2020): 1023–33. http://dx.doi.org/10.5713/ajas.19.0654.
Full textAbstract:
Objective: The efficiency of the knock-in process is very important to successful gene editing in domestic animals. Recently, it was reported that transient loosening of the nucleosomal folding of transcriptionally inactive chromatin might have the potential to enhance homologous recombination efficiency. The objective of this study was to determine whether histone deacetylases (HDAC) inhibitor and RAD51 recombinase (RAD51) expression were associated with increased knock-in efficiency on the β-casein (<i>bCSN2</i>) gene locus in mammary alveolar-large T antigen (MAC-T) cells using the transcription activator-like effector nucleases (TALEN) system.Methods: MAC-T cells were treated with HDAC inhibitors, valproic acid, trichostatin A, or sodium butyrate for 24 h, then transfected with a knock-in vector, RAD51 expression vector and TALEN to target the <i>bCSN2</i> gene. After 3 days of transfection, the knock-in efficiency was confirmed by polymerase chain reaction and DNA sequencing of the target site.Results: The level of HDAC 2 protein in MAC-T cells was decreased by treatment with HDAC inhibitors. The knock-in efficiency in MAC-T cells treated with HDAC inhibitors was higher than in cells not treated with inhibitors. However, the length of the homologous arm of the knock-in vector made no difference in the knock-in efficiency. Furthermore, DNA sequencing confirmed that the precision of the knock-in was more efficient in MAC-T cells treated with sodium butyrate.Conclusion: These results indicate that chromatin modification by HDAC inhibition and RAD51 expression enhanced the homologous recombination efficiency on the <i>bCSN2</i> gene locus in MAC-T cells.
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Yamanouchi, Jun, Takaaki Hato, Hiroshi Fujiwara, Yoshihiro Yakushijin, and Masaki Yasukawa. "Integrin β3 Membrane-Proximal and -Distal Residues Cooperatively Regulate Talin-Mediated αIIbβ3 Activation." Blood 112, no. 11 (November 16, 2008): 1848. http://dx.doi.org/10.1182/blood.v112.11.1848.1848.
Full textAbstract:
Abstract Integrin αIIbβ3 exists in a low affinity state in resting platelets and requires activation for high affinity binding with soluble ligands. Activation of αIIbβ3 is tightly linked to structural rearrangements of the αIIbβ3 molecule that is initiated from the cytoplasmic tails of the αIIb and β3 subunits. The β3 membrane-distal region has been shown to interact with many signaling and cytoskeletal molecules, and considered as a trigger point of integrin activation. The interaction of the β3 tail with a cytoplasmic protein, talin, largely contributes to integrin activation. In view of the link between integrin activation and allosteric structural rearrangements of integrins, one would expect that structural changes in the β3 membrane-distal region containing binding sites for intracellular proteins would be relayed to the membrane-proximal region, leading to αIIbβ3 activation. However, there has been no evidence that structural rearrangement of the β3 membrane-distal region is directly linked to integrin activation. No activating mutation has so far been reported in the β3 membrane-distal region despite numerous reports of loss-of-function mutants in this region. In this context, a previously reported αIIbβ3 mutant in which the β3 tail was replaced by the β1 tail was noteworthy. This chimeric integrin, αIIbβ3/β1, was constitutively active. Because the β1 and β3 subunits have relatively high sequence homology in their membrane-proximal regions, we reasoned that the residues differing between the β1 and β3 membrane-distal regions may be responsible for αIIbβ3 activation. To identify such residues, we produced 13 αIIbβ3 mutants in which the individual or group residues in the β3 tail were substituted with the corresponding β1 tail residues. The αIIbβ3 mutants were expressed on the surface of CHO cells by cotransfection of mutant β3 and wild-type αIIb cDNAs, and were tested for binding of fibrinogen and PAC1, a ligand-mimetic anti-αIIbβ3 antibody. Among them, only β3I719M and E749S mutants bound significant PAC1 and fibrinogen binding without any stimulation and the RGDS peptide abolished binding of these ligands, indicating a constitutively active state. The similar effect was observed with I719A and E749A mutants. Moreover, the I719M/E749S double mutant showed more PAC1 binding than the single mutants, reaching the same ligand binding activity as αIIbβ3/β1. These β3 mutations also induced αVβ3 activation. Conversely, substitution of M719 or S749 in the β1 tail with the corresponding β3 tail residue (M719I or S749E) inhibited αIIbβ3/β1 activation, and the M719I/S749E double mutant inhibited ligand binding to a level comparable with that of the wild-type αIIbβ3. Knock down of talin by short hairpin RNA inhibited the I719M- and E749S-induced αIIbβ3 activation, indicating talin-mediated activation of mutant integrins. Since I719 is located at the β3 membrane-proximal region, it is likely that the I719 mutation disrupts the well-known membrane-proximal clasp to maintain integrins at a low affinity state. On the other hand, E749 is located at the β3 membrane-distal region. This result provides experimental evidence that structural perturbation of the β3 membrane-distal region is linked to integrin activation. Moreover, our result showed that the mutational effects of the membrane-proximal I719 and the membrane-distal E749 residues were additive and talin-dependent, suggesting that the β3 membrane-proximal and –distal regions cooperatively regulate talin-mediated αIIbβ3 activation. This finding is consistent with a recent model of talin-induced αIIbβ3 activation in which talin cooperatively interacts with the β3 membrane-proximal and distal regions.