Calpain- and talin-dependent control of microvascular pericyte contractility and cellular stiffness

Kotecki, Maciej; Zeiger, Adam S.; Vliet, Krystyn J. Van; Herman, Ira M.

doi:10.1016/j.mvr.2010.07.012

Cited by 37 publications

(38 citation statements)

References 75 publications

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“…4G). The slightly lower C-terminal z-positions due to the L432G mutation may reflect the reduction in calpain-mediated cellular contractility documented previously (37). Consistent with this, the C-terminal z-position of wild-type T100 was similarly lowered upon pharmacological inhibition of calpain (z = 62.2 nm, Fig.…”

Section: Probing the Structural Role Of Talin By Recombinant Minitalinsupporting

confidence: 84%

“…This could be due to the force-induced unzipping of the R2-R3 four-helical bundles, which has been shown in vitro to occur at ∼5-7 pN; further experiments using R2-R3 stabilizing mutations (45) may help to verify this. Because calpain-mediated proteolysis of talin is required for FA turnover and the up-regulation of promigratory signaling pathways such as Src, the increased length of wild-type talin likely reflects a relatively higher talin tension resulting from greater contractility (36,37,46,47). In vitro force-extension experiments on talin rod domains allow this tension to be estimated, suggesting that wildtype talin sustains greater than 5 pN but less than 15 pN of tension, because another unfolding (hence lengthening) transition occurs at that level of force.…”

Section: Discussionmentioning

confidence: 99%

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Talin determines the nanoscale architecture of focal adhesions

Liu

Wang

Goh

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

182

169

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Insight into how molecular machines perform their biological functions depends on knowledge of the spatial organization of the components, their connectivity, geometry, and organizational hierarchy. However, these parameters are difficult to determine in multicomponent assemblies such as integrin-based focal adhesions (FAs). We have previously applied 3D superresolution fluorescence microscopy to probe the spatial organization of major FA components, observing a nanoscale stratification of proteins between integrins and the actin cytoskeleton. Here we combine superresolution imaging techniques with a protein engineering approach to investigate how such nanoscale architecture arises. We demonstrate that talin plays a key structural role in regulating the nanoscale architecture of FAs, akin to a molecular ruler. Talin diagonally spans the FA core, with its N terminus at the membrane and C terminus demarcating the FA/stress fiber interface. In contrast, vinculin is found to be dispensable for specification of FA nanoscale architecture. Recombinant analogs of talin with modified lengths recapitulated its polarized orientation but altered the FA/stress fiber interface in a linear manner, consistent with its modular structure, and implicating the integrin-talin-actin complex as the primary mechanical linkage in FAs. Talin was found to be ∼97 nm in length and oriented at ∼15°relative to the plasma membrane. Our results identify talin as the primary determinant of FA nanoscale organization and suggest how multiple cellular forces may be integrated at adhesion sites.superresolution microscopy | focal adhesions | talin | mechanobiology | nanoscale architecture C ell adhesion to the ECM is a highly coordinated process that involves ECM-specific recognition by integrin transmembrane receptors, and their aggregation with numerous cytoplasmic proteins into dense supramolecular complexes called focal adhesions (FAs) (1). Actin stress fibers terminate at FAs where actomyosin contractility is transmitted to the ECM, generating traction (2-5). Mechanical tension impinging on each FA is implicated in key steps including the elongation, reinforcement, and maintenance of the FA structures (6). FA mechanotransduction is a major aspect of cellular microenvironment sensing with wide-ranging consequences in physiological and pathological processes (7-10). However, molecular-scale spatial parameters that specify FA nanoscale organization have been difficult to access experimentally. Nevertheless, these are essential to understand how mechanosensitivity arises within such complex molecular machines (11-15).Previously 3D superresolution fluorescence microscopy has unveiled the nanoscale organization of major FA components, whereby a core region of ∼30 nm interposes between the integrin and the actin cytoskeleton along the vertical (z) axis (16). The FA core consists of a membrane-proximal layer that contains signaling proteins such as FAK (focal adhesion kinase) and paxillin, an intermediate zone that contains force-transduction proteins s...

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Section: Probing the Structural Role Of Talin By Recombinant Minitalinsupporting

confidence: 84%

Section: Discussionmentioning

confidence: 99%

Talin determines the nanoscale architecture of focal adhesions

Liu

Wang

Goh

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

182

169

View full text Add to dashboard Cite

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“…In a recent study, it was found that among the various cell types of the neurovascular unit, pericytes exhibited the highest level of MMP-9 secretion when challenged with thrombin that is known to induce barrier dysfunctions in endothelial cells (30). Furthermore, pericytes features such as contractility and cellular stiffness have been regulated by cellular calpains (31). Similarly, astrocytes are an integral component of the BBB that may be compromised by TBI or ischemic brain injury, and increased MMP-9 expression has been observed in astrocytes following trauma (32).…”

Section: Discussionmentioning

confidence: 99%

Attenuation of Blood-Brain Barrier Breakdown and Hyperpermeability by Calpain Inhibition

Alluri

Grimsley²,

Shaji

et al. 2016

Journal of Biological Chemistry

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Edited by Paul FraserBlood-brain barrier (BBB) breakdown and the associated microvascular hyperpermeability followed by brain edema are hallmark features of several brain pathologies, including traumatic brain injuries (TBI). Recent studies indicate that pro-inflammatory cytokine interleukin-1␤ (IL-1␤) that is upregulated following traumatic injuries also promotes BBB dysfunction and hyperpermeability, but the underlying mechanisms are not clearly known. The objective of this study was to determine the role of calpains in mediating BBB dysfunction and hyperpermeability and to test the effect of calpain inhibition on the BBB following traumatic insults to the brain. In these studies, rat brain microvascular endothelial cell monolayers exposed to calpain inhibitors (calpain inhibitor III and calpastatin) or transfected with calpain-1 siRNA demonstrated attenuation of IL-1␤-induced monolayer hyperpermeability. Calpain inhibition led to protection against IL-1␤-induced loss of zonula occludens-1 (ZO-1) at the tight junctions and alterations in F-actin cytoskeletal assembly. IL-1␤ treatment had no effect on ZO-1 gene (tjp1) or protein expression. Calpain inhibition via calpain inhibitor III and calpastatin decreased IL-1␤-induced calpain activity significantly (p < 0.05). IL-1␤ had no detectable effect on intracellular calcium mobilization or endothelial cell viability. Furthermore, calpain inhibition preserved BBB integrity/permeability in a mouse controlled cortical impact model of TBI when studied using Evans blue assay and intravital microscopy. These studies demonstrate that calpain-1 acts as a mediator of IL-1␤-induced loss of BBB integrity and permeability by altering tight junction integrity, promoting the displacement of ZO-1, and disorganization of cytoskeletal assembly. IL-1␤-mediated alterations in permeability are neither due to the changes in ZO-1 expression nor cell viability. Calpain inhibition has beneficial effects against TBI-induced BBB hyperpermeability.The blood-brain barrier (BBB) 2 plays an important role in maintaining the homeostasis of the brain. Blood-brain barrier breakdown and the associated hyperpermeability are hallmark features of several brain pathologies and injuries. The BBB is mainly composed of the cerebral endothelial cells and the tight junctions (TJs) between them (1). TJs between the neighboring endothelial cells include transmembrane TJs, i.e. occludin, claudins, junctional adhesion molecules, etc., and membranebound TJs, i.e. zonula occludens (1). Zonula occludens play an important role in regulating BBB permeability by binding to both transmembrane tight junctions and actin cytoskeleton intracellularly (2). Various mediators of inflammation are shown to modulate BBB breakdown and permeability in a variety of pathologies (3). Blood-brain barrier breakdown and the associated hyperpermeability is the leading cause of brain edema and elevated intracranial pressure followed by decreased perfusion pressure leading to poor clinical outcomes in traumatic brain injury (TBI) (4).I...

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“…Focal adhesion protein modification is an essential step in myocardial remodeling during heart failure (29), and interference with focal adhesion remodeling by calpain results in impaired contractile function in isolated myocytes (30) and even pericytes (31). In contrast to its obligatory role in normal cellular development, however, mechanical stress can activate calpain and cause an abnormal loss of talin signal in isolated muscle cells, according to other investigators (32).…”

Section: Discussionmentioning

confidence: 94%

Calpain Inhibition Preserves Talin and Attenuates Right Heart Failure in Acute Pulmonary Hypertension

Ahmad

Lu²,

Ye³

et al. 2012

Am J Respir Cell Mol Biol

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Right heart failure from right ventricular (RV) pressure overload is a major cause of morbidity and mortality, but its mechanism is incompletely understood. We tested the hypothesis that right heart failure during 4 hours of RV pressure overload is associated with alterations of the focal adhesion protein talin, and that the inhibition of calpain attenuates RV dysfunction and preserves RV talin. Anesthetized open-chest pigs treated with the calpain inhibitor MDL-28170 (n ¼ 20) or inactive vehicle (n ¼ 23) underwent 4 hours of RV pressure overload by pulmonary artery constriction (initial RV systolic pressure, 64 6 1 and 66 6 1 mm Hg in MDL-28170 and vehicle-treated pigs, respectively). Progressive RV contractile dysfunction was attenuated by MDL-28170: after 4 hours of RV pressure overload, RV systolic pressure was 44 6 4 mm Hg versus 49 6 6 mm Hg (P ¼ 0.011), and RV stroke work was 72 6 5% of baseline versus 90 6 5% of baseline, (P ¼ 0.027), in vehicle-treated versus MDL-28170-treated pigs, respectively. MDL-28170 reduced the incidence of hemodynamic instability (death or systolic blood pressure of , 85 mm Hg) by 46% (P ¼ 0.013). RV pressure overload disrupted talin organization. MDL-28170 preserved talin abundance in the RV free wall (P ¼ 0.039), and talin abundance correlated with the maintenance of RV free wall stroke work (r ¼ 0.58, P ¼ 0.0039). a-actinin and vinculin showed similar changes according to immunohistology. Right heart failure from acute RV pressure overload is associated with reduced talin abundance and disrupted talin organization. Calpain inhibition preserves the abundance and organization of talin and RV function. Calpain inhibition may offer clinical utility in treating acute cor pulmonale.Keywords: right ventricular dysfunction; pulmonary hypertension; calpain; talin; heart failure Right ventricular (RV) failure from acute right ventricular pressure overload (RVPO) is a major cause of morbidity and mortality in conditions such as pulmonary embolism and hypoxic pulmonary vasoconstriction, and in the early period after cardiopulmonary bypass or cardiac transplantation (1, 2). In the past, the conventional understanding of the pathophysiology of acute RV failure attributed it either to RV ischemia or to abnormal loading conditions during RVPO. However, we and others (3-5) previously demonstrated that the pathophysiology is more complex: a brief period of RVPO without RV ischemia results in intrinsic RV contractile dysfunction that persists beyond the period of RVPO itself, despite the restoration of normal loading conditions. Thus, the mechanism of intrinsic contractile dysfunction provoked by RVPO remains uncertain.Current clinical treatments for acute RV failure from RVPO depend almost exclusively on inotropic therapy or measures intended to relieve the underlying RVPO, such as thrombolysis, pulmonary thromboendarterectomy, and pulmonary vasodilators. These measures often result in serious complications, do not fully mitigate the high mortality, and do not directly address the mechanisms o...

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Calpain- and talin-dependent control of microvascular pericyte contractility and cellular stiffness

Cited by 37 publications

References 75 publications

Talin determines the nanoscale architecture of focal adhesions

Talin determines the nanoscale architecture of focal adhesions

Attenuation of Blood-Brain Barrier Breakdown and Hyperpermeability by Calpain Inhibition

Calpain Inhibition Preserves Talin and Attenuates Right Heart Failure in Acute Pulmonary Hypertension

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