Rho-kinase regulates myosin II activation in MDCK cells during recovery after ATP depletion.
ABSTRACT Alterations in the actin cytoskeleton of renal tubular epithelial cells during periods of ischemic injury and recovery have important consequences for normal cell and kidney function. Myosin II has been demonstrated to be an important effector in organizing basal actin structures in some cell types. ATP depletion in vitro has been demonstrated to recapitulate alterations of the actin cytoskeleton in renal tubular epithelial cells observed during renal ischemia in vivo. We utilized this reversible cell culture model of ischemia to examine the correlation of the activation state and cellular distribution of myosin II with disruption of actin stress fibers in Madin-Darby canine kidney (MDCK) cells during ATP depletion and recovery from ATP depletion. We found that myosin II inactivation occurs rapidly and precedes dissociation of myosin II from actin stress fibers during ATP depletion. Myosin II activation temporally correlates with colocalization of myosin II to reorganizing stress fibers during recovery from ATP depletion. Furthermore, myosin activation and actin stress fiber formation were found to be Rho-associated Ser/Thr protein kinase dependent during recovery from ATP depletion.
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ABSTRACT: Non-muscle myosin II (NM II) regulates a wide range of cellular functions, including neuronal differentiation, which requires precise spatio-temporal activation of Rho GTPases. The molecular mechanism underlying the NM II-mediated activation of Rho GTPases is poorly understood. The present study explored the possibility that NM II regulates neuronal differentiation, particularly morphological changes in growth cones and the distal axon, through guanine nucleotide exchange factors (GEFs) of the Dbl family. NM II colocalized with GEFs, such as βPIX, kalirin and intersectin, in growth cones. Inactivation of NM II by blebbistatin (BBS) led to the increased formation of short and thick filopodial actin structures at the periphery of growth cones. In line with these observations, FRET analysis revealed enhanced Cdc42 activity in BBS-treated growth cones. BBS treatment also induced aberrant targeting of various GEFs to the distal axon where GEFs were seldom observed under physiological conditions. As a result, numerous protrusions and branches were generated on the shaft of the distal axon. The disruption of the NM II-GEF interactions by overexpression of the DH domains of βPIX or Tiam1, or by βPIX depletion with specific siRNAs inhibited growth cone formation and induced slender axons concomitant with multiple branches in cultured hippocampal neurons. Finally, stimulation with nerve growth factor induced transient dissociation of the NM II-GEF complex, which was closely correlated with the kinetics of Cdc42 and Rac1 activation. Our results suggest that NM II maintains proper morphology of neuronal growth cones and the distal axon by regulating actin dynamics through the GEF-Rho GTPase signaling pathway.PLoS ONE 01/2014; 9(4):e95212. · 3.53 Impact Factor
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ABSTRACT: Neuronal cells change their growth properties in response to external physical stimuli such as variations in external temperature, stiffness of the growth substrate, or topographical guidance cues. Detailed knowledge of the mechanisms that control these biomechanical responses is necessary for understanding the basic principles that underlie neuronal growth and regeneration. Here, we present elasticity maps of living cortical neurons (embryonic rat) as a function of temperature, and correlate these maps to the locations of internal structural components of the cytoskeleton. Neurons display a significant increase in the average elastic modulus upon a decrease in ambient temperature from 37 to 25 °C. We demonstrate that the dominant mechanism by which the elasticity of the neurons changes in response to temperature is the stiffening of the actin components of the cytoskeleton induced by myosin II. We also report a reversible shift in the location and composition of the high-stiffness areas of the neuron cytoskeleton with temperature. At 37 °C the areas of the cell displaying high elastic modulus overlap with the tubulin-dense regions, while at 25 °C these high-stiffness areas correspond to the actin-dense regions of the cytoskeleton. These results demonstrate the importance of considering temperature effects when investigating cytoskeletal dynamics in cells.Physical Biology 08/2013; 10(5):056002. · 2.62 Impact Factor
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ABSTRACT: Ischemia-reperfusion injury (IRI) is a common cause of acute kidney injury (AKI) and is characterized by widespread tubular and microvascular damage. The tumor suppressor p53 is upregulated after IRI and contributes to renal injury in part by promoting apoptosis. Acute, short-term inhibition of p53 with pifithrin-alpha conveys significant protection after IRI. The hypoxia-inducible factor-1 (HIF-1) pathway is also activated after IRI and has opposing effects to those promoted by p53. The balance between the HIF-1 and p53 responses can determine the outcome of IRI. In this manuscript, we investigate whether p53 regulates the HIF-1 pathway in a rodent model of IRI. HIF-1alpha is principally expressed in the collecting tubules (CT) and thick ascending limbs (TAL) under physiological conditions. However, inhibition of p53 with pifithrin-alpha increases the faint expression of HIF-1alpha in proximal tubules (PT) under physiological conditions. Twenty-four hours after IRI, HIF-1alpha expression is decreased in both CT and TAL. HIF-1alpha expression in the PT is not significantly altered after IRI. Acute inhibition of p53 significantly increases HIF-1alpha expression in the PT after IRI. Additionally, pifithrin-alpha prevents the IRI-induced decrease in HIF-1alpha in the CT and TAL. Parallel changes are observed in the HIF-1alpha transcriptive target, carbonic anhydrase-9. Finally, inhibition of p53 prevents the dramatic changes in Von Hippel-Lindau protein morphology and expression after IRI. We conclude that activation of p53 after IRI mitigates the concomitant activation of the protective HIF-1 pathway. Modulating the interactions between the p53 and HIF-1 pathway can provide novel options in the treatment of AKI.American journal of physiology. Renal physiology 10/2008; 295(6):F1666-77. · 3.61 Impact Factor