Proximal tubule cells have a specialized apical membrane that mediates the selective and efficient reabsorption of molecules from the glomerular filtrate. Reabsorption requires rnicrovilli formation and delivery of ion channels, receptors, and regulatory molecules to their correct destination. Both of these processes depend upon actin-surface membrane interactions and vesicular transport mediated in part by actin-associated proteins. Myosin I's have been implicated in both vesicular transport and actin-membrane interactions, two processes affected by ischemia in proximal tubule cells. Myosin I beta is an ATPase that is important for both of these processes. It contains actin, membrane binding domains, and has three calmodulin light chains. This is the only myosin I isoform identified in the proximal tubule brush border and the vesicle rich region under the brush-border. This isoform has also been identified in a kidney high density endosome fraction. In addition, the applicant shows that myosin I beta's location in proximal tubule cells is severely altered by ischemia. This redistribution of myosin I is temporally and spatially related to the disruption of the apical plasma membrane during, ischemic injury. Together the data clearly implicate myosin I beta in both normal functions of proximal tubule cells and as a key mediator necessary to retarget membrane components and reorganize the brush border actin cytoskeleton following cell injury. The applicant proposes that myosin I beta transports vesicles through the terminal web region to the plasma membrane and participates in organizing and regulating the dynamic actin-membrane events in the brush border. Movement through cortical actin and regulation of membrane-actin events are impaired by ischemia with cell recovery requiring the normalization of these processes. The applicant proposes three Specific Aims to directly evaluate this hypothesis: 1. Evaluate the effect of ischemia on the cellular location of myosin 1 beta using light and electron microscopy; 2. Determine if ischemia modifies the normal regulation of myosin 1 beta, by analyzing phosphorylation changes and associated proteins including calmodulin; and 3. Evaluate the role of myosin 1 beta in rebuilding the brush border and retargeting membrane components by antibody and molecular disruption of myosin 1 beta's function. The applicant is in a unique position to establish the role of myosin 1 beta in the kidney proximal tubule under physiological conditions and following ischemic injury. The applicant has developed and been provided with essential tools to address the function of this protein, and is part of an excellent interactive group of renal cell and molecular biologists.
| Wagner, Mark C; Blazer-Yost, Bonnie L; Boyd-White, Judy et al. (2005) Expression of the unconventional myosin Myo1c alters sodium transport in M1 collecting duct cells. Am J Physiol Cell Physiol 289:C120-9 |
| Boyd-White, J; Srirangam, A; Goheen, M P et al. (2001) Ischemia disrupts myosin I beta in renal tubules. Am J Physiol Cell Physiol 281:C1326-35 |
| Chen, J; Wagner, M C (2001) Altered membrane-cytoskeleton linkage and membrane blebbing in energy-depleted renal proximal tubular cells. Am J Physiol Renal Physiol 280:F619-27 |
