Central to the vectorial transport of renal epithelium is the polarized distribution of surface membrane proteins in tubule cells. Recent evidence indicates that the spectrin based cortical cytoskeleton, which is often associated with certain integral membrane proteins, is also highly polarized, may play a fundamental role in , maintaining and guiding topographic membrane assembly, and is disrupted following even sublethal renal tubule ischemia. The overall goal of the proposed studies will be to understand how the cortical cytoskeleton achieves its polarized distribution, the relationship of this process to the sorting of basolaterally restricted integral membrane proteins, and the aberrations in this process that typify various pathologic states. Specifically, research will focus on the factors that target the assembly of the spectrin skeleton to the lateral margins of kidney epithelial cells, regulate its interactions with the integral membrane proteins L-CAM and Na+, K+-ATPase, and mediate the changes in the spectrin skeleton that accompany ischemia. The cytoskeletal proteins to be examined include the erythroid like and non-erythroid isoforms of spectrin (fodrin), ankyrin, adducin, and protein 4.1. Also of interest are certain new proteins which have tentatively been identified in the membrane of renal epithelial cells. The interaction between these proteins will be measured by in vitro assays; their distributions and rates of synthesis and assembly into the cortical cytoskeleton will be measured in cultured renal epithelial (MDCK) cells as a function of tight junction formation, epithelial monolayer formation, and in vitro ischemic injury. Recombinant DNA techniques will be used to characterize unique forms of spectrin and adducin in renal cells. Functional sites in these protein will be identified by deletional analysis in vitro and by the effects of expressed recombinant proteins in cultured epithelial cells. Sequence specific antibodies, Northern blot analysis, and in situ hybridization will be used to monitor changes in the distribution, composition, processing, and synthesis of these components after in vivo and in vitro ischemia.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK043812-04
Application #
2143316
Study Section
Pathology A Study Section (PTHA)
Project Start
1991-04-15
Project End
1996-03-31
Budget Start
1994-04-01
Budget End
1995-03-31
Support Year
4
Fiscal Year
1994
Total Cost
Indirect Cost
Name
Yale University
Department
Pathology
Type
Schools of Medicine
DUNS #
082359691
City
New Haven
State
CT
Country
United States
Zip Code
06520
Stankewich, Michael C; Moeckel, Gilbert W; Ji, Lan et al. (2016) Isoforms of Spectrin and Ankyrin Reflect the Functional Topography of the Mouse Kidney. PLoS One 11:e0142687
Kim, Jung H; Kwon, Soojung J; Stankewich, Michael C et al. (2016) Reactive protoplasmic and fibrous astrocytes contain high levels of calpain-cleaved alpha 2 spectrin. Exp Mol Pathol 100:1-7
Stankewich, Michael C; Cianci, Carol D; Stabach, Paul R et al. (2011) Cell organization, growth, and neural and cardiac development require ?II-spectrin. J Cell Sci 124:3956-66
La-Borde, Penelope J; Stabach, Paul R; Simonovic, Ivana et al. (2010) Ankyrin recognizes both surface character and shape of the 14-15 di-repeat of beta-spectrin. Biochem Biophys Res Commun 392:490-4
Cairo, Christopher W; Das, Raibatak; Albohy, Amgad et al. (2010) Dynamic regulation of CD45 lateral mobility by the spectrin-ankyrin cytoskeleton of T cells. J Biol Chem 285:11392-401
Stankewich, Michael C; Gwynn, Babette; Ardito, Thomas et al. (2010) Targeted deletion of betaIII spectrin impairs synaptogenesis and generates ataxic and seizure phenotypes. Proc Natl Acad Sci U S A 107:6022-7
Stabach, Paul R; Simonovi?, Ivana; Ranieri, Miranda A et al. (2009) The structure of the ankyrin-binding site of beta-spectrin reveals how tandem spectrin-repeats generate unique ligand-binding properties. Blood 113:5377-84
Stabach, Paul R; Devarajan, Prasad; Stankewich, Michael C et al. (2008) Ankyrin facilitates intracellular trafficking of alpha1-Na+-K+-ATPase in polarized cells. Am J Physiol Cell Physiol 295:C1202-14
Glantz, Susan B; Cianci, Carol D; Iyer, Rathna et al. (2007) Sequential degradation of alphaII and betaII spectrin by calpain in glutamate or maitotoxin-stimulated cells. Biochemistry 46:502-13
Nakayama, Yasuhiro; Stabach, Paul; Maher, Stephen E et al. (2006) A limited number of genes are involved in the differentiation of germinal center B cells. J Cell Biochem 99:1308-25

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