Central to the vectorial transport function of renal epithelium is the polarized distribution of surface membrane proteins in renal tubule cells. The spectrin-based membrane skeleton organizes mosaics of membrane proteins and lipids, and tethers them to cytoskeletal filament systems and motor proteins (e.g. dynein/dynactin). A working hypothesis that has emerged from recent data is that a novel Golgi-associated forms of spectrin and ankyrin, acting along the secretory and endocytic pathways, influences the efficiency of assembly for certain membrane proteins by forming a spectrin-ankyrin-adapter protein tethering system (SAATS). One such protein is alpha-Na,K-ATPase, which requires spectrin and ankyrin-binding early in the secretory pathway for its efficient and polarized delivery to the plasma membrane. Multiple recently discovered isoforms of spectrin and ankyrin also exist in the renal cell. The overall goal of the proposed studies is thus to understand the specific steps in the secretory pathway on which spectrin and or ankyrin act; understand which membrane proteins require spectrin or ankyrin for their efficient delivery to the membrane; and understand the aberrations in this process that typify cellular ATP depletion, a model of cellular ischemia. Research will focus on the role of beta-Ia nd beta-III spectrins and the role that two renal ankyrins (AnkG119 & AnkG190) play in the secretory pathway, as they interact with Na,K-ATPase.
Three specific aims are proposed. Time-lapse video microscopy and FRET analysis using eGFP tagged spectrin peptides expressed in MDCK, COS, and LLC-PK1 cells will be used to follow the in vivo movement and associations of spectrin and ankyrin with various organelle and cargo proteins. Specific interactions of ankyrin with alpha-Na,K-ATPase will be studied to determine if ankyrin acts like a chaperone or a chauffeur in the process of membrane biogenesis. The consequences of ATP depletion in model cell systems on the activities of spectrin or ankyrin will be examined. Collectively, these studies will answer important questions that have followed from the discovery of a heretofore-unanticipated role for the spectrin-ankyrin cytoskeleton in the secretory pathway, and will provide insights into the role of certain novel protein-protein interactions, interactions that may afford novel targets for therapeutic intervention.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK043812-14
Application #
7240527
Study Section
Pathology A Study Section (PTHA)
Program Officer
Mullins, Christopher V
Project Start
1991-04-15
Project End
2010-05-31
Budget Start
2007-06-01
Budget End
2010-05-31
Support Year
14
Fiscal Year
2007
Total Cost
$359,957
Indirect Cost
Name
Yale University
Department
Pathology
Type
Schools of Medicine
DUNS #
043207562
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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