understanding the structure and function of the spectrin-ankyrin-actin cytoskeleton
	Jon Morrow, Ph.D. Raymond Yesner Professor of Pathology; Chairman and Chief, Department of Pathology: Professor of Molecular, Cellular & Developmental Biology Email: jon.morrow@yale.edu Ph.D. Indiana University 1974; M.D. Yale University 1976

Central to the integrated function of multicellular organisms is cell contact mediated signaling and the spatial organization of specialized membrane-surface domains. While many factors contribute, recent evidence indicates that the spectrin based membrane skeleton plays a pivotal role in these processes, beginning with the transport of these proteins through the Golgi, all the way up to and including the control of their organization at the plasma membrane. Current research in the laboratory is aimed at understanding three aspects of the spectrin membrane skeleton in erythrocytes, epithelial cells, and neurons: 1) The factors that mediate its polarized assembly with specific surface membrane receptor domains; 2) the nature of the proteins that interact with spectrin and their role in signal transduction and vesicular trafficking; and 3) the molecular basis of diseases that involve spectrin or any of its associated proteins.

Our studies on the erythrocyte focus on a molecular understanding of how specific proteins bind to spectrin, how this binding is allosterically regulated, the role of post-translational regulation of spectrin and its associated proteins, and the molecular basis of hemolytic disorders involving the spectrin skeleton. We have used deletional analysis and in vitro binding assays to identify specific regions of beta-spectrin that bind ankyrin (Kennedy et al., 1991), the site of spectrin-spectrin self-association (Kennedy et al., 1993), and the site of calmodulin binding in adducin, a spectrin associated actin bundling protein (Mische et al., 1987; Scaramuzzino and Morrow, 1993). Using yeast two-hybrid assays, we have also recently identified several novel proteins that interact with spectrin? SH3 and PH domains (Cianci et al., submitted), as well as novel sites of direct membrane interaction (Lombardo et al.,1994). We are seeking candidate proteins that interact with stomatin, a 31kD protein absent in patients with the disease hereditary stomatocytosis (HSt). We hypothesize that stomatin functions as a transacting regulator of membrane channel conduc-tance. Current experimental approaches include both genetic selection strategies as well as sensitive biochemical methods seeking to directly identify proteins that bind stomatin.

The targeting of the spectrin skeleton to specific surface membrane domains is being studied in renal epithelial cells and in cultured neurons. We have identified novel forms of spectrin and ankyrin that contribute to a novel vesicular skeleton that appears to mediate protein trafficking from the ER through the Golgi and to the plasma membrane (Devarajan et al.,1996; Devarajan et al.,1997). We have also demonstrated that a region near the amino-terminus of beta-spectrin contains a highly specific cytoskeletal sorting signal that directs the compartmentation of the spectrin based skeleton to specific membrane domains. (Stabach et al., in preparation). We have also established that one of the proteins mediating this sorting is alpha-catenin, and that alpha catenin binds directly to spectrin and actin (Rimm et al.,1995; Roe et al.,1996). Our working hypothesis derived from these obser-vations is that E-cadherin and alpha-catenin define the regions of cell-cell contact during epithelial cell maturation, and guide spectrin to it. Experiments examining the role of specific post-translational changes on the function of the spectrin skeleton are also underway, utilizing assays and transgenic mice.

Selected Publications

Devarajan, P., P. R. Stabach, A. S. Mann, T. Ardito, M. Kashgarian and J. S. Morrow. (1996). Identification of a small cytoplasmic ankyrin (AnkG119) in kidney and muscle that binds bIS* spectrin and associates with the Golgi apparatus. J. Cell Biol. 133:819-830.

Devarajan, P., P. R. Stabach, M. A. De Matteis and J. S. Morrow. (1997). Na,K-ATPase transport from ER to Golgi requires an intact Golgi spectrin - ankyrin G119 skeleton in MDCK cells. Proc. Natl. Acad. Sci (USA) (in press).

Rimm, D. L., E. R. Koslov, P. Kebriaei, C. D. Cianci and J. S. Morrow. (1995). a1(E)-catenin is a novel actin binding and bundling protein mediating the attachment of F-actin to the membrane adhesion complex. Proc. Natl. Acad. Sci (USA) 92:8813-8817.

Roe, S., D. Pradhan, E. R. Koslov, J. S. Morrow and D. L. Rimm. (1996). Reduced binding of mutant alpha-catenin to spectrin in clone A cells is associated with a non-adhesive phenotype. Molec.Biol.Cell 7:285a.

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