[ Research Interests ] [ Representative Publications ]

Beverly Wendland, Ph.D. Professor Department of Biology Co-Director, CMDB Graduate Program B.S. University of California, San Diego Ph.D. Stanford University Postdoctoral Fellow University of California, San Diego

Department of Biology Johns Hopkins University 3400 North Charles Street Baltimore, MD 21218-2685 U.S.A. Office Telephone: Lab Telephone: Department Fax: Email: 410.516.0460 410.516.3875 410.516.5213 bwendland@jhu.edu  Office- Mudd 36B Lab- Mudd 48

Research Interests Eukaryotic cells contain intracellular membrane-bound organelles that segregate reactions that often are in opposition, such as protein biosynthesis and degradation. Furthermore, the protein and lipid composition of each organelle and membrane domain are distinct and specific for their individual functions. Selective membrane trafficking to and from these organelles occurs continually and is essential to maintain the discrete identities of each of these structures. The research goal of my lab is to gain a better understanding of the mechanisms of regulating membrane trafficking in eukaryotic cells. Specifically, the focus of my research is endocytosis, an essential process in which portions of the plasma membrane and extracellular fluid are internalized via coated pits. These coated vesicles fuse to endosomes, ultimately resulting in the delivery of some components to lysosomes. Endocytosis is a complex process required for such diverse functions as nutrient uptake, receptor down-regulation, membrane transporter activity, and regulation of signal transduction from the plasma membrane. The most well-characterized form of endocytosis is clathrin-dependent receptor-mediated endocytosis, although cells also utilize other modes of endocytosis. I have been using the yeast Saccharomyces cerevisiae and a novel FACS-based screening procedure to identify new gene products that function in endocytosis. By identifying important proteins and subjecting them to genetic and functional analyses in both yeast and mammalian systems, we will gain fundamental insights into the process and regulation of endocytosis. To accomplish these goals, we use a combination of yeast genetics (suppressor and synthetic lethal screens), biochemistry (protein interactions), and cell biology (microscopy, subcellular localization, phenotypic analyses). (Vida and Emr, JCB 128:779 (1995) A screen for novel endocytosis mutants using a fluorescent dye My lab uses a screen I developed that identified novel yeast mutants defective for the internalization of bulk membrane. Because this screen is very general, it should yield mutants defective for all endocytic pathways (i.e., clathrin-dependent, clathrin-independent, and novel uncharacterized pathways). My screen employs the fluorescent, lipophilic dye FM4-64, which labels the plasma membrane and is then endocytosed via endosomes to the lysosome-like vacuole in a time-, temperature-, and energy-dependent manner. Endocytosis mutants are thus "dim" due to reduced dye flow from the plasma membrane to the vacuole. Following EMS mutagenesis and FM4-64 labeling, FACS sorting was used to select for "dim" cells that are defective for internalization of the dye. Identification of a new endocytic protein complex My recent work has focused on understanding the function of the Pan1/Dim2 protein. Yeast cells with temperature-sensitive mutations in PAN1 display reduced internalization of a plasma membrane mating pheromone receptor, accumulate FM4-64 labeled aberrant endosome-like compartments, and contain elaborated plasma membrane invaginations. Pan1p is a yeast homologue of the mammalian protein eps15, which also has been implicated in endocytosis. To characterize further the function(s) of Pan1p and eps15 homology (EH) domains in endocytosis, genetic and biochemical studies were performed which identified two yeast proteins, yAP180A and yAP180B, which are homologous to mammalian proteins with clathrin cage assembly activity. In addition, genetic interactions between PAN1 and other genes whose products are implicated in endocytosis suggest that Pan1p may coordinate the functions of clathrin, ubiquitin, and actin-associated proteins to regulate endocytosis. Importantly, these recent studies of endocytosis in yeast have exemplified remarkable parallels in the endocytic machinery used by both lower and higher eukaryotes. For sample images of our work and others' see the Integrated Imaging Center. Representative Publications Barker, S.L. and Wendland, B. 2007. Interaction of the endocytic scaffold protein Pan1 with the type I myosins contributes to the late stages of endocytosis.. Mol. Biol. Cell . 18:2893-903. Aguilar RC, Longhi SA, Shaw JD, Yeh LY, Kim S, Schon A, Freire E, Hsu A, McCormick WK, Watson HA, Wendland B. (2006). Epsin N-terminal homology domains perform an essential function regulating Cdc42 through binding Cdc42 GTPase-activating proteins. Proc Natl Acad Sci U S A. 103(11):4116-21. Jahren, A.H., Kelm, K., Wendland, B. , Petersen, G., and Seberg, O. (2006). The carbon stable isotope composition of dsDNA isolated from an incipient paleosol. Geology. (in press). Katzmann, D.J. and Wendland, B. (2005). Analysis of ubiquitin-dependent protein sorting within the endocytic pathway in Saccharomyces cerevisiae . Methods Enzymol. 399:192-211 Aguilar R.C. and Wendland B. (2005). Endocytosis of membrane receptors: Two pathways are better than one. Proc Natl Acad Sci U S A . 102 :2679-80. Saiardi, A., Resnick, A.C., Snowman, A.M., Wendland, B. , and Snyder, S.H. (2005). Inositol pyrophosphates mediate cell death by regulating PI3-related protein kinases. Proc Natl Acad Sci USA. 102 :1911-1914. Miliaras, N.B., Park, J.-H., and Wendland, B. (2004). The function of the endocytic scaffold protein Pan1p depends on multiple domains . Traffic . 5 :963-978. Miliaras, N.B. and Wendland, B. (2004). EH-proteins: multivalent regulators of endocytosis (and other pathways). Cell Biochemistry and Biophysics. 41 :295-318. Watson, H.A., Von Zastrow, M., and Wendland, B. Endocytosis. (2004). In Encyclopedia of Molecular Cell Biology and Molecular Medicine . Second edition. Edited by Robert A. Myers. Wiley-VCH. Volume 4, 181-224. Baggett, J.J., Shaw, J.D., and Wendland, B. (2003). Fluorescent Labeling of Yeast. Edited by J. Lippincott-Schwartz and P. Matsudira. Current Protocols in Cell Biology , Unit 4.13. Baggett, J.J., D'Aquino, K.E., and Wendland, B. (2003). The Sla2p Talin domain plays a role in endocytosis in Saccharomyces cerevisiae . Genetics . 165 :1661-1674. Meriin, A.B., Zhang, X., Miliaras, N.B., Kazantsev, A., Chernoff, Y.O., McCaffery, J.M., Wendland, B. , and Sherman , M.Y. (2003). Aggregation of expanded polyglutamine domain in yeast leads to defects in endocytosis. Mol. Cell. Biol. 23 :7554-65. Sekiya-Kawasaki, M., Cope, M.J.T.V., Groen, A.C., Kaksonen, M., Zhang, C., Shokat, K.M., Wendland, B. , McDonald, K.L., McCaffery, J.M., and Drubin, D.G. (2003). Dynamic phosphoregulation of the cortical actin cytoskeleton and endocytic machinery revealed by real-time chemical genetic analysis. J. Cell Biol. 162 :765-72. Overstreet, E., Chen, X., Wendland, B. , and Fischer, J.A. (2003). Either portion of a severed Drosophila epsin (Liquid Facets) functions in the internalization of Delta in the developing eye. Curr Biol. 13 :854-60. Shaw, J.D., Hama , H., Sohrabi, F., DeWald, D.B., and Wendland, B. (2003). Phosphatidylinositol (3,5) bisphosphate is required for delivery of endocytic cargo into the multivesicular body. Traffic 4 :479-90. Aguilar, R.C. and Wendland, B. (2003). Ubiquitin: not just for proteasomes anymore. Curr. Opin. in Cell Biol . 15 : 184-90. Aguilar, R.C., Watson, H.A., and Wendland, B. (2003). The yeast epsin Ent1 is recruited to membranes through multiple independent interactions. J. Biol. Chem. 278 :10737-43. Wendland, B. Epsins: adaptors in endocytosis? (2002) Nature Rev. Mol. Cell Biol. 3 :971-7. Saiardi, A., Sciambi, C.J., McCaffery, J.M., Wendland, B. , and Snyder, S.H. (2002). Inositol pyrophosphates regulate endocytic trafficking. Proc. Natl. Acad. Sci. 99 :14206-11 . Hurley, J.H. and Wendland, B. (2002). Endocytosis: Driving membranes around the bend. Cell 111 :143-6 . Vida, T.A. and Wendland, B. (2002). Flow cytometry for selection of yeast membrane trafficking mutants. Edited by C. Guthrie and G. Fink. Methods in Enzymology 351 : 623-631. Babst, M., Katzmann, D.J., Snyder, W.B., Wendland, B. , and Emr , S.D. (2002). Endosome-Associated Complex, ESCRT-II, Recruits Transport Machinery for Protein Sorting at the Multivesicular Bodies. Dev. Cell. 3 :283-9. Watson, H.A., Cope, M.J.T.V., Groen, A.C., Drubin, D.G. and Wendland, B. (2001). In vivo role for Actin Regulating Kinases in endocytosis and yeast epsin phosphorylation. Mol. Biol. Cell . 12 (11):3668-79. Shaw, J.D., Cummings, K.B., Huyer, G., Michaelis, S., and Wendland, B. (2001). Yeast as a model system for studying endocytosis. Experimental Cell Research . 271 : 1-9. Duncan , M.C., Cope, M.J.T.V., Goode, B.L., Wendland, B. , and Drubin, D.G. (2001). Yeast Eps15-like endocytic protein, Pan1p, activates the Arp2/3 complex. Nature Cell Biology 3 (7):687-90. Wendland, B. (2001). Round-trip ticket: Re-cycling to the plasma membrane requires RME-1. News and Views in Nature Cell Biol. 3 (6):E133-5. Wang, G., McCaffery, J.M., Wendland, B. , Dupre, S., Haguenauer-Tsapis, R., and Huibregtse, J.M. (2001). Localization of the Rsp5p Ubiquitin-Protein Ligase at Multiple Sites within the Endocytic Pathway. Mol Cell Biol. 21 :3564-75.

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