Professor and Chair
36B Mudd Hall
Department of Biology
Johns Hopkins University
3400 N. Charles Street
Baltimore, MD 21218-2685
Office 410 516-0460
Lab 410 516-3875
Departmental fax 410 516-5213
B.S.University of California, San Diego
PostdoctoralUniversity of California, San Diego
Molecular Mechanisms and Regulation of Endocytic Vesicle Formation
Endocytosis regulates cell physiology and homeostasis by affecting nutrient uptake, signal transduction, and the population of receptors in the plasma membrane. Cellular outputs are in turn affected, including cell fate decisions, cell division/proliferation, and cell polarity. Understanding the mechanisms of endocytosis will therefore lead to a better understanding of its function in these fundamental cellular processes and reveal how endocytosis interfaces with other processes to affect the response of a cell to its environment. Because many components of the endocytic machinery are structurally and functionally conserved from yeast to humans, we are able to apply the many powerful tools available in the yeast system to develop new experimental approaches and generate more accurate models to decipher the endocytic pathway. We anticipate that our discoveries in yeast will inform studies of human diseases that arise from aberrant endocytic regulation, including some forms of cancer, and provide new targets for therapeutics, such as enhanced delivery of gene therapies.
We focus our studies on clathrin-dependent endocytosis, which can be divided into three stages. A) A specific subset of membrane proteins (cargo) is selected for incorporation into the endocytic vesicle. Endocytic proteins called “adaptors” regulate this cargo recruitment through binding to sorting motifs in the cytoplasmic domains of the cargo. B) The structural components of the endocytic vesicle are recruited, including the “coat” protein clathrin and the “scaffold” proteins that coordinate the proper association of adaptors and coats. We have recently shown that scaffolds link the early events of vesicle formation to the late events of vesicle scission, a new role for these proteins. C) The formed endocytic vesicle then separates from the plasma membrane (scission) and moves into the cytosol. Completion of both vesicle scission and movement require the actin cytoskeleton.
Our long-term goal is to understand the molecular mechanisms that
drive each step of endocytosis and to determine how these individual steps are
coordinated to occur with the proper order and timing. Toward this goal, our lab
has focused on two broad areas: 1) Defining which proteins act as adaptors, how
they regulate cargo selection, and if they have other functions in endocytosis.
2) Determining the role and mode of action of the endocytic scaffold Pan1 in
coordinating the sequential events in endocytosis.us (LIDL-COO-) mimics the canonical
‘clathrin box’ (LLDLD) of mammalian adaptors that binds the terminal domain of
In addition to binding clathrin, we and others showed that
the epsins contain multiple motifs with distinct binding activities,
including the lipid-binding ENTH domain, UIMs that bind ubiquitin, and NPF motifs that bind the
EH domains of the scaffolds Pan1 and Ede1. Using a novel in vitro membrane recruitment assay, we
obtained evidence that supports the view that the ENTH domain and UIM motifs act together through
their individual low affinity interactions to create a stable multivalent complex at endocytic sites.
These and other findings in the field led to the model that epsins can act as endocytic adaptors that
play a key role in initiating endocytic events.
Since adaptors likely play a major role in controlling cell physiology,
two key questions are how are adaptors regulated, and do they have additional functions?
Since phosphorylation is known to regulate the mammalian endocytic machinery, we asked if/how
phosphorylation regulates yeast endocytic adaptors. We determined that epsins are phosphorylated
in vivo by the conserved protein kinase Prk1, and that this modification inhibits epsin functions.
We also found that the epsins and the structurally related Yap180 proteins fulfill a redundant
function in endocytosis and that this function requires their NPF motifs, which bind EH domains
in scaffold proteins. Our exciting findings suggest a new function for adaptors, that were thought
to be primarily early-acting factors, in regulating the onset of the final stages of vesicle
scission (see below).
We next asked if the essential function of the epsins was connected to their role in support of endocytosis, or to some other process. We and others have shown that the conserved N-terminal 'ENTH' domains of the epsins bind phosphoinositides, while the epsin C-termini harbor the motifs for binding to other endocytic machinery components. Thus, we were surprised to find that the ENTH domain alone was necessary and sufficient to complement both endocytosis defects and inviability of ent1∆ent2∆ cells. A yeast two-hybrid screen with the ENTH domain led to our discovery of a novel essential role for ENTH domains that is independent of lipid-binding: down-regulation of the GTPase Cdc42 by binding to Cdc42 GAPs. Another recent screen in our lab has identified a genetic interaction between the adaptors and the GTPase Rho1. The Rho-family GTPases Cdc42 and Rho1 are critical regulators of cell polarity and the actin cytoskeleton. These findings connecting endocytic adaptor proteins and Cdc42/Rho1 may explain the correlation of sites of polarity cues, secretion, and endocytosis. Our future studies will define the mechanisms by which signaling pathways activated by these GTPases may regulate endocytosis, how the adaptors may serve as a critical interface, and if these interactions directly or indirectly impact endocytosis.
Endocytic scaffold protein functions: Our discovery of the scaffold protein Pan1 as a critical endocytic factor in yeast revealed the conserved, universal aspects of eukaryotic endocytic mechanisms. Subsequent data from our lab and others have led us to hypothesize that Pan1 is a central regulator or checkpoint protein that controls the transitions between early and late stages of endocytosis. Thus, understanding the molecular mechanisms of Pan1 function continues to be a major focus of our work. Specifically, we are testing the hypothesis that a Pan1:adaptor:cargo complex can sense the completion of cargo-loading, consequently triggering the recruitment and activation of the actin-based scission machinery. Consistent with this model, evidence from our lab and others shows that Pan1 interacts with factors important for both early stages (cargo collection) and late stages (vesicle scission) of endocytosis. Like Pan1, the EH domain-containing scaffold/adaptor protein Ede1 also binds the epsin and Yap180 adaptors; thus, we are studying the unique and shared functions of Pan1 vs. Ede1. We also demonstrated that Pan1 binds directly to the late-acting type I myosins (Myo3/5), and that Pan1 stimulates the actin-assembly activity of Myo5/Vrp1 at the time when vesicle invagination/scission commences. Additionally, we showed that Pan1 forms homo-oligomers and exhibits intra-molecular interactions between distinct domains that may also regulate Pan1 functions. Our ongoing and future studies are centered on clarifying the exchange of binding partners during the sequential formation and dissolution of Pan1-containing protein complexes, using biochemical, biophysical and cell biological appoaches. In this way we will test our model for how Pan1 may act as a checkpoint protein to control the progression from one endocytic stage to the next.
Matching cargos to adaptors: In our work on endocytic adaptors, we are identifying known and novel proteins that can act as adaptors and studying the mechanism(s) by which they fulfill their important functions. Adaptors have several characteristics, including binding directly to sorting signals in the tail of the transmembrane cargo that is to be endocytosed, promoting clathrin polymerization, and more. The wide variety of transmembrane cargo proteins suggests that a range of adaptors mediate cargo endocytosis, yet many of these adaptors remain unknown. Thus, we are trying to identify endocytic plasma membrane cargos and the sorting signals recognized by their cognate adaptors. For example, we have evidence that Yap1801 and Yap1802 (AP180/CALM homologs; recently implicated in Alzheimer’s Disease) are cargo-specific adaptors for the v-SNAREs Snc1/2 (VAMP/synaptobrevin homologs). Other candidate adaptors we are studying include the AP-2 complex, the ENTH domain protein Ent4, and the novel conserved protein Syp1. We have recently begun to apply structural biology tools to our questions, and found that Syp1 not only has a domain common to adaptor proteins, but it also has a homo-dimeric membrane tubulation domain. Additionally, while assessing the requirements and roles for adaptors in vivo using a genetic screen, we uncovered an allele of the gene encoding Sla2. Sla2 is critical for endocytosis (Riezman lab) and binds clathrin (Lemmon lab); these two results together with our findings are consistent with the model that Sla2 may be another endocytic adaptor.
Developing new tools to study endocytosis: To better define the functions and regulation of endocytic adaptors and scaffolds, we are applying tools used in studies of synaptic vesicle recycling to studies of yeast endocytosis. For example, to develop a cargo-specific, quantitative, high-throughput method to screen for endocytosis defects, we are using pH-sensitive GFP variants fused to endocytic cargo. This allows for quenching of the vacuolar GFP fluorescence derived from endocytosed receptors, so that the signal from uninternalized receptors can be selectively observed.
The combination of genetic, biochemical, cell biological and biophysical approaches used by our lab will allow us to uncover new factors and mechanism of endocytosis, and to test our model for Pan1 regulation of endocytosis by defining the sequential conformations and complexes that underlie Pan1’s functions. Our current and future directions will add clarity to mechanistic models of endocytic protein function. Overall, our lab’s work has contributed to elucidating the fundamental mechanisms of endocytosis in all eukaryotic cells. Our ongoing and future work will continue to shed light on the conserved functions and regulation of endocytosis and its role in cellular physiology, homeostasis and pathological conditions, including cancer, cardiovascular disease, lysosomal storage disorders, and infections by viral and bacterial pathogens.
Umasaker, P.K., Sanker, S., Thieman, J.R., Chakraborty, S., Wendland, B., Tsang, M., Traub, L.M. (2012). Distinct and separable activities of the endocytic clathrin-coat components Fcho1/2 and AP-2 in developmental patterning. Nature Cell Biology. In press.
Prosser, D.C. and B. Wendland. (2012). Conserved roles for yeast Rho1 and mammalian RhoA GTPases in clathrin-independent endocytosis. Small GTPases. (submitted).
Reider, A. and Wendland, B. (2011). Endocytic adaptors - social networking at the plasma membrane. Journal of Cell Science. 124:1613-22.
Prosser, D.C., Drivas, T.G., Maldonado-Báez, L., and Wendland, B. (2011). Existence of a novel clathrin-independent endocytic pathway in yeast that depends on Rho1 and formin. Journal of Cell Biology. 195:657-71.
Prosser, D.C., Whitworth, K., and Wendland, B. (2010). Quantitative analysis of endocytosis with cytoplasmic pHluorin chimeras. Traffic. 11:1141-50.
Prosser, D.C., Tran, D., Schooley, A., Wendland, B., and Ngsee, J.K (2010). A novel, retromer-independent role for sorting nexins 1 and 2 in RhoG-dependent membrane remodeling. Traffic. 11:1347-62.
Traub, L.M. and Wendland, B. (2010). Cell biology: How to don a coat. Nature. 465:556-557.
Dores, M.R., Schnell, J.D., Maldonado-Baez, L., Wendland, B., and Hicke, L. (2010). The function of yeast epsin and Ede1 ubiquitin-binding domains during receptor internalization. Traffic. 11:151-160.
Boettner, D.R., D’Agostino, J.L., Torres, O.T., Daugherty-Clarke, K., Uygur, A., Reider, A., Wendland, B., Lemmon, S.K., and Goode, B.L. (2009). The F-BAR domain protein Syp1 negatively regulates WASp-Arp2/3 complex activity during endocysptic patch formation. Current Biology. 19:1979-1987.
Burston, H.E., Maldonado-Báez, L., Davey, M., Montpetit, B., Schluter, C., Wendland, B., and Conibear, E. (2009). Regulators of Yeast Endocytosis Identified by Systematic Quantitative Analysis. J. Cell Biol. 185:1097-1110.
Pierce, B.D. and Wendland, B. (2009). Sequence of the yeast protein expression plasmid pEG(KT). Yeast. 26:349-353.
McPherson, P.S., Ritter, B., and Wendland, B. (2009). Clathrin-mediated endocytosis. Trafficking Inside Cells: Pathways, Mechanisms and Regulation. Molecular Biology Intelligence Unit, Landes Bioscience, Springer Science + Business Media, LLC. Editors: N. Segev, A. Alfonso, G. Payne and J. Donaldson. Chapter 9, pages 159-182.
Mukherjee, D., Coon, B.G., Edwards III, D.F., Hanna, C.B., Longhi, S.A., McCaffery, J.M., Wendland, B., Retegui, L.A., Bi, E., and Aguilar, R.C. (2009). The yeast endocytic protein Epsin-2 functions in a cell division signaling pathway. J. Cell Sci. 122:2543-2464.
Reider, A., Barker, S.L., Im, Y. J., Mishra, S., Hurley, J.M., Traub, L. and Wendland, B. Syp1 is a conserved endocytic adaptor that contains domains involved in cargo selection and membrane tubulation. EMBO J. 28:3103-3116.
Maldonado-Báez, L., Dores, M.R., Perkins, E.M., Drivas, T.G., Hicke, L., and Wendland, B. (2008). Interaction between Epsin/Yap180 Adaptors and the Scaffolds Ede1/Pan1 Is Required for Endocytosis. Mol Biol Cell. 19:2936-2948.
Barker, S.L., Lee, L., Pierce, B.D., Maldonado-Báez, L., Drubin, D., 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. 18:2893-2903.
Maldonado-Báez, L. and Wendland, B. (2006). Endocytic Adaptors: Recruiters, coordinators and regulators. Trends in Cell Biology. 16: 505-513.
Aguilar, R.C., Longhi, S.A., Shaw, J.D., Yeh, L.-Y., Kim, S., Schon, A., Freire, E., Hsu, A., Watson, H.A., McCormick, W.K., and Wendland, B. (2006). Epsin ENTH domains perform an essential function regulating Cdc42 through binding Cdc42 GTPase activating proteins. Proc Natl Acad Sci U S A. 103:4116-4121. [Cover highlight, and accompanying Commentary]
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.
Aguilar R.C. and Wendland B. (2005). Endocytosis of membrane receptors: Two pathways are better than one. Proc Natl Acad Sci U S A. 102:2679-2680.
Katzmann, D.J. and Wendland, B. (2005). Analysis of ubiquitin-dependent protein sorting within the endocytic pathway in Saccharomyces cerevisiae. Methods in Enzymology. 399:192-211.
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.
Miliaras, N.B. and Wendland, B. (2004). EH-proteins: multivalent regulators of endocytosis (and other pathways). Cell Biochemistry and Biophysics. 41:295-318.
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.
Aguilar, R.C., Watson, H.A., and Wendland, B. (2003). The yeast epsin Ent1 is recruited to membranes through multiple independent interactions. JBC 278(12):10737-43.
Aguilar, R.C. and Wendland, B. (2003). Ubiquitin: not just for proteasomes anymore. Curr. Opin. in Cell Biol. 15: 184-90.
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.
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.
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 Biology. 162:765-72.
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.
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.
Baggett, J.J., Shaw, J.D., and Wendland, B. (2003). Fluorescent Labeling of Yeast. Edited by J. Lippincott-Schwartz and P. Matsudaira. Current Protocols in Cell Biology, Unit 4.13.
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.
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.
Hurley, J.H. and Wendland, B. (2002). Endocytosis: Driving membranes around the bend. Cell. 111:143-6.
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.
Wendland, B. Epsins: adaptors in endocytosis? (2002) Nature Rev. Mol. Cell Biol. 3:971-7.
Baggett, J.J., and Wendland, B. (2001). Clathrin function in yeast endocytosis. Traffic. 2(5):297- 302.
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.
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.
Wendland, B. (2001). Everything you ever wanted to know about endocytosis. Review of: Endocytosis, edited by M. Marsh. Volume 36 in Frontiers in Molecular Biology series, edited by B.D. Hames and D.M. Glover. Oxford University Press, 2001. Nature Cell Biology. 3: E254.
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.
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.
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.
Wendland, B., Steece, K.E., and Emr, S.D. (1999) Yeast Epsins contain an essential N-terminal ENTH domain, bind clathrin, and are required for endocytosis. EMBO Journal. 18(16):4383-4393.
Kay, B.K., Yamabhai, M., Wendland, B., and Emr, S.D. (1998). Identification of a novel domain shared by putative components of the endocytic and cytoskeletal machinery. Protein Science 8: 435-438.
Wendland, B. and Emr, S.D. (1998). Pan1p, yeast eps15, functions as a multivalent adaptor that mediates protein-protein interactions essential for endocytosis. J. Cell Biol. 141: 71-84.
Wendland, B. (1998). For budding yeast investigators. Review of: Methods in Yeast Genetics by A. Adams, D.E. Gottschling, C.A. Kaiser, and T. Stearns. Cold Spring Harbor Laboratory Press, 1997. Trends Cell Biol. 8:462-463.
Babst, M., Wendland, B., Estepa, E.J., and Emr, S.D. (1998). The Vps4p AAA ATPase regulates membrane association of a Vps protein complex required for normal endosome function. EMBO J. 17: 2982-2993.
Wendland, B., Emr, S.D., and Riezman, H. (1998). Protein traffic in the yeast endocytic and vacuolar sorting pathways. Curr. Opin. in Cell Biol. 10: 513-522.
Wendland, B., McCaffery, J.M., Xiao, Q. and Emr, S.D. (1996). A novel fluorescence-activated cell sorter-based screen for yeast endocytosis mutants identifies a yeast homologue of mammalian eps15. J. Cell Biol. 135: 1485-1500.
Malgaroli, A., Ting, A.E., Wendland, B., Bergamaschi, A., Villa, A., Tsien, R.W., and Scheller, R.H. (1995). Presynaptic component of long-term potentiation visualized at individual hippocampal synapses. Science 268, 1624-1628.
Wendland, B., Schweizer, F.E., Ryan, T.A., Nakane, M., Murad, F., Scheller, R.H., and Tsien, R.W. (1994). Existence of nitric oxide synathase in rat hippocampal pyramidal cells. Proc. Natl. Acad. Sci. 91, 2151-2155.
Wendland, B. and Scheller, R.H. (1994). Molecular mechanism of synaptic vesicle docking and membrane fusion. Semin Neurosci 6: 167-76.
Wendland, B. and Scheller, R.H. (1994). Secretion in AtT-20 cells stably transfected with soluble synaptotagmins. Mol. Endocrinology 8, 1070-1082.
Miller, K.G., Wendland, B., and Scheller, R.H. (1993). Identification of a 34 kd synaptic vesicle and nervous system specific protein. Brain Res., 616, 99-104.
Ryan, T.A., Reuter, H., Wendland, B., Schweizer, F.E., Tsien, R.W., and Smith, S.J. (1993). The kinetics of synaptic vesicle recycling measured at single presynaptic boutons. Neuron 11: 713-724.
Wendland, B., Miller, K.G., Schilling, J. and Scheller, R.H. (1991). Differential expression of the p65 gene family. Neuron. 6, 993-1007.
Ngsee, J.K., Miller, K., Wendland, B., and Scheller, R.H. (1990). Multiple GTP-binding proteins from cholinergic synaptic vesicles. J. Neuroscience. 10, 317-322.
Ngsee J.K., Trimble W.S., Elferink L.A., Wendland B., Miller K., Calakos N., Scheller R.H. (1990). Molecular analysis of proteins associated with the synaptic vesicle membrane. Cold Spring Harb Symp Quant Biol. 55:111-8.