We are fascinated by the fundamental question of how the internal organization of cells is established, maintained and regulated. All cells have an intrinsic polarity, set up in response to extracellular and intracellular cues. [Just imagine what would happen if cells did not have an intrinsic polarity: they wouldn't know which axis to divide along, epithelial cells wouldn't be able to transport, secretory cells would secrete things to the wrong surface, cells wouldn't be able to migrate or chemotax, cells wouldn't be able to undergo asymmetric divisions that generate cellular diversity during development, etc!]. We study two model systems to delve into an understanding of the molecular basis of polarity. The first is to elucidate the structure and function of the apical aspect of polarized epithelial cells. These cells have distinct apical and basolateral domains that allow them to perform their transport function. Establishment of polarity is directed by cues supplied by the extracellular matrix and adjacent cells, which the cell interprets using signal transduction pathways to assemble a polarized cytoskeleton. The second system is the budding yeast Saccharomyces cerevisiae where we wish to determine how the cell establishes and sets up its polarity. During cell growth in yeast, the mother cell picks a region on the cell surface to which secretion is targeted for the assembly of a bud, and then all organelles have to be segregated along this axis during cell division. In both the epithelial cell and yeast systems, regulatory pathways organize microfilaments which then play a central role in this functional polarization.

These systems have lead us to study not only the components of microfilaments, actin and actin-binding proteins, but also their regulation by signal transduction pathways and how they participate in membrane traffick pathways. We use diverse biochemical, structural, genetic and cell biological approaches to investigate the molecular basis underlying microfilament regulation, structure and function.

Our Two Major Projects Are:

1. An investigation into the molecular organization and function of the microfilaments that make up the microvilli on the apical aspect of polarized cells.

Functionally polarized epithelial cells have a highly organized apical domain. To get insight into its functional construction, we are studying all the components of the cortical cytoskeleton of these cells. We have identified and characterized several microfilament-associated proteins (including villin, fimbrin, brush border myosin I, and ezrin) and, together with ultrastructural studies, have begun to piece together the functions of each component (reviewed in 1-3). Our current focus is on ezrin, a protein enriched in cell surface microvilli (Figure 1a and 1b), that we named in honor of Ezra Cornell.

Ezrin is the founding member of the ERM (ezrin-radixin-moesin) protein family, closely related to the tumor suppressor merlin and a member of the Band 4.1 superfamily (Figure 2). The established function of ezrin is to provide a regulated linkage between the actin cytoskeleton and the plasma membrane. Recent work suggests that ezrin and associated proteins have a much broader role: they contribute to both the morphology of the cell cortex and participate in signaling and membrane traffic pathways.

In addition, the first PDZ domain of EBP50 also binds the C-terminal tail of some cytoplasmic proteins, including EPI64 and nadrin (5) (Figure 4a). EPI64 has a TBC domain that is both a RabGAP and binds Arf6-GTP (Figure 4b) and regulates both membrane traffic and the presence of microvilli on the surface of epithelial cells (6). Nadrin is a BAR- and RhoGAP-containing protein that associates with EBP50, although the function of this interaction is not yet clear.

Ezrin (and radixin and moesin) is regulated by conformational masking (Figure 3): in the dormant form, a high affinity intra-molecular association exists between the FERM domain (also known as the N-ERMAD) and the C-ERMAD (8). Dissociation of this intramolecular interaction is necessary to unmask binding sites for several ligands of ezrin, including EBP50 (9) and F-actin (8).

Work from a number of labs has indicated that ezrin activation is regulated by signaling pathways, and that ezrin itself may be a hub of key signaling pathways (summarized diagrammatically in Figure 5). We are currently investigating how ezrin participates in regulation of microfilaments and membrane traffic on the apical aspect of epithelial cells.

Since ezrin undergoes conformational regulation and is implicated in multiple protein-protein interactions, it will be very revealing to understand these conformational changes and unmasking of binding sites at the atomic level. Towards this goal, we are collaborating with Dr. P. Andrew Karplus (formerly a colleague at Cornell, now at Oregon State University) and colleagues on the X-ray structure of various forms of ezrin alone and complexed with its ligands. So far, we have determined the structure of the associated N- (blue) and C-ERMADs (red) of moesin (Figure 6) in which the EBP50 and F-actin binding sites are masked (10), thus revealing the interface between these domains involved in masking binding protein interaction sites. We have also determined the structural basis of masking of the EBP50 binding site in dormant ezrin by determining the surface on the FERM domain to which EBP50 binds (11), and the structural changes that occur in the FERM domain upon release of the C-ERMAD (12).

Very recently, in work from the Tesmer lab at the University of Michigan, we have reported on the structure of full length dormant ERM protein from insect cells (13) (Figure 7 and moesin structure movie). As you can see, the central α-helical domain forms an anti-parallel coiled-coil structure. Based on biophysical studies on wild type and mutant proteins, we believe that the molecule is spring-loaded, so that upon activation when the C-ERMAD (red) dissociates from the FERM (blue) the central domain extends into a long α-helix spanning about 25nm between the membrane associated (FERM) domain and the F-actin binding site (located in the last 30 residues of the C-ERMAD).

An important finding was that yeast tropomyosins (encoded by the TPM1 and TPM2 genes specifying two related isoforms), are a specific component of actin cables and provide an essential function (15) (Figure 9). Using a conditional tropomyosin mutant (tpm2Δtpm1-2), we found that actin cables are highly dynamic and they, not cortical patches, determine the polarity of secretion (15).

We have also shown that the unconventional myosin V encoded by the MYO2 gene is the molecular motor that delivers secretory vesicles down actin cables to their site of exocytosis (18, 19). Remarkably, this same myosin is responsible for orienting the nucleus, as shown in Figure 10.

In collaboration with Tim Huffaker's lab in our Department, we have shown it does this by binding through its tail to a protein called Kar9p, which itself binds to Bim1p bound to the end of cytoplasmic microtubules (20). Myo2p is also responsible for segregation of the vacuole, peroxisomes and the trans-Golgi network (Figure 11). Myo2p has to transport these various cargos at the right time during the cell cycle; current work is aimed at understanding how this is achieved.

Our current studies are aimed at understanding the regulation of yeast formins (23) and the distinct contributions of yeast's two formins, Bni1p and Bnr1p (24), and how yeast distributes actin between different organizations (26).

References:

1. Bretscher, A. (1991). Microfilament structure and function in the cortical cytoskeleton. Ann. Rev. Cell Biol. 7, 337-374.

2. Bretscher, A., Chambers, D., Nguyen, R. & Reczek, D. (2000) ERM-merlin and EBP50 protein families in plasma membrane organization and function. Ann. Rev. Cell & Devel. Biol. 16, 113-143.

3. Bretscher, A., Edwards, K. & Fehon, R. (2002) ERM proteins and merlin: integrators at the cell cortex. Nature Reviews: Molecular and Cell Biology 3, 586-599.

4. Reczek, D., Berryman, M. & Bretscher, A. (1997) Identification of EBP50: a PDZ domain containing phosphoprotein that associates with members of the ERM family. J. Cell Biol. 139, 169-179.

5. Reczek, D. & Bretscher, A. (2001) Identification of EPI64, a TBC/rabGAP domain-containing microvillar protein that binds to the first PDZ domain of EBP50 and E3KARP. J. Cell Biol. 153, 191-206.

6. Hanono, A., Garbett, D., Reczek, D., Chambers, D. N. & Bretscher, A. (2006). EPI64 regulates microvillar sub-domains and structure. J. Cell Biol. 175, 803-813.

7. Ilani, T., Khanna, C., Zhou, M., Veenstra, T. D. & Bretscher, A. (2007). Immune synapse formation requires ZAP-70 recruitment by ezrin and CD43 removal by moesin. J. Cell Biol. 179, 733-746

8. Gary, R. & Bretscher, A. (1995). Ezrin self-association involves binding of an N-terminal domain to a normally masked C-terminal domain that includes the F-actin binding site. Mol. Biol. Cell 6, 1061-1075.

9. Reczek, D. & Bretscher, A. (1998). The carboxy-terminal region of EBP50 binds to a site in the amino-terminal domain of ezrin that is masked in the dormant monomer. J. Biol. Chem. 273, 18378-18384.

10. Pearson, M., Reczek, D., Bretscher, A. & Karplus, P. A. (2000). Structure of the ERM protein moesin reveals the FERM domain fold masked by an extended actin-binding tail domain Cell 101, 259-270.

11. Finnerty, C., Chambers, D., Ingraffea, J., Faber, H. R., Karplus, P. A. & Bretscher, A. (2004). The EBP50-moesin interaction: structural analysis of a binding site regulated by direct masking on the FERM domain. J. Cell Sci. 117, 1547-1552.

12. Smith, W.J., Nassar, N., Bretscher, A., Cerione, R. A. & Karplus, P.A. (2003). Structure of the active FERM Domain of Ezrin: conformational and mobility changes identify keystone interactions. J. Biol. Chem. 278, 4949-4956.

13. Li, Q., Nance, M. R., Kulikauskas, R., Nyberg, K., Fehon, R., P., Karplus, P. A., Bretscher, A. & Tesmer, J. J. G. (2006). Self-masking in an intact ERM-merlin protein: an active role for the central a-helical domain. J. Mol. Biol. 365,1446-59.

14. Nuygen, R., Reczek, D. & Bretscher, A. (2001). Heirarchy of N- and C-ERMAD associations and common ligands between ezrin and merlin. J. Biol Chem. 276, 7621-7629.

15. Pruyne, D., Schott, D. & Bretscher, A. (1998). Tropomyosin-containing actin cables are the primary cytoskeletal determinants of polarity in budding yeast. J. Cell Biol. 143, 1931-1945.

16. Schott, D., Huffaker, T. & Bretscher, A. (2002). Microfilaments and microtubules: the news from yeast. Curr. Opin. Microbiol. 5, 564-574.

17. Pruyne, D., Legesse-Miller, A., Gao, L., Dong, Y. & Bretscher, A. (2004). Mechanisms of polarized growth and organelle segregation in yeast. Ann. Rev. Cell Dev. Biol. 20, 559-591.

18. Schott, D., Ho, J., Pruyne, D. & Bretscher, A. (1999). The carboxyl-terminal domain of a yeast myosin V has a direct role in secretory vesicle targeting. J. Cell Biol. 147, 791-807.

19. Schott, D., Collins, R. N. & Bretscher, A. (2002). Secretory vesicle transport velocity in living cells depends on the myosin-V lever-arm length. J. Cell Biol. 156, 35-39.

20. Yin, H., Pruyne, D., Huffaker, T. & Bretscher, A. (2000). Myosin V orientates the mitotic spindle in yeast. Nature 406, 1013-1015.

21. Evangelista, M., Pruyne, D., Amberg, D., Boone, C. & Bretscher, A. (2002). Formins direct Arp2/3-independent actin filament assembly to polarize cell growth in yeast. Nature Cell Biology, 4, 32-41.

22. Pruyne, D., Evangelista, M., Yang, C., Bi., E., Zigmond, S., Bretscher, A. & Boone, C. (2002). Role of formins in actin assembly: nucleation and barbed end association. Science 297, 612-615.

23. Dong, Y., Pruyne, D. & Bretscher, A. (2003). Two Rho pathways converge to regulate formin-dependent actin assembly in yeast. J. Cell Biol. 161, 1081-1092.

24. Pruyne, D., Gao., L., Bi., E. & Bretscher A. (2004). Stable and dynamic axes of polarity utilize distinct forming isoforms in budding yeast. Mol. Biol. Cell, 15, 4971-4989.

25. Bretscher, A. (2003). Polarized growth and organelle segregation in yeast: the tracks, motors and receptors. J. Cell Biol. 160, 811-816

26. Gao, L., Bretscher, A. (2008). Analysis of Unregulated Formin Activity Reveals How Yeast Can Balance F-Actin Assembly between Different Microfilament-based Organizations. Mol. Biol. Cell, 19, 1474-84.

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