- Email: [email protected]
Understanding ERM proteins – the awesome power of genetics finally brought to bear
Understanding ERM proteins – the awesome power of genetics finally brought to bear Sarah C Hughes1 and Richard G Fehon2 In epithelial cells, the Ezrin, Radixin and Moesin (ERM) proteins are involved in many cellular functions, including regulation of actin cytoskeleton, control of cell shape, adhesion and motility, and modulation of signaling pathways. However, discerning the specific cellular roles of ERMs has been complicated by redundancy between these proteins. Recent genetic studies in model organisms have identified unique roles for ERM proteins. These include the regulation of morphogenesis and maintenance of integrity of epithelial cells, stabilization of intercellular junctions, and regulation of the Rho small GTPase. These studies also suggest that ERMs have roles in actomyosin contractility and vesicular trafficking in the apical domain of epithelial cells. Thus, genetic analysis has enhanced our understanding of these widely expressed membraneassociated proteins. Addresses 1 Department of Cell Biology, University of Alberta, Edmonton, Alberta, T6G 2H7, Canada 2 Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, IL 60637, USA Corresponding author: Fehon, Richard G ([email protected])
Current Opinion in Cell Biology 2007, 19:51–56 This review comes from a themed issue on Cell structure and dynamics Edited by Daniel P Kiehart and Kerry Bloom Available online 18th December 2006 0955-0674/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. DOI 10.1016/j.ceb.2006.12.004
Introduction Apical/basal polarity, the ability to tightly adhere to one another, and a highly organized cytoskeleton are interdependent properties of all epithelial cells. Intrinsic and extrinsic cues coordinate these properties in developing epithelia, primarily via modulation of interactions between transmembrane proteins and underlying protein complexes, most notably those associated with the cytoskeleton. Cell biological studies have highlighted the importance of three paralogous proteins, Ezrin, Radixin and Moesin (ERM), in these crucial components of epithelial morphogenesis. These widely expressed proteins control the structure and function of specific subdomains of the cell cortex, such as apical microvilli, and participate in a variety of cellular processes, including motility, cell adhesion, the determination of cell shape www.sciencedirect.com
and intercellular signaling [1]. ERM proteins have an Nterminal FERM domain and a C-terminal actin-binding domain, and intramolecular interaction between these domains is believed to regulate ERM activity. These interactions in turn are regulated via phosphorylation of the C-terminal domain and binding of the FERM domain to phospholipids (for an in-depth review see [1]). Until quite recently, studies of ERM function have concentrated on their biochemical and biophysical properties, complemented by cell biological analysis in cultured mammalian cells. In addition, use of genetic approaches to characterize ERM function has been hampered by functional redundancy between these proteins in most mammalian cells. Recent studies using model systems, including the mouse, Drosophila and C. elegans, have circumvented this problem, providing important advances and several surprises in our understanding of the role of ERM proteins in epithelial integrity, apical cell surface morphogenesis and intercellular signaling. In this short review, we highlight progress in understanding ERM function made possible by genetic analysis. These studies have cemented the fundamental importance of ERM proteins in maintaining epithelial architecture and integrity, and raised new questions regarding the cellular and molecular mechanisms that provide a basis for these functions.
Morphogenesis of specialized epithelial structures: organization of intestinal epithelia The mammalian intestinal epithelium, with its extensive folding to form villi and elaborate apical microvilli known as the brush border, is one of the most highly organized and well studied epithelia. In mid-gestational mice, the intestinal lumen is less complex, consisting of a simple lumen lined by several layers of flat stratified epithelia surrounded by mesenchyme. As the intestine develops, apical junctions form between cells that line the primary lumen, with separate secondary junctional complexes forming between cells deeper within the epithelium (Figure 1a). Prior to birth, the secondary junctions enlarge, via fusion of vesicles containing apical membrane, forming a secondary lumen. The secondary lumen expands along with the junctional complexes and eventually fuses with the primary lumen. This results in a continuous structure lined with brush border microvilli extending from the epithelial cells [2,3]. Although the ERM proteins are functionally redundant when co-expressed in mammalian cells, recent work has shown that only Ezrin is expressed in the developing Current Opinion in Cell Biology 2007, 19:51–56
52 Cell structure and dynamics
Figure 1
Schematic representations of intestinal epithelial morphogenesis in the fetal mouse and C. elegans. Both require ERM proteins for the proper formation of the epithelial apical surface and junctional remodeling. (a) In the wild type mouse small secondary lumens, surrounded by apical membrane and bordered by adherens junctions, begin to form at day 15 (adherens junctions in red, apical membrane in blue). These expand to form lumens that then coalesce with the primary lumen to form the mature villi by day 19. In Ezrin knockout mice the secondary lumens fail to expand and do not join with the primary lumen resulting in shortened, fused villi. Based on data from [6]. Primary (18) and secondary (28) lumens are indicated. Mesenchymal cells are shown as red cells. (b) Intestinal morphogenesis in the worm. Paired drawings depict longitudinal (upper) and cross (lower) sections of the intestine in wild type and erm-1 mutant worms. Intercellular junctions (red) outline the apical (blue) membrane in both views. The longitudinal views depict only a late stage intestine. In the wild type, the cross sections show early, mid and late stage views. In the mutant, cross-sections represent early and late stages. Lines marked A, B, and C indicate points at which the corresponding cross-sections are taken. Following polarization the apical membrane and junctional complex accumulate at the future apical cortex of the developing intestinal cells. Junctions then remodel from an apical to an apical–lateral position, with a concomitant increase in the apical membrane to form the lumen of the future intestine. In erm-1 RNAi animals the junctions fail to remodel properly resulting in disorganization of the apical membrane and a twisted and occluded intestine. Based on data from [8,9].
intestinal epithelium [4,5,6]. Ezrin knockout mice appear normal at birth but fail to thrive and do not survive past weaning [6]. In these mice, adjacent villi are fused as a result of incomplete separation of apical cell surfaces during morphogenesis (Figure 1b). Surprisingly, Ezrin is not required for the formation of the microvilli, but rather for the organization and integrity of the apical terminal web from which the brush border microvilli project [6]. Current Opinion in Cell Biology 2007, 19:51–56
Also, both tight and adherens cell junctions are malformed and elongated. Interestingly, Crumbs, a transmembrane protein that identifies the apical surface of epithelial cells, is localized normally, indicating that apical/basal polarity is not grossly disrupted. These observations suggest that Ezrin functions to organize the apical cytoskeleton, which is necessary for proper elongation of apical microvilli and organization of the junctional www.sciencedirect.com
Understanding ERM proteins Hughes and Fehon 53
complex. The authors propose that apical tension may be important in villar morphogenesis, and that in the absence of Ezrin function abnormal apical tension results in incomplete secondary lumen formation [6]. The mechanism behind this defect is not yet clear, but studies in flies (see below) indicate that ERMs are important regulators of the small GTPase Rho, which is well known to regulate actomyosin contractility. A strikingly similar story emerges from genetic studies of ERM function in C. elegans. The worm intestine consists of a polarized cell monolayer with apical microvilli, similar to that of other animals. It forms from a repeated series of apposed pairs of cells that fold over to form a tube [7]. This requires dramatic changes in junctional organization and apical membrane organization both within and between the cells forming the intestine (Figure 1b). The C. elegans single ERM orthologue, ERM-1, is localized to the apical surfaces of a variety of epithelial cells. RNAi knockdown analysis in two laboratories found that loss of erm-1 is lethal and causes architectural defects in the morphogenesis of the digestive, reproductive and excretory systems [8,9]. Over- or under-expression of erm-1 results in a twisted, partially occluded lumen, and formation of large luminal cysts (Figure 1b). In the mutant intestine, the apical surfaces contain fewer microvilli and the apical junctions, while present, are distorted in shape. Notably, erm-1 defects are not caused by a general loss of polarity or integrity of the epithelium, but rather by failure of apical domain remodeling, including the junctional complex during gut morphogenesis. Consistent with this idea, genetic interactions were observed between ERM-1 and E-Cadherin, a junctional component [8]. In addition, reduction of erm-1 causes loss of F-actin accumulation in the apical cortex of intestinal cells [8], similar to what is observed in Drosophila Moesin mutants [10], and there appear to be functional similarities between erm-1 mutants and mutants in cytoskeletal components such as actin and b-H-Spectrin [8,9]. For example, mutations in genes encoding these proteins in worms display phenotypes, including the formation of luminal cysts, that resemble erm-1 phenotypes [9]. Taken together, these results indicate roles for ERM-1 in organizing the apical cytoskeleton and in modulating apical membrane morphogenesis and junctional remodeling. These phenotypes could reflect a role for ERM-1 in regulating actomyosin contractility in the apical cortex [8,9] as proposed for the mouse intestine [6], or a structural role for ERM-1 in linking to the actin cytoskeleton [8,9].
Organization of apical microvilli: photoreceptor morphogenesis Differentiation of photoreceptors in mammals and Drosophila involves large increases in the apical membrane via directed membrane traffic and subsequent membrane www.sciencedirect.com
specialization in association with the cytoskeleton. The apical microvilli of the retinal pigmented epithelium (RPE) interdigitate with the outer segments of the photoreceptor cells, providing both structural support and specific functions including vectorial transport and secretion. In mice, in addition to its role in the intestinal epithelium, Ezrin is required for proper morphogenesis of apical microvilli in the RPE [11]. Loss of Ezrin causes reduced apical microvilli, formation of microvillar inclusions and a reduction in the Muller cell apical microvilli, which face the RPE. Photoreceptors in the RPE develop more slowly in Ezrin knockout mice, but are still able to differentiate [11]. In addition, EBP50, one of many apical membrane proteins required for ion transport, is mislocalized [11,12]. Thus, although Ezrin is not required for formation or maintenance of the retinal pigment epithelium, it is critical for proper morphogenesis of the apical microvilli and other aspects of the photoreceptor apical membrane. As in C. elegans, there is a single ERM orthologue in Drosophila known as Moesin [13], which also has a critical role in photoreceptor morphogenesis. During final photoreceptor differentiation, the enlarged apical membrane is folded into compact columns of actin-rich photosensitive microvilli called rhabdomeres. These sit upon a stalk supported by an actin-rich terminal web at the base of the rhabdomere. Moesin is localized to this region, which is an area of active membrane trafficking and actin reorganization. Lowering Moesin protein levels in rhabdomere cells results in disorganization of this region, although the junctional complex and overall apical/basal polarity remain intact [14]. In addition, Moesin may have a role in regulating the transient receptor potential (TRP) calcium channel trafficking that is required for maintenance of adult photoreceptors [15]. In normal photoreceptors, TRP rapidly translocates from the rhabdomere membrane to the cytoplasm in response to light activation. In dark adapted photoreceptors, Moesin coimmunoprecipitates with TRP channels, suggesting they form a complex. In response to light, Moesin rapidly dephosphorylates and no longer associates with TRP. Furthermore, mutations that block phosphorylation at Thr 559, a residue that has been shown to be crucial for ERM function, cause retinal degeneration in response to light [15]. Taken together, these results suggest that Moesin may play a role in TRP translocation from the rhabdomere membrane in response to light activation, though the mechanism for this is still unclear. Consistent with the notion that ERM proteins function in vesicle trafficking in the fly eye, genetic studies in the mouse indicate that Ezrin also functions during the morphogenesis of tubulovesicular membrane compartments in parietal cells [16]. These H+, K+-ATPase rich intracellular vesicles fuse with the apical canalicular secretory membrane to generate and secrete gastric acid, Current Opinion in Cell Biology 2007, 19:51–56
54 Cell structure and dynamics
a process that requires dynamic changes within the actin cytoskeleton [17,18]. Adult mice with reduced Ezrin expression exhibited severe achlorhydria (loss of gastric acid secretion), increased cytoplasmic tubulovesicles, and decreased apical plasma membrane infoldings called canaliculae [16]. The authors concluded that Ezrin is essential for fusion of these tubulovesicles with the apical plasma membrane. It is striking that in the mouse, worm and flies, ERMs appear necessary for apical membrane remodeling, perhaps functioning in vesicle trafficking.
Mechanistic insights into ERM function from Drosophila Several groups have taken advantage of the lack of functional redundancy and powerful genetic tools available in Drosophila to analyze ERM function in a variety of tissues and developmental stages. This work has been reviewed in detail recently [19], so we will only consider the highlights here. Perhaps the most striking finding from phenotypic analyses of Moesin mutations in the fly is the wide range of developmental and morphological defects that have been observed. For example, female flies that express reduced amounts of Moesin protein in their germline produce offspring with dramatic anterior–posterior body axis defects that are remarkably similar to those caused by ‘posterior group’ mutations such as nanos and oskar [20,21]. In Moesin-deficient eggs, Oskar, which functions as a part of a complex that specifies posterior structures in the early embryo, fails to localize in the posterior cortex of the embryo. At the same time, the cortical actin network normally found in the egg is severely disrupted, suggesting that Moesin functions either to anchor or in some other way to organize the cortical cytoskeleton in the posterior pole. Another dramatic Moesin phenotype occurs in the imaginal discs, which are monolayered polarized epithelial structures that produce adult appendages such as wings and legs. Imaginal disc cells severely compromised for Moesin function lose contact with neighboring cells and migrate basally from the epithelium [10]. In these cells the apical cystoskeletal network and expression of junctional markers is also disrupted. Surprisingly, these phenotypes are strongly suppressed by reducing activity of the Rho small GTPase, suggesting that the cytoskeletal and epithelial integrity defects observed are due to misregulation of Rho activity [10,22]. Consistent with this hypothesis, overexpression of Rho in the imaginal epithelium produces phenotypes similar to loss of Moesin function [10]. This model differs significantly from the conventional view, derived from studies of mammalian ERM proteins, that all ERM proteins provide a structural link between the apical membrane and the actin cytoskeleton. However, a function in Rho regulation seems evolutionarily conserved, because cultured mammalian Current Opinion in Cell Biology 2007, 19:51–56
cells expressing dominant-negative Ezrin have increased levels of GTP-bound, activated Rho protein [10]. Studies in Drosophila have also provided key insights into how ERM functions are regulated. Studies in mammalian cells have shown that phosphorylation of a conserved Thr residue near the C terminus is a key factor in regulating head to tail intramolecular interactions and binding activity of ERM proteins. Control of this phosphorylation appears to be regulated by activity of the Rho signaling pathway (potentially forming a negative feedback loop — see above), though the responsible kinase is unknown and the possibility of other regulatory mechanisms remains open. Using a modular misexpression screen, a Sterile-20 family kinase, Slik, was identified and appears to regulate Moesin phosphorylation and function in a Rho-independent fashion [10,22]. Overexpression of Slik causes overproliferation in imaginal epithelia, while loss-of-function slik mutations have phenotypes remarkably similar to those caused by Moesin mutations. Phosphorylated Moesin protein, which is normally quite abundant in the apical domain of epithelial cells, is absent in slik mutant cells, indicating that Slik, either directly or through an intermediary kinase, is necessary for phosphorylation of the Cterminal Thr residue [22]. These experiments clearly identify a mechanism by which ERM activity is regulated in developing epithelia and suggests that dynamic regulation of ERM function is important in developing tissues, though it remains to be seen how Slik activity itself is regulated.
Conclusions ERM function is required in animals as divergent as mice and worms for remodeling of adherens junctions and the apical membrane during epithelial morphogenesis. Similarly, Moesin stabilizes adherens junctions in the early Drosophila embryo [23]. Clearly these phenotypes suggest an important role for ERM proteins in adherens junction formation and maintenance. This function may in part be mediated by the ability of ERM proteins to bind to and stabilize the actin cytoskeleton, which in turn is believed to be important for adherens junction stability. However, the events associated with apical remodeling undoubtedly require coordination of a variety of cellular processes and structures, for example vesicle trafficking and actomyosin contractility (Figure 2). At the moment it is unclear precisely which of these processes ERM proteins regulate, but given their ability to control activity of the small GTPase Rho, it seems likely that ERM functions could be quite complex. In this regard, a potential link between Moesin function and vesicle trafficking is particularly interesting. In addition to possible roles in this process described earlier [11,14,16], the recent demonstration of functional interactions between Moesin and the synaptotagmin-like protein Bitesize also suggests this possibility as these www.sciencedirect.com
Understanding ERM proteins Hughes and Fehon 55
Figure 2
3.
Mathan M, Moxey PC, Trier JS: Morphogenesis of fetal rat duodenal villi. Am J Anat 1976, 146:73-92.
4.
Berryman M, Franck Z, Bretscher A: Ezrin is concentrated in the apical microvilli of a wide variety of epithelial cells whereas moesin is found primarily in endothelial cells. J Cell Sci 1993, 105:1025-1043.
5.
Ingraffea J, Reczek D, Bretscher A: Distinct cell type-specific expression of scaffolding proteins EBP50 and E3KARP: EBP50 is generally expressed with ezrin in specific epithelia, whereas E3KARP is not. Eur J Cell Biol 2002, 81:61-68.
6.
Saotome I, Curto M, McClatchey AI: Ezrin is essential for epithelial organization and villus morphogenesis in the developing intestine. Dev Cell 2004, 6:855-864. This study generates and characterizes an Ezrin knockout mutation in the mouse. It shows that Ezrin has unique functions in the morphogenesis of the villi and remodeling of the apical membrane and junctional complex in the developing intestine. 7.
Leung B, Hermann GJ, Priess JR: Organogenesis of the Caenorhabditis elegans intestine. Dev Biol 1999, 216:114-134.
8.
Van Furden D, Johnson K, Segbert C, Bossinger O: The C. elegans ezrin-radixin-moesin protein ERM-1 is necessary for apical junction remodelling and tubulogenesis in the intestine. Dev Biol 2004, 272:262-276. This study uses the RNAi technique to demonstrate that the ERM homologue in worms (ERM-1) is required for junctional positioning through interaction with the actin cytoskeleton. Additionally, this paper shows that ERM-1 functionally interacts with the E-cadherin homologue (HMR-1).
9.
This cartoon depicts possible roles of ERM proteins in the apical domains of epithelial cells suggested by recent genetic analysis in model organisms. These functions include regulating vesicle trafficking, adherens junction stability, actomyosin contractility and activity of the small GTPase Rho, in addition to the classical model for ERMs linking the actin cytoskeleton to the apical plasma membrane. It is currently unclear if all of these functions are linked via a single molecular mechanism (such as regulation of Rho activity), or whether ERM proteins have multiple, independent functions.
proteins are believed to regulate exocytosis [23]. In addition, Ezrin has been implicated in b-adrenergic receptor recycling [24,25]. Determining how ERM proteins regulate vesicle trafficking will be an important and exciting next step.
Acknowledgements The authors would like to thank the members of the Fehon laboratory for helpful comments and discussions. This work is supported by NIH R01 NS034783 to RGF.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest
Gobel V, Barrett PL, Hall DH, Fleming JT: Lumen morphogenesis in C. elegans requires the membrane-cytoskeleton linker erm-1. Dev Cell 2004, 6:865-873. As in [8], here the authors use RNAi to show that ERM-1 is required for apical membrane morphogenesis in the adult worm. They also show that ERM-1 phenotypes resemble those in actin knockdown worms. 10. Speck O, Hughes SC, Noren NK, Kulikauskas RM, Fehon RG: Moesin functions antagonistically to the Rho pathway to maintain epithelial integrity. Nature 2003, 421:83-87. 11. Bonilha VL, Rayborn ME, Saotome I, McClatchey AI, Hollyfield JG: Microvilli defects in retinas of ezrin knockout mice. Exp Eye Res 2006, 82:720-729. This study demonstrates a role for Ezrin in the development and homeostasis of the retina and photoreceptors in mice. 12. Bonilha VL, Rodriguez-Boulan E: Polarity and developmental regulation of two PDZ proteins in the retinal pigment epithelium. Invest Ophthalmol Vis Sci 2001, 42:3274-3282. 13. McCartney BM, Fehon RG: Distinct cellular and subcellular patterns of expression imply distinct functions for the Drosophila homologues of moesin and the neurofibromatosis 2 tumor suppressor, merlin. J Cell Biol 1996, 133:843-852. 14. Karagiosis SA, Ready DF: Moesin contributes an essential structural role in Drosophila photoreceptor morphogenesis. Development 2004, 131:725-732. 15. Chorna-Ornan I, Tzarfaty V, Ankri-Eliahoo G, Joel-Almagor T, Meyer NE, Huber A, Payre F, Minke B: Light-regulated interaction of Dmoesin with TRP and TRPL channels is required for maintenance of photoreceptors. J Cell Biol 2005, 171:143-152. 16. Tamura A, Kikuchi S, Hata M, Katsuno T, Matsui T, Hayashi H, Suzuki Y, Noda T, Tsukita S: Achlorhydria by ezrin knockdown: defects in the formation/expansion of apical canaliculi in gastric parietal cells. J Cell Biol 2005, 169:21-28. Using RNAi mediated knockdown of Ezrin expression, the authors show that Ezrin plays a role in membrane trafficking and tubulovesicle formation in parietal cells.
1.
Bretscher A, Edwards K, Fehon RG: ERM proteins and Merlin: integrators at the cell cortex. Nat Rev Mol Cell Biol 2002, 3:586-599.
17. Forte JG, Yao X: The membrane-recruitment-and-recycling hypothesis of gastric HCl secretion. Trends Cell Biol 1996, 6:45-48.
2.
Madara JL, Neutra MR, Trier JS: Junctional complexes in fetal rat small intestine during morphogenesis. Dev Biol 1981, 86:170-178.
18. Forte JG, Ly B, Rong Q, Ogihara S, Ramilo M, Agnew B, Yao X: State of actin in gastric parietal cells. Am J Physiol 1998, 274:C97-C104.
www.sciencedirect.com
Current Opinion in Cell Biology 2007, 19:51–56
56 Cell structure and dynamics
19. Polesello C, Payre F: Small is beautiful: what flies tell us about ERM protein function in development. Trends Cell Biol 2004, 14:294-302. 20. Jankovics F, Sinka R, Lukacsovich T, Erdelyi M: MOESIN crosslinks actin and cell membrane in drosophila oocytes and is required for OSKAR anchoring. Curr Biol 2002, 12:2060-2065. 21. Polesello C, Delon I, Valenti P, Ferrer P, Payre F: Dmoesin controls actin-based cell shape and polarity during Drosophila melanogaster oogenesis. Nat Cell Biol 2002, 4:782-789. 22. Hipfner DR, Keller N, Cohen SM: Slik Sterile-20 kinase regulates Moesin activity to promote epithelial integrity during tissue growth. Genes Dev 2004, 18:2243-2248. This paper shows that the Sterile-20 family kinase Slik is necessary for phosphorylation of Thr 559 near the C-terminus of Moesin, and thereby regulates its activity. The authors also show that Rho signaling does not seem to control phosphorylation of this site in cultured S2 cells.
Current Opinion in Cell Biology 2007, 19:51–56
23. Pilot F, Philippe JM, Lemmers C, Lecuit T: Spatial control of actin organization at adherens junctions by a synaptotagmin-like protein Btsz. Nature 2006, 442:580-584. In this study the authors demonstrate that Bitesize, a synaptotagmin like protein, functions to stabilize adherens junctions in the early Drosophila embryo via its interactions with Moesin. The results suggest that Moesin regulates the actin cytoskeleton, thereby promoting adherens junction stability. 24. Cao TT, Deacon HW, Reczek D, Bretscher A, von Zastrow M: A kinase-regulated PDZ-domain interaction controls endocytic sorting of the b2-adrenergic receptor. Nature 1999, 401:286-290. 25. Stanasila L, Abuin L, Diviani D, Cotecchia S: Ezrin directly interacts with the a1b-adrenergic receptor and plays a role in receptor recycling. J Biol Chem 2006, 281:4354-4363.
www.sciencedirect.com
Current Opinion in Cell Biology 2007, 19:51–56 This review comes from a themed issue on Cell structure and dynamics Edited by Daniel P Kiehart and Kerry Bloom Available online 18th December 2006 0955-0674/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. DOI 10.1016/j.ceb.2006.12.004
Introduction Apical/basal polarity, the ability to tightly adhere to one another, and a highly organized cytoskeleton are interdependent properties of all epithelial cells. Intrinsic and extrinsic cues coordinate these properties in developing epithelia, primarily via modulation of interactions between transmembrane proteins and underlying protein complexes, most notably those associated with the cytoskeleton. Cell biological studies have highlighted the importance of three paralogous proteins, Ezrin, Radixin and Moesin (ERM), in these crucial components of epithelial morphogenesis. These widely expressed proteins control the structure and function of specific subdomains of the cell cortex, such as apical microvilli, and participate in a variety of cellular processes, including motility, cell adhesion, the determination of cell shape www.sciencedirect.com
and intercellular signaling [1]. ERM proteins have an Nterminal FERM domain and a C-terminal actin-binding domain, and intramolecular interaction between these domains is believed to regulate ERM activity. These interactions in turn are regulated via phosphorylation of the C-terminal domain and binding of the FERM domain to phospholipids (for an in-depth review see [1]). Until quite recently, studies of ERM function have concentrated on their biochemical and biophysical properties, complemented by cell biological analysis in cultured mammalian cells. In addition, use of genetic approaches to characterize ERM function has been hampered by functional redundancy between these proteins in most mammalian cells. Recent studies using model systems, including the mouse, Drosophila and C. elegans, have circumvented this problem, providing important advances and several surprises in our understanding of the role of ERM proteins in epithelial integrity, apical cell surface morphogenesis and intercellular signaling. In this short review, we highlight progress in understanding ERM function made possible by genetic analysis. These studies have cemented the fundamental importance of ERM proteins in maintaining epithelial architecture and integrity, and raised new questions regarding the cellular and molecular mechanisms that provide a basis for these functions.
Morphogenesis of specialized epithelial structures: organization of intestinal epithelia The mammalian intestinal epithelium, with its extensive folding to form villi and elaborate apical microvilli known as the brush border, is one of the most highly organized and well studied epithelia. In mid-gestational mice, the intestinal lumen is less complex, consisting of a simple lumen lined by several layers of flat stratified epithelia surrounded by mesenchyme. As the intestine develops, apical junctions form between cells that line the primary lumen, with separate secondary junctional complexes forming between cells deeper within the epithelium (Figure 1a). Prior to birth, the secondary junctions enlarge, via fusion of vesicles containing apical membrane, forming a secondary lumen. The secondary lumen expands along with the junctional complexes and eventually fuses with the primary lumen. This results in a continuous structure lined with brush border microvilli extending from the epithelial cells [2,3]. Although the ERM proteins are functionally redundant when co-expressed in mammalian cells, recent work has shown that only Ezrin is expressed in the developing Current Opinion in Cell Biology 2007, 19:51–56
52 Cell structure and dynamics
Figure 1
Schematic representations of intestinal epithelial morphogenesis in the fetal mouse and C. elegans. Both require ERM proteins for the proper formation of the epithelial apical surface and junctional remodeling. (a) In the wild type mouse small secondary lumens, surrounded by apical membrane and bordered by adherens junctions, begin to form at day 15 (adherens junctions in red, apical membrane in blue). These expand to form lumens that then coalesce with the primary lumen to form the mature villi by day 19. In Ezrin knockout mice the secondary lumens fail to expand and do not join with the primary lumen resulting in shortened, fused villi. Based on data from [6]. Primary (18) and secondary (28) lumens are indicated. Mesenchymal cells are shown as red cells. (b) Intestinal morphogenesis in the worm. Paired drawings depict longitudinal (upper) and cross (lower) sections of the intestine in wild type and erm-1 mutant worms. Intercellular junctions (red) outline the apical (blue) membrane in both views. The longitudinal views depict only a late stage intestine. In the wild type, the cross sections show early, mid and late stage views. In the mutant, cross-sections represent early and late stages. Lines marked A, B, and C indicate points at which the corresponding cross-sections are taken. Following polarization the apical membrane and junctional complex accumulate at the future apical cortex of the developing intestinal cells. Junctions then remodel from an apical to an apical–lateral position, with a concomitant increase in the apical membrane to form the lumen of the future intestine. In erm-1 RNAi animals the junctions fail to remodel properly resulting in disorganization of the apical membrane and a twisted and occluded intestine. Based on data from [8,9].
intestinal epithelium [4,5,6]. Ezrin knockout mice appear normal at birth but fail to thrive and do not survive past weaning [6]. In these mice, adjacent villi are fused as a result of incomplete separation of apical cell surfaces during morphogenesis (Figure 1b). Surprisingly, Ezrin is not required for the formation of the microvilli, but rather for the organization and integrity of the apical terminal web from which the brush border microvilli project [6]. Current Opinion in Cell Biology 2007, 19:51–56
Also, both tight and adherens cell junctions are malformed and elongated. Interestingly, Crumbs, a transmembrane protein that identifies the apical surface of epithelial cells, is localized normally, indicating that apical/basal polarity is not grossly disrupted. These observations suggest that Ezrin functions to organize the apical cytoskeleton, which is necessary for proper elongation of apical microvilli and organization of the junctional www.sciencedirect.com
Understanding ERM proteins Hughes and Fehon 53
complex. The authors propose that apical tension may be important in villar morphogenesis, and that in the absence of Ezrin function abnormal apical tension results in incomplete secondary lumen formation [6]. The mechanism behind this defect is not yet clear, but studies in flies (see below) indicate that ERMs are important regulators of the small GTPase Rho, which is well known to regulate actomyosin contractility. A strikingly similar story emerges from genetic studies of ERM function in C. elegans. The worm intestine consists of a polarized cell monolayer with apical microvilli, similar to that of other animals. It forms from a repeated series of apposed pairs of cells that fold over to form a tube [7]. This requires dramatic changes in junctional organization and apical membrane organization both within and between the cells forming the intestine (Figure 1b). The C. elegans single ERM orthologue, ERM-1, is localized to the apical surfaces of a variety of epithelial cells. RNAi knockdown analysis in two laboratories found that loss of erm-1 is lethal and causes architectural defects in the morphogenesis of the digestive, reproductive and excretory systems [8,9]. Over- or under-expression of erm-1 results in a twisted, partially occluded lumen, and formation of large luminal cysts (Figure 1b). In the mutant intestine, the apical surfaces contain fewer microvilli and the apical junctions, while present, are distorted in shape. Notably, erm-1 defects are not caused by a general loss of polarity or integrity of the epithelium, but rather by failure of apical domain remodeling, including the junctional complex during gut morphogenesis. Consistent with this idea, genetic interactions were observed between ERM-1 and E-Cadherin, a junctional component [8]. In addition, reduction of erm-1 causes loss of F-actin accumulation in the apical cortex of intestinal cells [8], similar to what is observed in Drosophila Moesin mutants [10], and there appear to be functional similarities between erm-1 mutants and mutants in cytoskeletal components such as actin and b-H-Spectrin [8,9]. For example, mutations in genes encoding these proteins in worms display phenotypes, including the formation of luminal cysts, that resemble erm-1 phenotypes [9]. Taken together, these results indicate roles for ERM-1 in organizing the apical cytoskeleton and in modulating apical membrane morphogenesis and junctional remodeling. These phenotypes could reflect a role for ERM-1 in regulating actomyosin contractility in the apical cortex [8,9] as proposed for the mouse intestine [6], or a structural role for ERM-1 in linking to the actin cytoskeleton [8,9].
Organization of apical microvilli: photoreceptor morphogenesis Differentiation of photoreceptors in mammals and Drosophila involves large increases in the apical membrane via directed membrane traffic and subsequent membrane www.sciencedirect.com
specialization in association with the cytoskeleton. The apical microvilli of the retinal pigmented epithelium (RPE) interdigitate with the outer segments of the photoreceptor cells, providing both structural support and specific functions including vectorial transport and secretion. In mice, in addition to its role in the intestinal epithelium, Ezrin is required for proper morphogenesis of apical microvilli in the RPE [11]. Loss of Ezrin causes reduced apical microvilli, formation of microvillar inclusions and a reduction in the Muller cell apical microvilli, which face the RPE. Photoreceptors in the RPE develop more slowly in Ezrin knockout mice, but are still able to differentiate [11]. In addition, EBP50, one of many apical membrane proteins required for ion transport, is mislocalized [11,12]. Thus, although Ezrin is not required for formation or maintenance of the retinal pigment epithelium, it is critical for proper morphogenesis of the apical microvilli and other aspects of the photoreceptor apical membrane. As in C. elegans, there is a single ERM orthologue in Drosophila known as Moesin [13], which also has a critical role in photoreceptor morphogenesis. During final photoreceptor differentiation, the enlarged apical membrane is folded into compact columns of actin-rich photosensitive microvilli called rhabdomeres. These sit upon a stalk supported by an actin-rich terminal web at the base of the rhabdomere. Moesin is localized to this region, which is an area of active membrane trafficking and actin reorganization. Lowering Moesin protein levels in rhabdomere cells results in disorganization of this region, although the junctional complex and overall apical/basal polarity remain intact [14]. In addition, Moesin may have a role in regulating the transient receptor potential (TRP) calcium channel trafficking that is required for maintenance of adult photoreceptors [15]. In normal photoreceptors, TRP rapidly translocates from the rhabdomere membrane to the cytoplasm in response to light activation. In dark adapted photoreceptors, Moesin coimmunoprecipitates with TRP channels, suggesting they form a complex. In response to light, Moesin rapidly dephosphorylates and no longer associates with TRP. Furthermore, mutations that block phosphorylation at Thr 559, a residue that has been shown to be crucial for ERM function, cause retinal degeneration in response to light [15]. Taken together, these results suggest that Moesin may play a role in TRP translocation from the rhabdomere membrane in response to light activation, though the mechanism for this is still unclear. Consistent with the notion that ERM proteins function in vesicle trafficking in the fly eye, genetic studies in the mouse indicate that Ezrin also functions during the morphogenesis of tubulovesicular membrane compartments in parietal cells [16]. These H+, K+-ATPase rich intracellular vesicles fuse with the apical canalicular secretory membrane to generate and secrete gastric acid, Current Opinion in Cell Biology 2007, 19:51–56
54 Cell structure and dynamics
a process that requires dynamic changes within the actin cytoskeleton [17,18]. Adult mice with reduced Ezrin expression exhibited severe achlorhydria (loss of gastric acid secretion), increased cytoplasmic tubulovesicles, and decreased apical plasma membrane infoldings called canaliculae [16]. The authors concluded that Ezrin is essential for fusion of these tubulovesicles with the apical plasma membrane. It is striking that in the mouse, worm and flies, ERMs appear necessary for apical membrane remodeling, perhaps functioning in vesicle trafficking.
Mechanistic insights into ERM function from Drosophila Several groups have taken advantage of the lack of functional redundancy and powerful genetic tools available in Drosophila to analyze ERM function in a variety of tissues and developmental stages. This work has been reviewed in detail recently [19], so we will only consider the highlights here. Perhaps the most striking finding from phenotypic analyses of Moesin mutations in the fly is the wide range of developmental and morphological defects that have been observed. For example, female flies that express reduced amounts of Moesin protein in their germline produce offspring with dramatic anterior–posterior body axis defects that are remarkably similar to those caused by ‘posterior group’ mutations such as nanos and oskar [20,21]. In Moesin-deficient eggs, Oskar, which functions as a part of a complex that specifies posterior structures in the early embryo, fails to localize in the posterior cortex of the embryo. At the same time, the cortical actin network normally found in the egg is severely disrupted, suggesting that Moesin functions either to anchor or in some other way to organize the cortical cytoskeleton in the posterior pole. Another dramatic Moesin phenotype occurs in the imaginal discs, which are monolayered polarized epithelial structures that produce adult appendages such as wings and legs. Imaginal disc cells severely compromised for Moesin function lose contact with neighboring cells and migrate basally from the epithelium [10]. In these cells the apical cystoskeletal network and expression of junctional markers is also disrupted. Surprisingly, these phenotypes are strongly suppressed by reducing activity of the Rho small GTPase, suggesting that the cytoskeletal and epithelial integrity defects observed are due to misregulation of Rho activity [10,22]. Consistent with this hypothesis, overexpression of Rho in the imaginal epithelium produces phenotypes similar to loss of Moesin function [10]. This model differs significantly from the conventional view, derived from studies of mammalian ERM proteins, that all ERM proteins provide a structural link between the apical membrane and the actin cytoskeleton. However, a function in Rho regulation seems evolutionarily conserved, because cultured mammalian Current Opinion in Cell Biology 2007, 19:51–56
cells expressing dominant-negative Ezrin have increased levels of GTP-bound, activated Rho protein [10]. Studies in Drosophila have also provided key insights into how ERM functions are regulated. Studies in mammalian cells have shown that phosphorylation of a conserved Thr residue near the C terminus is a key factor in regulating head to tail intramolecular interactions and binding activity of ERM proteins. Control of this phosphorylation appears to be regulated by activity of the Rho signaling pathway (potentially forming a negative feedback loop — see above), though the responsible kinase is unknown and the possibility of other regulatory mechanisms remains open. Using a modular misexpression screen, a Sterile-20 family kinase, Slik, was identified and appears to regulate Moesin phosphorylation and function in a Rho-independent fashion [10,22]. Overexpression of Slik causes overproliferation in imaginal epithelia, while loss-of-function slik mutations have phenotypes remarkably similar to those caused by Moesin mutations. Phosphorylated Moesin protein, which is normally quite abundant in the apical domain of epithelial cells, is absent in slik mutant cells, indicating that Slik, either directly or through an intermediary kinase, is necessary for phosphorylation of the Cterminal Thr residue [22]. These experiments clearly identify a mechanism by which ERM activity is regulated in developing epithelia and suggests that dynamic regulation of ERM function is important in developing tissues, though it remains to be seen how Slik activity itself is regulated.
Conclusions ERM function is required in animals as divergent as mice and worms for remodeling of adherens junctions and the apical membrane during epithelial morphogenesis. Similarly, Moesin stabilizes adherens junctions in the early Drosophila embryo [23]. Clearly these phenotypes suggest an important role for ERM proteins in adherens junction formation and maintenance. This function may in part be mediated by the ability of ERM proteins to bind to and stabilize the actin cytoskeleton, which in turn is believed to be important for adherens junction stability. However, the events associated with apical remodeling undoubtedly require coordination of a variety of cellular processes and structures, for example vesicle trafficking and actomyosin contractility (Figure 2). At the moment it is unclear precisely which of these processes ERM proteins regulate, but given their ability to control activity of the small GTPase Rho, it seems likely that ERM functions could be quite complex. In this regard, a potential link between Moesin function and vesicle trafficking is particularly interesting. In addition to possible roles in this process described earlier [11,14,16], the recent demonstration of functional interactions between Moesin and the synaptotagmin-like protein Bitesize also suggests this possibility as these www.sciencedirect.com
Understanding ERM proteins Hughes and Fehon 55
Figure 2
3.
Mathan M, Moxey PC, Trier JS: Morphogenesis of fetal rat duodenal villi. Am J Anat 1976, 146:73-92.
4.
Berryman M, Franck Z, Bretscher A: Ezrin is concentrated in the apical microvilli of a wide variety of epithelial cells whereas moesin is found primarily in endothelial cells. J Cell Sci 1993, 105:1025-1043.
5.
Ingraffea J, Reczek D, Bretscher A: Distinct cell type-specific expression of scaffolding proteins EBP50 and E3KARP: EBP50 is generally expressed with ezrin in specific epithelia, whereas E3KARP is not. Eur J Cell Biol 2002, 81:61-68.
6.
Saotome I, Curto M, McClatchey AI: Ezrin is essential for epithelial organization and villus morphogenesis in the developing intestine. Dev Cell 2004, 6:855-864. This study generates and characterizes an Ezrin knockout mutation in the mouse. It shows that Ezrin has unique functions in the morphogenesis of the villi and remodeling of the apical membrane and junctional complex in the developing intestine. 7.
Leung B, Hermann GJ, Priess JR: Organogenesis of the Caenorhabditis elegans intestine. Dev Biol 1999, 216:114-134.
8.
Van Furden D, Johnson K, Segbert C, Bossinger O: The C. elegans ezrin-radixin-moesin protein ERM-1 is necessary for apical junction remodelling and tubulogenesis in the intestine. Dev Biol 2004, 272:262-276. This study uses the RNAi technique to demonstrate that the ERM homologue in worms (ERM-1) is required for junctional positioning through interaction with the actin cytoskeleton. Additionally, this paper shows that ERM-1 functionally interacts with the E-cadherin homologue (HMR-1).
9.
This cartoon depicts possible roles of ERM proteins in the apical domains of epithelial cells suggested by recent genetic analysis in model organisms. These functions include regulating vesicle trafficking, adherens junction stability, actomyosin contractility and activity of the small GTPase Rho, in addition to the classical model for ERMs linking the actin cytoskeleton to the apical plasma membrane. It is currently unclear if all of these functions are linked via a single molecular mechanism (such as regulation of Rho activity), or whether ERM proteins have multiple, independent functions.
proteins are believed to regulate exocytosis [23]. In addition, Ezrin has been implicated in b-adrenergic receptor recycling [24,25]. Determining how ERM proteins regulate vesicle trafficking will be an important and exciting next step.
Acknowledgements The authors would like to thank the members of the Fehon laboratory for helpful comments and discussions. This work is supported by NIH R01 NS034783 to RGF.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest
Gobel V, Barrett PL, Hall DH, Fleming JT: Lumen morphogenesis in C. elegans requires the membrane-cytoskeleton linker erm-1. Dev Cell 2004, 6:865-873. As in [8], here the authors use RNAi to show that ERM-1 is required for apical membrane morphogenesis in the adult worm. They also show that ERM-1 phenotypes resemble those in actin knockdown worms. 10. Speck O, Hughes SC, Noren NK, Kulikauskas RM, Fehon RG: Moesin functions antagonistically to the Rho pathway to maintain epithelial integrity. Nature 2003, 421:83-87. 11. Bonilha VL, Rayborn ME, Saotome I, McClatchey AI, Hollyfield JG: Microvilli defects in retinas of ezrin knockout mice. Exp Eye Res 2006, 82:720-729. This study demonstrates a role for Ezrin in the development and homeostasis of the retina and photoreceptors in mice. 12. Bonilha VL, Rodriguez-Boulan E: Polarity and developmental regulation of two PDZ proteins in the retinal pigment epithelium. Invest Ophthalmol Vis Sci 2001, 42:3274-3282. 13. McCartney BM, Fehon RG: Distinct cellular and subcellular patterns of expression imply distinct functions for the Drosophila homologues of moesin and the neurofibromatosis 2 tumor suppressor, merlin. J Cell Biol 1996, 133:843-852. 14. Karagiosis SA, Ready DF: Moesin contributes an essential structural role in Drosophila photoreceptor morphogenesis. Development 2004, 131:725-732. 15. Chorna-Ornan I, Tzarfaty V, Ankri-Eliahoo G, Joel-Almagor T, Meyer NE, Huber A, Payre F, Minke B: Light-regulated interaction of Dmoesin with TRP and TRPL channels is required for maintenance of photoreceptors. J Cell Biol 2005, 171:143-152. 16. Tamura A, Kikuchi S, Hata M, Katsuno T, Matsui T, Hayashi H, Suzuki Y, Noda T, Tsukita S: Achlorhydria by ezrin knockdown: defects in the formation/expansion of apical canaliculi in gastric parietal cells. J Cell Biol 2005, 169:21-28. Using RNAi mediated knockdown of Ezrin expression, the authors show that Ezrin plays a role in membrane trafficking and tubulovesicle formation in parietal cells.
1.
Bretscher A, Edwards K, Fehon RG: ERM proteins and Merlin: integrators at the cell cortex. Nat Rev Mol Cell Biol 2002, 3:586-599.
17. Forte JG, Yao X: The membrane-recruitment-and-recycling hypothesis of gastric HCl secretion. Trends Cell Biol 1996, 6:45-48.
2.
Madara JL, Neutra MR, Trier JS: Junctional complexes in fetal rat small intestine during morphogenesis. Dev Biol 1981, 86:170-178.
18. Forte JG, Ly B, Rong Q, Ogihara S, Ramilo M, Agnew B, Yao X: State of actin in gastric parietal cells. Am J Physiol 1998, 274:C97-C104.
www.sciencedirect.com
Current Opinion in Cell Biology 2007, 19:51–56
56 Cell structure and dynamics
19. Polesello C, Payre F: Small is beautiful: what flies tell us about ERM protein function in development. Trends Cell Biol 2004, 14:294-302. 20. Jankovics F, Sinka R, Lukacsovich T, Erdelyi M: MOESIN crosslinks actin and cell membrane in drosophila oocytes and is required for OSKAR anchoring. Curr Biol 2002, 12:2060-2065. 21. Polesello C, Delon I, Valenti P, Ferrer P, Payre F: Dmoesin controls actin-based cell shape and polarity during Drosophila melanogaster oogenesis. Nat Cell Biol 2002, 4:782-789. 22. Hipfner DR, Keller N, Cohen SM: Slik Sterile-20 kinase regulates Moesin activity to promote epithelial integrity during tissue growth. Genes Dev 2004, 18:2243-2248. This paper shows that the Sterile-20 family kinase Slik is necessary for phosphorylation of Thr 559 near the C-terminus of Moesin, and thereby regulates its activity. The authors also show that Rho signaling does not seem to control phosphorylation of this site in cultured S2 cells.
Current Opinion in Cell Biology 2007, 19:51–56
23. Pilot F, Philippe JM, Lemmers C, Lecuit T: Spatial control of actin organization at adherens junctions by a synaptotagmin-like protein Btsz. Nature 2006, 442:580-584. In this study the authors demonstrate that Bitesize, a synaptotagmin like protein, functions to stabilize adherens junctions in the early Drosophila embryo via its interactions with Moesin. The results suggest that Moesin regulates the actin cytoskeleton, thereby promoting adherens junction stability. 24. Cao TT, Deacon HW, Reczek D, Bretscher A, von Zastrow M: A kinase-regulated PDZ-domain interaction controls endocytic sorting of the b2-adrenergic receptor. Nature 1999, 401:286-290. 25. Stanasila L, Abuin L, Diviani D, Cotecchia S: Ezrin directly interacts with the a1b-adrenergic receptor and plays a role in receptor recycling. J Biol Chem 2006, 281:4354-4363.
www.sciencedirect.com