Organizing the cell cortex: the role of ERM proteins.

Specialized membrane domains are an important feature of almost all cells. In particular, they are essential to tissues that have a highly organized cell cortex, such as the intestinal brush border epithelium. The ERM proteins (ezrin, radixin and moesin) have a crucial role in organizing membrane domains through their ability to interact with transmembrane proteins and the cytoskeleton. In doing so, they can provide structural links to strengthen the cell cortex and regulate the activities of signal transduction pathways. Recent studies examining the structure and in vivo functions of ERMs have greatly advanced our understanding of the importance of membrane-cytoskeleton interactions.

The transmembrane protein Crumbs displays complex dynamics during follicular morphogenesis and is regulated competitively by Moesin and aPKC.

The transmembrane protein Crumbs (Crb) functions in apical polarity and epithelial integrity. To better understand its role in epithelial morphogenesis, we examined Crb localization and dynamics in the late follicular epithelium of Drosophila. Crb was unexpectedly dynamic during middle-to-late stages of egg chamber development, being lost from the marginal zone (MZ) in stage 9 before abruptly returning at the end of stage 10b, then undergoing a pulse of endocytosis in stage 12. The reappearance of MZ Crb is necessary to maintain an intact adherens junction and MZ. Although Crb has been proposed to interact through its juxtamembrane domain with Moesin (Moe), a FERM domain protein that regulates the cortical actin cytoskeleton, the functional significance of this interaction is poorly understood. We found that whereas the Crb juxtamembrane domain was not required for adherens junction integrity, it was necessary for MZ localization of Moe, aPKC and F-actin. Furthermore, Moe and aPKC functioned antagonistically, suggesting that Moe limits Crb levels by reducing its interactions with the apical Par network. Additionally, Moe mutant cells lost Crb from the apical membrane and accumulated excess Crb at the MZ, suggesting that Moe regulates Crb distribution at the membrane. Together, these studies reveal reciprocal interactions between Crb, Moe and aPKC during cellular morphogenesis.

Macroglobulin complement-related encodes a protein required for septate junction organization and paracellular barrier function in Drosophila.

Polarized epithelia play crucial roles as barriers to the outside environment and enable the formation of specialized compartments for organs to carry out essential functions. Barrier functions are mediated by cellular junctions that line the lateral plasma membrane between cells, principally tight junctions in vertebrates and septate junctions (SJs) in invertebrates. Over the last two decades, more than 20 genes have been identified that function in SJ biogenesis in Drosophila, including those that encode core structural components of the junction such as Neurexin IV, Coracle and several claudins, as well as proteins that facilitate the trafficking of SJ proteins during their assembly. Here we demonstrate that Macroglobulin complement-related (Mcr), a gene previously implicated in innate immunity, plays an essential role during embryonic development in SJ organization and function. We show that Mcr colocalizes with other SJ proteins in mature ectodermally derived epithelial cells, that it shows interdependence with other SJ proteins for SJ localization, and that Mcr mutant epithelia fail to form an effective paracellular barrier. Tissue-specific RNA interference further demonstrates that Mcr is required cell-autonomously for SJ organization. Finally, we show a unique interdependence between Mcr and Nrg for SJ localization that provides new insights into the organization of the SJ. Together, these studies demonstrate that Mcr is a core component of epithelial SJs and also highlight an interesting relationship between innate immunity and epithelial barrier functions.

Understanding ERM proteins--the awesome power of genetics finally brought to bear.

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 membrane-associated proteins.

Dachsous and Fat coordinately repress the Dachs-Dlish-Approximated complex to control growth.

Two protocadherins, Dachsous (Ds) and Fat (Ft), regulate organ growth in Drosophila via the Hippo pathway. Ds and Ft bind heterotypically to regulate the abundance and subcellular localization of a 'core complex' consisting of Dachs, Dlish and Approximated. This complex localizes to the junctional cortex where it promotes growth by repressing the pathway kinase Warts. Ds is believed to promote growth by recruiting and stabilizing the core complex at the junctional cortex, while Ft represses growth by promoting degradation of core complex components. Here, we examine the functions of intracellular domains of Ds and Ft and their relationship to the core complex. While Ds promotes accumulation of the core complex proteins in cortical puncta, it is not required for core complex assembly. Indeed, the core complex assembles maximally in the absence of both Ds and Ft. Furthermore, while Ds promotes growth in the presence of Ft, it represses growth in the absence of Ft by removing the core complex from the junctional cortex. Ft similarly recruits core complex components, however it normally promotes their degradation. Our findings reveal that Ds and Ft constrain tissue growth by repressing the default 'on' state of the core complex.

Dachsous and Fat coordinately repress the Dachs-Dlish-Approximated complex to control growth.

Two protocadherins, Dachsous and Fat, regulate organ growth in Drosophila via the Hippo pathway. Dachsous and Fat bind heterotypically to regulate the abundance and subcellular localization of a "core complex" consisting of Dachs, Dlish, and Approximated. This complex localizes to the junctional cortex where it represses Warts. Dachsous is believed to promote growth by recruiting and stabilizing this complex, while Fat represses growth by promoting its degradation. Here, we examine the functional relationships between the intracellular domains of Dachsous and Fat and the core complex. While Dachsous promotes the accumulation of core complex proteins in puncta, it is not required for their assembly. Indeed, the core complex accumulates maximally in the absence of both Dachsous and Fat. Furthermore, Dachsous represses growth in the absence of Fat by removing the core complex from the junctional cortex. Fat similarly recruits core complex components but promotes their degradation. Our findings reveal that Dachsous and Fat coordinately constrain tissue growth by repressing the core complex.

Yellow and oxidation-resistant derivatives of a monomeric superfolder GFP.

Fluorescent proteins (FPs) are essential tools in biology. The utility of FPs depends on their brightness, photostability, efficient folding, monomeric state, and compatibility with different cellular environments. Despite the proliferation of available FPs, derivatives of the originally identified Aequorea victoria GFP often show superior behavior as fusion tags. We recently generated msGFP2, an optimized monomeric superfolder variant of A. victoria GFP. Here, we describe two derivatives of msGFP2. The monomeric variant msYFP2 is a yellow superfolder FP with high photostability. The monomeric variant moxGFP2 lacks cysteines but retains significant folding stability, so it works well in the lumen of the secretory pathway. These new FPs are useful for common imaging applications.

Yellow and oxidation-resistant derivatives of a monomeric superfolder GFP.

Fluorescent proteins (FPs) are essential tools in biology. The utility of FPs depends on their brightness, photostability, efficient folding, monomeric state, and compatibility with different cellular environments. Despite the proliferation of available FPs, derivatives of the originally identified Aequorea victoria green fluorescent protein often show superior behavior as fusion tags. We recently generated msGFP2, an optimized monomeric superfolder variant of A. victoria GFP. Here, we describe two derivatives of msGFP2. The monomeric variant msYFP2 is a yellow superfolder FP with high photostability. The monomeric variant moxGFP2 lacks cysteines but retains significant folding stability, so it works well in the lumen of the secretory pathway. These new FPs are useful for common imaging applications.

Cortical tension promotes Kibra degradation via Par-1.

The Hippo pathway is an evolutionarily conserved regulator of tissue growth. Multiple Hippo signaling components are regulated via proteolytic degradation. However, how these degradation mechanisms are themselves modulated remains unexplored. Kibra is a key upstream pathway activator that promotes its own ubiquitin-mediated degradation upon assembling a Hippo signaling complex. Here, we demonstrate that Hippo complex-dependent Kibra degradation is modulated by cortical tension. Using classical genetic, osmotic, and pharmacological manipulations of myosin activity and cortical tension, we show that increasing cortical tension leads to Kibra degradation, whereas decreasing cortical tension increases Kibra abundance. Our study also implicates Par-1 in regulating Kib abundance downstream of cortical tension. We demonstrate that Par-1 promotes ubiquitin-mediated Kib degradation in a Hippo complex-dependent manner and is required for tension-induced Kib degradation. Collectively, our results reveal a previously unknown molecular mechanism by which cortical tension affects Hippo signaling and provide novel insights into the role of mechanical forces in growth control.

FERMing up the plasma membrane.

As cells enter mitosis, shape changes occur that involve rearrangements of the actin cytoskeleton and an increase in cortical stiffness. In a recent article in Current Biology, Kunda et al. describe a new role for ERM proteins in regulating rearrangements of the cortical cytoskeleton during mitosis.

Analysis of protein dynamics within the septate junction reveals a highly stable core protein complex that does not include the basolateral polarity protein Discs large.

Barrier junctions prevent pathogen invasion and restrict paracellular leakage across epithelial sheets. To understand how one barrier junction, the septate junction (SJ), is regulated in vivo, we used fluorescence recovery after photobleaching (FRAP) to examine SJ protein dynamics in Drosophila. Most SJ-associated proteins, including Coracle, Neurexin IV and Nervana 2, displayed similar, extremely immobile kinetics. Loss of any of these components resulted in dramatically increased mobility of all others, suggesting that they form a single, highly interdependent core complex. Immobilization of SJ core components coincided with formation of the morphological SJ but occurred after their known role in maintaining epithelial polarity, suggesting that these functions are independent. In striking contrast to the core components, the tumor suppressor protein Discs large was much more mobile and its loss did not affect mobility of core SJ proteins, suggesting that it is not a member of this complex, even though it colocalizes with the SJ. Similarly, disruption of endocytosis affected localization of SJ core components, but did not affect their mobility. These results indicate that formation of a stable SJ core complex is separable from its proper subcellular localization, and provide new insights into the complex processes that regulate epithelial polarity and assembly of the SJ.

Crooked, coiled and crimpled are three Ly6-like proteins required for proper localization of septate junction components.

Cellular junction formation is an elaborate process that is dependent on the regulated synthesis, assembly and membrane targeting of constituting components. Here, we report on three Drosophila Ly6-like proteins essential for septate junction (SJ) formation. SJs provide a paracellular diffusion barrier and appear molecularly and structurally similar to vertebrate paranodal septate junctions. We show that Crooked (Crok), a small GPI-anchored Ly6-like protein, is required for septa formation and barrier functions. In embryos that lack Crok, SJ components are produced but fail to accumulate at the plasma membrane. Crok is detected in intracellular puncta and acts tissue-autonomously, which suggests that it resides in intracellular vesicles to assist the cell surface localization of SJ components. In addition, we demonstrate that two related Ly6 proteins, Coiled (Cold) and Crimpled (Crim), are required for SJ formation and function in a tissue-autonomous manner, and that Cold also localizes to intracellular vesicles. Specifically, Crok and Cold are required for correct membrane trafficking of Neurexin IV, a central SJ component. The non-redundant requirement for Crok, Cold, Crim and Boudin (Bou; another Ly6 protein that was recently shown to be involved in SJ formation) suggests that members of this conserved family of proteins cooperate in the assembly of SJ components, possibly by promoting core SJ complex formation in intracellular compartments associated with membrane trafficking.

Microtubule-mediated transport of the tumor-suppressor protein Merlin and its mutants.

The neurofibromatosis type 2 (NF2) tumor-suppressor protein Merlin is a member of the ERM family of proteins that links the cytoskeleton to the plasma membrane. In humans, mutations in the NF2 gene cause neurofibromatosis type-2 (NF2), a cancer syndrome characterized by the development of tumors of the nervous system. Previous reports have suggested that the subcellular distribution of Merlin is critical to its function, and that several NF2 mutants that lack tumor-suppressor activity present improper localization. Here we used a Drosophila cell culture model to study the distribution and mechanism of intracellular transport of Merlin and its mutants. We found that Drosophila Merlin formed cytoplasmic particles that move bidirectionally along microtubules. A single NF2-causing amino acid substitution in the FERM domain dramatically inhibited Merlin particle movement. Surprisingly, the presence of this immotile Merlin mutant also inhibited trafficking of the WT protein. Analysis of the movement of WT protein using RNAi and pull-downs showed that Merlin particles are associated with and moved by microtubule motors (kinesin-1 and cytoplasmic dynein), and that binding of motors and movement is regulated by Merlin phosphorylation. Inhibition of Merlin transport by expression of the dominant-negative mutant or depletion of kinesin-1 results in increased nuclear accumulation of the transcriptional coactivator Yorkie. These results demonstrate the requirement of microtubule-dependent transport for Merlin function.

Apical polarity and actomyosin dynamics control Kibra subcellular localization and function in Drosophila Hippo signaling.

The Hippo pathway is an evolutionarily conserved regulator of tissue growth that integrates inputs from both polarity and actomyosin networks. An upstream activator of the Hippo pathway, Kibra, localizes at the junctional and medial regions of the apical cortex in epithelial cells, and medial accumulation promotes Kibra activity. Here, we demonstrate that cortical Kibra distribution is controlled by a tug-of-war between apical polarity and actomyosin dynamics. We show that while the apical polarity network, in part via atypical protein kinase C (aPKC), tethers Kibra at the junctional cortex to silence its activity, medial actomyosin flows promote Kibra-mediated Hippo complex formation at the medial cortex, thereby activating the Hippo pathway. This study provides a mechanistic understanding of the relationship between the Hippo pathway, polarity, and actomyosin cytoskeleton, and it offers novel insights into how fundamental features of epithelial tissue architecture can serve as inputs into signaling cascades that control tissue growth, patterning, and morphogenesis.

Sip1, the Drosophila orthologue of EBP50/NHERF1, functions with the sterile 20 family kinase Slik to regulate Moesin activity.

Organization of the plasma membrane in polarized epithelial cells is accomplished by the specific localization of transmembrane or membrane-associated proteins, which are often linked to cytoplasmic protein complexes, including the actin cytoskeleton. In this study, we identified Sip1 as a Drosophila orthologue of the ezrin-radixin-moesin (ERM) binding protein 50 (EBP50; also known as the Na(+)/H(+) exchanger regulatory factor NHERF1). In mammals, EBP50/NHERF1 is a scaffold protein required for the regulation of several transmembrane receptors and downstream signal transduction activity. In Drosophila, loss of Sip1 leads to a reduction in Slik kinase protein abundance, loss of Moesin phosphorylation and changes in epithelial structure, including mislocalization of E-cadherin and F-actin. Consistent with these findings, Moesin and Sip1 act synergistically in genetic-interaction experiments, and Sip1 protein abundance is dependent on Moesin. Co-immunoprecipitation experiments indicate that Sip1 forms a complex with both Moesin and Slik. Taken together, these data suggest that Sip1 promotes Slik-dependent phosphorylation of Moesin, and suggests a mechanism for the regulation of Moesin activity within the cell to maintain epithelial integrity.

Phosphorylation and activity of the tumor suppressor Merlin and the ERM protein Moesin are coordinately regulated by the Slik kinase.

Merlin and Moesin are closely related members of the 4.1 Ezrin/Radixin/Moesin domain superfamily implicated in regulating proliferation and epithelial integrity, respectively. The activity of both proteins is regulated by head to tail folding that is controlled, in part, by phosphorylation. Few upstream regulators of these phosphorylation events are known. In this study, we demonstrate that in Drosophila melanogaster, Slik, a Ste20 kinase, controls subcellular localization and phosphorylation of Merlin, resulting in the coordinate but opposite regulation of Merlin and Moesin. These results suggest the existence of a novel mechanism for coordinate regulation of cell proliferation and epithelial integrity in developing tissues.

Negative feedback couples Hippo pathway activation with Kibra degradation independent of Yorkie-mediated transcription.

The Hippo (Hpo) pathway regulates tissue growth in many animals. Multiple upstream components promote Hpo pathway activity, but the organization of these different inputs, the degree of crosstalk between them, and whether they are regulated in a distinct manner is not well understood. Kibra (Kib) activates the Hpo pathway by recruiting the core Hpo kinase cassette to the apical cortex. Here, we show that the Hpo pathway downregulates Drosophila Kib levels independently of Yorkie-mediated transcription. We find that Hpo signaling complex formation promotes Kib degradation via SCFSlimb-mediated ubiquitination, that this effect requires Merlin, Salvador, Hpo, and Warts, and that this mechanism functions independently of other upstream Hpo pathway activators. Moreover, Kib degradation appears patterned by differences in mechanical tension across the wing. We propose that Kib degradation mediated by Hpo pathway components and regulated by cytoskeletal tension serves to control Kib-driven Hpo pathway activation and ensure optimally scaled and patterned tissue growth.

Rho1 activation recapitulates early gastrulation events in the ventral, but not dorsal, epithelium of Drosophila embryos.

Ventral furrow formation, the first step in Drosophila gastrulation, is a well-studied example of tissue morphogenesis. Rho1 is highly active in a subset of ventral cells and is required for this morphogenetic event. However, it is unclear whether spatially patterned Rho1 activity alone is sufficient to recapitulate all aspects of this morphogenetic event, including anisotropic apical constriction and coordinated cell movements. Here, using an optogenetic probe that rapidly and robustly activates Rho1 in Drosophila tissues, we show that Rho1 activity induces ectopic deformations in the dorsal and ventral epithelia of Drosophila embryos. These perturbations reveal substantial differences in how ventral and dorsal cells, both within and outside the zone of Rho1 activation, respond to spatially and temporally identical patterns of Rho1 activation. Our results demonstrate that an asymmetric zone of Rho1 activity is not sufficient to recapitulate ventral furrow formation and reveal that additional, ventral-specific factors contribute to the cell- and tissue-level behaviors that emerge during ventral furrow formation.

The tumor suppressors Merlin and Expanded function cooperatively to modulate receptor endocytosis and signaling.

The precise coordination of signals that control proliferation is a key feature of growth regulation in developing tissues . While much has been learned about the basic components of signal transduction pathways, less is known about how receptor localization, compartmentalization, and trafficking affect signaling in developing tissues. Here we examine the mechanism by which the Drosophila Neurofibromatosis 2 (NF2) tumor suppressor ortholog Merlin (Mer) and the related tumor suppressor expanded (ex) regulate proliferation and differentiation in imaginal epithelia. Merlin and Expanded are members of the FERM (Four-point one, Ezrin, Radixin, Moesin) domain superfamily, which consists of membrane-associated cytoplasmic proteins that interact with transmembrane proteins and may function as adapters that link to protein complexes and/or the cytoskeleton . We demonstrate that Merlin and Expanded function to regulate the steady-state levels of signaling and adhesion receptors and that loss of these proteins can cause hyperactivation of associated signaling pathways. In addition, pulse-chase labeling of Notch in living tissues indicates that receptor levels are upregulated at the plasma membrane in Mer; ex double mutant cells due to a defect in receptor clearance from the cell surface. We propose that these proteins control proliferation by regulating the abundance, localization, and turnover of cell-surface receptors and that misregulation of these processes may be a key component of tumorigenesis.