[Show abstract][Hide abstract] ABSTRACT: The proper control of tissue growth is essential during normal development and an important problem in human disease. Merlin, the product of the Neurofibromatosis 2 tumor suppressor gene, has been extensively studied to understand its functions in growth control. Here we describe experiments in which we used Drosophila as an in vivo system to test the functions of the normal human NF2 gene products and patient-derived mutant alleles. Although the predominant NF2 gene isoform, isoform 1, could functionally replace the Drosophila Merlin gene, a second isoform with a distinct C-terminal tail could not. Immunofluorescence studies show that the two isoforms have distinct subcellular localizations when expressed in the polarized imaginal epithelium, and function in genetic rescue assays correlates with apical localization of the NF2 protein. Interestingly, we found that a patient-derived missense allele, NF2L64P, appears to be temperature sensitive. These studies highlight the utility of Drosophila for in vivo functional analysis of highly conserved human disease genes.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: RhoA, a small GTPase, regulates epithelial integrity and morphogenesis by controlling filamentous actin assembly and actomyosin contractility. Another important cytoskeletal regulator, the ERM protein Moesin, has the ability to bind to and organize cortical F-actin as well as the ability to regulate RhoA activity. ERM proteins have been shown previously to interact both with RhoGEFs and RhoGAPs, proteins that control the activation state of RhoA, but the functions of these interactions remain unclear. We demonstrate that Moesin interacts with an unusual RhoGAP, Conundrum, and recruits it to the cell cortex to negatively regulate RhoA activity. In addition, we show that cortically localized Conundrum can promote cell proliferation and that this function requires RhoGAP activity. Surprisingly, Conundrum's ability to promote growth also appears dependent on increased Rac activity. Our results reveal a molecular mechanism by which ERM proteins control RhoA activity, and suggest a novel linkage between the small GTPases RhoA and Rac in growth control.
Full-text · Article · Mar 2013 · Molecular biology of the cell
[Show abstract][Hide abstract] ABSTRACT: Over the past decade, the Hippo tumor suppressor pathway has emerged as a central regulator of growth in epithelial tissues. Research in Drosophila and in mammals has shown that this kinase signaling cascade regulates the activity of the transcriptional coactivator and oncoprotein Yorkie/Yap. In this review, we discuss recent findings that emphasize the cell cortex-specifically the actin cytoskeleton, intercellular junctions, and protein complexes that determine cell polarity-as a key site for Hippo pathway regulation. We also highlight where additional research is needed to integrate known functional interactions between Hippo pathway components.
[Show abstract][Hide abstract] ABSTRACT: Recent studies have shown that the Hippo-Salvador-Warts (HSW) pathway restrains tissue growth by phosphorylating and inactivating the oncoprotein Yorkie. How growth-suppressive signals are transduced upstream of Hippo remains unclear. We show that the Sterile 20 family kinase, Tao-1, directly phosphorylates T195 in the Hippo activation loop and that, like other HSW pathway genes, Tao-1 functions to restrict cell proliferation in developing imaginal epithelia. This relationship appears to be evolutionarily conserved, because mammalian Tao-1 similarly affects MST kinases. In S2 cells, Tao-1 mediates the effects of the upstream HSW components Merlin and Expanded, consistent with the idea that Tao-1 functions in tissues to regulate Hippo phosphorylation. These results demonstrate that one family of Ste20 kinases can activate another and identify Tao-1 as a component of the regulatory network controlling HSW pathway signaling, and therefore tissue growth, during development.
[Show abstract][Hide abstract] ABSTRACT: 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.
No preview · Article · Aug 2011 · Journal of Cell Science
[Show abstract][Hide abstract] ABSTRACT: The cell cortex serves as a critical nexus between the extracellular environment/cell membrane and the underlying cytoskeleton and cytoplasm. In many cells, the cell cortex is organized and maintained by the Ezrin, Radixin and Moesin (ERM) proteins, which have the ability to interact with both the plasma membrane and filamentous actin. Although this membrane-cytoskeletal linkage function is critical to stability of the cell cortex, recent studies indicate that this is only a part of what ERMs do in many cells. In addition to their role in binding filamentous actin, ERMs regulate signaling pathways through their ability to bind transmembrane receptors and link them to downstream signaling components. In this review we discuss recent evidence in a variety of cells indicating that ERMs serve as scaffolds to facilitate efficient signal transduction on the cytoplasmic face of the plasma membrane.
Preview · Article · May 2011 · Current opinion in cell biology
[Show abstract][Hide abstract] ABSTRACT: When testing antiserum quality, it is essential to determine first whether the antiserum recognizes the fusion protein against which it was raised. A dot blot will provide a qualitative estimate of antibody titer, as described in this protocol. The same test can be done quantitatively by enzyme-linked immunosorbent assay (ELISA), but the technically simpler dot blot gives adequate information, uses less protein, and does not require an ELISA plate reader. If the same protein has been expressed in two different fusion-protein vectors, the test should be performed with the protein not used for immunization. If there is only one fusion protein-e.g., a glutathione-S-transferase (GST)-fusion-a second dot blot can be done in parallel with GST alone to compare the titers. If the antiserum recognizes the protein, there should be a lower titer of antibodies against GST alone as compared to the entire fusion protein. A preimmune control is always advisable. If a positive result is not obtained against the fusion protein, it is futile to proceed to secondary tests. Continue boosting and retesting until a positive response is achieved, or try a different approach.
No preview · Article · Jan 2011 · Cold Spring Harbor Protocols
[Show abstract][Hide abstract] ABSTRACT: Once antibodies against a Drosophila protein have been produced and an antiserum is determined to have a high titer against the fusion protein used for immunization, the next step is to ask whether the antiserum recognizes the appropriate antigen in Drosophila tissues. This protocol describes staining of transfected S2 cells for testing antisera. There are several advantages to using transfected S2 cells as a first tissue test. First, the antibody only needs to penetrate a single cell. In addition, these cells are easy to fix and stain and require no dissection. Second, the overexpressed protein will be easier to detect than lower levels of endogenous protein. Third, background is generally less of a problem in S2 cells than in embryonic or imaginal tissues, allowing lower dilutions of the antiserum to be tested if the response is marginal. Finally, the untransfected cells form a useful built-in control. S2 cell stainings are also an easy way to determine the subcellular localization of a particular protein.
No preview · Article · Jan 2011 · Cold Spring Harbor Protocols
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Precisely controlled growth and morphogenesis of developing epithelial tissues require coordination of multiple factors, including proliferation, adhesion, cell shape, and apoptosis. RhoA, a small GTPase, is known to control epithelial morphogenesis and integrity through its ability to regulate the cytoskeleton. In this study, we examine a less well-characterized RhoA function in cell survival. We demonstrate that the Drosophila melanogaster RhoA, Rho1, promotes apoptosis independently of Rho kinase through its effects on c-Jun NH(2)-terminal kinase (JNK) signaling. In addition, Rho1 forms a complex with Slipper (Slpr), an upstream activator of the JNK pathway. Loss of Moesin (Moe), an upstream regulator of Rho1 activity, results in increased levels of Rho1 at the plasma membrane and cortical accumulation of Slpr. Together, these results suggest that Rho1 functions at the cell cortex to regulate JNK activity and implicate Rho1 and Moe in epithelial cell survival.
Full-text · Article · Apr 2010 · The Journal of Cell Biology
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
Full-text · Article · Apr 2010 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: 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.
No preview · Article · Mar 2010 · Journal of Cell Science
[Show abstract][Hide abstract] ABSTRACT: A wide variety of plasmid vectors are commercially available for the production of fusion proteins in bacterial cells. Most are also designed to incorporate a tag that allows affinity purification of the expressed fusion protein from bacterial cell extracts. The most commonly used are vectors that incorporate a portion of the glutathione-S-transferase (GST) enzyme that is able to bind to immobilized glutathione and vectors that use a polyhistidine tag which binds immobilized nickel ions with high affinity. This protocol describes preparation of a soluble GST fusion protein, isolated using glutathione agarose beads.
No preview · Article · Nov 2009 · Cold Spring Harbor Protocols
[Show abstract][Hide abstract] ABSTRACT: A wide variety of plasmid vectors are commercially available for the production of fusion proteins in bacterial cells. Most are also designed to incorporate a tag that allows affinity purification of the expressed fusion protein from bacterial cell extracts. The most commonly used are vectors that incorporate a portion of the glutathione-S-transferase (GST) enzyme that is able to bind to immobilized glutathione and vectors that use a polyhistidine tag which binds immobilized nickel ions with high affinity. This protocol describes preparation of an insoluble GST fusion protein, isolated using glutathione agarose beads.
No preview · Article · Nov 2009 · Cold Spring Harbor Protocols
[Show abstract][Hide abstract] ABSTRACT: Recent studies highlight the importance of the distribution of membrane receptors in controlling receptor output and in contributing to complex biological processes. The cortical cytoskeleton is known to affect membrane protein distribution but the molecular basis of this is largely unknown. Here, we discuss the functions of Merlin and the ERM proteins both in linking membrane proteins to the underlying cortical cytoskeleton and in controlling the distribution of and signaling from membrane receptors. We also propose a model that could account for the intricacies of Merlin function across model organisms.
Preview · Article · May 2009 · Trends in cell biology