Septate junctions (SJs) serve as occluding barriers in invertebrate epithelia. In Drosophila, at least 30 genes are required for the formation or maintenance of SJs. Interestingly, loss-of-function mutations in core SJ components are embryonic lethal, with defects in developmental events such as head involution and dorsal closure (DC) that occur prior to the formation of a mature SJ, indicating a role for these proteins in mid-embryogenesis independent of their occluding function. To understand this novel function in development, we examined loss-of-function mutations in three core SJ proteins during the process of DC. DC occurs during mid-embryogenesis to seal a dorsal gap in the epidermis following germ band retraction. Closure is driven by contraction of the extraembryonic amnioserosa cells that temporarily cover the dorsal surface and by cell shape changes (elongation) of lateral epidermal cells that bring the contralateral sheets together at the dorsal midline. Using live imaging and examination of fixed tissues, we show that early events in DC occur normally in SJ mutant embryos, but during later closure, coracle, Macroglobulin complement-related and Neurexin-IV mutant embryos exhibit slower rates of closure and display aberrant cells shapes in the dorsolateral epidermis, including dorsoventral length and apical surface area. SJ mutant embryos also show mild defects in actomyosin structures along the leading edge, but laser cutting experiments suggest similar tension and viscoelastic properties in SJ mutant versus wild type epidermis. In a high percentage of SJ mutant embryos, the epidermis tears free from the amnioserosa near the end of DC and live imaging and immunostaining reveal reduced levels of E-cadherin, suggesting that defective adhesion may be responsible for these tears. Supporting this notion, reducing E-cadherin by half significantly enhances the penetrance of DC defects in coracle mutant embryos.

The septate junction (SJ) provides an occluding function for epithelial tissues in invertebrate organisms. This ability to seal the paracellular route between cells allows internal tissues to create unique compartments for organ function and endows the epidermis with a barrier function to restrict the passage of pathogens. Over the past twenty-five years, numerous investigators have identified more than 30 proteins that are required for the formation or maintenance of the SJs in Drosophila melanogaster, and have determined many of the steps involved in the biogenesis of the junction. Along the way, it has become clear that SJ proteins are also required for a number of developmental events that occur throughout the life of the organism. Many of these developmental events occur prior to the formation of the occluding junction, suggesting that SJ proteins possess non-occluding functions. In this review, we will describe the composition of SJs, taking note of which proteins are core components of the junction versus resident or accessory proteins, and the steps involved in the biogenesis of the junction. We will then elaborate on the functions that core SJ proteins likely play outside of their role in forming the occluding junction and describe studies that provide some cell biological perspectives that are beginning to provide mechanistic understanding of how these proteins function in developmental contexts.

Protein components of the invertebrate occluding junction-known as the septate junction (SJ)-are required for morphogenetic developmental events during embryogenesis in Drosophila melanogaster. In order to determine whether SJ proteins are similarly required for morphogenesis during other developmental stages, we investigated the localization and requirement of four representative SJ proteins during oogenesis: Contactin, Macroglobulin complement-related, Neurexin IV, and Coracle. A number of morphogenetic processes occur during oogenesis, including egg elongation, formation of dorsal appendages, and border cell (BC) migration. We found that all four SJ proteins are expressed in egg chambers throughout oogenesis, with the highest and the most sustained levels in the follicular epithelium (FE). In the FE, SJ proteins localize along the lateral membrane during early and mid-oogenesis, but become enriched in an apical-lateral domain (the presumptive SJ) by stage 11. SJ protein relocalization requires the expression of other SJ proteins, as well as Rab5 and Rab11 like SJ biogenesis in the embryo. Knocking down the expression of these SJ proteins in follicle cells throughout oogenesis results in egg elongation defects and abnormal dorsal appendages. Similarly, reducing the expression of SJ genes in the BC cluster results in BC migration defects. Together, these results demonstrate an essential requirement for SJ genes in morphogenesis during oogenesis, and suggest that SJ proteins may have conserved functions in epithelial morphogenesis across developmental stages.

Imaginal disc morphogenesis during metamorphosis in Drosophila melanogaster provides an excellent model to uncover molecular mechanisms by which hormonal signals effect physical changes during development. The broad (br) Z2 isoform encodes a transcription factor required for disc morphogenesis in response to 20-hydroxyecdysone, yet how it accomplishes this remains largely unknown. Here, we use functional studies of amorphic br(5) mutants and a transcriptional target approach to identify processes driven by br and its regulatory targets in leg imaginal discs. br(5) mutants fail to properly remodel their basal extracellular matrix (ECM) between 4 and 7 hr after puparium formation. Additionally, br(5) mutant discs do not undergo the cell shape changes necessary for leg elongation and fail to elongate normally when exposed to the protease trypsin. RNA-sequencing of wild-type and br(5) mutant leg discs identified 717 genes differentially regulated by br, including a large number of genes involved in glycolysis, and genes that encode proteins that interact with the ECM. RNA interference-based functional studies reveal that several of these genes are required for adult leg formation, particularly those involved in remodeling the ECM. Additionally, br Z2 expression is abruptly shut down at the onset of metamorphosis, and expressing it beyond this time results in failure of leg development during the late prepupal and pupal stages. Taken together, our results suggest that br Z2 is required to drive ECM remodeling, change cell shape, and maintain metabolic activity through the midprepupal stage, but must be switched off to allow expression of pupation genes.

Imaginal disc morphogenesis during metamorphosis in Drosophila melanogaster provides an excellent model to uncover molecular mechanisms by which hormonal signals effect physical changes during development. The broad ( br ) Z2 isoform encodes a transcription factor required for disc morphogenesis in response to 20-hydroxyecdysone, yet how it accomplishes this remains largely unknown. Here, we show that amorphic br5 mutant discs fail to remodel their basal extracellular matrix (ECM) after puparium formation and do not undergo necessary cell shape changes. RNA sequencing of wild type and mutant leg discs identified 717 genes differentially regulated by br ; functional studies reveal that several are required for adult leg formation, particularly those involved in remodeling the ECM. Additionally, br Z2 expression is abruptly shut down at the onset of metamorphosis, and expressing it beyond this time results in failure of leg development during the late prepupal and pupal stages. Taken together, our results suggest that br Z2 is required to drive ECM remodeling, change cell shape, and maintain metabolic activity through the mid prepupal stage, but must be switched off to allow expression of pupation genes.Summary Statement The Drosophila melanogaster ecdysone-responding transcription factor broad controls morphogenetic processes in leg imaginal discs during metamorphosis through regulation of genes involved in extracellular matrix remodeling, metabolism, and cell shape changes and rearrangements.

The Kansas-IDeA Network of Biomedical Research Excellence (K-INBRE) is an infrastructure-building program funded by the National Institute of General Medical Sciences. Undergraduate education, through undergraduate research, is a key component of the program. The K-INBRE network includes 10 higher education institutions in Kansas and northern Oklahoma, with over 1,000 student participants in 16 yr. Since 2003, the K-INBRE has held an annual state-wide research symposium that includes national and regional speakers and provides a forum for undergraduates to give platform and poster presentations. The symposium is well attended by K-INBRE participants and has grown to a size of over 300 participants per year from all 10 K-INBRE schools. Two surveys were distributed to students and mentors to assess the impact of the symposium on student learning. Surveys (153) were distributed to students who participated in K-INBRE from 2013 through 2015 with a 51% response rate. Mentors were surveyed with a response of 111 surveys out of 161. Survey results indicate that students and mentors alike find the symposium to be beneficial and enriching of the student experience. Almost 80% of student respondents indicated that their participation in the symposium fostered appreciation of research. In short, the K-INBRE symposium provides a unique opportunity for students to gain experience in collecting, preparing, and communicating research in a professional environment. The collaborative experience of the annual K-INBRE symposium, the impact it has on student learning, and how it has influenced the research culture at our 10 institutions will be described.

The septate junction (SJ) is the occluding junction found in the ectodermal epithelia of invertebrate organisms, and is essential to maintain chemically distinct compartments in epithelial organs, to provide the blood-brain barrier in the nervous system, and to provide an important line of defense against invading pathogens. More than 20 genes have been identified to function in the establishment or maintenance of SJs in Drosophila melanogaster Numerous studies have demonstrated the cell biological function of these proteins in establishing the occluding junction, whereas very few studies have examined further developmental roles for them. Here we examined embryos with mutations in nine different core SJ genes and found that all nine result in defects in embryonic development as early as germ band retraction, with the most penetrant defect observed in head involution. SJ genes are also required for cell shape changes and cell rearrangements that drive the elongation of the salivary gland during midembryogenesis. Interestingly, these developmental events occur at a time prior to the formation of the occluding junction, when SJ proteins localize along the lateral membrane and have not yet coalesced into the region of the SJ. Together, these observations reveal an underappreciated role for a large group of SJ genes in essential developmental events during embryogenesis, and suggest that the function of these proteins in facilitating cell shape changes and rearrangements is independent of their role in the occluding junction.

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.

Preface The editors Fermented beverages Chapter 1: Beer Tom Shellhammer Chapter 2: Wine David Jeffery and Kerry Wilkinson Chapter 3: Cider and Perry AGH Lea Distilled fermented beverages Chapter 4: Whisky Frances Jack Chapter 5: Brandy/Cognac/Armagnac N.V. Bougas, P. van Rensburg, C.L.C. Snyman & M.G. Lambrechts Chapter 6: Rum L Fahrasmane Chapter 7: Liqueurs M.L. Gonzalez-Sanjose Foods and other beverages Chapter 8: Vinegar Maria Plessi and Giulia Papotti Chapter 9: Other dairy products-yogurt, kiefer, koumiss Nagendra Shah Chapter 10: Vegetables BJB Wood Chapter 11: Breads and other baked goods Zannini E., Moroni A., Belz M., Faltermaier A., and Arendt K. E. Chapter 12: Meat FK Lucke Chapter 13: Fish Jirawat Yongsawatdigul, Dong-Soo Kim, Somjintana Tungkawachara and Jae W. Park Chapter 14: Asian Foods-natto, fish sauce, tofu, kimchi R Nout, Bei-Zhong Han and Cherl-Ho Lee Chapter 15: Cocoa Rosane Freitas Schwan and Disney Ribeiro Dias Chapter 16: Tea P Owuor Chapter 17: Coffee Rosane Freitas Schwan, Cristina Ferreira Silva and Luis Roberto Batista Biotech Chapter 18: Fermentations to produce flavors and other components for use in foods R. Ward Chapter 19: Microbial biomass protein Mauricio Mora-Pale, Mariano Garcia-Garibay and Eduardo Barzana Appendices 1. Other fermented beverages CW Bamforth 2. Other distilled beverages CW Bamforth 3. Cheese CW Bamforth

Nonmuscle myosin II (myosin hereafter) has well-established roles in generating contractile force on actin filaments during morphogenetic processes in all metazoans. Myosin activation is regulated by phosphorylation of the myosin regulatory light chain (MRLC, encoded by spaghetti squash or sqh in Drosophila) first on Ser21 and subsequently on Thr20. These phosphorylation events are positively controlled by a variety of kinases including myosin light chain kinase, Rho kinase, citron kinase, and AMP kinase and are negatively regulated by myosin phosphatase. The activation of myosin is thus highly regulated and likely developmentally controlled. In order to monitor the activity of myosin during development, we have generated antibodies against the monophosphorylated (Sqh1P) and diphosphorylated (Sqh2P) forms of Sqh. We first show that the antibodies are highly specific. We then used these antibodies to monitor myosin activation in wild type Drosophila tissues. Interestingly, Sqh1P and Sqh2P show distinct patterns of expression in embryos. Sqh1P is expressed nearly ubiquitously and outlines cells consistent with a junctional localization, whereas Sqh2P is strongly expressed on the apical surfaces and in filopodia of tissues undergoing extensive cell shape change or cell movements including the invaginating fore- and hindgut, the invaginating tracheal system, the dorsal pouch and the dorsal most row of epidermal (DME) cells during dorsal closure. In imaginal discs, Sqh1P predominantly localizes in the adherens junction, whereas Sqh2P locates to the apical domain. These antibodies thus have the potential to be very useful in monitoring myosin activation for functional studies of morphogenesis in Drosophila.

During dorsal closure in Drosophila , signaling events in the dorsalmost row of epidermal cells (DME cells) direct the migration of lateral epidermal sheets towards the dorsal midline where they fuse to enclose the embryo. A Jun amino‐terminal kinase (JNK) cascade in the DME cells induces the expression of Decapentaplegic (Dpp). Dpp signaling then regulates the cytoskeleton in the DME cells and amnioserosa to affect the cell shape changes necessary to complete dorsal closure. We identified a mutation in Sec61 α that specifically perturbs dorsal closure. Sec61 α encodes the main subunit of the translocon complex for co‐translational import of proteins into the ER. JNK signaling is normal in Sec61 α mutant embryos, but Dpp signaling is attenuated and the DME cells fail to maintain an actinomyosin cable as epithelial migration fails. Consistent with this model, dorsal closure is rescued in Sec61 α mutant embryos by an activated form of the Dpp receptor Thick veins. Developmental Dynamics 239:784–797, 2010. © 2010 Wiley‐Liss, Inc.

Background Rho1 is a small GTPase of the Ras superfamily that serves as the central component in a highly conserved signaling pathway that regulates tissue morphogenesis during development in all animals. Since there is tremendous diversity in the upstream signals that can activate Rho1 as well as the effector molecules that carry out its functions, it is important to define relevant Rho1-interacting genes for each morphogenetic event regulated by this signaling pathway. Previous work from our lab and others has shown that Rho signaling is necessary for the morphogenesis of leg imaginal discs during metamorphosis in Drosophila, although a comprehensive identification of Rho1-interacting genes has not been attempted for this process. Methodology/Principal Findings We characterized an amorphic allele of Rho1 that displays a poorly penetrant dominant malformed leg phenotype and is capable of being strongly enhanced by Rho1-interacting heterozygous mutations. We then used this allele in a second-site noncomplementation screen with the Exelixis collection of molecularly defined deficiencies to identify Rho1-interacting genes necessary for leg morphogenesis. In a primary screen of 461 deficiencies collectively uncovering ∼50% of the Drosophila genome, we identified twelve intervals harboring Rho1-interacting genes. Through secondary screening we identified six Rho1-interacting genes including three that were previously identified (RhoGEF2, broad, and stubbloid), thereby validating the screen. In addition, we identified Cdc42, Rheb and Sc2 as novel Rho1-interacting genes involved in adult leg development. Conclusions/Significance This screen identified well-known and novel Rho1-interacting genes necessary for leg morphogenesis, thereby increasing our knowledge of this important signaling pathway. We additionally found that Rheb may have a unique function in leg morphogenesis that is independent of its regulation of Tor.

The tracheal system of Drosophila melanogaster has proven to be an excellent model system for studying the development of branched tubular organs. Mechanisms regulating the patterning and initial maturation of the tracheal system have been largely worked out, yet important questions remain regarding how the mature tubes inflate with air at the end of embryogenesis, and how the tracheal system grows in response to the oxygen needs of a developing larva that increases nearly 1000-fold in volume over a four day period. Here we describe the cloning and characterization of uninflatable (uif), a gene that encodes a large transmembrane protein containing carbohydrate binding and cell signaling motifs in its extracellular domain. Uif is highly conserved in insect species, but does not appear to have a true ortholog in vertebrate species. uif is expressed zygotically beginning in stage 5 embryos, and Uif protein localizes to the apical plasma membrane in all ectodermally derived epithelia, most notably in the tracheal system. uif mutant animals show defects in tracheal inflation at the end of embryogenesis, and die primarily as larvae. Tracheal tubes in mutant larvae are often crushed or twisted, although tracheal patterning and maturation appear normal during embryogenesis. uif mutant larvae also show defects in tracheal growth and molting of their tracheal cuticle.

Background As we move into the post genome-sequencing era, an immediate challenge is how to make best use of the large amount of high-throughput experimental data to assign functions to currently uncharacterized proteins. We here describe CSIDOP, a new method for protein function assignment based on shared interacting domain patterns extracted from cross-species protein-protein interaction data. Methodology/Principal Findings The proposed method is assessed both biologically and statistically over the genome of H. sapiens. The CSIDOP method is capable of making protein function prediction with accuracy of 95.42% using 2,972 gene ontology (GO) functional categories. In addition, we are able to assign novel functional annotations for 181 previously uncharacterized proteins in H. sapiens. Furthermore, we demonstrate that for proteins that are characterized by GO, the CSIDOP may predict extra functions. This is attractive as a protein normally executes a variety of functions in different processes and its current GO annotation may be incomplete. Conclusions/Significance It can be shown through experimental results that the CSIDOP method is reliable and practical in use. The method will continue to improve as more high quality interaction data becomes available and is readily scalable to a genome-wide application.

Coracle is a member of the Protein 4.1 superfamily of proteins, whose members include Protein 4.1, the Neurofibromatosis 2 tumor suppressor Merlin, Expanded, the ERM proteins, protein tyrosine phosphatases, and unconventional myosins. Recent evidence suggests that members of this family participate in cell signaling events, including those that regulate cell proliferation and the cytoskeleton. Previously, we demonstrated that Coracle protein is localized to the septate junction in epithelial cells and is required for septate junction integrity. Loss of coracle function leads to defects in embryonic development, including failure in dorsal closure, and to proliferation defects. In addition, we determined that the N-terminal 383 amino acids define an essential functional domain possessing membrane-organizing properties. Here we investigate the full range of functions provided by this highly conserved domain and find that it is sufficient to rescue all embryonic defects associated with loss of coracle function. In addition, this domain is sufficient to rescue the reduced cell proliferation defect in imaginal discs, although it is incapable of rescuing null mutants to the adult stage. This result suggests the presence of a second functional domain within Coracle, a notion supported by molecular characterization of a series of coracle alleles.

The protein 4.1 superfamily is comprised of a diverse group of cytoplasmic proteins, many of which have been shown to associate with the plasma membrane via binding to specific transmembrane proteins, Coracle, a Drosophila protein 4.1 homologue, is required during embryogenesis and is localized to the cytoplasmic face of the septate junction in epithelial cells, Using in vitro mutagenesis, we demonstrate that the amino-terminal 383 amino acids of Coracle define a functional domain that is both necessary and sufficient for proper septate junction localization in transgenic embryos, Genetic mutations within this domain disrupt the subcellular localization of Coracle and severely affect its genetic function, indicating that correct subcellular localization is essential for Coracle function. Furthermore, the localization of Coracle and the transmembrane protein Neurexin to the septate junction display an interdependent relationship, suggesting that Coracle and Neurexin interact with one another at the cytoplasmic face of the septate junction. Consistent with this notion, immunoprecipitation and in vitro binding studies demonstrate that the amino-terminal 383 amino acids of Coracle and cytoplasmic domain of Neurexin interact directly. Together these results indicate that Coracle provides essential membrane-organizing functions at the septate junction, and that these functions are carried out by an amino-terminal domain that is conserved in all protein 4.1 superfamily members.

Although extensively studied biochemically, members of the Protein 4. 1 superfamily have not been as well characterized genetically. Studies of coracle, a Drosophila Protein 4.1 homologue, provide an opportunity to examine the genetic functions of this gene family. coracle was originally identified as a dominant suppressor of EgfrElp, a hypermorphic form of the Drosophila Epidermal growth factor receptor gene. In this article, we present a phenotypic analysis of coracle, one of the first for a member of the Protein 4. 1 superfamily. Screens for new coracle alleles confirm the null coracle phenotype of embryonic lethality and failure in dorsal closure, and they identify additional defects in the embryonic epidermis and salivary glands. Hypomorphic coracle alleles reveal functions in many imaginal tissues. Analysis of coracle mutant cells indicates that Coracle is a necessary structural component of the septate junction required for the maintenance of the transepithelial barrier but is not necessary for apical-basal polarity, epithelial integrity, or cytoskeletal integrity. In addition, coracle phenotypes suggest a specific role in cell signaling events. Finally, complementation analysis provides information regarding the functional organization of Coracle and possibly other Protein 4.1 superfamily members. These studies provide insights into a range of in vivo functions for coracle in developing embryos and adults.