Gross morphology and adhesion-associated physical properties of Drosophila larval salivary gland glue secretion.

One of the major functions of the larval salivary glands (SGs) of many Drosophila species is to produce a massive secretion during puparium formation. This so-called proteinaceous glue is exocytosed into the centrally located lumen, and subsequently expectorated, serving as an adhesive to attach the puparial case to a solid substrate during metamorphosis. Although this was first described almost 70 years ago, a detailed description of the morphology and mechanical properties of the glue is largely missing. Its main known physical property is that it is released as a watery liquid that quickly hardens into a solid cement. Here, we provide a detailed morphological and topological analysis of the solidified glue. We demonstrated that it forms a distinctive enamel-like plaque that is composed of a central fingerprint surrounded by a cascade of laterally layered terraces. The solidifying glue rapidly produces crystals of KCl on these alluvial-like terraces. Since the properties of the glue affect the adhesion of the puparium to its substrate, and so can influence the success of metamorphosis, we evaluated over 80 different materials for their ability to adhere to the glue to determine which properties favor strong adhesion. We found that the alkaline Sgs-glue adheres strongly to wettable and positively charged surfaces but not to neutral or negatively charged and hydrophobic surfaces. Puparia formed on unfavored materials can be removed easily without leaving fingerprints or cascading terraces. For successful adhesion of the Sgs-glue, the material surface must display a specific type of triboelectric charge. Interestingly, the expectorated glue can move upwards against gravity on the surface of freshly formed puparia via specific, unique and novel anatomical structures present in the puparial's lateral abdominal segments that we have named bidentia.

Apocrine secretion in the salivary glands of Drosophilidae and other dipterans is evolutionarily conserved.

Apocrine secretion is a transport and secretory mechanism that remains only partially characterized, even though it is evolutionarily conserved among all metazoans, including humans. The excellent genetic model organism Drosophila melanogaster holds promise for elucidating the molecular mechanisms regulating this fundamental metazoan process. Two prerequisites for such investigations are to clearly define an experimental system to investigate apocrine secretion and to understand the evolutionarily and functional contexts in which apocrine secretion arose in that system. To this end, we recently demonstrated that, in D. melanogaster, the prepupal salivary glands utilize apocrine secretion prior to pupation to deliver innate immune and defense components to the exuvial fluid that lies between the metamorphosing pupae and its chitinous case. This finding provided a unique opportunity to appraise how this novel non-canonical and non-vesicular transport and secretory mechanism is employed in different developmental and evolutionary contexts. Here we demonstrate that this apocrine secretion, which is mechanistically and temporarily separated from the exocytotic mechanism used to produce the massive salivary glue secretion (Sgs), is shared across Drosophilidae and two unrelated dipteran species. Screening more than 30 species of Drosophila from divergent habitats across the globe revealed that apocrine secretion is a widespread and evolutionarily conserved cellular mechanism used to produce exuvial fluid. Species with longer larval and prepupal development than D. melanogaster activate apocrine secretion later, while smaller and more rapidly developing species activate it earlier. In some species, apocrine secretion occurs after the secretory material is first concentrated in cytoplasmic structures of unknown origin that we name "collectors." Strikingly, in contrast to the widespread use of apocrine secretion to provide exuvial fluid, not all species use exocytosis to produce the viscid salivary glue secretion that is seen in D. melanogaster. Thus, apocrine secretion is the conserved mechanism used to realize the major function of the salivary gland in fruitflies and related species: it produces the pupal exuvial fluid that provides an active defense against microbial invasion during pupal metamorphosis.

An apocrine mechanism delivers a fully immunocompetent exocrine secretion.

Apocrine secretion is a recently discovered widespread non-canonical and non-vesicular secretory mechanism whose regulation and purpose is only partly defined. Here, we demonstrate that apocrine secretion in the prepupal salivary glands (SGs) of Drosophila provides the sole source of immune-competent and defense-response proteins to the exuvial fluid that lies between the metamorphosing pupae and its pupal case. Genetic ablation of its delivery from the prepupal SGs to the exuvial fluid decreases the survival of pupae to microbial challenges, and the isolated apocrine secretion has strong antimicrobial effects in "agar-plate" tests. Thus, apocrine secretion provides an essential first line of defense against exogenously born infection and represents a highly specialized cellular mechanism for delivering components of innate immunity at the interface between an organism and its external environment.

Fine infrastructure of released and solidified Drosophila larval salivary secretory glue using SEM.

The Golgi-derived large secretory granules of Drosophila salivary glands (SGs) constitute the components of the salivary glue secretion (Sgs). The Sgs represents a highly special and unique extracellular composite glue matrix that has not yet been identified outside of Cyclorrhaphous Dipterans. For over half a century, the only major and unambiguously documented function of the larval salivary glands was to produce a large amount of mucinous glue-containing secretory granules that, when released during pupariation, serves to affix the freshly formed puparia to a substrate. Besides initial biochemical characterization of the Sgs proteins and cloning of their corresponding Sgs genes, very little is known about other properties and functions of the Sgs glue. We report here observations on the fine SEM-ultrastructure of the Sgs glue released into to the lumen of SGs, and after it has been expectorated and solidified into the external environment. Surprisingly, in contrast to long held expectations, it appears to be a highly structured bioadhesive mass with an internal spongious to trabecular infrastructure, reflecting the state of its hydratation. We also found that in addition to its cementing properties, it is highly efficient at glueing and trapping microorganisms, and thus may serve a potentially very important immune and defense role. High hydration capacity, the speed by which this glue can dry, uniqueness of its protein composition and spongious infrastructure can provide inspiration for development of potential biomimetics that can attach completely different or incompatible surfaces with high efficiency and strength.

A protocol for processing the delicate larval and prepupal salivary glands of Drosophila for scanning electron microscopy.

Although scanning electron microscopy (SEM) has been broadly used for the examination of fixed whole insects or their hard exoskeleton-derived structures, including model organisms such as Drosophila, the routine use of SEM to evaluate vulnerable soft internal organs and tissues was often hampered by their fragile nature and frequent surface contamination. Here, we describe a simple four-step protocol that allows for the reliable and reproducible preparation of the larval and prepupal salivary glands (SGs) of Drosophila for SEM devoid of any surface contamination. The steps are to: first, proteolytically digest the adhering fat body; second, use detergent washes to remove contaminating coarse tissue fragments, including sticky remnants of the fat body; third, use nonionic emulsifying polysorbate emulsifiers to remove fine contaminants from the SGs surface; and fourth, use aminopolycarboxylate-based chelating agents to detach sessile hemocytes. Short but repeated rinses in 100 µL of a saline-based buffer between steps ensure efficient removal of remnants removed by each treatment. After these steps, the SGs are fixed in glutaraldehyde, postfixed in osmium tetroxide, dehydrated, critically point-dried, mounted on aluminum stubs, sputter coated with gold-palladium alloy and examined in the SEM.

Endosomal vacuoles of the prepupal salivary glands of Drosophila play an essential role in the metabolic reallocation of iron.

In the recent past, we demonstrated that a great deal is going on in the salivary glands of Drosophila in the interval after they release their glycoprotein-rich secretory glue during pupariation. The early-to-mid prepupal salivary glands undergo extensive endocytosis with widespread vacuolation of the cytoplasm followed by massive apocrine secretion. Here, we describe additional novel properties of these endosomes. The use of vital pH-sensitive probes provided confirmatory evidence that these endosomes have acidic contents and that there are two types of endocytosis seen in the prepupal glands. The salivary glands simultaneously generate mildly acidic, small, basally-derived endosomes and strongly acidic, large and apical endosomes. Staining of the large vacuoles with vital acidic probes is possible only after there is ambipolar fusion of both basal and apical endosomes, since only basally-derived endosomes can bring fluorescent probes into the vesicular system. We obtained multiple lines of evidence that the small basally-derived endosomes are chiefly involved in the uptake of dietary Fe3+ iron. The fusion of basal endosomes with the larger and strongly acidic apical endosomes appears to facilitate optimal conditions for ferrireductase activity inside the vacuoles to release metabolic Fe2+ iron. While iron was not detectable directly due to limited staining sensitivity, we found increasing fluorescence of the glutathione-sensitive probe CellTracker Blue CMAC in large vacuoles, which appeared to depend on the amount of iron released by ferrireductase. Moreover, heterologous fluorescently-labeled mammalian iron-bound transferrin is actively taken up, providing direct evidence for active iron uptake by basal endocytosis. In addition, we serendipitously found that small (basal) endosomes were uniquely recognized by PNA lectin, whereas large (apical) vacuoles bound DBA lectin.

Massive excretion of calcium oxalate from late prepupal salivary glands of Drosophila melanogaster demonstrates active nephridial-like anion transport.

The Drosophila salivary glands (SGs) were well known for the puffing patterns of their polytene chromosomes and so became a tissue of choice to study sequential gene activation by the steroid hormone ecdysone. One well-documented function of these glands is to produce a secretory glue, which is released during pupariation to fix the freshly formed puparia to the substrate. Over the past two decades SGs have been used to address specific aspects of developmentally-regulated programmed cell death (PCD) as it was thought that they are doomed for histolysis and after pupariation are just awaiting their fate. More recently, however, we have shown that for the first 3-4 h after pupariation SGs undergo tremendous endocytosis and vacuolation followed by vacuole neutralization and membrane consolidation. Furthermore, from 8 to 10 h after puparium formation (APF) SGs display massive apocrine secretion of a diverse set of cellular proteins. Here, we show that during the period from 11 to 12 h APF, the prepupal glands are very active in calcium oxalate (CaOx) extrusion that resembles renal or nephridial excretory activity. We provide genetic evidence that Prestin, a Drosophila homologue of the mammalian electrogenic anion exchange carrier SLC26A5, is responsible for the instantaneous production of CaOx by the late prepupal SGs. Its positive regulation by the protein kinases encoded by fray and wnk lead to increased production of CaOx. The formation of CaOx appears to be dependent on the cooperation between Prestin and the vATPase complex as treatment with bafilomycin A1 or concanamycin A abolishes the production of detectable CaOx. These data demonstrate that prepupal SGs remain fully viable, physiologically active and engaged in various cellular activities at least until early pupal period, that is, until moments prior to the execution of PCD.

Vacuole dynamics in the salivary glands of Drosophila melanogaster during prepupal development.

A central function of the Drosophila salivary glands (SGs), historically known for their polytene chromosomes, is to produce and then release during pupariation the secretory glue used to affix a newly formed puparium to a substrate. This essential event in the life history of Drosophila is regulated by the steroid hormone ecdysone in the late-larval period. Ecdysone triggers a cascade of sequential gene activation that leads to glue secretion and initiates the developmentally-regulated programmed cell death (PCD) of the larval salivary glands, which culminates 16 h after puparium formation (APF). We demonstrate here that, even after the larval salivary glands have completed what is perceived to be one of their major biological functions--glue secretion during pupariation--they remain dynamic and physiologically active up until the execution phase of PCD. We have used specific metabolic inhibitors and genetic tools, including mutations or transgenes for shi, Rab5, Rab11, vha55, vha68-2, vha36-1, syx1A, syx4, and Vps35 to characterize the dramatic series of cellular changes occurring in the SG cells between pupariation and 7-8 h APF. Early in the prepupal period, they are remarkably active in endocytosis, forming acidic vacuoles. Midway through the prepupal period, there is abundant late endosomal trafficking and vacuole growth, which is followed later by vacuole neutralization and disappearance via membrane consolidation. This work provides new insights into the function of Drosophila SGs during the early- to mid-prepupal period.

Requirement for highly efficient pre-mRNA splicing during Drosophila early embryonic development.

Drosophila syncytial nuclear divisions limit transcription unit size of early zygotic genes. As mitosis inhibits not only transcription, but also pre-mRNA splicing, we reasoned that constraints on splicing were likely to exist in the early embryo, being splicing avoidance a possible explanation why most early zygotic genes are intronless. We isolated two mutant alleles for a subunit of the NTC/Prp19 complexes, which specifically impaired pre-mRNA splicing of early zygotic but not maternally encoded transcripts. We hypothesized that the requirements for pre-mRNA splicing efficiency were likely to vary during development. Ectopic maternal expression of an early zygotic pre-mRNA was sufficient to suppress its splicing defects in the mutant background. Furthermore, a small early zygotic transcript with multiple introns was poorly spliced in wild-type embryos. Our findings demonstrate for the first time the existence of a developmental pre-requisite for highly efficient splicing during Drosophila early embryonic development and suggest in highly proliferative tissues a need for coordination between cell cycle and gene architecture to ensure correct gene expression and avoid abnormally processed transcripts. DOI: http://dx.doi.org/10.7554/eLife.02181.001.

Apocrine secretion in Drosophila salivary glands: subcellular origin, dynamics, and identification of secretory proteins.

In contrast to the well defined mechanism of merocrine exocytosis, the mechanism of apocrine secretion, which was first described over 180 years ago, remains relatively uncharacterized. We identified apocrine secretory activity in the late prepupal salivary glands of Drosophila melanogaster just prior to the execution of programmed cell death (PCD). The excellent genetic tools available in Drosophila provide an opportunity to dissect for the first time the molecular and mechanistic aspects of this process. A prerequisite for such an analysis is to have pivotal immunohistochemical, ultrastructural, biochemical and proteomic data that fully characterize the process. Here we present data showing that the Drosophila salivary glands release all kinds of cellular proteins by an apocrine mechanism including cytoskeletal, cytosolic, mitochondrial, nuclear and nucleolar components. Surprisingly, the apocrine release of these proteins displays a temporal pattern with the sequential release of some proteins (e.g. transcription factor BR-C, tumor suppressor p127, cytoskeletal ß-tubulin, non-muscle myosin) earlier than others (e.g. filamentous actin, nuclear lamin, mitochondrial pyruvate dehydrogenase). Although the apocrine release of proteins takes place just prior to the execution of an apoptotic program, the nuclear DNA is never released. Western blotting indicates that the secreted proteins remain undegraded in the lumen. Following apocrine secretion, the salivary gland cells remain quite vital, as they retain highly active transcriptional and protein synthetic activity.

Molecular determinants of juvenile hormone action as revealed by 3D QSAR analysis in Drosophila.

BACKGROUND: Postembryonic development, including metamorphosis, of many animals is under control of hormones. In Drosophila and other insects these developmental transitions are regulated by the coordinate action of two principal hormones, the steroid ecdysone and the sesquiterpenoid juvenile hormone (JH). While the mode of ecdysone action is relatively well understood, the molecular mode of JH action remains elusive. METHODOLOGY/PRINCIPAL FINDINGS: To gain more insights into the molecular mechanism of JH action, we have tested the biological activity of 86 structurally diverse JH agonists in Drosophila melanogaster. The results were evaluated using 3D QSAR analyses involving CoMFA and CoMSIA procedures. Using this approach we have generated both computer-aided and species-specific pharmacophore fingerprints of JH and its agonists, which revealed that the most active compounds must possess an electronegative atom (oxygen or nitrogen) at both ends of the molecule. When either of these electronegative atoms are replaced by carbon or the distance between them is shorter than 11.5 A or longer than 13.5 A, their biological activity is dramatically decreased. The presence of an electron-deficient moiety in the middle of the JH agonist is also essential for high activity. CONCLUSIONS/SIGNIFICANCE: The information from 3D QSAR provides guidelines and mechanistic scope for identification of steric and electrostatic properties as well as donor and acceptor hydrogen-bonding that are important features of the ligand-binding cavity of a JH target protein. In order to refine the pharmacophore analysis and evaluate the outcomes of the CoMFA and CoMSIA study we used pseudoreceptor modeling software PrGen to generate a putative binding site surrogate that is composed of eight amino acid residues corresponding to the defined molecular interactions.

Exploring some of the physico-chemical properties of the LAMMER protein kinase DOA of Drosophila.

Members of the highly conserved LAMMER family of protein kinases have been described in all eukaryotes. LAMMER kinases possess markedly similar peptide motifs in their kinase catalytic subdomains that are responsible for phosphotransfer and substrate interaction, suggesting that family members serve similar functions in widely diverged species. This hypothesis is supported by their phosphorylation of SR and SR-related proteins in diverged species. Here we describe a 3-dimensional homology model of the catalytic domain of DOA, a representative LAMMER kinase, encoded by the Drosophila locus Darkener of apricot (Doa). Homology modeling of DOA based on a Sky1p template revealed a highly conserved structural framework within conserved core regions. These adopt typical kinase folding like that of other protein kinases. However, in contrast to Sky1p, some structural features, such as those in helix alphaC suggest that the DOA kinase is not a constitutively active enzyme but requires activation. This may occur by phosphorylation within an activation loop that forms a broad turn and in which interactions between the side chains occur across the loop. The fold of the activation loop is stabilized through interactions with residues in the C-terminal tail, which is not part of the conserved kinase core and is variable among protein kinases. Immediately following the activation loop in the segment between the beta9 sheet and helix alphaF is a P + 1 loop. The electrostatic surface potential of the DOA substrate-binding groove is largely negative, as it is in other known SR protein kinases, suggesting that DOA substrates must be basic. All differences between D. melanogaster and other Drosophila species are single amino acid changes situated in regions outside of any alpha-helices or beta-sheets, and after modeling these had absolutely no visible effect on protein structure. The absence of evolved amino acid changes among 12 Drosophila species that would cause at least predictable changes in DOA structure indicate that evolution has already selected evolved mutations for having minimal effect on kinase structure.

Processing of soft pupae and uneclosed pharate adults of Drosophila for scanning electron microscopy.

For over four decades, scanning electron microscopy (SEM) has been used in research involving Drosophila genetics and developmental biology. It allows for observation and documentation of the gross morphology of exoskeletal structures as well as their characterization at very high resolution. In most cases, SEM in Drosophila has been limited to imaging adult heads, thoraces, appendages, and embryos, as these structures are relatively hard and/or easy to process for SEM. In contrast, the structures of the pharate adult stages are difficult to prepare for SEM because their integument is quite soft, they are extremely dirty and they are resistant to the usual processing methods. Here, we present an innovative method to prepare these types of structures. Our protocol efficiently removes extraneous material originating from the exuvial fluid of pharate adults and uses a hydrophobic expansion step to keep the soft exoskeleton of the body inflated. In addition to using immersion fixation, it utilizes fixation within the body that occurs via a reaction between osmium tetroxide and alcohols that are infiltrated into the body during a hydrophobic expansion step. This novel approach results in a properly inflated integument that retains its shape in subsequent procedures. Our method provides a useful, general alternative for processing difficult samples, including soft, biological "whole-mount" specimens and samples that are extremely dirty or resistant to fixative penetration.