Drosophila Larval Salivary Glands Research Articles

Structured RhoGEF recruitment drives myosin II organization on large exocytic vesicles.

The Rho family of GTPases plays a crucial role in cellular mechanics by regulating actomyosin contractility through the parallel induction of actin and myosin assembly and function. Using exocytosis of large vesicles in the Drosophila larval salivary gland as a model, we followed the spatiotemporal regulation of Rho1, which in turn creates distinct organization patterns of actin and myosin. After vesicle fusion, low levels of activated Rho1 reach the vesicle membrane and drive actin nucleation in an uneven, spread-out pattern. Subsequently, the Rho1 activator RhoGEF2 distributes as an irregular meshwork on the vesicle membrane, activating Rho1 in a corresponding punctate pattern and driving local myosin II recruitment, resulting in vesicle constriction. Vesicle membrane buckling and subsequent crumpling occur at local sites of high myosin II concentrations. These findings indicate that distinct thresholds for activated Rho1 create a biphasic mode of actomyosin assembly, inducing anisotropic membrane crumpling during exocrine secretion.

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.

The Ykt6–Snap29–Syx13 SNARE complex promotes crinophagy via secretory granule fusion with Lamp1 carrier vesicles

In the Drosophila larval salivary gland, developmentally programmed fusions between lysosomes and secretory granules (SGs) and their subsequent acidification promote the maturation of SGs that are secreted shortly before puparium formation. Subsequently, ongoing fusions between non-secreted SGs and lysosomes give rise to degradative crinosomes, where the superfluous secretory material is degraded. Lysosomal fusions control both the quality and quantity of SGs, however, its molecular mechanism is incompletely characterized. Here we identify the R-SNARE Ykt6 as a novel regulator of crinosome formation, but not the acidification of maturing SGs. We show that Ykt6 localizes to Lamp1+ carrier vesicles, and forms a SNARE complex with Syntaxin 13 and Snap29 to mediate fusion with SGs. These Lamp1 carriers represent a distinct vesicle population that are functionally different from canonical Arl8+, Cathepsin L+ lysosomes, which also fuse with maturing SGs but are controlled by another SNARE complex composed of Syntaxin 13, Snap29 and Vamp7. Ykt6- and Vamp7-mediated vesicle fusions also determine the fate of SGs, as loss of either of these SNAREs prevents crinosomes from acquiring endosomal PI3P. Our results highlight that fusion events between SGs and different lysosome-related vesicle populations are critical for fine regulation of the maturation and crinophagic degradation of SGs.

Fusion pore dynamics of large secretory vesicles define a distinct mechanism of exocytosis.

Exocrine cells utilize large secretory vesicles (LSVs) up to 10 μm in diameter. LSVs fuse with the apical surface, often recruiting actomyosin to extrude their content through dynamic fusion pores. The molecular mechanism regulating pore dynamics remains largely uncharacterized. We observe that the fusion pores of LSVs in the Drosophila larval salivary glands expand, stabilize, and constrict. Arp2/3 is essential for pore expansion and stabilization, while myosin II is essential for pore constriction. We identify several Bin-Amphiphysin-Rvs (BAR) homology domain proteins that regulate fusion pore expansion and stabilization. We show that the I-BAR protein Missing-in-Metastasis (MIM) localizes to the fusion site and is essential for pore expansion and stabilization. The MIM I-BAR domain is essential but not sufficient for localization and function. We conclude that MIM acts in concert with actin, myosin II, and additional BAR-domain proteins to control fusion pore dynamics, mediating a distinct mode of exocytosis, which facilitates actomyosin-dependent content release that maintains apical membrane homeostasis during secretion.

Cytological Approaches to Visualize Intracellular Dynamics of RNA-Binding Proteins at Active Genes in Drosophila.

Heterogeneous nuclear ribonucleoproteins (hnRNPs) are a family of RNA-binding proteins that modulate multiple aspects of gene activity and RNA processing, including transcription, splicing, localization, translation, and decay of RNA. Interaction of hnRNPs with RNA is a highly dynamic but regulated process. Poly(ADP-ribose) polymerase (PARP)-dependent PARylation of different hnRNPs is a well-known posttranslational modification that affects their interactions with RNA. Here, we described a protocol for in situ localization of RNA-binding proteins (RBPs) on giant polytene chromosomes in Drosophila larval salivary glands, which have been widely used to visualize the dynamic binding profiles of various RBPs and other transcription-related proteins at specific loci on chromosomes. This chapter also includes a stepwise description of RNA:RNA in situ hybridization, in conjunction with immunostaining, using polytene chromosome squashes or intact tissues. We also highlight advanced live cell imaging methods, including FRAP and FLIP, using transgenic lines that express fluorescent-tagged hnRNPs. These cytological approaches can be used to visualize the localization of RNA-binding proteins and their interacting RNAs under different cellular conditions.

Immunofluorescence as a Method to Study Golgi Organization in Larval Salivary Glands of Drosophila melanogaster.

Immunofluorescence is an important research tool in cell biology that reveals structural organization of subcellular organelles by detecting their associated constituents. Here, we describe an antibody staining method to detect Golgi-associated proteins in Drosophila larval salivary glands, using the cis-Golgi protein Lava lamp and the clathrin adaptor AP-1 as a suitable example. Golgi bodies immunostained using this protocol can be visualized using confocal or structured illumination microscopy.

Using Single Molecule RNA FISH to Determine Nuclear Export and Transcription Phenotypes in Drosophila Tissues.

Single molecule RNA fluorescence in situ hybridization (smRNA FISH) is a widely used method for examining cellular localization of RNA and assessing gene expression outputs. The Nuclear Pore Complex (NPC) is a nuclear macro-complex known to both mediate nucleocytoplasmic transport and influence transcription via interactions with chromatin. Consequently, depletion of NPC proteins can result in defects in either transcription or nuclear export of mRNA. To distinguish between these two different functions of NPC components, it is preferable to analyze transcription and mRNA export simultaneously or in the same cell. Here, we present a smRNA FISH protocol with downstream custom MATLAB image analysis for application in Drosophila larval salivary gland tissues. This method can detect both nuclear export and transcriptional phenotypes in the same cell and as a single assay, and can be adapted to many other cell types and organisms.

In Vivo Analysis of Pathways Regulating Epithelial Polarity and Secretion Using Drosophila Salivary Glands.

The experimental versatility of the fruit fly has helped to uncover the molecular basis of epithelial cell polarity. In this chapter, we provide protocols to dissect Drosophila larval salivary glands (SGs) for ex vivo culture and live imaging, and for fixing and immunostaining for analysis by fluorescence microscopy. We describe how to combine these approaches with genetic and pharmacological assays. These techniques can be applied to study signaling pathways regulating epithelial cell polarity, membrane trafficking, gland secretion, and their impacts on animal feeding behavior.

The Hob proteins are novel and conserved lipid-binding proteins at ER-PM contact sites.

Membrane contact sites are critical junctures for organelle signaling and communication. Endoplasmic reticulum-plasma membrane (ER-PM) contact sites were the first membrane contact sites to be described; however, the protein composition and molecular function of these sites is still emerging. Here, we leverage yeast and Drosophila model systems to uncover a novel role for the Hobbit (Hob) proteins at ER-PM contact sites. We find that Hobbit localizes to ER-PM contact sites in both yeast cells and the Drosophila larval salivary glands, and this localization is mediated by an N-terminal ER membrane anchor and conserved C-terminal sequences. The C-terminus of Hobbit binds to plasma membrane phosphatidylinositols, and the distribution of these lipids is altered in hobbit mutant cells. Notably, the Hobbit protein is essential for viability in Drosophila, providing one of the first examples of a membrane contact site-localized lipid binding protein that is required for development.

A novel function for Rab1 and Rab11 during secretory granule maturation.

Regulated exocytosis is an essential process whereby specific cargo proteins are secreted in a stimulus-dependent manner. Cargo-containing secretory granules are synthesized in the trans-Golgi network (TGN); after budding from the TGN, granules undergo modifications, including an increase in size. These changes occur during a poorly understood process called secretory granule maturation. Here, we leverage the Drosophila larval salivary glands as a model to characterize a novel role for Rab GTPases during granule maturation. We find that secretory granules increase in size ∼300-fold between biogenesis and release, and loss of Rab1 or Rab11 reduces granule size. Surprisingly, we find that Rab1 and Rab11 localize to secretory granule membranes. Rab11 associates with granule membranes throughout maturation, and Rab11 recruits Rab1. In turn, Rab1 associates specifically with immature granules and drives granule growth. In addition to roles in granule growth, both Rab1 and Rab11 appear to have additional functions during exocytosis; Rab11 function is necessary for exocytosis, while the presence of Rab1 on immature granules may prevent precocious exocytosis. Overall, these results highlight a new role for Rab GTPases in secretory granule maturation.

Fine structure of Drosophila larval salivary gland ducts as revealed by laser confocal microscopy and SEM

The functions of the larval salivary glands (SGs) of Drosophila are traditionally associated with the production of a massive secretion during puparium formation; it is exocytosed into a centrally located lumen and subsequently expectorated via ducts, the pharynx and mouth. This so-called proteinaceous glue serves as an adhesive to attach the puparial case to a solid substrate. Great attention has been paid to the secretory cells of SGs, which are famous for their giant polytene chromosomes. However, substantially less attention has been devoted to individual or common ducts that form the most proximal portion of the SG organ via which the glue is released into the pharynx. In the present paper, we describe the organization and fine structure of the taenidia, highly specialized circumferential ring-like extracellular (cuticular) components on the internal side of these tubes. Two chitin-specific probes that have previously been used to recognize taenidia in Drosophila tracheae, Calcofluor White M2R (also known as Fluorescent Brightener 28) and the novel vital fluorescent dye SiR-COOH, show positively stained ductal taenidia in late larval SGs. As seen using scanning electron microscopy (SEM), the interior of the ductal tube contains regular and densely-arranged ridge-like circumferential rings which represent local thickenings of the cuticle in various geometries. The microtubular arrays that optically colocalize with taenidia in both the trachea and SG ducts are specifically and strongly recognized by fluorescently-conjugated colchicine as well as anti-tubulin antibody. In contrast to taenidia in the tracheae, the analogous structures in SG ducts cannot be detected by fluorescently-labeled phalloidin or even actin-GFP fusion protein, suggesting that the ducts lack a cortical network made of filamentous actin. We speculate that these taenidia may serve to reinforce the duct during the secretory processes that SGs undergo during late larval and late prepupal stages.

Quantitation of Secretory Granule Size in Drosophila Larval Salivary Glands.

Maturation of secretory granules is a crucial process that ensures the bioactivity of cargo proteins undergoing regulated secretion. In Drosophila melanogaster, the larval salivary glands produce secretory granules that are up to four-fold larger in cross-sectional area after maturation. Therefore, we developed a live imaging microscopy approach to quantitate the size of secretory granules with a view to identifying genes involved in their maturation. Here, we describe the procedures of larval salivary gland dissection and sample preparation for live imaging with a fluorescence confocal microscope. Furthermore, we describe the workflow for measuring the size of secretory granules by cross-sectional surface area and statistical analysis. Our live imaging microscopy method provides a reliable read-out for the status of secretory granule maturation in Drosophila larval salivary glands.

Mistargeting of secretory cargo in retromer-deficient cells.

ABSTRACTIntracellular trafficking is a basic and essential cellular function required for delivery of proteins to the appropriate subcellular destination; this process is especially demanding in professional secretory cells, which synthesize and secrete massive quantities of cargo proteins via regulated exocytosis. The Drosophila larval salivary glands are composed of professional secretory cells that synthesize and secrete mucin proteins at the onset of metamorphosis. Using the larval salivary glands as a model system, we have identified a role for the highly conserved retromer complex in trafficking of secretory granule membrane proteins. We demonstrate that retromer-dependent trafficking via endosomal tubules is induced at the onset of secretory granule biogenesis, and that recycling via endosomal tubules is required for delivery of essential secretory granule membrane proteins to nascent granules. Without retromer function, nascent granules do not contain the proper membrane proteins; as a result, cargo from these defective granules is mistargeted to Rab7-positive endosomes, where it progressively accumulates to generate dramatically enlarged endosomes. Retromer complex dysfunction is strongly associated with neurodegenerative diseases, including Alzheimer's disease, characterized by accumulation of amyloid β (Aβ). We show that ectopically expressed amyloid precursor protein (APP) undergoes regulated exocytosis in salivary glands and accumulates within enlarged endosomes in retromer-deficient cells. These results highlight recycling of secretory granule membrane proteins as a critical step during secretory granule maturation and provide new insights into our understanding of retromer complex function in secretory cells. These findings also suggest that missorting of secretory cargo, including APP, may contribute to the progressive nature of neurodegenerative disease.

Tango1 Coordinates the Formation of ER/Golgi Docking Sites to Mediate Secretory Granule Formation

Regulated secretion is a conserved process occurring across diverse cells and tissues. Current models suggest that the conserved cargo receptor Tango1 mediates the packaging of collagen into large coat protein complex II (COPII) vesicles that move from the endoplasmic reticulum (ER) to the Golgi apparatus. However, how Tango1 regulates the formation of COPII carriers and influences the secretion of other cargo remains unknown. Here, through high‐resolution imaging of Tango1, COPII, Golgi and secretory cargo (mucins) in Drosophila larval salivary glands, we found that Tango1 forms ring‐like structures that mediate the formation of COPII rings, rather than vesicles. These COPII rings act as docking sites for the cis‐Golgi. Moreover, we observed nascent secretory mucins emerging from the Golgi side of these Tango1/COPII/Golgi complexes, suggesting that these structures represent functional docking sites/fusion points between the ER exit sites and the Golgi. Loss of Tango1 disrupted the formation of COPII rings, the association of COPII with the cis‐Golgi, mucin O‐glycosylation and secretory granule biosynthesis. Additionally, we identified a Tango1 self‐association domain that is essential for formation of this structure. Our results provide evidence that Tango1 organizes an interaction site where secretory cargo is efficiently transferred from the ER to Golgi and then to secretory vesicles. These findings may explain how the loss of Tango1 can influence Golgi/ER morphology and affect the secretion of diverse proteins across many tissues.

Tango1 coordinates the formation of endoplasmic reticulum/Golgi docking sites to mediate secretory granule formation

Regulated secretion is a conserved process occurring across diverse cells and tissues. Current models suggest that the conserved cargo receptor Tango1 mediates the packaging of collagen into large coat protein complex II (COPII) vesicles that move from the endoplasmic reticulum (ER) to the Golgi apparatus. However, how Tango1 regulates the formation of COPII carriers and influences the secretion of other cargo remains unknown. Here, through high-resolution imaging of Tango1, COPII, Golgi, and secretory cargo (mucins) in Drosophila larval salivary glands, we found that Tango1 forms ring-like structures that mediate the formation of COPII rings rather than vesicles. These COPII rings act as docking sites for the cis-Golgi. Moreover, we observed nascent secretory mucins emerging from the Golgi side of these Tango1-COPII-Golgi complexes, suggesting that these structures represent functional docking sites/fusion points between the ER exit sites and the Golgi. Loss of Tango1 disrupted the formation of COPII rings, the association of COPII with the cis-Golgi, mucin O-glycosylation, and secretory granule biosynthesis. Additionally, we identified a Tango1 self-association domain that is essential for formation of this structure. Our results provide evidence that Tango1 organizes an interaction site where secretory cargo is efficiently transferred from the ER to Golgi and then to secretory vesicles. These findings may explain how the loss of Tango1 can influence Golgi/ER morphology and affect the secretion of diverse proteins across many tissues.

The NF-κB Factor Relish Regulates Atg1 Expression and Controls Autophagy

SUMMARYMacroautophagy and cell death both contribute to innate immunity, but little is known about how these processes integrate. Drosophila larval salivary glands require autophagy for developmentally programmed cell death, and innate immune signaling factors increase in these dying cells. Here, we show that the nuclear factor κB (NF-κB) factor Relish, a component of the immune deficiency (Imd) pathway, is required for salivary gland degradation. Surprisingly, of the classic Imd pathway components, only Relish and the PGRP receptors were involved in salivary gland degradation. Significantly, Relish controls salivary gland degradation by regulating autophagy but not caspases. In addition, expression of either Relish or PGRP-LC causes premature autophagy induction and subsequent gland degradation. Relish controls autophagy by regulating the expression of Atg1, a core component and activator of the autophagy pathway. Together these findings demonstrate that a NF-κB pathway regulates autophagy during developmentally programmed cell death.

Revisiting Point FRAP to Quantitatively Characterize Anomalous Diffusion in Live Cells

Fluorescence recovery after photobleaching (FRAP) is widely used to interrogate diffusion and binding of proteins in live cells. Herein, we apply two-photon excited FRAP with a diffraction limited bleaching and observation volume to study anomalous diffusion of unconjugated green fluorescence protein (GFP) in vitro and in cells. Experiments performed on dilute solutions of GFP reveal that reversible fluorophore bleaching can be mistakenly interpreted as anomalous diffusion. We derive a reaction-diffusion FRAP model that includes reversible photobleaching, and demonstrate that it properly accounts for these photophysics. We then apply this model to investigate the diffusion of GFP in HeLa cells and polytene cells of Drosophila larval salivary glands. GFP exhibits anomalous diffusion in the cytoplasm of both cell types and in HeLa nuclei. Polytene nuclei contain optically resolvable chromosomes, permitting FRAP experiments that focus separately on chromosomal or interchrosomal regions. We find that GFP exhibits anomalous diffusion in chromosomal regions but diffuses normally in regions devoid of chromatin. This observation indicates that obstructed transport through chromatin and not crowding by macromolecules is a source of anomalous diffusion in polytene nuclei. This behavior is likely true in other cells, so it will be important to account for this type of transport physics and for reversible photobleaching to properly interpret future FRAP experiments on DNA-binding proteins.

Identification of factors that function in Drosophila salivary gland cell death during development using proteomics

Proteasome inhibitors induce cell death and are used in cancer therapy, but little is known about the relationship between proteasome impairment and cell death under normal physiological conditions. Here, we investigate the relationship between proteasome function and larval salivary gland cell death during development in Drosophila. Drosophila larval salivary gland cells undergo synchronized programmed cell death requiring both caspases and autophagy (Atg) genes during development. Here, we show that ubiquitin proteasome system (UPS) function is reduced during normal salivary gland cell death, and that ectopic proteasome impairment in salivary gland cells leads to early DNA fragmentation and salivary gland condensation in vivo. Shotgun proteomic analyses of purified dying salivary glands identified the UPS as the top category of proteins enriched, suggesting a possible compensatory induction of these factors to maintain proteolysis during cell death. We compared the proteome following ectopic proteasome impairment to the proteome during developmental cell death in salivary gland cells. Proteins that were enriched in both populations of cells were screened for their function in salivary gland degradation using RNAi knockdown. We identified several factors, including trol, a novel gene CG11880, and the cop9 signalsome component cop9 signalsome 6, as required for Drosophila larval salivary gland degradation.

Fhos encodes a Drosophila Formin‐Like Protein participating in autophagic programmed cell death

Larval tissues undergo programmed cell death (PCD) during Drosophila metamorphosis. PCD is triggered in a stage and tissue-specific fashion in response to ecdysone pulses. The understanding of how ecdysone induces the stage and tissue-specificity of cell death remains obscure. Several steroid-regulated primary response genes have been shown to act as key regulators of cellular responses to ecdysone by inducing a cascade of transcriptional regulation of late responsive genes. In this article, the authors identify Fhos as a gene that is required for Drosophila larval salivary gland destruction. Animals with a P-element mutation in Fhos possess persistent larval salivary glands, and precise excisions of this P-element insertion resulted in reversion of this salivary gland mutant phenotype. Fhos encodes the Drosophila homolog of mammalian Formin Fhos. Fhos is differentially transcribed during development and responds to ecdysone in a method that is similar to other cell death genes. Similarly to what has been shown for its mammalian counterpart, FHOS protein is translocated to the nucleus at later stages of cell death. Fhos mutants posses disrupted actin cytoskeleton dynamics in persistent salivary glands. Together, our data indicate that Fhos is a new ecdysone-regulated gene that is crucial for changes in the actin cytoskeleton during salivary gland elimination in Drosophila.

Dynein light chain 1 is required for autophagy, protein clearance, and cell death inDrosophila

Autophagy is a catabolic pathway that is important for turnover of long-lived proteins and organelles, and has been implicated in cell survival, tumor progression, protection from infection, neurodegeneration, and cell death. Autophagy and caspases are required for type II autophagic cell death of Drosophila larval salivary glands during development, but the mechanisms that regulate these degradation pathways are not understood. We conducted a forward genetic screen for genes that are required for salivary gland cell death, and here we describe the identification of Drosophila dynein light chain 1 (ddlc1) as a gene that is required for type II cell death. Autophagy is attenuated in ddlc1 mutants, but caspases are active in these cells. ddlc1 mutant salivary glands develop large fibrillar protein inclusions that stain positive for amyloid-specific dyes and ubiquitin. Ectopic expression of Atg1 is sufficient to induce autophagy, clear protein inclusions, and rescue degradation of ddlc1 mutant salivary glands. Furthermore, ddlc1 mutant larvae have decreased motility, and mutations in ddlc1 enhance the impairment of motility that is observed in a Drosophila model of neurodegenerative disease. Significantly, this decrease in larval motility is associated with decreased clearance of protein with polyglutamine expansion, the accumulation of p62 in neurons and muscles, and fewer synaptic boutons. These results indicate that DDLC1 is required for protein clearance by autophagy that is associated with autophagic cell death and neurodegeneration.