Crinophagy mechanisms and its potential role in human health and disease.

Autophagic-lysosomal degradation is essential for the maintenance of normal homeostasis in eukaryotic cells. Several types of such self-degradative and recycling pathways have been identified. From these, probably the least known autophagic process is crinophagy, during which unnecessary or obsolete secretory granules directly fuse with late endosomes/lysosomes as a means of rapid elimination of unused secretory material from the cytoplasm. This process was identified in 1966, but we are only beginning to understand the molecular mechanisms and regulation of crinophagy. In this review, we summarize the current examination methods and possible model systems, discuss the recently identified factors that are required for crinophagy, and give an overview of the potential medical relevance of this process.

Hobbit regulates intracellular trafficking to drive insulin-dependent growth during Drosophila development.

All animals must coordinate growth rate and timing of maturation to reach the appropriate final size. Here, we describe hobbit, a novel and conserved gene identified in a forward genetic screen for Drosophila animals with small body size. hobbit is highly conserved throughout eukaryotes, but its function remains unknown. We demonstrate that hobbit mutant animals have systemic growth defects because they fail to secrete insulin. Other regulated secretion events also fail in hobbit mutant animals, including mucin-like 'glue' protein secretion from the larval salivary glands. hobbit mutant salivary glands produce glue-containing secretory granules that are reduced in size. Importantly, secretory granules in hobbit mutant cells lack essential membrane fusion machinery required for exocytosis, including Synaptotagmin 1 and the SNARE SNAP-24. These membrane fusion proteins instead accumulate inside enlarged late endosomes. Surprisingly, however, the Hobbit protein localizes to the endoplasmic reticulum. Our results suggest that Hobbit regulates a novel step in intracellular trafficking of membrane fusion proteins. Our studies also suggest that genetic control of body size, as a measure of insulin secretion, is a sensitive functional readout of the secretory machinery.

Molecular mechanisms of developmentally programmed crinophagy in Drosophila.

At the onset of metamorphosis, Drosophila salivary gland cells undergo a burst of glue granule secretion to attach the forming pupa to a solid surface. Here, we show that excess granules evading exocytosis are degraded via direct fusion with lysosomes, a secretory granule-specific autophagic process known as crinophagy. We find that the tethering complex HOPS (homotypic fusion and protein sorting); the small GTPases Rab2, Rab7, and its effector, PLEKHM1; and a SNAP receptor complex consisting of Syntaxin 13, Snap29, and Vamp7 are all required for the fusion of secretory granules with lysosomes. Proper glue degradation within lysosomes also requires the Uvrag-containing Vps34 lipid kinase complex and the v-ATPase proton pump, whereas Atg genes involved in macroautophagy are dispensable for crinophagy. Our work establishes the molecular mechanism of developmentally programmed crinophagy in Drosophila and paves the way for analyzing this process in metazoans.

Glue protein production can be triggered by steroid hormone signaling independent of the developmental program in Drosophila melanogaster.

Steroid hormones regulate life stage transitions, allowing animals to appropriately follow a developmental timeline. During insect development, the steroid hormone ecdysone is synthesized and released in a regulated manner by the prothoracic gland (PG) and then hydroxylated to the active molting hormone, 20-hydroxyecdysone (20E), in peripheral tissues. We manipulated ecdysteroid titers, through temporally controlled over-expression of the ecdysteroid-inactivating enzyme, CYP18A1, in the PG using the GeneSwitch-GAL4 system in the fruit fly Drosophila melanogaster. We monitored expression of a 20E-inducible glue protein gene, Salivary gland secretion 3 (Sgs3), using a Sgs3:GFP fusion transgene. In wild type larvae, Sgs3-GFP expression is activated at the midpoint of the third larval instar stage in response to the rising endogenous level of 20E. By first knocking down endogenous 20E levels during larval development and then feeding 20E to these larvae at various stages, we found that Sgs3-GFP expression could be triggered at an inappropriate developmental stage after a certain time lag. This stage-precocious activation of Sgs3 required expression of the Broad-complex, similar to normal Sgs3 developmental regulation, and a small level of nutritional input. We suggest that these studies provide evidence for a tissue-autonomic regulatory system for a metamorphic event independent from the primary 20E driven developmental progression.

Actin puts the squeeze on Drosophila glue secretion.

An actin filament coat promotes cargo expulsion from large exocytosing vesicles, but the mechanisms of coat formation and force generation have been poorly characterized. Elegant imaging studies of the Drosophila melanogaster salivary gland now reveal how actin and myosin are recruited, and show that myosin II forms a contractile 'cage' that facilitates exocytosis.

Orchestrated content release from Drosophila glue-protein vesicles by a contractile actomyosin network.

Releasing content from large vesicles measuring several micrometres in diameter poses exceptional challenges to the secretory system. An actomyosin network commonly coats these vesicles, and is thought to provide the necessary force mediating efficient cargo release. Here we describe the spatial and temporal dynamics of the formation of this actomyosin coat around large vesicles and the resulting vesicle collapse, in live Drosophila melanogaster salivary glands. We identify the Formin family protein Diaphanous (Dia) as the main actin nucleator involved in generating this structure, and uncover Rho as an integrator of actin assembly and contractile machinery activation comprising this actomyosin network. High-resolution imaging reveals a unique cage-like organization of myosin II on the actin coat. This myosin arrangement requires branched-actin polymerization, and is critical for exerting a non-isotropic force, mediating efficient vesicle contraction.

Type II phosphatidylinositol 4-kinase regulates trafficking of secretory granule proteins in Drosophila.

Type II phosphatidylinositol 4-kinase (PI4KII) produces the lipid phosphatidylinositol 4-phosphate (PI4P), a key regulator of membrane trafficking. Here, we generated genetic models of the sole Drosophila melanogaster PI4KII gene. A specific requirement for PI4KII emerged in larval salivary glands. In PI4KII mutants, mucin-containing glue granules failed to reach normal size, with glue protein aberrantly accumulating in enlarged Rab7-positive late endosomes. Presence of PI4KII at the Golgi and on dynamic tubular endosomes indicated two distinct foci for its function. First, consistent with the established role of PI4P in the Golgi, PI4KII is required for sorting of glue granule cargo and the granule-associated SNARE Snap24. Second, PI4KII also has an unforeseen function in late endosomes, where it is required for normal retromer dynamics and for formation of tubular endosomes that are likely to be involved in retrieving Snap24 and Lysosomal enzyme receptor protein (Lerp) from late endosomes to the trans-Golgi network. Our genetic analysis of PI4KII in flies thus reveals a novel role for PI4KII in regulating the fidelity of granule protein trafficking in secretory tissues.

Members of the salivary gland surface protein (SGS) family are major immunogenic components of mosquito saliva.

Mosquitoes transmit Plasmodium and certain arboviruses during blood feeding, when they are injected along with saliva. Mosquito saliva interferes with the host's hemostasis and inflammation response and influences the transmission success of some pathogens. One family of mosquito salivary gland proteins, named SGS, is composed of large bacterial-type proteins that in Aedes aegypti were implicated as receptors for Plasmodium on the basal salivary gland surface. Here, we characterize the biology of two SGSs in the malaria mosquito, Anopheles gambiae, and demonstrate their involvement in blood feeding. Western blots and RT-PCR showed that Sgs4 and Sgs5 are produced exclusively in female salivary glands, that expression increases with age and after blood feeding, and that protein levels fluctuate in a circadian manner. Immunohistochemistry showed that SGSs are present in the acinar cells of the distal lateral lobes and in the salivary ducts of the proximal lobes. SDS-PAGE, Western blots, bite blots, and immunization via mosquito bites showed that SGSs are highly immunogenic and form major components of mosquito saliva. Last, Western and bioinformatic analyses suggest that SGSs are secreted via a non-classical pathway that involves cleavage into a 300-kDa soluble fragment and a smaller membrane-bound fragment. Combined, these data strongly suggest that SGSs play an important role in blood feeding. Together with their role in malaria transmission, we propose that SGSs could be used as markers of human exposure to mosquito bites and in the development of disease control strategies.

Drosophila suppressor of sable protein [Su(s)] promotes degradation of aberrant and transposon-derived RNAs.

RNA-binding proteins act at various stages of gene expression to regulate and fine-tune patterns of mRNA accumulation. One protein in this class is Drosophila Su(s), a nuclear protein that has been previously shown to inhibit the accumulation of mutant transcripts by an unknown mechanism. Here, we have identified several additional RNAs that are downregulated by Su(s). These Su(s) targets include cryptic wild-type transcripts from the developmentally regulated Sgs4 and ng1 genes, noncoding RNAs derived from tandemly repeated alphabeta/alphagamma elements within an Hsp70 locus, and aberrant transcripts induced by Hsp70 promoter transgenes inserted at ectopic sites. We used the alphabeta RNAs to investigate the mechanism of Su(s) function and obtained evidence that these transcripts are degraded by the nuclear exosome and that Su(s) promotes this process. Furthermore, we showed that the RNA binding domains of Su(s) are important for this effect and mapped the sequences involved to a 267-nucleotide region of an alphabeta element. Taken together, these results suggest that Su(s) binds to certain nascent transcripts and stimulates their degradation by the nuclear exosome.

A novel ecdysone receptor mediates steroid-regulated developmental events during the mid-third instar of Drosophila.

The larval salivary gland of Drosophila melanogaster synthesizes and secretes glue glycoproteins that cement developing animals to a solid surface during metamorphosis. The steroid hormone 20-hydroxyecdysone (20E) is an essential signaling molecule that modulates most of the physiological functions of the larval gland. At the end of larval development, it is known that 20E--signaling through a nuclear receptor heterodimer consisting of EcR and USP--induces the early and late puffing cascade of the polytene chromosomes and causes the exocytosis of stored glue granules into the lumen of the gland. It has also been reported that an earlier pulse of hormone induces the temporally and spatially specific transcriptional activation of the glue genes; however, the receptor responsible for triggering this response has not been characterized. Here we show that the coordinated expression of the glue genes midway through the third instar is mediated by 20E acting to induce genes of the Broad Complex (BRC) through a receptor that is not an EcR/USP heterodimer. This result is novel because it demonstrates for the first time that at least some 20E-mediated, mid-larval, developmental responses are controlled by an uncharacterized receptor that does not contain an RXR-like component.

Genes and biological processes controlled by the Drosophila FOXA orthologue Fork head.

The larval salivary glands of Drosophila express the FOXA transcription factor Fork head (Fkh) before, but not after, puparium formation. Forced expression of Fkh in late prepupae prevents the programmed destruction of the tissue, which normally occurs in the early pupa. Using Affymetrix GeneChips, we analysed changes in gene expression brought about by Fkh when expressed shortly before the normal time of salivary gland death. Genes identified as responsive to Fkh include not only cell death genes, but also genes involved in autophagy, phospholipid metabolism and hormone-controlled signalling pathways. In addition, Fkh changed the expression of genes involved in glucose and fatty acid metabolism that are known to be target genes of the FOXAs in vertebrates. Premature loss of fkh induced by RNAi and gain of Fkh by ectopic expression at earlier times of development confirmed that genes identified in the microarray study are under normal developmental control by Fkh. These genes include Eip63F-1, which is expressed in both salivary glands and Malpighian tubules, suggesting that Fkh controls common aspects of the secretory function of the two organs. Eip63F-1 is one of many genes controlled by the steroid hormone 20-hydroxyecdysone that appear to be co-regulated by Fkh.

Ecdysone-regulated puff genes 2000.

The Ashburner model for the hormonal control of polytene chromosome puffing has provided a strong foundation for understanding the basic mechanisms of steroid-regulated gene expression (Cold Spring Harbor Symp. Quant. Biol. 38 (1974) 655). According to this model, the steroid hormone 20-hydroxyecdysone (referred here as ecdysone) directly induces the expression of a small set of early regulatory genes. These genes, in turn, induce a much larger set of late target genes that play a more direct role in controlling the biological responses to the hormone. The recent characterization of two early puff genes, E63-1 and E23, and three late puff genes, D-spinophilin, L63, and L82, provide further confirmation of the Ashburner model. In addition, these studies provide exciting new directions for our understanding of ecdysone signaling. Overexpression studies of E63-1 implicate this gene in directing calcium-dependent salivary gland glue secretion. In contrast, overexpression of E23 indicates that this ABC transporter family member may negatively regulate ecdysone signaling by actively transporting the hormone out of target cells. Finally, genetic studies of the L63 and L82 late genes reveal unexpected possible functions for ecdysone in controlling developmental timing and growth. This review surveys the recent characterization of these ecdysone-inducible genes and provides an overview of how they expand our understanding of ecdysone functions during development.

Downregulation of the tissue-specific transcription factor Fork head by Broad-Complex mediates a stage-specific hormone response.

Drosophila development is coordinated by pulses of the steroid hormone 20-hydroxyecdysone (20E). During metamorphosis, the 20E-inducible Broad-Complex (BR-C) gene plays a key role in the genetic hierarchies that transduce the hormone signal, being required for the destruction of larval tissues and numerous aspects of adult development. Most of the known BR-C target genes, including the salivary gland secretion protein (Sgs) genes, are terminal differentiation genes that are thought to be directly regulated by BR-C-encoded transcription factors. Here, we show that repression of Sgs expression is indirectly controlled by the BR-C through transcriptional down-regulation of fork head, a tissue-specific gene that plays a central role in salivary gland development and is required for Sgs expression. Our results demonstrate that integration of a tissue-specific regulatory gene into a 20E-controlled genetic hierarchy provides a mechanism for hormonal repression. Furthermore, they suggest that the BR-C is placed at a different position within the 20E-controlled hierarchies than previously assumed, and that at least part of its pleiotropic functions are mediated by tissue-specific regulators.

Spt5 and spt6 are associated with active transcription and have characteristics of general elongation factors in D. melanogaster.

The Spt4, Spt5, and Spt6 proteins are conserved throughout eukaryotes and are believed to play critical and related roles in transcription. They have a positive role in transcription elongation in Saccharomyces cerevisiae and in the activation of transcription by the HIV Tat protein in human cells. In contrast, a complex of Spt4 and Spt5 is required in vitro for the inhibition of RNA polymerase II (Pol II) elongation by the drug DRB, suggesting also a negative role in vivo. To learn more about the function of the Spt4/Spt5 complex and Spt6 in vivo, we have identified Drosophila homologs of Spt5 and Spt6 and characterized their localization on Drosophila polytene chromosomes. We find that Spt5 and Spt6 localize extensively with the phosphorylated, actively elongating form of Pol II, to transcriptionally active sites during salivary gland development and upon heat shock. Furthermore, Spt5 and Spt6 do not colocalize widely with the unphosphorylated, nonelongating form of Pol II. These results strongly suggest that Spt5 and Spt6 play closely related roles associated with active transcription in vivo.

The fork head product directly specifies the tissue-specific hormone responsiveness of the Drosophila Sgs-4 gene.

Here we describe the identification of four binding sites of secretion enhancer binding protein 2 (SEBP2) in the regulatory region of the Drosophila salivary gland secretion protein gene 4 (Sgs-4) and show that despite these sites' correspondence with previously described Broad-Complex protein binding sites, SEBP2 is a Broad-Complex-independent factor encoded by the region-specific homeotic gene fork head (fkh). Two of the Fork head/SEBP2 binding sites are located within an ecdysone response unit which controls the tissue- and stage-specific responses of Sgs-4 to the steroid hormone 20-hydroxyecdysone. We demonstrate that these binding sites are relevant to the transcriptional activation of Sgs-4 and show that Fork head also binds to the Sgs-4 ecdysone response unit in vivo. Aside from being involved in the control of decisions during embryonic development, fkh thus participates directly in the control of specialized functions of differentiated cells at later stages of development.

GEBF-I in Drosophila species and hybrids: the co-evolution of an enhancer and its cognate factor.

The activation of the Drosophila melanogaster salivary gland secretion protein gene Sgs-3 is marked by important changes in chromatin structure in the distal regulatory region at -600 bp from the Sgs-3 start site. A stage- and tissue-specific glue enhancer binding factor, GEBF-I, binds in vitro to sequences from this region. Previous studies have revealed considerable variation in the DNA sequences of comparable regions in the related Drosophila species, D. simulans, D. erecta and D. yakuba. We detected GEBF-I-like proteins in these species, which appear to evolve as rapidly as the corresponding DNA sequences, and studied in detail the binding characteristics of the GEBF-I proteins of the two most closely related species, D. melanogaster and D. simulans. In crosses between these species, certain strains produce hybrid larvae which, unexpectedly, synthesised a single intermediate form of the protein. This suggests that the factor is subject to species-specific post-transcriptional modifications. In these hybrid larvae, which carry one D. melanogaster and one D. simulans Sgs-3 gene, the hybrid GEBF-I protein appears equally effective in the induction of both target genes.

Drosophila Sgs3 TATA: effects of point mutations on expression in vivo and protein binding in vitro with staged nuclear extracts.

The Drosophila salivary gland secretion protein gene, Sgs3, has a consensus TATA sequence and gives rise to abundant stage and tissue-specific transcripts. Two TATA point mutations (TAAA and TAGA) reduce transcript levels approximately 50-fold when assayed in transgenic flies. This effect is reflected in vitro, in DNase I footprint and gel retardation assays where we observed TATA-probe-specific complexes that are not seen with TAAA, TAGA or non-specific probes. The binding patterns observed when using nuclear extracts from 0-2- and 0-20-h embryos (Sgs3 inactive) differ from those seen with extracts from third instar salivary glands (Sgs3 active). There are also differences in in vitro binding when using an hsp70 TATA fragment, previously shown to substitute in vivo for the Sgs3 TATA sequence, as probe. Together these observations suggest the possibility that more than one TATA box factor may be present in these extracts. We conclude that a wild-type TATA motif is crucial for the binding of a TATA box factor and all subsequent interactions with other factors bound to the proximal and distal regulatory sequences that are necessary for the normal expression of Sgs3.