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1.
PLoS Genet ; 19(10): e1010979, 2023 Oct.
Article in English | MEDLINE | ID: mdl-37844085

ABSTRACT

Secretory cells in glands and the nervous system frequently package and store proteins destined for regulated secretion in dense-core granules (DCGs), which disperse when released from the cell surface. Despite the relevance of this dynamic process to diseases such as diabetes and human neurodegenerative disorders, our mechanistic understanding is relatively limited, because of the lack of good cell models to follow the nanoscale events involved. Here, we employ the prostate-like secondary cells (SCs) of the Drosophila male accessory gland to dissect the cell biology and genetics of DCG biogenesis. These cells contain unusually enlarged DCGs, which are assembled in compartments that also form secreted nanovesicles called exosomes. We demonstrate that known conserved regulators of DCG biogenesis, including the small G-protein Arf1 and the coatomer complex AP-1, play key roles in making SC DCGs. Using real-time imaging, we find that the aggregation events driving DCG biogenesis are accompanied by a change in the membrane-associated small Rab GTPases which are major regulators of membrane and protein trafficking in the secretory and endosomal systems. Indeed, a transition from trans-Golgi Rab6 to recycling endosomal protein Rab11, which requires conserved DCG regulators like AP-1, is essential for DCG and exosome biogenesis. Our data allow us to develop a model for DCG biogenesis that brings together several previously disparate observations concerning this process and highlights the importance of communication between the secretory and endosomal systems in controlling regulated secretion.


Subject(s)
Drosophila Proteins , Exosomes , Animals , Humans , Male , Dense Core Vesicles , Drosophila , Drosophila Proteins/genetics , Exosomes/genetics , Proteins , rab GTP-Binding Proteins/genetics , Transcription Factor AP-1
2.
PLoS Genet ; 19(6): e1010815, 2023 06.
Article in English | MEDLINE | ID: mdl-37363926

ABSTRACT

In prostate cancer, loss of the tumour suppressor gene, Retinoblastoma (Rb), and consequent activation of transcription factor E2F1 typically occurs at a late-stage of tumour progression. It appears to regulate a switch to an androgen-independent form of cancer, castration-resistant prostate cancer (CRPC), which frequently still requires androgen receptor (AR) signalling. We have previously shown that upon mating, binucleate secondary cells (SCs) of the Drosophila melanogaster male accessory gland (AG), which share some similarities with prostate epithelial cells, switch their growth regulation from a steroid-dependent to a steroid-independent form of Ecdysone Receptor (EcR) control. This physiological change induces genome endoreplication and allows SCs to rapidly replenish their secretory compartments, even when ecdysone levels are low because the male has not previously been exposed to females. Here, we test whether the Drosophila Rb homologue, Rbf, and E2F1 regulate this switch. Surprisingly, we find that excess Rbf activity reversibly suppresses binucleation in adult SCs. We also demonstrate that Rbf, E2F1 and the cell cycle regulators, Cyclin D (CycD) and Cyclin E (CycE), are key regulators of mating-dependent SC endoreplication, as well as SC growth in both virgin and mated males. Importantly, we show that the CycD/Rbf/E2F1 axis requires the EcR, but not ecdysone, to trigger CycE-dependent endoreplication and endoreplication-associated growth in SCs, mirroring changes seen in CRPC. Furthermore, Bone Morphogenetic Protein (BMP) signalling, mediated by the BMP ligand Decapentaplegic (Dpp), intersects with CycD/Rbf/E2F1 signalling to drive endoreplication in these fly cells. Overall, our work reveals a signalling switch, which permits rapid growth of SCs and increased secretion after mating, independently of previous exposure to females. The changes observed share mechanistic parallels with the pathological switch to hormone-independent AR signalling seen in CRPC, suggesting that the latter may reflect the dysregulation of a currently unidentified physiological process.


Subject(s)
Drosophila Proteins , Prostatic Neoplasms, Castration-Resistant , Humans , Animals , Female , Male , Drosophila/metabolism , Drosophila melanogaster/metabolism , Prostate/pathology , Prostatic Neoplasms, Castration-Resistant/metabolism , Prostatic Neoplasms, Castration-Resistant/pathology , Endoreduplication , Ecdysone/genetics , Ecdysone/metabolism , E2F1 Transcription Factor/genetics , Transcription Factors/genetics , Retinoblastoma Protein/genetics , Retinoblastoma Protein/metabolism , Drosophila Proteins/genetics , Drosophila Proteins/metabolism
3.
J Extracell Vesicles ; 12(3): e12311, 2023 03.
Article in English | MEDLINE | ID: mdl-36872252

ABSTRACT

Exosomes are secreted nanovesicles with potent signalling activity that are initially formed as intraluminal vesicles (ILVs) in late Rab7-positive multivesicular endosomes, and also in recycling Rab11a-positive endosomes, particularly under some forms of nutrient stress. The core proteins of the Endosomal Sorting Complex Required for Transport (ESCRT) participate in exosome biogenesis and ILV-mediated destruction of ubiquitinylated cargos. Accessory ESCRT-III components have reported roles in ESCRT-III-mediated vesicle scission, but their precise functions are poorly defined. They frequently only appear essential under stress. Comparative proteomics analysis of human small extracellular vesicles revealed that accessory ESCRT-III proteins, CHMP1A, CHMP1B, CHMP5 and IST1, are increased in Rab11a-enriched exosome preparations. We show that these proteins are required to form ILVs in Drosophila secondary cell recycling endosomes, but unlike core ESCRTs, they are not involved in degradation of ubiquitinylated proteins in late endosomes. Furthermore, CHMP5 knockdown in human HCT116 colorectal cancer cells selectively inhibits Rab11a-exosome production. Accessory ESCRT-III knockdown suppresses seminal fluid-mediated reproductive signalling by secondary cells and the growth-promoting activity of Rab11a-exosome-containing EVs from HCT116 cells. We conclude that accessory ESCRT-III components have a specific, ubiquitin-independent role in Rab11a-exosome generation, a mechanism that might be targeted to selectively block pro-tumorigenic activities of these vesicles in cancer.


Subject(s)
Exosomes , Extracellular Vesicles , Humans , Endosomes , Biological Transport , Endosomal Sorting Complexes Required for Transport
4.
Proc Natl Acad Sci U S A ; 118(5)2021 02 02.
Article in English | MEDLINE | ID: mdl-33495334

ABSTRACT

Seminal fluid plays an essential role in promoting male reproductive success and modulating female physiology and behavior. In the fruit fly, Drosophila melanogaster, Sex Peptide (SP) is the best-characterized protein mediator of these effects. It is secreted from the paired male accessory glands (AGs), which, like the mammalian prostate and seminal vesicles, generate most of the seminal fluid contents. After mating, SP binds to spermatozoa and is retained in the female sperm storage organs. It is gradually released by proteolytic cleavage and induces several long-term postmating responses, including increased ovulation, elevated feeding, and reduced receptivity to remating, primarily signaling through the SP receptor (SPR). Here, we demonstrate a previously unsuspected SPR-independent function for SP. We show that, in the AG lumen, SP and secreted proteins with membrane-binding anchors are carried on abundant, large neutral lipid-containing microcarriers, also found in other SP-expressing Drosophila species. These microcarriers are transferred to females during mating where they rapidly disassemble. Remarkably, SP is a key microcarrier assembly and disassembly factor. Its absence leads to major changes in the seminal proteome transferred to females upon mating. Males expressing nonfunctional SP mutant proteins that affect SP's binding to and release from sperm in females also do not produce normal microcarriers, suggesting that this male-specific defect contributes to the resulting widespread abnormalities in ejaculate function. Our data therefore reveal a role for SP in formation of seminal macromolecular assemblies, which may explain the presence of SP in Drosophila species that lack the signaling functions seen in Dmelanogaster.


Subject(s)
Drosophila Proteins/metabolism , Drosophila melanogaster/metabolism , Intercellular Signaling Peptides and Proteins/metabolism , Lipids/chemistry , Microspheres , Semen/chemistry , Animals , Drosophila Proteins/genetics , Female , Intercellular Signaling Peptides and Proteins/genetics , Male , Mutation/genetics , Proteome/metabolism , Sexual Behavior, Animal , Species Specificity
5.
J Cell Biol ; 206(5): 671-88, 2014 Sep 01.
Article in English | MEDLINE | ID: mdl-25154396

ABSTRACT

Male reproductive glands secrete signals into seminal fluid to facilitate reproductive success. In Drosophila melanogaster, these signals are generated by a variety of seminal peptides, many produced by the accessory glands (AGs). One epithelial cell type in the adult male AGs, the secondary cell (SC), grows selectively in response to bone morphogenetic protein (BMP) signaling. This signaling is involved in blocking the rapid remating of mated females, which contributes to the reproductive advantage of the first male to mate. In this paper, we show that SCs secrete exosomes, membrane-bound vesicles generated inside late endosomal multivesicular bodies (MVBs). After mating, exosomes fuse with sperm (as also seen in vitro for human prostate-derived exosomes and sperm) and interact with female reproductive tract epithelia. Exosome release was required to inhibit female remating behavior, suggesting that exosomes are downstream effectors of BMP signaling. Indeed, when BMP signaling was reduced in SCs, vesicles were still formed in MVBs but not secreted as exosomes. These results demonstrate a new function for the MVB-exosome pathway in the reproductive tract that appears to be conserved across evolution.


Subject(s)
Bone Morphogenetic Proteins/physiology , Drosophila Proteins/physiology , Exosomes/physiology , Genitalia, Male/metabolism , Animals , Drosophila melanogaster , Epithelial Cells/metabolism , Female , Genitalia, Female/cytology , Lysosomes/metabolism , Male , Membrane Fusion , Membrane Microdomains/metabolism , Multivesicular Bodies/metabolism , Protein Transport , Secretory Vesicles/metabolism , Sexual Behavior, Animal , Signal Transduction , Spermatozoa/metabolism , Tetraspanin 30/metabolism , Vacuoles/metabolism
6.
G3 (Bethesda) ; 4(4): 749-60, 2014 Apr 16.
Article in English | MEDLINE | ID: mdl-24531791

ABSTRACT

We have screened chromosome arm 3L for ethyl methanesulfonate-induced mutations that disrupt localization of fluorescently labeled gurken (grk) messenger (m)RNA, whose transport along microtubules establishes both major body axes of the developing Drosophila oocyte. Rapid identification of causative mutations by single-nucleotide polymorphism recombinational mapping and whole-genomic sequencing allowed us to define nine complementation groups affecting grk mRNA localization and other aspects of oogenesis, including alleles of elg1, scaf6, quemao, nudE, Tsc2/gigas, rasp, and Chd5/Wrb, and several null alleles of the armitage Piwi-pathway gene. Analysis of a newly induced kinesin light chain allele shows that kinesin motor activity is required for both efficient grk mRNA localization and oocyte centrosome integrity. We also show that initiation of the dorsoanterior localization of grk mRNA precedes centrosome localization, suggesting that microtubule self-organization contributes to breaking axial symmetry to generate a unique dorsoventral axis.


Subject(s)
Centrosome/metabolism , Drosophila Proteins/genetics , Drosophila/metabolism , RNA/metabolism , Transforming Growth Factor alpha/genetics , Animals , Chromosome Mapping , Drosophila/growth & development , Drosophila Proteins/antagonists & inhibitors , Drosophila Proteins/metabolism , Female , Genetic Testing , High-Throughput Nucleotide Sequencing , Kinesins/genetics , Kinesins/metabolism , Male , Oocytes/metabolism , Oogenesis , Polymorphism, Single Nucleotide , RNA/chemistry , RNA, Messenger/analysis , RNA, Messenger/metabolism , RNA, Small Interfering/metabolism , Sequence Analysis, DNA , Transforming Growth Factor alpha/metabolism
7.
EMBO Rep ; 9(1): 76-83, 2008 Jan.
Article in English | MEDLINE | ID: mdl-18034187

ABSTRACT

The Groucho (Gro)/transducin-like enhancer of split family of transcriptional corepressors are implicated in many signalling pathways that are important in development and disease, including those mediated by Notch, Wnt and Hedgehog. Here, we describe a genetic screen in Drosophila that yielded 50 new gro alleles, including the first protein-null allele, and has two mutations in the conserved Q oligomerization domain that have been proposed to have an essential role in corepressor activity. One of these latter mutations, encoding an amino-terminal protein truncation that lacks part of the Q domain, abolishes oligomerization in vitro and renders the protein unstable in vivo. Nevertheless, the mutation is not a null: maternal mutant embryos have intermediate segmentation phenotypes and relatively normal terminal patterning suggesting that the mutant protein retains partial corepressor activity. Our results show that homo-oligomerization of Gro is not obligatory for its action in vivo, and that Gro represses transcription through more than one molecular mechanism.


Subject(s)
Basic Helix-Loop-Helix Transcription Factors/chemistry , Basic Helix-Loop-Helix Transcription Factors/metabolism , Drosophila melanogaster/metabolism , Repressor Proteins/chemistry , Repressor Proteins/metabolism , Alleles , Amino Acid Sequence , Animals , Eye/cytology , Eye/metabolism , Gene Deletion , Molecular Sequence Data , Mutant Proteins/chemistry , Mutant Proteins/metabolism , Phenotype , Protein Structure, Quaternary , Protein Structure, Tertiary , Transcription, Genetic , beta-Galactosidase/metabolism
8.
Mol Cell ; 22(5): 645-55, 2006 Jun 09.
Article in English | MEDLINE | ID: mdl-16762837

ABSTRACT

The Groucho (Gro)/TLE/Grg family of corepressors operates in many signaling pathways (including Notch and Wnt). Gro/TLE proteins recognize a wide range of transcriptional repressors by binding to divergent short peptide sequences, including a C-terminal WRPW/Y motif (Hairy/Hes/Runx) and internal eh1 motifs (FxIxxIL; Engrailed/Goosecoid/Pax/Nkx). Here, we identify several missense mutations in Drosophila Gro, which demonstrate peptide binding to the central pore of the WD (WD40) beta propeller domain in vitro and in vivo. We define these interactions at the molecular level with crystal structures of the WD domain of human TLE1 bound to either WRPW or eh1 peptides. The two distinct peptide motifs adopt markedly different bound conformations but occupy overlapping sites across the central pore of the beta propeller. Our structural and functional analysis explains the rigid conservation of the WRPW motif, the sequence flexibility of eh1 motifs, and other aspects of repressor recognition by Gro in vivo.


Subject(s)
Basic Helix-Loop-Helix Transcription Factors/chemistry , Repressor Proteins/chemistry , Amino Acid Motifs/genetics , Animals , Basic Helix-Loop-Helix Transcription Factors/genetics , Basic Helix-Loop-Helix Transcription Factors/metabolism , Binding Sites , Co-Repressor Proteins , Drosophila/embryology , Drosophila/genetics , Drosophila/metabolism , Gene Expression Regulation, Developmental , Humans , Models, Molecular , Mutation, Missense , Protein Structure, Secondary , Protein Structure, Tertiary/genetics , Repressor Proteins/genetics , Repressor Proteins/metabolism , Signal Transduction , Structure-Activity Relationship
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