RESUMO
Drosophila sperm development is characterized by extensive post-transcriptional regulation whereby thousands of transcripts are preserved for translation during later stages. A key step in translation initiation is the binding of eukaryotic initiation factor 4E (eIF4E) to the 5' mRNA cap. In addition to canonical eIF4E-1, Drosophila has multiple eIF4E paralogs, including four (eIF4E-3, -4, -5, and -7) that are highly expressed in the testis. Among these, only eIF4E-3 has been characterized genetically. Here, using CRISPR/Cas9 mutagenesis, we determined that eIF4E-5 is essential for male fertility. eIF4E-5 protein localizes to the distal ends of elongated spermatid cysts, and eIF4E-5 mutants exhibit defects during post-meiotic stages, including a mild defect in spermatid cyst polarization. eIF4E-5 mutants also have a fully penetrant defect in individualization, resulting in failure to produce mature sperm. Indeed, our data indicate that eIF4E-5 regulates non-apoptotic caspase activity during individualization by promoting local accumulation of the E3 ubiquitin ligase inhibitor Soti. Our results further extend the diversity of non-canonical eIF4Es that carry out distinct spatiotemporal roles during spermatogenesis.
Assuntos
Drosophila melanogaster , Sêmen , Animais , Masculino , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Sêmen/metabolismo , Drosophila/metabolismo , Espermatogênese/genética , Fator de Iniciação 4E em Eucariotos/genética , Fator de Iniciação 4E em Eucariotos/metabolismoRESUMO
Epithelial patterning in the developing Drosophila melanogaster eye requires the Neph1 homolog Roughest (Rst), an immunoglobulin family cell surface adhesion molecule expressed in interommatidial cells (IOCs). Here, using a novel temperature-sensitive (ts) allele, we show that the phosphoinositide phosphatase Sac1 is also required for IOC patterning. Sac1ts mutants have rough eyes and retinal patterning defects that resemble rst mutants. Sac1ts retinas exhibit elevated levels of phosphatidylinositol 4-phosphate (PI4P), consistent with the role of Sac1 as a PI4P phosphatase. Indeed, genetic rescue and interaction experiments reveal that restriction of PI4P levels by Sac1 is crucial for normal eye development. Rst is delivered to the cell surface in Sac1ts mutants. However, Sac1ts mutant IOCs exhibit severe defects in microtubule organization, associated with accumulation of Rst and the exocyst subunit Sec8 in enlarged intracellular vesicles upon cold fixation ex vivo Together, our data reveal a novel requirement for Sac1 in promoting microtubule stability and suggest that Rst trafficking occurs in a microtubule- and exocyst-dependent manner.
Assuntos
Moléculas de Adesão Celular Neuronais/genética , Forma Celular/fisiologia , Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Proteínas do Olho/genética , Microtúbulos/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatases de Fosfoinositídeos/genética , Animais , Diferenciação Celular/fisiologia , Proteínas de Drosophila/metabolismo , Olho/embriologia , Fosfatases de Fosfoinositídeos/metabolismo , Transporte Proteico/fisiologia , Temperatura , Proteínas de Transporte Vesicular/metabolismoRESUMO
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.
Assuntos
1-Fosfatidilinositol 4-Quinase/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Vesículas Secretórias/metabolismo , 1-Fosfatidilinositol 4-Quinase/genética , Animais , Animais Geneticamente Modificados , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Endossomos/metabolismo , Proteínas do Grude Salivar de Drosophila/metabolismo , Complexo de Golgi/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Modelos Biológicos , Transporte Proteico , Glândulas Salivares/crescimento & desenvolvimento , Glândulas Salivares/metabolismo , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7RESUMO
Cell proliferation is dependent on growth factors insulin and IGF1. We sought to identify interactors of IRS1, the most proximal mediator of insulin/IGF1 signaling, that regulate cell proliferation. Using proximity-dependent biotin identification (BioID), we detected 40 proteins displaying proximal interactions with IRS1, including DCAF7 and its interacting partners DYRK1A and DYRK1B. In HepG2 cells, DCAF7 knockdown attenuated cell proliferation by inducing cell cycle arrest at G2. DCAF7 expression was required for insulin-stimulated AKT phosphorylation, and its absence promoted nuclear localization of the transcription factor FOXO1. DCAF7 knockdown induced expression of FOXO1-target genes implicated in G2 cell cycle inhibition, correlating with G2 cell cycle arrest. In Drosophila melanogaster, wing-specific knockdown of DCAF7/wap caused smaller wing size and lower wing cell number; the latter recovered upon double knockdown of wap and dfoxo. We propose that DCAF7 regulates cell proliferation and cell cycle via IRS1-FOXO1 signaling, of relevance to whole organism growth.
RESUMO
[This corrects the article DOI: 10.1016/j.isci.2022.105188.].
RESUMO
Regulated secretion is a fundamental cellular process in which biologically active molecules stored in long-lasting secretory granules (SGs) are secreted in response to external stimuli. Many studies have described mechanisms responsible for biogenesis and secretion of SGs, but how SGs mature remains poorly understood. In a genetic screen, we discovered a large number of endolysosomal trafficking genes required for proper SG maturation, indicating that maturation of SGs might occur in a manner similar to lysosome-related organelles (LROs). CD63, a tetraspanin known to decorate LROs, also decorates SG membranes and facilitates SG maturation. Moreover, CD63-mediated SG maturation requires type II phosphatidylinositol 4 kinase (PI4KII)-dependent early endosomal sorting and accumulation of phosphatidylinositol 4-phosphate (PI4P) on SG membranes. In addition, the PI4P effector Past1 is needed for formation of stable PI4KII-containing endosomal tubules associated with this process. Our results reveal that maturation of post-Golgi-derived SGs requires trafficking via the endosomal system, similar to mechanisms employed by LROs.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Endossomos/metabolismo , Glândulas Salivares/metabolismo , Vesículas Secretórias/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Animais Geneticamente Modificados , Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Endossomos/genética , Antígenos de Histocompatibilidade Menor/genética , Antígenos de Histocompatibilidade Menor/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Transporte Proteico , Glândulas Salivares/embriologia , Vesículas Secretórias/genética , Tetraspanina 30/genética , Tetraspanina 30/metabolismo , Fatores de TempoRESUMO
The role of calcium (Ca(2+)) in cytokinesis is controversial, and the precise pathways that lead to its release during cleavage are not well understood. Ca(2+) is released from intracellular stores by binding of inositol trisphosphate (IP3) to the IP3 receptor (IP3R), yet no clear role in cytokinesis has been established for the precursor of IP3, phosphatidylinositol 4,5-bisphosphate (PIP2). Here, using transgenic flies expressing PLCdelta-PH-GFP, which specifically binds PIP2, we identify PIP2 in the plasma membrane and cleavage furrows of dividing Drosophila melanogaster spermatocytes, and we establish that this phospholipid is required for continued ingression but not for initiation of cytokinesis. In addition, by inhibiting phospholipase C, we show that PIP2 must be hydrolyzed to maintain cleavage furrow stability. Using an IP3R antagonist and a Ca(2+) chelator to examine the roles of IP3R and Ca(2+) in cytokinesis, we demonstrate that both of these factors are required for cleavage furrow stability, although Ca(2+) is dispensable for cleavage plane specification and initiation of furrowing. Strikingly, providing cells with Ca(2+) obviates the need to hydrolyze PIP2. Thus, PIP2, PIP2 hydrolysis, and Ca(2+) are required for the normal progression of cytokinesis in these cells.
Assuntos
Cálcio/metabolismo , Citocinese/fisiologia , Fosfatidilinositol 4,5-Difosfato/metabolismo , Transdução de Sinais/fisiologia , Espermatócitos/citologia , Animais , Animais Geneticamente Modificados , Drosophila melanogaster , Proteínas de Fluorescência Verde , Hidrólise , Isoenzimas/metabolismo , Masculino , Microscopia de Fluorescência , Fosfolipase C delta , Transgenes/genética , Fosfolipases Tipo C/metabolismoRESUMO
Gaucher disease is a lysosomal storage disorder caused by a defect in the degradation of glucosylceramide catalyzed by the lysosomal enzyme ß-glucocerebrosidase (GBA). GBA reaches lysosomes via association with its receptor, lysosomal integral membrane protein type 2 (LIMP-2). We found that distinct phosphatidylinositol 4-kinases (PI4Ks) play important roles at multiple steps in the trafficking pathway of the LIMP-2/GBA complex. Acute depletion of phosphatidylinositol 4-phosphate in the Golgi caused accumulation of LIMP-2 in this compartment, and PI4KIIIß was found to be responsible for controlling the exit of LIMP-2 from the Golgi. In contrast, depletion of PI4KIIα blocked trafficking at a post-Golgi compartment, leading to accumulation of LIMP-2 in enlarged endosomal vesicles. PI4KIIα depletion also caused secretion of missorted GBA into the medium, which was attenuated by limiting LIMP-2/GBA exit from the Golgi by PI4KIIIß inhibitors. These studies identified PI4KIIIß and PI4KIIα as important regulators of lysosomal delivery of GBA, revealing a new element of control to sphingolipid homeostasis by phosphoinositides.
Assuntos
1-Fosfatidilinositol 4-Quinase/metabolismo , Glucosilceramidase/metabolismo , Proteína 2 de Membrana Associada ao Lisossomo/metabolismo , Lisossomos/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , 1-Fosfatidilinositol 4-Quinase/genética , Animais , Células COS , Chlorocebus aethiops , Endossomos/metabolismo , Doença de Gaucher/enzimologia , Doença de Gaucher/metabolismo , Complexo de Golgi/enzimologia , Complexo de Golgi/metabolismo , Células HEK293 , Humanos , Antígenos de Histocompatibilidade Menor , Fosfotransferases (Aceptor do Grupo Álcool)/deficiência , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Transporte Proteico , Interferência de RNA , RNA Interferente PequenoRESUMO
During spermiogenesis, Drosophila melanogaster spermatids coordinate their elongation in interconnected cysts that become highly polarized, with nuclei localizing to one end and sperm tail growth occurring at the other. Remarkably little is known about the signals that drive spermatid polarity and elongation. Here we identify phosphoinositides as critical regulators of these processes. Reduction of plasma membrane phosphatidylinositol 4,5-bisphosphate (PIP(2)) by low-level expression of the PIP(2) phosphatase SigD or mutation of the PIP(2) biosynthetic enzyme Skittles (Sktl) results in dramatic defects in spermatid cysts, which become bipolar and fail to fully elongate. Defects in polarity are evident from the earliest stages of elongation, indicating that phosphoinositides are required for establishment of polarity. Sktl and PIP(2) localize to the growing end of the cysts together with the exocyst complex. Strikingly, the exocyst becomes completely delocalized when PIP(2) levels are reduced, and overexpression of Sktl restores exocyst localization and spermatid cyst polarity. Moreover, the exocyst is required for polarity, as partial loss of function of the exocyst subunit Sec8 results in bipolar cysts. Our data are consistent with a mechanism in which localized synthesis of PIP(2) recruits the exocyst to promote targeted membrane delivery and polarization of the elongating cysts.
Assuntos
Polaridade Celular , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Espermátides/metabolismo , Animais , Animais Geneticamente Modificados , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Células Cultivadas , Proteínas de Drosophila/genética , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Exocitose , Immunoblotting , Infertilidade Masculina/genética , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , Microscopia Eletrônica , Microscopia de Fluorescência , Mutação , Fosfatidilinositol 4,5-Difosfato , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Espermátides/citologia , Testículo/citologia , Testículo/metabolismo , Testículo/ultraestruturaRESUMO
Successful completion of cytokinesis relies on addition of new membrane, and requires the recycling endosome regulator Rab11, which localizes to the midzone. Despite the critical role of Rab11 in this process, little is known about the formation and composition of Rab11-containing organelles. Here, we identify the phosphatidylinositol (PI) 4-kinase III beta four wheel drive (Fwd) as a key regulator of Rab11 during cytokinesis in Drosophila melanogaster spermatocytes. We show Fwd is required for synthesis of PI 4-phosphate (PI4P) on Golgi membranes and for formation of PI4P-containing secretory organelles that localize to the midzone. Fwd binds and colocalizes with Rab11 on Golgi membranes, and is required for localization of Rab11 in dividing cells. A kinase-dead version of Fwd also binds Rab11 and partially restores cytokinesis to fwd mutant flies. Moreover, activated Rab11 partially suppresses loss of fwd. Our data suggest Fwd plays catalytic and noncatalytic roles in regulating Rab11 during cytokinesis.
Assuntos
1-Fosfatidilinositol 4-Quinase/metabolismo , Citocinese , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Espermatócitos/enzimologia , Proteínas rab de Ligação ao GTP/metabolismo , 1-Fosfatidilinositol 4-Quinase/biossíntese , 1-Fosfatidilinositol 4-Quinase/genética , Animais , Células COS , Chlorocebus aethiops , Drosophila melanogaster/genética , Fertilidade , Complexo de Golgi/enzimologia , Isoenzimas , Masculino , Microscopia de Fluorescência , Ligação Proteica , Proteínas Recombinantes de Fusão/metabolismo , Vesículas Secretórias/enzimologia , Transfecção , Técnicas do Sistema de Duplo-Híbrido , Proteínas rab de Ligação ao GTP/genéticaRESUMO
Axonemes are microtubule-based organelles of crucial importance in the structure and function of eukaryotic cilia and flagella. Despite great progress in understanding how axonemes are assembled, the signals that initiate axoneme outgrowth remain unknown. Here, we identified phosphatidylinositol phosphates (phosphoinositides) as key regulators of early stages of axoneme outgrowth in Drosophila melanogaster spermatogenesis. In a study of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] function in developing Drosophila male germ cells, we depleted PtdIns(4,5)P2 by expression of a potent phosphoinositide phosphatase. Phosphatase expression dramatically inhibited sperm tail formation and perturbed microtubule organization in a manner reversible by co-expression of a PtdIns 4-phosphate 5-kinase. Depletion of PtdIns(4,5)P2 caused increased levels of basal body gamma-tubulin and altered the distribution of proteins known to be required for axoneme assembly. Examination of PtdIns(4,5)P2-depleted spermatids by transmission electron microscopy revealed defects in basal body docking to the nuclear envelope, and in axoneme architecture and integrity of the developing flagellar axoneme and axial sheath. Our results provide the first evidence that phosphoinositides act at several steps during flagellar biogenesis, coordinately regulating microtubule and membrane organization. They further suggest that phosphoinositides play evolutionarily conserved roles in flagella and cilia, across phyla and in structurally diverse cell types.