Spatially revealed roles for lncRNAs in Drosophila spermatogenesis, Y chromosome function and evolution.

Unlike coding genes, the number of lncRNA genes in organism genomes is relatively proportional to organism complexity. From plants to humans, the tissues with highest numbers and levels of lncRNA gene expression are the male reproductive organs. To learn why, we initiated a genome-wide analysis of Drosophila lncRNA spatial expression patterns in these tissues. The numbers of genes and levels of expression observed greatly exceed those previously reported, due largely to a preponderance of non-polyadenylated transcripts. In stark contrast to coding genes, the highest numbers of lncRNAs expressed are in post-meiotic spermatids. Correlations between expression levels, localization and previously performed genetic analyses indicate high levels of function and requirement. More focused analyses indicate that lncRNAs play major roles in evolution by controlling transposable element activities, Y chromosome gene expression and sperm construction. A new type of lncRNA-based particle found in seminal fluid may also contribute to reproductive outcomes.

Emergent dynamics of adult stem cell lineages from single nucleus and single cell RNA-Seq of Drosophila testes.

Proper differentiation of sperm from germline stem cells, essential for production of the next generation, requires dramatic changes in gene expression that drive remodeling of almost all cellular components, from chromatin to organelles to cell shape itself. Here, we provide a single nucleus and single cell RNA-seq resource covering all of spermatogenesis in Drosophila starting from in-depth analysis of adult testis single nucleus RNA-seq (snRNA-seq) data from the Fly Cell Atlas (FCA) study. With over 44,000 nuclei and 6000 cells analyzed, the data provide identification of rare cell types, mapping of intermediate steps in differentiation, and the potential to identify new factors impacting fertility or controlling differentiation of germline and supporting somatic cells. We justify assignment of key germline and somatic cell types using combinations of known markers, in situ hybridization, and analysis of extant protein traps. Comparison of single cell and single nucleus datasets proved particularly revealing of dynamic developmental transitions in germline differentiation. To complement the web-based portals for data analysis hosted by the FCA, we provide datasets compatible with commonly used software such as Seurat and Monocle. The foundation provided here will enable communities studying spermatogenesis to interrogate the datasets to identify candidate genes to test for function in vivo.

Eukaryotic translation initiation factor eIF4E-5 is required for spermiogenesis in Drosophila melanogaster.

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.

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.

Erratum: DCAF7 regulates cell proliferation through IRS1-FOXO1 signaling.

[This corrects the article DOI: 10.1016/j.isci.2022.105188.].

DCAF7 regulates cell proliferation through IRS1-FOXO1 signaling.

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.

A lipid primes the final cut in dividing cells.

A newly described pathway activates separation of lens cells at the end of cytokinesis.

Sperm Cyst "Looping": A Developmental Novelty Enabling Extreme Male Ornament Evolution.

Postcopulatory sexual selection is credited as a principal force behind the rapid evolution of reproductive characters, often generating a pattern of correlated evolution between interacting, sex-specific traits. Because the female reproductive tract is the selective environment for sperm, one taxonomically widespread example of this pattern is the co-diversification of sperm length and female sperm-storage organ dimension. In Drosophila, having testes that are longer than the sperm they manufacture was believed to be a universal physiological constraint. Further, the energetic and time costs of developing long testes have been credited with underlying the steep evolutionary allometry of sperm length and constraining sperm length evolution in Drosophila. Here, we report on the discovery of a novel spermatogenic mechanism-sperm cyst looping-that enables males to produce relatively long sperm in short testis. This phenomenon (restricted to members of the saltans and willistoni species groups) begins early during spermatogenesis and is potentially attributable to heterochronic evolution, resulting in growth asynchrony between spermatid tails and the surrounding spermatid and somatic cyst cell membranes. By removing the allometric constraint on sperm length, this evolutionary innovation appears to have enabled males to evolve extremely long sperm for their body mass while evading delays in reproductive maturation time. On the other hand, sperm cyst looping was found to exact a cost by requiring greater total energetic investment in testes and a pronounced reduction in male lifespan. We speculate on the ecological selection pressures underlying the evolutionary origin and maintenance of this unique adaptation.

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.

Maturing secretory granules: Where secretory and endocytic pathways converge.

Secretory granules (SGs) are specialized organelles responsible for the storage and regulated release of various biologically active molecules from the endocrine and exocrine systems. Thus, proper SG biogenesis is critical to normal animal physiology. Biogenesis of SGs starts at the trans-Golgi network (TGN), where immature SGs (iSGs) bud off and undergo maturation before fusing with the plasma membrane (PM). How iSGs mature is unclear, but emerging studies have suggested an important role for the endocytic pathway. The requirement for endocytic machinery in SG maturation blurs the line between SGs and another class of secretory organelles called lysosome-related organelles (LROs). Therefore, it is important to re-evaluate the differences and similarities between SGs and LROs.

Endosomal Rab GTPases regulate secretory granule maturation in Drosophila larval salivary glands.

Secretory granules (SGs) are organelles responsible for regulated exocytosis of biologically active molecules in professional secretory cells. Maturation of SGs is a crucial process in which cargoes of SGs are processed and activated, allowing them to exert their function upon secretion. Nonetheless, the intracellular trafficking pathways required for SG maturation are not well defined. We recently performed an RNA interference (RNAi) screen in Drosophila larval salivary glands to identify trafficking components needed for SG maturation. From the screen, we identified several Rab GTPases (Rabs) that affect SG maturation. Expression of constitutively active (CA) and dominant-negative (DN) forms narrowed down the Rabs important for this process to Rab5, Rab9 and Rab11. However, none of these Rabs localizes to the limiting membrane of SGs. In contrast, examination of endogenously YFP-tagged Rabs (YRabs) in larval salivary glands revealed that YRab1 and YRab6 localize to the limiting membrane of immature SGs (iSGs) and SGs. These findings provide new insights into how Rab GTPases contribute to the process of SG maturation.

The NEMP family supports metazoan fertility and nuclear envelope stiffness.

Human genome-wide association studies have linked single-nucleotide polymorphisms (SNPs) in NEMP1 (nuclear envelope membrane protein 1) with early menopause; however, it is unclear whether NEMP1 has any role in fertility. We show that whole-animal loss of NEMP1 homologs in Drosophila, Caenorhabditis elegans, zebrafish, and mice leads to sterility or early loss of fertility. Loss of Nemp leads to nuclear shaping defects, most prominently in the germ line. Biochemical, biophysical, and genetic studies reveal that NEMP proteins support the mechanical stiffness of the germline nuclear envelope via formation of a NEMP-EMERIN complex. These data indicate that the germline nuclear envelope has specialized mechanical properties and that NEMP proteins play essential and conserved roles in fertility.

Cellular homeostasis in the Drosophila retina requires the lipid phosphatase Sac1.

The complex functions of cellular membranes, and thus overall cell physiology, depend on the distribution of crucial lipid species. Sac1 is an essential, conserved, ER-localized phosphatase whose substrate, phosphatidylinositol 4-phosphate (PI4P), coordinates secretory trafficking and plasma membrane function. PI4P from multiple pools is delivered to Sac1 by oxysterol-binding protein and related proteins in exchange for other lipids and sterols, which places Sac1 at the intersection of multiple lipid distribution pathways. However, much remains unknown about the roles of Sac1 in subcellular homeostasis and organismal development. Using a temperature-sensitive allele (Sac1ts), we show that Sac1 is required for structural integrity of the Drosophila retinal floor. The ßps-integrin Myospheroid, which is necessary for basal cell adhesion, is mislocalized in Sac1ts retinas. In addition, the adhesion proteins Roughest and Kirre, which coordinate apical retinal cell patterning at an earlier stage, accumulate within Sac1ts retinal cells due to impaired endo-lysosomal degradation. Moreover, Sac1 is required for ER homeostasis in Drosophila retinal cells. Together, our data illustrate the importance of Sac1 in regulating multiple aspects of cellular homeostasis during tissue development.

An early endosome-derived retrograde trafficking pathway promotes secretory granule maturation.

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.

The expanding spectrum of neurological disorders of phosphoinositide metabolism.

Phosphoinositides (PIPs) are a ubiquitous group of seven low-abundance phospholipids that play a crucial role in defining localized membrane properties and that regulate myriad cellular processes, including cytoskeletal remodeling, cell signaling cascades, ion channel activity and membrane traffic. PIP homeostasis is tightly regulated by numerous inositol kinases and phosphatases, which phosphorylate and dephosphorylate distinct PIP species. The importance of these phospholipids, and of the enzymes that regulate them, is increasingly being recognized, with the identification of human neurological disorders that are caused by mutations in PIP-modulating enzymes. Genetic disorders of PIP metabolism include forms of epilepsy, neurodegenerative disease, brain malformation syndromes, peripheral neuropathy and congenital myopathy. In this Review, we provide an overview of PIP function and regulation, delineate the disorders associated with mutations in genes that modulate or utilize PIPs, and discuss what is understood about gene function and disease pathogenesis as established through animal models of these diseases.