The yeast endocytic early/sorting compartment exists as an independent sub-compartment within the trans-Golgi network.

Although budding yeast has been extensively used as a model organism for studying organelle functions and intracellular vesicle trafficking, whether it possesses an independent endocytic early/sorting compartment that sorts endocytic cargos to the endo-lysosomal pathway or the recycling pathway has long been unclear. The structure and properties of the endocytic early/sorting compartment differ significantly between organisms; in plant cells, the trans-Golgi network (TGN) serves this role, whereas in mammalian cells a separate intracellular structure performs this function. The yeast syntaxin homolog Tlg2p, widely localizing to the TGN and endosomal compartments, is presumed to act as a Q-SNARE for endocytic vesicles, but which compartment is the direct target for endocytic vesicles remained unanswered. Here we demonstrate by high-speed and high-resolution 4D imaging of fluorescently labeled endocytic cargos that the Tlg2p-residing compartment within the TGN functions as the early/sorting compartment. After arriving here, endocytic cargos are recycled to the plasma membrane or transported to the yeast Rab5-residing endosomal compartment through the pathway requiring the clathrin adaptors GGAs. Interestingly, Gga2p predominantly localizes at the Tlg2p-residing compartment, and the deletion of GGAs has little effect on another TGN region where Sec7p is present but suppresses dynamics of the Tlg2-residing early/sorting compartment, indicating that the Tlg2p- and Sec7p-residing regions are discrete entities in the mutant. Thus, the Tlg2p-residing region seems to serve as an early/sorting compartment and function independently of the Sec7p-residing region within the TGN.

Eps15/Pan1p is a master regulator of the late stages of the endocytic pathway.

Endocytosis is a multistep process involving the sequential recruitment and action of numerous proteins. This process can be divided into two phases: an early phase, in which sites of endocytosis are formed, and a late phase in which clathrin-coated vesicles are formed and internalized into the cytosol, but how these phases link to each other remains unclear. In this study, we demonstrate that anchoring the yeast Eps15-like protein Pan1p to the peroxisome triggers most of the events occurring during the late phase at the peroxisome. At this ectopic location, Pan1p recruits most proteins that function in the late phases-including actin nucleation promoting factors-and then initiates actin polymerization. Pan1p also recruited Prk1 kinase and actin depolymerizing factors, thereby triggering disassembly immediately after actin assembly and inducing dissociation of endocytic proteins from the peroxisome. These observations suggest that Pan1p is a key regulator for initiating, processing, and completing the late phase of endocytosis.

Cell division in tissues enables macrophage infiltration.

Cells migrate through crowded microenvironments within tissues during normal development, immune response, and cancer metastasis. Although migration through pores and tracks in the extracellular matrix (ECM) has been well studied, little is known about cellular traversal into confining cell-dense tissues. We find that embryonic tissue invasion by Drosophila macrophages requires division of an epithelial ectodermal cell at the site of entry. Dividing ectodermal cells disassemble ECM attachment formed by integrin-mediated focal adhesions next to mesodermal cells, allowing macrophages to move their nuclei ahead and invade between two immediately adjacent tissues. Invasion efficiency depends on division frequency, but reduction of adhesion strength allows macrophage entry independently of division. This work demonstrates that tissue dynamics can regulate cellular infiltration.

A translation control module coordinates germline stem cell differentiation with ribosome biogenesis during Drosophila oogenesis.

Ribosomal defects perturb stem cell differentiation, and this is the cause of ribosomopathies. How ribosome levels control stem cell differentiation is not fully known. Here, we discover that three DExD/H-box proteins govern ribosome biogenesis (RiBi) and Drosophila oogenesis. Loss of these DExD/H-box proteins, which we name Aramis, Athos, and Porthos, aberrantly stabilizes p53, arrests the cell cycle, and stalls germline stem cell (GSC) differentiation. Aramis controls cell-cycle progression by regulating translation of mRNAs that contain a terminal oligo pyrimidine (TOP) motif in their 5' UTRs. We find that TOP motifs confer sensitivity to ribosome levels that are mediated by La-related protein (Larp). One such TOP-containing mRNA codes for novel nucleolar protein 1 (Non1), a conserved p53 destabilizing protein. Upon a sufficient ribosome concentration, Non1 is expressed, and it promotes GSC cell-cycle progression via p53 degradation. Thus, a previously unappreciated TOP motif in Drosophila responds to reduced RiBi to co-regulate the translation of ribosomal proteins and a p53 repressor, coupling RiBi to GSC differentiation.

Macrophage mitochondrial bioenergetics and tissue invasion are boosted by an Atossa-Porthos axis in Drosophila.

Cellular metabolism must adapt to changing demands to enable homeostasis. During immune responses or cancer metastasis, cells leading migration into challenging environments require an energy boost, but what controls this capacity is unclear. Here, we study a previously uncharacterized nuclear protein, Atossa (encoded by CG9005), which supports macrophage invasion into the germband of Drosophila by controlling cellular metabolism. First, nuclear Atossa increases mRNA levels of Porthos, a DEAD-box protein, and of two metabolic enzymes, lysine-α-ketoglutarate reductase (LKR/SDH) and NADPH glyoxylate reductase (GR/HPR), thus enhancing mitochondrial bioenergetics. Then Porthos supports ribosome assembly and thereby raises the translational efficiency of a subset of mRNAs, including those affecting mitochondrial functions, the electron transport chain, and metabolism. Mitochondrial respiration measurements, metabolomics, and live imaging indicate that Atossa and Porthos power up OxPhos and energy production to promote the forging of a path into tissues by leading macrophages. Since many crucial physiological responses require increases in mitochondrial energy output, this previously undescribed genetic program may modulate a wide range of cellular behaviors.

The Solute Carrier MFSD1 Decreases the Activation Status of ß1 Integrin and Thus Tumor Metastasis.

Solute carriers are increasingly recognized as participating in a plethora of pathologies, including cancer. We describe here the involvement of the orphan solute carrier Major Facilitator Superfamily Domain-containing protein 1 (MFSD1) in the regulation of tumor cell migration. Loss of MFSD1 enabled higher levels of metastasis in experimental and spontaneous metastasis mouse models. We identified an increased migratory potential in MFSD1-/- tumor cells which was mediated by increased focal adhesion turnover, reduced stability of mature inactive ß1 integrin, and the resulting increased integrin activation index. We show that MFSD1 promoted recycling to the cell surface of endocytosed inactive ß1 integrin and thereby protected ß1 integrin from proteolytic degradation; this led to dampening of the integrin activation index. Furthermore, downregulation of MFSD1 expression was observed during the early steps of tumorigenesis, and higher MFSD1 expression levels correlate with a better cancer patient prognosis. In sum, we describe a requirement for endolysosomal MFSD1 in efficient ß1 integrin recycling to suppress tumor cell dissemination.

Optogenetic delivery of trophic signals in a genetic model of Parkinson's disease.

Optogenetics has been harnessed to shed new mechanistic light on current and future therapeutic strategies. This has been to date achieved by the regulation of ion flow and electrical signals in neuronal cells and neural circuits that are known to be affected by disease. In contrast, the optogenetic delivery of trophic biochemical signals, which support cell survival and are implicated in degenerative disorders, has never been demonstrated in an animal model of disease. Here, we reengineered the human and Drosophila melanogaster REarranged during Transfection (hRET and dRET) receptors to be activated by light, creating one-component optogenetic tools termed Opto-hRET and Opto-dRET. Upon blue light stimulation, these receptors robustly induced the MAPK/ERK proliferative signaling pathway in cultured cells. In PINK1B9 flies that exhibit loss of PTEN-induced putative kinase 1 (PINK1), a kinase associated with familial Parkinson's disease (PD), light activation of Opto-dRET suppressed mitochondrial defects, tissue degeneration and behavioral deficits. In human cells with PINK1 loss-of-function, mitochondrial fragmentation was rescued using Opto-dRET via the PI3K/NF-кB pathway. Our results demonstrate that a light-activated receptor can ameliorate disease hallmarks in a genetic model of PD. The optogenetic delivery of trophic signals is cell type-specific and reversible and thus has the potential to inspire novel strategies towards a spatio-temporal regulation of tissue repair.

Muscle function and homeostasis require cytokine inhibition of AKT activity in Drosophila.

Unpaired ligands are secreted signals that act via a GP130-like receptor, domeless, to activate JAK/STAT signalling in Drosophila. Like many mammalian cytokines, unpaireds can be activated by infection and other stresses and can promote insulin resistance in target tissues. However, the importance of this effect in non-inflammatory physiology is unknown. Here, we identify a requirement for unpaired-JAK signalling as a metabolic regulator in healthy adult Drosophila muscle. Adult muscles show basal JAK-STAT signalling activity in the absence of any immune challenge. Plasmatocytes (Drosophila macrophages) are an important source of this tonic signal. Loss of the dome receptor on adult muscles significantly reduces lifespan and causes local and systemic metabolic pathology. These pathologies result from hyperactivation of AKT and consequent deregulation of metabolism. Thus, we identify a cytokine signal that must be received in muscle to control AKT activity and metabolic homeostasis.

A conserved major facilitator superfamily member orchestrates a subset of O-glycosylation to aid macrophage tissue invasion.

Aberrant display of the truncated core1 O-glycan T-antigen is a common feature of human cancer cells that correlates with metastasis. Here we show that T-antigen in Drosophila melanogaster macrophages is involved in their developmentally programmed tissue invasion. Higher macrophage T-antigen levels require an atypical major facilitator superfamily (MFS) member that we named Minerva which enables macrophage dissemination and invasion. We characterize for the first time the T and Tn glycoform O-glycoproteome of the Drosophila melanogaster embryo, and determine that Minerva increases the presence of T-antigen on proteins in pathways previously linked to cancer, most strongly on the sulfhydryl oxidase Qsox1 which we show is required for macrophage tissue entry. Minerva's vertebrate ortholog, MFSD1, rescues the minerva mutant's migration and T-antigen glycosylation defects. We thus identify a key conserved regulator that orchestrates O-glycosylation on a protein subset to activate a program governing migration steps important for both development and cancer metastasis.

The Repo Homeodomain Transcription Factor Suppresses Hematopoiesis in Drosophila and Preserves the Glial Fate.

Despite their different origins, Drosophila glia and hemocytes are related cell populations that provide an immune function. Drosophila hemocytes patrol the body cavity and act as macrophages outside the nervous system, whereas glia originate from the neuroepithelium and provide the scavenger population of the nervous system. Drosophila glia are hence the functional orthologs of vertebrate microglia, even though the latter are cells of immune origin that subsequently move into the brain during development. Interestingly, the Drosophila immune cells within (glia) and outside (hemocytes) the nervous system require the same transcription factor glial cells deficient/glial cells missing (Glide/Gcm) for their development. This raises the issue of how do glia specifically differentiate in the nervous system, and hemocytes in the procephalic mesoderm. The Repo homeodomain transcription factor and panglial direct target of Glide/Gcm is known to ensure glial terminal differentiation. Here we show that Repo also takes center stage in the process that discriminates between glia and hemocytes. First, Repo expression is repressed in the hemocyte anlagen by mesoderm-specific factors. Second, Repo ectopic activation in the procephalic mesoderm is sufficient to repress the expression of hemocyte-specific genes. Third, the lack of Repo triggers the expression of hemocyte markers in glia. Thus, a complex network of tissue-specific cues biases the potential of Glide/Gcm. These data allow us to revise the concept of fate determinants and help us to understand the bases of cell specification. Both sexes were analyzed.SIGNIFICANCE STATEMENT Distinct cell types often require the same pioneer transcription factor, raising the issue of how one factor triggers different fates. In Drosophila, glia and hemocytes provide a scavenger activity within and outside the nervous system, respectively. While they both require the glial cells deficient/glial cells missing (Glide/Gcm) transcription factor, glia originate from the ectoderm, and hemocytes from the mesoderm. Here we show that tissue-specific factors inhibit the gliogenic potential of Glide/Gcm in the mesoderm by repressing the expression of the homeodomain protein Repo, a major glial-specific target of Glide/Gcm. Repo expression in turn inhibits the expression of hemocyte-specific genes in the nervous system. These cell-specific networks secure the establishment of the glial fate only in the nervous system and allow cell diversification.

Drosophila TNF Modulates Tissue Tension in the Embryo to Facilitate Macrophage Invasive Migration.

Migrating cells penetrate tissue barriers during development, inflammatory responses, and tumor metastasis. We study if migration in vivo in such three-dimensionally confined environments requires changes in the mechanical properties of the surrounding cells using embryonic Drosophila melanogaster hemocytes, also called macrophages, as a model. We find that macrophage invasion into the germband through transient separation of the apposing ectoderm and mesoderm requires cell deformations and reductions in apical tension in the ectoderm. Interestingly, the genetic pathway governing these mechanical shifts acts downstream of the only known tumor necrosis factor superfamily member in Drosophila, Eiger, and its receptor, Grindelwald. Eiger-Grindelwald signaling reduces levels of active Myosin in the germband ectodermal cortex through the localization of a Crumbs complex component, Patj (Pals-1-associated tight junction protein). We therefore elucidate a distinct molecular pathway that controls tissue tension and demonstrate the importance of such regulation for invasive migration in vivo.

Tools Allowing Independent Visualization and Genetic Manipulation of Drosophila melanogaster Macrophages and Surrounding Tissues.

Drosophila melanogaster plasmatocytes, the phagocytic cells among hemocytes, are essential for immune responses, but also play key roles from early development to death through their interactions with other cell types. They regulate homeostasis and signaling during development, stem cell proliferation, metabolism, cancer, wound responses, and aging, displaying intriguing molecular and functional conservation with vertebrate macrophages. Given the relative ease of genetics in Drosophila compared to vertebrates, tools permitting visualization and genetic manipulation of plasmatocytes and surrounding tissues independently at all stages would greatly aid a fuller understanding of these processes, but are lacking. Here, we describe a comprehensive set of transgenic lines that allow this. These include extremely brightly fluorescing mCherry-based lines that allow GAL4-independent visualization of plasmatocyte nuclei, the cytoplasm, or the actin cytoskeleton from embryonic stage 8 through adulthood in both live and fixed samples even as heterozygotes, greatly facilitating screening. These lines allow live visualization and tracking of embryonic plasmatocytes, as well as larval plasmatocytes residing at the body wall or flowing with the surrounding hemolymph. With confocal imaging, interactions of plasmatocytes and inner tissues can be seen in live or fixed embryos, larvae, and adults. They permit efficient GAL4-independent Fluorescence-Activated Cell Sorting (FACS) analysis/sorting of plasmatocytes throughout life. To facilitate genetic studies of reciprocal signaling, we have also made a plasmatocyte-expressing QF2 line that, in combination with extant GAL4 drivers, allows independent genetic manipulation of both plasmatocytes and surrounding tissues, and GAL80 lines that block GAL4 drivers from affecting plasmatocytes, all of which function from the early embryo to the adult.

A Moving Source of Matrix Components Is Essential for De Novo Basement Membrane Formation.

The basement membrane (BM) is a thin layer of extracellular matrix (ECM) beneath nearly all epithelial cell types that is critical for cellular and tissue function. It is composed of numerous components conserved among all bilaterians [1]; however, it is unknown how all of these components are generated and subsequently constructed to form a fully mature BM in the living animal. Although BM formation is thought to simply involve a process of self-assembly [2], this concept suffers from a number of logistical issues when considering its construction in vivo. First, incorporation of BM components appears to be hierarchical [3-5], yet it is unclear whether their production during embryogenesis must also be regulated in a temporal fashion. Second, many BM proteins are produced not only by the cells residing on the BM but also by surrounding cell types [6-9], and it is unclear how large, possibly insoluble protein complexes [10] are delivered into the matrix. Here we exploit our ability to live image and genetically dissect de novo BM formation during Drosophila development. This reveals that there is a temporal hierarchy of BM protein production that is essential for proper component incorporation. Furthermore, we show that BM components require secretion by migrating macrophages (hemocytes) during their developmental dispersal, which is critical for embryogenesis. Indeed, hemocyte migration is essential to deliver a subset of ECM components evenly throughout the embryo. This reveals that de novo BM construction requires a combination of both production and distribution logistics allowing for the timely delivery of core components.

Drosophila immune cell migration and adhesion during embryonic development and larval immune responses.

The majority of immune cells in Drosophila melanogaster are plasmatocytes; they carry out similar functions to vertebrate macrophages, influencing development as well as protecting against infection and cancer. Plasmatocytes, sometimes referred to with the broader term of hemocytes, migrate widely during embryonic development and cycle in the larvae between sessile and circulating positions. Here we discuss the similarities of plasmatocyte developmental migration and its functions to that of vertebrate macrophages, considering the recent controversy regarding the functions of Drosophila PDGF/VEGF related ligands. We also examine recent findings on the significance of adhesion for plasmatocyte migration in the embryo, as well as proliferation, trans-differentiation, and tumor responses in the larva. We spotlight parallels throughout to vertebrate immune responses.

In vivo migration: a germ cell perspective.

The basic concepts of the molecular machinery that mediates cell migration have been gleaned from cell culture systems. However, the three-dimensional environment within an organism presents migrating cells with a much greater challenge. They must move between and among other cells while interpreting multiple attractive and repulsive cues to choose their proper path. They must coordinate their cell adhesion with their surroundings and know when to start and stop moving. New insights into the control of these remaining mysteries have emerged from genetic dissection and live imaging of germ cell migration in Drosophila, zebrafish, and mouse embryos. In this review, we first describe germ cell migration in cellular and mechanistic detail in these different model systems. We then compare these systems to highlight the emerging principles. Finally, we contrast the migration of germ cells with that of immune and cancer cells to outline the conserved and different mechanisms.

Spontaneous receptor-independent heterotrimeric G-protein signalling in an RGS mutant.

Tripartite G-protein-coupled receptors (GPCRs) represent one of the largest groups of signal transducers, transmitting signals from hormones, neuropeptides, odorants, food and light. Ligand-bound receptors catalyse GDP/GTP exchange on the G-protein alpha-subunit (Galpha), leading to alpha-GTP separation from the betagamma subunits and pathway activation. Activating mutations in the receptors or G proteins underlie many human diseases, including some cancers, dwarfism and premature puberty. Regulators of G-protein signalling (RGS proteins) are known to modulate the level and duration of ligand-induced signalling by accelerating the intrinsic GTPase activity of the Galpha subunit, and thus reformation of the inactive GDP-bound Galpha. Here we find that even in the absence of receptor, mutation of the RGS family member Sst2 (refs 6-9) permits spontaneous activation of the G-protein-coupled mating pathway in Saccharomyces cerevisiae at levels normally seen only in the presence of ligand. Our work demonstrates the occurrence of spontaneous tripartite G-protein signalling in vivo and identifies a requirement for RGS proteins in preventing such receptor-independent activation.

amontillado, the Drosophila homolog of the prohormone processing protease PC2, is required during embryogenesis and early larval development.

Biosynthesis of most peptide hormones and neuropeptides requires proteolytic excision of the active peptide from inactive proprotein precursors, an activity carried out by subtilisin-like proprotein convertases (SPCs) in constitutive or regulated secretory pathways. The Drosophila amontillado (amon) gene encodes a homolog of the mammalian PC2 protein, an SPC that functions in the regulated secretory pathway in neuroendocrine tissues. We have identified amon mutants by isolating ethylmethanesulfonate (EMS)-induced lethal and visible mutations that define two complementation groups in the amon interval at 97D1 of the third chromosome. DNA sequencing identified the amon complementation group and the DNA sequence change for each of the nine amon alleles isolated. amon mutants display partial embryonic lethality, are defective in larval growth, and arrest during the first to second instar larval molt. Mutant larvae can be rescued by heat-shock-induced expression of the amon protein. Rescued larvae arrest at the subsequent larval molt, suggesting that amon is also required for the second to third instar larval molt. Our data indicate that the amon proprotein convertase is required during embryogenesis and larval development in Drosophila and support the hypothesis that AMON acts to proteolytically process peptide hormones that regulate hatching, larval growth, and larval ecdysis.