No abstract

Toll-like receptor (TLR) recruitment to phagosomes in dendritic cells (DCs) and downstream TLR signaling are essential to initiate antimicrobial immune responses. However, the mechanisms underlying TLR localization to phagosomes are poorly characterized. We show herein that phosphatidylinositol-4-kinase IIα (PI4KIIα) plays a key role in initiating phagosomal TLR4 responses in murine DCs by generating a phosphatidylinositol-4-phosphate (PtdIns4P) platform conducive to the binding of the TLR sorting adaptor Toll-IL1 receptor (TIR) domain-containing adaptor protein (TIRAP). PI4KIIα is recruited to maturing lipopolysaccharide (LPS)-containing phagosomes in an adaptor protein-3 (AP-3)-dependent manner, and both PI4KIIα and PtdIns4P are detected on phagosomal membrane tubules. Knockdown of PI4KIIα—but not the related PI4KIIβ—impairs TIRAP and TLR4 localization to phagosomes, reduces proinflammatory cytokine secretion, abolishes phagosomal tubule formation, and impairs major histocompatibility complex II (MHC-II) presentation. Phagosomal TLR responses in PI4KIIα-deficient DCs are restored by reexpression of wild-type PI4KIIα, but not of variants lacking kinase activity or AP-3 binding. Our data indicate that PI4KIIα is an essential regulator of phagosomal TLR signaling in DCs by ensuring optimal TIRAP recruitment to phagosomes.

Lysosomes degrade macromolecules and recycle their nutrient content to support cell function and survival over a broad range of metabolic conditions. Yet, the machineries involved in lysosomal recycling of many essential nutrients remain to be discovered, with a notable example being choline, an essential metabolite liberated in large quantities within the lysosome via the degradation of choline-containing lipids. To identify critical lysosomal choline transport pathways, we engineered metabolic dependency on lysosome-derived choline in pancreatic cancer cells. We then exploited this dependency to perform an endolysosome-focused CRISPR-Cas9 negative selection screen for genes mediating lysosomal choline recycling. Our screen identified the orphan lysosomal transmembrane protein SPNS1, whose loss leads to neurodegeneration-like disease in animal models, as critical for cell survival under free choline limitation. We find that SPNS1 loss leads to massive accumulation of lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE) within the lysosome. Mechanistically, we revealed that SPNS1 is required for the efflux of LPC species from the lysosome to enable their re-esterification into choline-containing phospholipids in the cytosol. Using cell-based lipid uptake assays, we determine that SPNS1 functions as a proton gradient-dependent transporter of LPC. Collectively, our work defines a novel lysosomal phospholipid salvage pathway that is required for cell survival under conditions of choline limitation, and more broadly, provides a robust platform to deorphan lysosomal gene functions.

The role of the endoplasmic reticulum (ER) in phagocytosis has been the subject of debate for over a decade. Proteomic determinations and dynamic microscopy of live cells led to conflicting conclusions. Recent insights into the existence of a variety of membrane contact sites (MCS) may help reconcile the seemingly disparate views. Specifically, earlier results can be rationalized considering that the ER forms specialized MCS with nascent and maturing phagosomes, without undergoing fusion. The composition and function of documented ER‐to‐phagosome contact sites is described. In addition, we speculate about the possible existence of additional phagosomal contact sites, based on available knowledge of interactions between the ER and other endocytic compartments. The interaction between phagosomes and the ER has been the subject of debate. Earlier observations that led to the suggestion that the ER fuses with the phagosomal membrane can now be explained in the light of recent evidence that intimate contacts form between the two organelles.

Lysosomes degrade macromolecules and recycle their nutrient content to support cell function and survival. However, the machineries involved in lysosomal recycling of many nutrients remain to be discovered, with a notable example being choline, an essential metabolite liberated via lipid degradation. Here, we engineered metabolic dependency on lysosome-derived choline in pancreatic cancer cells to perform an endolysosome-focused CRISPR-Cas9 screen for genes mediating lysosomal choline recycling. We identified the orphan lysosomal transmembrane protein SPNS1 as critical for cell survival under choline limitation. SPNS1 loss leads to intralysosomal accumulation of lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE). Mechanistically, we reveal that SPNS1 is a proton gradient–dependent transporter of LPC species from the lysosome for their re-esterification into phosphatidylcholine in the cytosol. Last, we establish that LPC efflux by SPNS1 is required for cell survival under choline limitation. Collectively, our work defines a lysosomal phospholipid salvage pathway that is essential under nutrient limitation and, more broadly, provides a robust platform to deorphan lysosomal gene function.

No abstract

Phagocytosis is an essential mechanism for immunity and homeostasis, performed by a subset of cells known as phagocytes. Upon target engulfment, de novo formation of specialized compartments termed phagosomes takes place. Phagosomes then undergo a series of fusion and fission events as they interact with the endolysosomal system and other organelles, in a dynamic process known as phagosome maturation. Because phagocytes play a key role in tissue patrolling and immune surveillance, phagosome maturation is associated with signaling pathways that link phagocytosis to antigen presentation and the development of adaptive immune responses. In addition, and depending on the nature of the cargo, phagosome integrity may be compromised, triggering additional cellular mechanisms including inflammation and autophagy. Upon completion of maturation, phagosomes enter a recently described phase: phagosome resolution, where catabolites from degraded cargo are metabolized, phagosomes are resorbed, and vesicles of phagosomal origin are recycled. Finally, phagocytes return to homeostasis and become ready for a new round of phagocytosis. Altogether, phagosome maturation and resolution encompass a series of dynamic events and organelle crosstalk that can be measured by biochemical, imaging, photoluminescence, cytometric, and immune-based assays that will be described in this guide.

Early-stage drug discovery has been limited by initial hit identification and lead optimization and their associated costs. Ultra-large virtual screens (ULVSs), which involve the virtual evaluation of massive numbers of molecules to engage a macromolecular target, have the ability to significantly alleviate these problems, as was recently demonstrated in multiple studies. Despite their potential, ULVSs have so far only explored a tiny fraction of the chemical space and of available docking programs. Here, we present VirtualFlow 2.0, the next generation of the first open-source drug discovery platform dedicated to ultra-large virtual screenings. VirtualFlow 2.0 provides the REAL Space from Enamine containing 69 billion drug-like molecules in a "ready-to-dock" format, the largest library of its kind available to date. We provide an 18-dimensional matrix for intuitive exploration of the library through a web interface, where each dimension corresponds to a molecular property of the ligands. Additionally, VirtualFlow 2.0 supports multiple techniques that dramatically reduce computational costs, including a new method called Adaptive Target-Guided Virtual Screening (ATG-VS). By sampling a representative sparse version of the library, ATG-VS identifies the sections of the ultra-large chemical space that harbors the highest potential to engage the target site, leading to substantially reduced computational costs by up to a factor of 1000. In addition, VirtualFlow 2.0 supports the latest deep learning- and GPU-based docking methods, allowing further speed-ups by up to two orders of magnitude. VirtualFlow 2.0 supports 1500 unique docking methods providing target-specific and consensus docking options to increase accuracy and has the ability to screen new types of ligands (such as peptides) and target receptors (including RNA and DNA). Moreover, VirtualFlow 2.0 has many advanced new features, such as enhanced AI and cloud support. We demonstrate a perfectly linear scaling behavior up to 5.6 million CPUs in the AWS Cloud, a new global record for parallel cloud computing. Due to its open-source nature and versatility, we expect that VirtualFlow 2.0 will play a key role in the future of early-stage drug discovery.