Development of Genetically Encoded Fluorescent KSR1-Based Probes to Track Ceramides during Phagocytosis.

Ceramides regulate phagocytosis; however, their exact function remains poorly understood. Here, we sought (1) to develop genetically encoded fluorescent tools for imaging ceramides, and (2) to use them to examine ceramide dynamics during phagocytosis. Fourteen enhanced green fluorescent protein (EGFP) fusion constructs based on four known ceramide-binding domains were generated and screened. While most constructs localized to the nucleus or cytosol, three based on the CA3 ceramide-binding domain of kinase suppressor of ras 1 (KSR1) localized to the plasma membrane or autolysosomes. C-terminally tagged CA3 with a vector-based (C-KSR) or glycine-serine linker (C-KSR-GS) responded sensitively and similarly to ceramide depletion and accumulation using a panel of ceramide modifying drugs, whereas N-terminally tagged CA3 (N-KSR) responded differently to a subset of treatments. Lipidomic and liposome microarray analysis suggested that, instead, N-KSR may preferentially bind glucosyl-ceramide. Additionally, the three probes showed distinct dynamics during phagocytosis. Despite partial autolysosomal degradation, C-KSR and C-KSR-GS accumulated at the plasma membrane during phagocytosis, whereas N-KSR did not. Moreover, the weak recruitment of C-KSR-GS to the endoplasmic reticulum and phagosomes was enhanced through overexpression of the endoplasmic reticulum proteins stromal interaction molecule 1 (STIM1) and Sec22b, and was more salient in dendritic cells. The data suggest these novel probes can be used to analyze sphingolipid dynamics and function in living cells.

Sec22b regulates phagosome maturation by promoting ORP8-mediated lipid exchange at endoplasmic reticulum-phagosome contact sites.

Phagosome maturation is critical for immune defense, defining whether ingested material is destroyed or converted into antigens. Sec22b regulates phagosome maturation, yet how has remained unclear. Here we show Sec22b tethers endoplasmic reticulum-phagosome membrane contact sites (MCS) independently of the known tether STIM1. Sec22b knockdown increases calcium signaling, phagolysosome fusion and antigen degradation and alters phagosomal phospholipids PI(3)P, PS and PI(4)P. Levels of PI(4)P, a lysosome docking lipid, are rescued by Sec22b re-expression and by expression of the artificial tether MAPPER but not the MCS-disrupting mutant Sec22b-P33. Moreover, Sec22b co-precipitates with the PS/PI(4)P exchange protein ORP8. Wild-type, but not mutant ORP8 rescues phagosomal PI(4)P and reduces antigen degradation. Sec22b, MAPPER and ORP8 but not P33 or mutant-ORP8 restores phagolysosome fusion in knockdown cells. These findings clarify an alternative mechanism through which Sec22b controls phagosome maturation and beg a reassessment of the relative contribution of Sec22b-mediated fusion versus tethering to phagosome biology.

The lipid transfer proteins Nir2 and Nir3 sustain phosphoinositide signaling and actin dynamics during phagocytosis.

Changes in membrane phosphoinositides and local Ca2+ elevations at sites of particle capture coordinate the dynamic remodeling of the actin cytoskeleton during phagocytosis. Here, we show that the phosphatidylinositol (PI) transfer proteins PITPNM1 (Nir2) and PITPNM2 (Nir3) maintain phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] homeostasis at phagocytic cups, thereby promoting actin contractility and the sealing of phagosomes. Nir3 and to a lesser extent Nir2 accumulated on endoplasmic reticulum (ER) cisternae juxtaposed to phagocytic cups when expressed in phagocytic COS-7 cells. CRISPR-Cas9 editing of Nir2 and Nir3 genes decreased plasma membrane PI(4,5)P2 levels, store-operated Ca2+ entry (SOCE) and receptor-mediated phagocytosis, stalling particle capture at the cup stage. Re-expression of either Nir2 or Nir3 restored phagocytosis, but not SOCE, proportionally to the PM PI(4,5)P2 levels. Phagosomes forming in Nir2 and Nir3 (Nir2/3) double-knockout cells had decreased overall PI(4,5)P2 levels but normal periphagosomal Ca2+ signals. Nir2/3 depletion reduced the density of contractile actin rings at sites of particle capture, causing repetitive low-intensity contractile events indicative of abortive phagosome closure. We conclude that Nir proteins maintain phosphoinositide homeostasis at phagocytic cups, thereby sustaining the signals that initiate the remodeling of the actin cytoskeleton during phagocytosis.

Control of lysosomal-mediated cell death by the pH-dependent calcium channel RECS1.

Programmed cell death is regulated by the balance between activating and inhibitory signals. Here, we have identified RECS1 (responsive to centrifugal force and shear stress 1) [also known as TMBIM1 (transmembrane BAX inhibitor motif containing 1)] as a proapoptotic member of the TMBIM family. In contrast to other proteins of the TMBIM family, RECS1 expression induces cell death through the canonical mitochondrial apoptosis pathway. Unbiased screening indicated that RECS1 sensitizes cells to lysosomal perturbations. RECS1 localizes to lysosomes, where it regulates their acidification and calcium content, triggering lysosomal membrane permeabilization. Structural modeling and electrophysiological studies indicated that RECS1 is a pH-regulated calcium channel, an activity that is essential to trigger cell death. RECS1 also sensitizes whole animals to stress in vivo in Drosophila melanogaster and zebrafish models. Our results unveil an unanticipated function for RECS1 as a proapoptotic component of the TMBIM family that ignites cell death programs at lysosomes.

Simultaneous determination of intraluminal lysosomal calcium and pH by dextran-conjugated fluorescent dyes.

The lysosome is the main catabolic organelle in the cell, also serving as a signaling platform. Lysosomes maintain a low intraluminal pH where dozens of hydrolytic enzymes degrade a wide variety of macromolecules. Besides degradation of polymers, the lysosome is involved in various cellular processes, including energy metabolism, plasma membrane repair and antigen presentation. Recent work has shown that the lysosome is an important calcium store, modulating diverse cellular functions such as membrane fusion and fission, autophagy and lysosomal biogenesis. Precise measurement of free lysosomal calcium concentration has been hampered by its low luminal pH, since the affinity of most calcium probes decreases with higher proton concentration. Here we detailed an adapted protocol for the simultaneous measurement of lysosomal pH and calcium using dextran-conjugated ratiometric fluorescent dyes. As compared with indirect measurements of lysosomal calcium release using genetically-encoded calcium indicators (GECIs), the present method offers the possibility of obtaining pH-corrected, intraluminal calcium concentrations at single lysosome resolution. It also enables simultaneous temporal resolution of lysosomal calcium and pH.

Interorganelle Tethering to Endocytic Organelles Determines Directional Cytokine Transport in CD4+ T Cells.

Delivery of vesicles to their desired destinations plays a central role in maintaining proper cell functionality. In certain scenarios, depending on loaded cargos, the vesicles have spatially distinct destinations. For example, in T cells, some cytokines (e.g., IL-2) are polarized to the T cell-target cell interface, whereas the other cytokines are delivered multidirectionally (e.g., TNF-α). In this study, we show that in primary human CD4+ T cells, both TNF-α+ and IL-2+ vesicles can tether with endocytic organelles (lysosomes/late endosomes) by forming membrane contact sites. Tethered cytokine-containing vesicle (CytV)-endocytic organelle pairs are released sequentially. Only endocytic organelle-tethered CytVs are preferentially transported to their desired destination. Mathematical models suggest that endocytic organelle tethering could regulate the direction of cytokine transport by selectively attaching different microtubule motor proteins (such as kinesin and dynein) to the corresponding CytVs. These findings establish the previously unknown interorganelle tethering to endocytic organelles as a universal solution for directional cytokine transport in CD4+ T cells. Modulating tethering to endocytic organelles can, therefore, coordinately control directionally distinct cytokine transport.

ORP5 and ORP8: Sterol Sensors and Phospholipid Transfer Proteins at Membrane Contact Sites?

Oxysterol binding related proteins 5 and 8 (ORP5 and ORP8) are two close homologs of the larger oxysterol binding protein (OSBP) family of sterol sensors and lipid transfer proteins (LTP). Early studies indicated these transmembrane proteins, anchored to the endoplasmic reticulum (ER), bound and sensed cholesterol and oxysterols. They were identified as important for diverse cellular functions including sterol homeostasis, vesicular trafficking, proliferation and migration. In addition, they were implicated in lipid-related diseases such as atherosclerosis and diabetes, but also cancer, although their mechanisms of action remained poorly understood. Then, alongside the increasing recognition that membrane contact sites (MCS) serve as hubs for non-vesicular lipid transfer, added to their structural similarity to other LTPs, came discoveries showing that ORP5 and 8 were in fact phospholipid transfer proteins that rather sense and exchange phosphatidylserine (PS) for phosphoinositides, including phosphatidylinositol-4-phosphate (PI(4)P) and potentially phosphatidylinositol-(4,5)-bisphosphate (PI(4,5)P2). Evidence now points to their action at MCS between the ER and various organelles including the plasma membrane, lysosomes, mitochondria, and lipid droplets. Dissecting exactly how this unexpected phospholipid transfer function connects with sterol regulation in health or disease remains a challenge for future studies.

Molecular Mechanisms of Calcium Signaling During Phagocytosis.

Calcium (Ca2+) is a ubiquitous second messenger involved in the regulation of numerous cellular functions including vesicular trafficking, cytoskeletal rearrangements and gene transcription. Both global as well as localized Ca2+ signals occur during phagocytosis, although their functional impact on the phagocytic process has been debated. After nearly 40 years of research, a consensus may now be reached that although not strictly required, Ca2+ signals render phagocytic ingestion and phagosome maturation more efficient, and their manipulation make an attractive avenue for therapeutic interventions. In the last decade many efforts have been made to identify the channels and regulators involved in generating and shaping phagocytic Ca2+ signals. While molecules involved in store-operated calcium entry (SOCE) of the STIM and ORAI family have taken center stage, members of the canonical, melastatin, mucolipin and vanilloid transient receptor potential (TRP), as well as purinergic P2X receptor families are now recognized to play significant roles. In this chapter, we review the recent literature on research that has linked specific Ca2+-permeable channels and regulators to phagocytic function. We highlight the fact that lipid mediators are emerging as important regulators of channel gating and that phagosomal ionic homeostasis and Ca2+ release also play essential parts. We predict that improved methodologies for measuring these factors will be critical for future advances in dissecting the intricate biology of this fascinating immune process.

Ultrastructural Characterization of Flashing Mitochondria.

Mitochondria undergo spontaneous transient elevations in matrix pH associated with drops in mitochondrial membrane potential. These mitopHlashes require a functional respiratory chain and the profusion protein optic atrophy 1, but their mechanistic basis is unclear. To gain insight on the origin of these dynamic events, we resolved the ultrastructure of flashing mitochondria by correlative light and electron microscopy. HeLa cells expressing the matrix-targeted pH probe mitoSypHer were screened for mitopHlashes and fixed immediately after the occurrence of a flashing event. The cells were then processed for imaging by serial block face scanning electron microscopy using a focused ion beam to generate ~1,200 slices of 10 nm thickness from a 28 µm × 15 µm cellular volume. Correlation of live/fixed fluorescence and electron microscopy images allowed the unambiguous identification of flashing and nonflashing mitochondria. Three-dimensional reconstruction and surface mapping revealed that each tomogram contained two flashing mitochondria of unequal sizes, one being much larger than the average mitochondrial volume. Flashing mitochondria were 10-fold larger than silent mitochondria but with a surface to volume ratio and a cristae volume similar to nonflashing mitochondria. Flashing mitochondria were connected by tubular structures, formed more membrane contact sites, and a constriction was observed at a junction between a flashing mitochondrion and a nonflashing mitochondrion. These data indicate that flashing mitochondria are structurally preserved and bioenergetically competent but form numerous membrane contact sites and are connected by tubular structures, consistent with our earlier suggestion that mitopHlashes might be triggered by the opening of fusion pores between contiguous mitochondria.

Oxidized lipids keep heat shock chaperones busy: new insights on the deficiencies of tumour-associated dendritic cells.

In a recent publication in Nature Communications the group of Dr. Dmitry Gabrilovich takes us one step closer to understanding why lipid accumulation impairs the function of tumour-associated dendritic cells (DCs). In this study, the authors present two surprising and significant findings. First, they show that in mouse DCs oxidized lipids function as a sink that traps the heat shock chaperone HSP70, a molecular target of emerging anti-cancer strategies. Secondly, they find that HSP70 in turn regulates the trafficking of peptide-loaded major histocompatibility complex class I (pMHC-I) molecules, a complex that triggers the proliferation of cancer-killing T cells. These observations are discussed briefly in the context of lipid droplet function and pMHC-I trafficking in tumour-associated DCs, as well as HSP70's pleiotropic and incompletely understood roles - and what they mean for future cancer therapy designs.

The Bicarbonate Transporter SLC4A7 Plays a Key Role in Macrophage Phagosome Acidification.

Macrophages represent the first line of immune defense against pathogens, and phagosome acidification is a necessary step in pathogen clearance. Here, we identified the bicarbonate transporter SLC4A7, which is strongly induced upon macrophage differentiation, as critical for phagosome acidification. Loss of SLC4A7 reduced acidification of phagocytosed beads or bacteria and impaired the intracellular microbicidal capacity in human macrophage cell lines. The phenotype was rescued by wild-type SLC4A7, but not by SLC4A7 mutants, affecting transport capacity or cell surface localization. Loss of SLC4A7 resulted in increased cytoplasmic acidification during phagocytosis, suggesting that SLC4A7-mediated, bicarbonate-driven maintenance of cytoplasmic pH is necessary for phagosome acidification. Altogether, we identify SLC4A7 and bicarbonate-driven cytoplasmic pH homeostasis as an important element of phagocytosis and the associated microbicidal functions in macrophages.

STIM1 promotes migration, phagosomal maturation and antigen cross-presentation in dendritic cells.

Antigen cross-presentation by dendritic cells (DC) stimulates cytotoxic T cell activation to promote immunity to intracellular pathogens, viruses and cancer. Phagocytosed antigens generate potent T cell responses, but the signalling and trafficking pathways regulating their cross-presentation are unclear. Here, we show that ablation of the store-operated-Ca2+-entry regulator STIM1 in mouse myeloid cells impairs cross-presentation and DC migration in vivo and in vitro. Stim1 ablation reduces Ca2+ signals, cross-presentation, and chemotaxis in mouse bone-marrow-derived DCs without altering cell differentiation, maturation or phagocytic capacity. Phagosomal pH homoeostasis and ROS production are unaffected by STIM1 deficiency, but phagosomal proteolysis and leucyl aminopeptidase activity, IRAP recruitment, as well as fusion of phagosomes with endosomes and lysosomes are all impaired. These data suggest that STIM1-dependent Ca2+ signalling promotes the delivery of endolysosomal enzymes to phagosomes to enable efficient cross-presentation.

UNC93B1 interacts with the calcium sensor STIM1 for efficient antigen cross-presentation in dendritic cells.

Dendritic cells (DC) have the unique ability to present exogenous antigens via the major histocompatibility complex class I pathway to stimulate naive CD8+ T cells. In DCs with a non-functional mutation in Unc93b1 (3d mutation), endosomal acidification, phagosomal maturation, antigen degradation, antigen export to the cytosol and the function of the store-operated-Ca2+-entry regulator STIM1 are impaired. These defects result in compromised antigen cross-presentation and anti-tumor responses in 3d-mutated mice. Here, we show that UNC93B1 interacts with the calcium sensor STIM1 in the endoplasmic reticulum, a critical step for STIM1 oligomerization and activation. Expression of a constitutively active STIM1 mutant, which no longer binds UNC93B1, restores antigen degradation and cross-presentation in 3d-mutated DCs. Furthermore, ablation of STIM1 in mouse and human cells leads to a decrease in cross-presentation. Our data indicate that the UNC93B1 and STIM1 cooperation is important for calcium flux and antigen cross-presentation in DCs.

The ER phagosome connection in the era of membrane contact sites.

Phagocytosis is an essential mechanism through which innate immune cells ingest foreign material that is either destroyed or used to generate and present antigens and initiate adaptive immune responses. While a role for the ER during phagosome biogenesis has been recognized, whether fusion with ER cisternae or vesicular derivatives occurs has been the source of much contention. Membrane contact sites (MCS) are tight appositions between ER membranes and various organelles that coordinate multiple functions including localized signalling, lipid transfer and trafficking. The discovery that MCS form between the ER and phagosomes now begs the question of whether MCS play a role in connecting the ER to phagosomes under different contexts. In this review, we consider the implications of MCS between the ER and phagosomes during cross-presentation and infection with intracellular pathogens. We also discuss the similarities between these contacts and those between the ER and plasma membrane and acidic organelles such as endosomes and lysosomes. This article is part of a Special Issue entitled: Membrane Contact Sites edited by Christian Ungermann and Benoit Kornmann.