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.

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.

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.

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.

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.

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.

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.

Molecular Mechanisms of Calcium Signaling During Phagocytosis.

Calcium (Ca) 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 Ca 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, Ca signals render phagocytic ingestion and phagosome maturation more efficient, and their manipulation make an attractive avenue for therapeutic interventions.

Ultrastructural Characterization of Flashing Mitochondria.

Mitochondria undergo spontaneous transient elevations in matrix pH associated with drops in mitochondrial membrane potential. These 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.

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.

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.

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-Ca-entry regulator STIM1 in mouse myeloid cells impairs cross-presentation and DC migration in vivo and in vitro.

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-Ca-entry regulator STIM1 are impaired. These defects result in compromised antigen cross-presentation and anti-tumor responses in 3d-mutated mice.

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.