TRP Channels as Interior Designers: Remodeling the Endolysosomal Compartment in Natural Killer Cells.

Cytotoxic lymphocytes, including natural killer (NK) cells and T cells are distinguished by their ability to eliminate target cells through release of secretory lysosomes. Conventional lysosomes and secretory lysosomes are part of the pleomorphic endolysosomal system and characterized by its highly dynamic nature. Several calcium-permeable TRP calcium channels play an essential role in endolysosomal calcium signaling to ensure proper function of these organelles. In NK cells, the expression of self MHC-specific inhibitory receptors dynamically tunes their secretory potential in a non-transcriptional, calcium-dependent manner. New insights suggest that TRPML1-mediated lysosomal calcium fluxes are tightly interconnected to NK cell functionality through modulation of granzyme B and perforin content of the secretory lysosome. Lysosomal TRP channels show a subset-specific expression pattern during NK differentiation, which is paralleled with gradually increased loading of effector molecules in secretory lysosomes. Methodological advances, including organellar patch-clamping, specific pharmacological modulators, and genetically-encoded calcium indicators open up new possibilities to investigate how TRP channels influence communication between intracellular organelles in immune cells. This review discusses our current understanding of lysosome biogenesis in NK cells with an emphasis on the TRP mucolipin family and the implications for NK cell functionality and cancer immunotherapy.

Evolution, role in inflammation, and redox control of leaderless secretory proteins.

Members of the interleukin (IL)-1 family are key determinants of inflammation. Despite their role as intercellular mediators, most lack the leader peptide typically required for protein secretion. This lack is a characteristic of dozens of other proteins that are actively and selectively secreted from living cells independently of the classical endoplasmic reticulum-Golgi exocytic route. These proteins, termed leaderless secretory proteins (LLSPs), comprise proteins directly or indirectly involved in inflammation, including cytokines such as IL-1ß and IL-18, growth factors such as fibroblast growth factor 2 (FGF2), redox enzymes such as thioredoxin, and proteins most expressed in the brain, some of which participate in the pathogenesis of neurodegenerative disorders. Despite much effort, motifs that promote LLSP secretion remain to be identified. In this review, we summarize the mechanisms and pathophysiological significance of the unconventional secretory pathways that cells use to release LLSPs. We place special emphasis on redox regulation and inflammation, with a focus on IL-1ß, which is secreted after processing of its biologically inactive precursor pro-IL-1ß in the cytosol. Although LLSP externalization remains poorly understood, some possible mechanisms have emerged. For example, a common feature of LLSP pathways is that they become more active in response to stress and that they involve several distinct excretion mechanisms, including direct plasma membrane translocation, lysosome exocytosis, exosome formation, membrane vesiculation, autophagy, and pyroptosis. Further investigations of unconventional secretory pathways for LLSP secretion may shed light on their evolution and could help advance therapeutic avenues for managing pathological conditions, such as diseases arising from inflammation.

Membrane trafficking in osteoclasts and implications for osteoporosis.

Osteoclasts are large multinucleated cells exquisitely adapted to resorb bone matrix. Like other eukaryotes, osteoclasts possess an elaborate ensemble of intracellular organelles through which solutes, proteins and other macromolecules are trafficked to their target destinations via membrane-bound intermediaries. During bone resorption, membrane trafficking must be tightly regulated to sustain the structural and functional polarity of the osteoclasts' membrane domains. Of these, the ruffled border (RB) is most characteristic, functioning as the osteoclasts' secretory apparatus. This highly convoluted organelle is classically considered to be formed by the targeted fusion of acidic vesicles with the bone-facing plasma membrane. Emerging findings disclose new evidence that the RB is far more complex than previously envisaged, possessing discrete subdomains that are serviced by several intersecting endocytic, secretory, transcytotic and autophagic pathways. Bone-resorbing osteoclasts therefore serve as a unique model system for studying polarized membrane trafficking. Recent advances in high-resolution microscopy together with the convergence of genetic and cell biological studies in humans and in mice have helped illuminate the major membrane trafficking pathways in osteoclasts and unmask the core molecular machinery that governs these distinct vesicle transport routes. Among these, small Rab GTPases, their binding partners and members of the endocytic sorting nexin family have emerged as critical regulators. This mini review summarizes our current understanding of membrane trafficking in osteoclasts, the key molecular participants, and discusses how these transport machinery may be exploited for the development of new therapies for metabolic disorders of bone-like osteoporosis.

Cyclodextrin triggers MCOLN1-dependent endo-lysosome secretion in Niemann-Pick type C cells.

In specialized cell types, lysosome-related organelles support regulated secretory pathways, whereas in nonspecialized cells, lysosomes can undergo fusion with the plasma membrane in response to a transient rise in cytosolic calcium. Recent evidence also indicates that lysosome secretion can be controlled transcriptionally and promote clearance in lysosome storage diseases. In addition, evidence is also accumulating that low concentrations of cyclodextrins reduce the cholesterol-storage phenotype in cells and animals with the cholesterol storage disease Niemann-Pick type C, via an unknown mechanism. Here, we report that cyclodextrin triggers the secretion of the endo/lysosomal content in nonspecialized cells and that this mechanism is responsible for the decreased cholesterol overload in Niemann-Pick type C cells. We also find that the secretion of the endo/lysosome content occurs via a mechanism dependent on the endosomal calcium channel mucolipin-1, as well as FYCO1, the AP1 adaptor, and its partner Gadkin. We conclude that endo-lysosomes in nonspecialized cells can acquire secretory functions elicited by cyclodextrin and that this pathway is responsible for the decrease in cholesterol storage in Niemann-Pick C cells.

Granulysin species segregate to different lysosome-related effector vesicles (LREV) and get mobilized by either classical or non-classical degranulation.

Granulysin (GNLY) is a cationic antimicrobial, proinflammatory, and cytotoxic effector protein primarily expressed in human cytotoxic T and NK cells. Its two variants, the 15 kDa precursor and the mature 9 kDa protein processed by proteolysis, act on different microbes or infected and transformed target cells and utilize mechanistically different effector activities. In human peripheral blood lymphocytes of healthy individuals, both forms of GNLY are detected in TCR αß+ (CD4+ and CD8+) T cells, TCR γδ+ T cells, and CD3-CD56+ NK cells. In general, classical cytotoxic cells (i.e. CD8+ TCR αß+ T cells, TCR γδ+ T cells, and NK cells) contain effector proteins in higher abundance in more cells of the subset as compared to TCR αß+ CD4+ T cells. Imaging flow cytometry analyses demonstrate that the subcellular localization and internal pools of 9 kDa and 15 kDa GNLY are virtually non-overlapping. The 9 kDa form is enriched in dense granules that also contain granzymes (Grz) and carry CD107a, whereas 15 kDa GNLY is associated with CD107a-negative lysosome-related effector vesicles. We further demonstrate that 15 kDa GNLY serves as an additional indicator for non-classical, PKC-dependent degranulation while the liberation of granules containing 9 kDa GNLY requires calcium mobilization. Our studies provide a deeper insight into the subcellular localization and release mechanisms of the individual GNLY species. This information will not only be useful for the interpretation of GNLY-related pathophysiologies, but also for the development of therapeutic interventions employing distinct GNLY effector functions for microbial targeting or immunoregulation.

The unconventional secretion of IL-1ß: Handling a dangerous weapon to optimize inflammatory responses.

Interleukin 1ß (IL-1ß) is a major mediator of inflammation, with a causative role in many diseases. Unlike most other cytokines, however, it lacks a secretory signal sequence, raising intriguing mechanistic, functional and evolutionary questions. Despite decades of strenuous efforts in many laboratories, how IL-1ß is secreted is still a matter of intense debate. Here, we summarize the different mechanisms and pathways that have been proposed for IL-1ß secretion. At least two of them, namely the endolysosomal vesicle-based and gasdermin D-dependent pathways (types III and I in the recent Rabouille's classification of unconventional protein secretion), can be triggered in monocytes, the main source of IL-1ß in humans, according to the type and strength of the pro-inflammatory stimuli. As during the escalation of human conflicts, monocytes deploy secretory mechanisms of increasing efficiency and dangerousness, shifting from the specific and controlled type III pathway to the much faster release of type I. Thus, the different mechanisms are activated depending on the severity of the conditions, from the self-limiting type III pathways in response of low pathogen load or small trauma, to the uncontrolled responses that underlie autoinflammatory disorders and sepsis.

Subcellular localization and activation of ADAM proteases in the context of FasL shedding in T lymphocytes.

The "A Disintegrin And Metalloproteinases" (ADAMs) form a subgroup of the metzincin endopeptidases. Proteolytically active members of this protein family act as sheddases and govern key processes in development and inflammation by regulating cell surface expression and release of cytokines, growth factors, adhesion molecules and their receptors. In T lymphocytes, ADAM10 sheds the death factor Fas Ligand (FasL) and thereby regulates T cell activation, death and effector function. Although FasL shedding by ADAM10 was confirmed in several studies, its regulation is still poorly defined. We recently reported that ADAM10 is highly abundant on T cells whereas its close relative ADAM17 is expressed at low levels and transiently appears at the cell surface upon stimulation. Since FasL is also stored intracellularly and brought to the plasma membrane upon stimulation, we addressed where the death factor gets exposed to ADAM proteases. We report for the first time that both ADAM10 and ADAM17 are associated with FasL-containing secretory lysosomes. Moreover, we demonstrate that TCR/CD3/CD28-stimulation induces a partial positioning of both proteases and FasL to lipid rafts and only the activation-induced raft-positioning results in FasL processing. TCR/CD3/CD28-induced FasL proteolysis is markedly affected by reducing both ADAM10 and ADAM17 protein levels, indicating that in human T cells also ADAM17 is implicated in FasL processing. Since FasL shedding is affected by cholesterol depletion and by inhibition of Src kinases or palmitoylation, we conclude that it requires mobilization and co-positioning of ADAM proteases in lipid raft-like platforms associated with an activation of raft-associated Src-family kinases.

Pathophysiology and spectrum of diseases caused by defects in lymphocyte cytotoxicity.

In experimental settings, lymphocyte cytotoxicity has been recognized as a central mechanism for immune defense against infected and neoplastic cells. More recently, molecular determinants of lymphocyte cytotoxicity have been identified through studies of rare, inherited hyperinflammatory and lymphoproliferative syndromes that include hemophagocytic lymphohistiocytosis (HLH). These studies have unraveled a set of genes pivotal for the biogenesis and directed release of perforin-containing lysosomes that mediate target cell killing, in addition to other pathways including Fas that also contribute to induction of cell death. Furthermore, studies of such human primary immunodeficiencies have highlighted non-redundant roles of perforin for maintenance of immune homeostasis. Besides providing mechanistic insights to lymphocyte cytotoxicity, studies of individuals with rare hyperinflammatory diseases are highlighting the relevance of lymphocyte cytotoxicity to more common human diseases. It is increasingly recognized that mutations abrogating lymphocyte cytotoxicity not only cause HLH, but also are associated with susceptibility to cancer and autoimmune syndromes. In addition, patients may initially be present with neurological symptoms or severe infectious disease masquerading as variable immunodeficiency syndrome. Here, we highlight new knowledge regarding the molecular mechanisms regulating lymphocyte cytotoxicity and review how mutations in genes associated with HLH cause disease. We also discuss the wider implications of impairments in lymphocyte cytotoxicity for human disease predisposition.

Late steps in secretory lysosome exocytosis in cytotoxic lymphocytes.

Natural Killer cells are a subset of cytotoxic lymphocytes that are important in host defense against infections and transformed cells. They exert this function through recognition of target cells by cell surface receptors, which triggers a signaling program that results in a re-orientation of the microtubule organizing center and secretory lysosomes toward the target cell. Upon movement of secretory lysosomes to the plasma membrane and subsequent fusion, toxic proteins are released by secretory lysosomes in the immunological synapse which then enter and kill the target cell. In this minireview we highlight recent progress in our knowledge of late steps in this specialized secretion pathway and address important open questions.

Syntaxin binding mechanism and disease-causing mutations in Munc18-2.

Mutations in either syntaxin 11 (Stx11) or Munc18-2 abolish cytotoxic T lymphocytes (CTL) and natural killer cell (NK) cytotoxicity, and give rise to familial hemophagocytic lymphohistiocytosis (FHL4 or FHL5, respectively). Although Munc18-2 is known to interact with Stx11, little is known about the molecular mechanisms governing the specificity of this interaction or how in vitro IL-2 activation leads to compensation of CTL and NK cytotoxicity. To understand how mutations in Munc18-2 give rise to disease, we have solved the structure of human Munc18-2 at 2.6 Å resolution and mapped 18 point mutations. The four surface mutations identified (R39P, L130S, E132A, P334L) map exclusively to the predicted syntaxin and soluble N-ethylmaleimide-sensitive factor accessory protein receptor binding sites of Munc18-2. We find that Munc18-2 binds the N-terminal peptide of Stx11 with a ~20-fold higher affinity than Stx3, suggesting a potential role in selective binding. Upon IL-2 activation, levels of Stx3 are increased, favoring Munc18-2 binding when Stx11 is absent. Similarly, Munc18-1, expressed in IL-2-activated CTL, is capable of binding Stx11. These findings provide potential explanations for restoration of Munc18-Stx function and cytotoxicity in IL-2-activated cells.

The secretion of IL-1ß and options for release.

Unlike most cytokines, IL-1ß lacks a secretory signal sequence raising the question of how is this cytokine processed and delivered outside the producing cells. After the seminal observation that IL-1ß is actively secreted by human monocytes through a route alternative to the classic endoplasmic reticulum-Golgi, several different pathways have been proposed for IL-1ß secretion in different cell types and culture conditions, some of which are unique to macrophage cell lines. Here we describe the most credited of these pathways. In particular, we will focus on IL-1ß secretion from primary human blood monocytes. In fact, although data from macrophages or macrophage cell lines are predominant, secretion of IL-1ß by monocytes is the most clinically relevant.

Labeling and exocytosis of secretory compartments in RBL mastocytes by polystyrene and mesoporous silica nanoparticles.

BACKGROUND: For a safe 'in vivo' biomedical utilization of nanoparticles, it is essential to assess not only biocompatibility, but also the potential to trigger unwanted side effects at both cellular and tissue levels. Mastocytes (cells having secretory granules containing cytokines, vasoactive amine, and proteases) play a pivotal role in the immune and inflammatory responses against exogenous toxins. Mastocytes are also recruited in the tumor stroma and are involved in tumor vascularization and growth. AIM AND METHODS: In this work, mastocyte-like rat basophilic leukemia (RBL) cells were used to investigate whether carboxyl-modified 30 nm polystyrene (PS) nanoparticles (NPs) and naked mesoporous silica (MPS) 10 nm NPs are able to label the secretory inflammatory granules, and possibly induce exocytosis of these granules. Uptake, cellular retention and localization of fluorescent NPs were analyzed by cytofluorometry and microscope imaging. RESULTS: OUR FINDINGS WERE THAT: (1) secretory granules of mastocytes are accessible by NPs via endocytosis; (2) PS and MPS silica NPs label two distinct subpopulations of inflammatory granules in RBL mastocytes; and (3) PS NPs induce calcium-dependent exocytosis of inflammatory granules. CONCLUSION: These findings highlight the value of NPs for live imaging of inflammatory processes, and also have important implications for the clinical use of PS-based NPs, due to their potential to trigger the unwanted activation of mastocytes.

Roles for ca(2+) mobilization and its regulation in mast cell functions.

Mobilization of Ca(2+) in response to IgE receptor-mediated signaling is a key process in many aspects of mast cell function. Here we summarize our current understanding of the molecular bases for this process and the roles that it plays in physiologically relevant mast cell biology. Activation of IgE receptor signaling by antigen that crosslinks these complexes initiates Ca(2+) mobilization as a fast wave that is frequently followed by a series of Ca(2+) oscillations which are dependent on Ca(2+) influx-mediated by coupling of the endoplasmic reticulum luminal Ca(2+) sensor STIM1 to the calcium release activated calcium channel protein Orai1. Granule exocytosis depends on this process, together with the activation of protein kinase C isoforms, and specific roles for these signaling steps are beginning to be understood. Ca(2+) mobilization also plays important roles in stimulated exocytosis of recycling endosomes and newly synthesized cytokines, as well as in antigen-mediated chemotaxis of rat mucosal mast cells. Phosphoinositide metabolism plays key roles in all of these processes, and we highlight these roles in several cases.

Munc13-4*rab27 complex tethers secretory lysosomes at the plasma membrane.

Natural Killer (NK) cells and Cytotoxic T lymphocytes (CTL) are critical for the immune response against virus infections or transformed cells. They kill target cells via polarized exocytosis of lytic proteins from secretory lysosomes (SL). Rab27a and munc13-4 interact directly and are required for target cell killing. How they cooperate in the intricate degranulation process is not known. We identified critical residues in munc13-4 for rab27 interaction and tested binding mutants in several complementation assays. In a rat mast cell line we replaced endogenous munc13-4 with ectopically expressed munc13-4 constructs. Unlike wild type munc13-4, binding mutants fail to rescue ß-hexosaminidase secretion. In accord, expression of binding mutants in CTL of Familial Hemophagocytic Lymphohistiocytosis type 3 patients, does not rescue CD107 appearance on the plasma membrane. Total Internal Reflection Fluorescence (TIRF) imaging shows that munc13-4*rab27a restricts motility of SL in the subapical cytoplasm. We propose that rab27*munc13-4 tethers SL to the plasma membrane, a requirement for formation of a cognate SNARE complex for fusion.