LYST Affects Lysosome Size and Quantity,but not Trafficking or Degradation Through Autophagy or Endocytosis

Mutations in the large BEACH domain‐containing protein LYST causes Chediak–Higashi syndrome. The diagnostic hallmark is enlarged lysosomes and lysosome‐related organelles in various cell types. Dysfunctional secretion of enlarged lysosome‐related organelles has been observed in cells with mutations in LYST, but the capacity of the enlarged lysosomes to degrade endogenous proteins has not been studied. Here, we show for the first time that small interfering RNA‐depletion of LYST in human cell lines recapitulates the LYST mutant phenotype of enlarged lysosomes. We found no evidence for an effect of LYST depletion on autophagy or endocytic degradation. Autophagosomes are formed in normal size and quantities and are able to fuse to the enlarged lysosomes, leading to normal rates of degradation. Degradation of the epidermal growth factor receptor (EGFR) was similarly not affected, indicating that the enlarged lysosomes are fully functional in degrading endogenous proteins. Retrograde trafficking of toxins as well as the localization of transporters of lysosomal proteins, adaptor protein‐3 (AP‐3) and cation‐independent mannose‐6‐phosphate receptor (CI‐MPR), were all found to be unaffected by LYST. Quantitative analysis of the enlarged lysosomes shows that LYST depletion causes a reduction in vesicle quantity per cell, while the total enzymatic content and vesicular pH are unaffected, supporting a role for LYST in lysosomal fission and/or fusion events.      

Syntaxin 4 mediates endosome recycling for lytic granule exocytosis in cytotoxic T‐lymphocytes

Adaptive and innate immunity utilize the perforin‐killing pathway to eliminate virus‐infected or cancer cells. Cytotoxic T‐lymphocytes (CTLs) and natural killer cells mediate this process by releasing toxic proteins at the contact area with target cells known as immunological synapse (IS). Formation of a stable IS and exocytosis of toxic proteins requires persistent fusion of Rab11a recycling endosomes with the plasma membrane (PM) that may assure the delivery of key effector proteins. Despite the importance of the recycling endosomal compartment, the membrane fusion proteins that control this process at the IS remain elusive. Here, by performing knockdown experiments we found that syntaxin 4 (STX4) is necessary for cytotoxic activity and CD107a degranulation against target cells in a similar fashion to syntaxin 11, which is involved in lytic granule (LG) exocytosis and immunodeficiency when it is mutated. Using total internal reflection fluorescent microscopy we identified that STX4 mediates fusion of EGFP‐Rab11a vesicles at the IS. Immunoprecipitation experiments in lysates of activated CTLs indicate that endogenous STX4 may drive this fusion step by interacting with cognate proteins: Munc18‐3/SNAP23/VAMP7 and/or VAMP8. These results reveal the role of STX4 in mediating fusion of Rab11a endosomes upstream of lytic granules (LGs) exocytosis and further demonstrate the importance of this pathway in controlling CTL‐mediated cytotoxicity.      

Drosophila mauve Mutants Reveal a Role of LYST Homologs Late in the Maturation of Phagosomes and Autophagosomes

Chediak–Higashi syndrome (CHS) is a lethal disease caused by mutations that inactivate the lysosomal trafficking regulator protein (LYST). Patients suffer from diverse symptoms including oculocutaneous albinism, recurrent infections, neutropenia and progressive neurodegeneration. These defects have been traced back to over‐sized lysosomes and lysosome‐related organelles (LROs) in different cell types. Here, we explore mutants in the Drosophila mauve gene as a new model system for CHS. The mauve gene (CG42863) encodes a large BEACH domain protein of 3535 amino acids similar to LYST. This reflects a functional homology between these proteins as mauve mutants also display enlarged LROs, such as pigment granules. This Drosophila model also replicates the enhanced susceptibility to infections and we show a defect in the cellular immune response. Early stages of phagocytosis proceed normally in mauve mutant hemocytes but, unlike in wild type, late phagosomes fuse and generate large vacuoles containing many bacteria. Autophagy is similarly affected in mauve fat bodies as starvation‐induced autophagosomes grow beyond their normal size. Together these data suggest a model in which Mauve functions to restrict homotypic fusion of different pre‐lysosomal organelles and LROs.     

Secretory lysosome biogenesis in cytotoxic T lymphocytes from normal and Chediak Higashi syndrome patients

The lytic proteins mediating target cell killing are stored in the lysosomes of activated cytotoxic T lymphocytes (CTL) and are secreted upon recognition of a target cell. These secretory lysosomes cannot be detected in resting T lymphocytes. Interaction of a resting cell with a target cell activates de novo formation of secretory lysosomes. CTL clones in culture mimic this behaviour, and so provide an ideal system for studying secretory lysosome biogenesis and maturation. In the genetic disease, Chediak Higashi syndrome (CHS), all lysosomes in the cells are enlarged and reduced in number compared with wild-type (WT) cells. We have used CTL from this disease to study secretory lysosome biogenesis and maturation. We show that at early stages after activation the secretory lysosomes are identical in WT and mutant cells, and that delivery of proteins to the secretory lysosome along the biosynthetic and endocytic pathways is normal in the mutant cells. With time, the lysosomes in the mutant cells aggregate, become larger and fewer in number and eventually form giant structures. Our results show that the initial steps of secretory lysosome formation are normal in CHS, but that the organelles subsequently fuse together during cell maturation to form the giant secretory lysosomes.     

The Rab27a effectors JFC1/Slp1 and Munc13-4 regulate exocytosis of neutrophil granules

Neutrophil granules contain secretory molecules that contribute to the implementation of all neutrophil functions. The molecular components that regulate the exocytosis of neutrophil granules have not been characterized. In this study, using small interfering RNA gene-targeting approaches and granulocytes from genetically modified mice, we characterized the Rab27a effectors JFC1/Slp1 and Munc13-4 as components of the exocytic machinery of granulocytes. Using total internal reflection fluorescence microscopy analysis, we show that Rab27a and JFC1 colocalize in predocked and docked vesicles in granulocytes. Next, we demonstrate that JFC1-downregulated granulocytes have impaired myeloperoxidase secretion. Using immunological interference, we confirm that JFC1 plays an important role in azurophilic granule exocytosis in human neutrophils. Interference with Rab27a but not with JFC1 impaired gelatinase B secretion in neutrophils, suggesting that a different Rab27a effector modulates this process. In similar studies, we confirmed that Munc13-4 regulates gelatinase secretion. Immunofluorescence analysis indicates that Munc13-4 localizes at secretory organelles in neutrophils. Using neutrophils from a Munc13-4-deficient mouse model (Jinx), we demonstrate that Munc13-4 plays a central role in the regulation of exocytosis of various sets of secretory organelles. However, mobilization of CD11b was not affected in Munc13-4-deficient neutrophils, indicating that secretory defects in these cells are limited to a selective group of exocytosable organelles.     

Two patients walk into a clinic...a genomics perspective on the future of schizophrenia

Background

Cytolytic cells of the immune system destroy pathogen-infected cells by polarised exocytosis of secretory lysosomes containing the pore-forming protein perforin. Precise delivery of this lethal hit is essential to ensuring that only the target cell is destroyed. In cytotoxic T lymphocytes (CTLs), this is accomplished by an unusual movement of the centrosome to contact the plasma membrane at the centre of the immunological synapse formed between killer and target cells. Secretory lysosomes are directed towards the centrosome along microtubules and delivered precisely to the point of target cell recognition within the immunological synapse, identified by the centrosome. We asked whether this mechanism of directing secretory lysosome release is unique to CTL or whether natural killer (NK) and invariant NKT (iNKT) cytolytic cells of the innate immune system use a similar mechanism to focus perforin-bearing lysosome release.

Results

NK cells were conjugated with B-cell targets lacking major histocompatibility complex class I 721.221 cells, and iNKT cells were conjugated with glycolipid-pulsed CD1-bearing targets, then prepared for thin-section electron microscopy. High-resolution electron micrographs of the immunological synapse formed between NK and iNKT cytolytic cells with their targets revealed that in both NK and iNKT cells, the centrioles could be found associated (or 'docked') with the plasma membrane within the immunological synapse. Secretory clefts were visible within the synapses formed by both NK and iNKT cells, and secretory lysosomes were polarised along microtubules leading towards the docked centrosome. The Golgi apparatus and recycling endosomes were also polarised towards the centrosome at the plasma membrane within the synapse.

Conclusions

These results reveal that, like CTLs of the adaptive immune system, the centrosomes of NK and iNKT cells (cytolytic cells of the innate immune system) direct secretory lysosomes to the immunological synapse. Morphologically, the overall structure of the immunological synapses formed by NK and iNKT cells are very similar to those formed by CTLs, with both exocytic and endocytic organelles polarised towards the centrosome at the plasma membrane, which forms a focal point for exocytosis and endocytosis within the immunological synapse. We conclude that centrosomal polarisation provides a rapid, responsive and precise mechanism for secretory lysosome delivery to the immunological synapse in CTLs, NK cells and iNKT cells.     

Normal lytic granule secretion by cytotoxic T lymphocytes deficient in BLOC-1, -2 and -3 and myosins Va, VIIa and XV

Melanocytes and cells of the immune system share an unusual secretory mechanism which uses the lysosome as a regulated secretory organelle. Recently, a number of the proteins required for these 'secretory lysosomes' to undergo exocytosis have been identified. These include Rab27a, Lyst, Rab geranyl geranyl transferase and the adapter protein complex AP-3. Patients lacking any of these proteins are characterized by the rare combination of albinism and immunodeficiency, revealing roles for these proteins in both melanocyte and immune cell secretion. In order to ask how far the link between albinism and immunodeficiency extends we have examined cytotoxic T-lymphocyte (CTL) secretion from two BLOC-3-deficient patients and seven different mouse models of Hermansky-Pudlak syndrome, all of which display defects in pigmentation and platelet function. We find that CTL function is normal in HPS patients and pale-ear mice deficient in BLOC-3, pallid, muted and sandy mice deficient in BLOC-1, ruby-eye mice deficient in BLOC-2 and buff mice deficient in Vps33a. Similarly, the unconventional myosins, Va, VIIa and XV, which can act as effectors for Rab27a in some cell types, are not required in CTL. These results reveal differences in the protein machinery required for biogenesis and/or secretion of lysosome-related organelles in CTL and melanocytes.     

Regulation of perforin activation and pre‐synaptic toxicity through C‐terminal glycosylation

Perforin is a highly cytotoxic pore‐forming protein essential for immune surveillance by cytotoxic lymphocytes. Prior to delivery to target cells by exocytosis, perforin is stored in acidic secretory granules where it remains functionally inert. However, how cytotoxic lymphocytes remain protected from their own perforin prior to its export to secretory granules, particularly in the Ca²⁺‐rich endoplasmic reticulum, remains unknown. Here, we show that N‐linked glycosylation of the perforin C‐terminus at Asn549 within the endoplasmic reticulum inhibits oligomerisation of perforin monomers and thus protects the host cell from premature pore formation. Subsequent removal of this glycan occurs through proteolytic processing of the C‐terminus within secretory granules and is imperative for perforin activation prior to secretion. Despite evolutionary conservation of the C‐terminus, we found that processing is carried out by multiple proteases, which we attribute to the unstructured and exposed nature of the region. In sum, our studies reveal a post‐translational regulatory mechanism essential for maintaining perforin in an inactive state until its secretion from the inhibitory acidic environment of the secretory granule.     

Dictyostelium LvsB mutants model the lysosomal defects associated with Chediak-Higashi syndrome

Chediak-Higashi syndrome is a genetic disorder caused by mutations in a gene encoding a protein named LYST in humans ("lysosomal trafficking regulator") or Beige in mice. A prominent feature of this disease is the accumulation of enlarged lysosome-related granules in a variety of cells. The genome of Dictyostelium discoideum contains six genes encoding proteins that are related to LYST/Beige in amino acid sequence, and disruption of one of these genes, lvsA (large volume sphere), results in profound defects in cytokinesis. To better understand the function of this family of proteins in membrane trafficking, we have analyzed mutants disrupted in lvsA, lvsB, lvsC, lvsD, lvsE, and lvsF. Of all these, only lvsA and lvsB mutants displayed interesting phenotypes in our assays. lvsA-null cells exhibited defects in phagocytosis and contained abnormal looking contractile vacuole membranes. Loss of LvsB, the Dictyostelium protein most similar to LYST/Beige, resulted in the formation of enlarged vesicles that by multiple criteria appeared to be acidic lysosomes. The rates of endocytosis, phagocytosis, and fluid phase exocytosis were normal in lvsB-null cells. Also, the rates of processing and the efficiency of targeting of lysosomal alpha-mannosidase were normal, although lvsB mutants inefficiently retained alpha-mannosidase, as well as two other lysosomal cysteine proteinases. Finally, results of pulse-chase experiments indicated that an increase in fusion rates accounted for the enlarged lysosomes in lvsB-null cells, suggesting that LvsB acts as a negative regulator of fusion. Our results support the notion that LvsB/LYST/Beige function in a similar manner to regulate lysosome biogenesis.     

Different Munc13 Isoforms Function as Priming Factors in Lytic Granule Release from Murine Cytotoxic T Lymphocytes

In order to fuse lytic granules (LGs) with the plasma membrane at the immunological synapse, cytotoxic T lymphocytes (CTLs) have to render these LGs fusion‐competent through the priming process. In secretory tissues such as brain and neuroendocrine glands, this process is mediated by members of the Munc13 protein family. In human CTLs, mutations in the Munc13‐4 gene cause a severe loss in killing efficiency, resulting in familial hemophagocytic lymphohistiocytosis type 3, suggesting a similar role of other Munc13 isoforms in the immune system. Here, we investigate the contribution of different Munc13 isoforms to the priming process of murine CTLs at both the mRNA and protein level. We demonstrate that Munc13‐1 and Munc13‐4 are the only Munc13 isoforms present in mouse CTLs. Both isoforms rescue the drastical secretion defect of CTLs derived from Munc13‐4‐deficient Jinx mice. Mobility studies using total internal reflection fluorescence microscopy indicate that Munc13‐4 and Munc13‐1 are responsible for the priming process of LGs. Furthermore, the domains of the Munc13 protein, which is responsible for functional fusion, could be identified. We conclude from these data that both isoforms of the Munc13 family, Munc13‐1 and Munc13‐4, are functionally redundant in murine CTLs .      

Secretory lysosomes - a special mechanism of regulated secretion in haemopoietic cells

The secretory granules o f cells derived from the haemopoietic lineage are 'secretory lysosomes' containing both lysosomal hydrolases and secretory proteins. Studies on cytotoxic T lymphocytes (CTLs) have elucidated several of the mechanisms that regulate protein sorting to, and secretion from, this unusual secretory organelle. In particular, recent findings from a CTL mutant have led to the hypothesis that CTLs, and other cells of the haemopoietic lineage, use specialized sorting and secretory mechanisms in which the lysosome functions as a regulated secretory granule.     

Cytotoxic T lymphocyte exocytosis: bring on the SNAREs!

Despite our general understanding of membrane traffic, the molecular machinery at the immunological synapse (IS) that regulates exocytosis of lytic granules from cytotoxic T lymphocytes (CTLs) remains elusive. The identification of disease-causing mutations in the small GTPase Rab27a, priming factor Munc13-4 and fusion protein syntaxin11 has defined an important role for these proteins in CTL exocytosis. In addition, the demonstration of a direct interaction in vitro between Rab27a and Munc13-4 suggests the possibility that the Rab27a-Munc13-4 cascade might regulate CTL exocytosis by engaging SNAREs such as syntaxin11. We propose that these SNAREs are likely to mediate the fusion of lytic granules with the plasma membrane of the IS.     

Rab27a is required for regulated secretion in cytotoxic T lymphocytes

Rab27a activity is affected in several mouse models of human disease including Griscelli (ashen mice) and Hermansky-Pudlak (gunmetal mice) syndromes. A loss of function mutation occurs in the Rab27a gene in ashen (ash), whereas in gunmetal (gm) Rab27a dysfunction is secondary to a mutation in the alpha subunit of Rab geranylgeranyl transferase, an enzyme required for prenylation and activation of Rabs. We show here that Rab27a is normally expressed in cytotoxic T lymphocytes (CTLs), but absent in ashen homozygotes (ash/ash). Cytotoxicity and secretion assays show that ash/ash CTLs are unable to kill target cells or to secrete granzyme A and hexosaminidase. By immunofluorescence and electron microscopy, we show polarization but no membrane docking of ash/ash lytic granules at the immunological synapse. In gunmetal CTLs, we show underprenylation and redistribution of Rab27a to the cytosol, implying reduced activity. Gunmetal CTLs show a reduced ability to kill target cells but retain the ability to secrete hexosaminidase and granzyme A. However, only some of the granules polarize to the immunological synapse, and many remain dispersed around the periphery of the CTLs. These results demonstrate that Rab27a is required in a final secretory step and that other Rab proteins also affected in gunmetal are likely to be involved in polarization of the granules to the immunological synapse.     

An evolutionary balance: conservation vs innovation in ciliate membrane trafficking

As most of eukaryotic diversity lies in single‐celled protists, they represent unique opportunities to ask questions about the balance of conservation and innovation in cell biological features. Among free‐living protists the ciliates offer ease of culturing, a rich array of experimental approaches, and versatile molecular tools, particularly in Tetrahymena thermophila and Paramecium tetraurelia. These attributes have been exploited by researchers to analyze a wealth of cellular structures in these large and complex cells. This mini‐review focuses on 3 aspects of ciliate membrane dynamics, all linked with endolysosomal trafficking. First is nutrition based on phagocytosis and maturation of food vacuoles. Secondly, we discuss regulated exocytosis from vesicles that have features of both dense core secretory granules but also lysosome‐related organelles. The third topic is the targeting, breakdown and resorption of parental nuclei in mating partners. For all 3 phenomena, it is clear that elements of the canonical membrane‐trafficking system have been retained and in some cases repurposed. In addition, there is evidence that recently evolved, lineage‐specific proteins provide determinants in these pathways.      

Expression and function of synaptotagmin VII in CTLs

The Ca(2+) sensor synaptotagmin (Syt) VII regulates the exocytosis of conventional lysosomes in several cell types. In CTLs, the Ca(2+)-regulated exocytosis of lytic granules/secretory lysosomes is responsible for the perforin/granzyme-mediated lysis of target cells. To investigate the role of Syt VII in CTL effector function, the expression and function of Syt VII were examined in wild-type and Syt VII-deficient mice. In comparison with Syt VII(+/+) controls, Syt VII(-/-) animals were impaired in their ability to clear an infection with the intracellular pathogen Listeria monocytogenes. When isolated CTLs were examined, we found that Syt VII is expressed upon CTL activation and localizes to granzyme A-containing lytic granules. Syt VII-deficient CTLs have no defects in proliferation and cytokine production, and their lytic granules contain normal amounts of perforin and granzyme A and polarize normally at the immunological synapse. However, despite normal conjugate formation with target cells, CTLs from Syt VII(-/-) mice exhibit reduced effector activity, when compared with controls. Treatment of Syt VII(+/+) or Syt VII(-/-) CTLs with an inhibitor of the perforin-mediated lytic pathway resulted in comparable levels of cytotoxic activity, suggesting that Syt VII regulates perforin-mediated cytolytic CTL responses.     

Polyamines are present in mast cell secretory granules and are important for granule homeostasis

Background

Mast cell secretory granules accommodate a large number of components, many of which interact with highly sulfated serglycin proteoglycan (PG) present within the granules. Polyamines (putrescine, spermidine and spermine) are absolutely required for the survival of the vast majority of living cells. Given the reported ability of polyamines to interact with PGs, we investigated the possibility that polyamines may be components of mast cell secretory granules.

Methodology/Principal Findings

Spermidine was released by mouse bone marrow derived mast cells (BMMCs) after degranulation induced by IgE/anti-IgE or calcium ionophore A23187. Additionally, both spermidine and spermine were detected in isolated mouse mast cell granules. Further, depletion of polyamines by culturing BMMCs with α-difluoromethylornithine (DFMO) caused aberrant secretory granule ultrastructure, impaired histamine storage, reduced serotonin levels and increased β-hexosaminidase content. A proteomic approach revealed that DFMO-induced polyamine depletion caused an alteration in the levels of a number of proteins, many of which are connected either with the regulated exocytosis or with the endocytic system.

Conclusions/Significance

Taken together, our results show evidence that polyamines are present in mast cell secretory granules and, furthermore, indicate an essential role of these polycations during the biogenesis and homeostasis of these organelles.     

Unique features of secretory granules observed in the pituitary growth hormone-secreting (GH) cells of the musk shrew (Suncus murinus L.)

Summary Unique rod-shaped secretory granules were observed among oval or spherical secretory granules in GH cells of the anterior pituitary gland of musk shrew using the protein A-gold procedure combined with electron microscopy. The rod-shaped and spherical secretory granules were both immunoreactive by the immuno-gold method using antiserum to sheep GH. The rod-shaped secretory granules, which seem to be formed directly from the Golgi vesicles, extend from several hundred to several thousand nm in length. They often touch each other and fuse. The spherical secretory granules are also unique in that they may also fuse with loss of dense contents to leave empty circular membrane profiles in the cytoplasm. Both the rodshaped and spherical secretory granules are secreted from the cell by exocytosis.     

VAMP8-dependent fusion of recycling endosomes with the plasma membrane facilitates T lymphocyte cytotoxicity

Cytotoxic T lymphocytes (CTLs) eliminate infected and neoplastic cells through directed release of cytotoxic granule contents. Although multiple SNARE proteins have been implicated in cytotoxic granule exocytosis, the role of vesicular SNARE proteins, i.e., vesicle-associated membrane proteins (VAMPs), remains enigmatic. VAMP8 was posited to represent the cytotoxic granule vesicular SNARE protein mediating exocytosis in mice. In primary human CTLs, however, VAMP8 colocalized with Rab11a-positive recycling endosomes. Upon stimulation, these endosomes rapidly trafficked to and fused with the plasma membrane, preceding fusion of cytotoxic granules. Knockdown of VAMP8 blocked both recycling endosome and cytotoxic granule fusion at immune synapses, without affecting activating signaling. Mechanistically, VAMP8-dependent recycling endosomes deposited syntaxin-11 at immune synapses, facilitating assembly of plasma membrane SNARE complexes for cytotoxic granule fusion. Hence, cytotoxic granule exocytosis is a sequential, multivesicle fusion process requiring VAMP8-mediated recycling endosome fusion before cytotoxic granule fusion. Our findings imply that secretory granule exocytosis pathways in other cell types may also be more complex than previously appreciated.     

Exophilin4/Slp2-a targets glucagon granules to the plasma membrane through unique Ca2+-inhibitory phospholipid-binding activity of the C2A domain

Rab27a and Rab27b have recently been recognized to play versatile roles in regulating the exocytosis of secretory granules and lysosome-related organelles by using multiple effector proteins. However, the precise roles of these effector proteins in particular cell types largely remain uncharacterized, except for those in pancreatic beta cells and in melanocytes. Here, we showed that one of the Rab27a/b effectors, exophilin4/Slp2-a, is specifically expressed in pancreatic alpha cells, in contrast to another effector, granuphilin, in beta cells. Like granuphilin toward insulin granules, exophilin4 promotes the targeting of glucagon granules to the plasma membrane. Although the interaction of granuphilin with syntaxin-1a is critical for the targeting activity, exophilin4 does this primarily through the affinity of its C2A domain toward the plasma membrane phospholipids phosphatidylserine and phosphatidylinositol-4,5-bisphosphate. Notably, the binding activity to phosphatidylserine is inhibited by a physiological range of the Ca(2+) concentration attained after secretagogue stimulation, which presents a striking contrast to the Ca(2+)-stimulatory activity of the C2A domain of synaptotagmin I. Analyses of the mutant suggested that this novel Ca(2+)-inhibitory phospholipid-binding activity not only mediates docking but also modulates the subsequent fusion of the secretory granules.