VAMP5 promotes Fcγ receptor-mediated phagocytosis and regulates phagosome maturation in macrophages.

Phagosome formation and maturation reportedly occur via sequential membrane fusion events mediated by synaptosomal-associated protein of 23 kDa (SNAP23), a plasma membrane-localized soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) family. Vesicle-associated membrane protein 5 (VAMP5), also a plasmalemma SNARE, interacts with SNAP23; however, its precise function in phagocytosis in macrophages remains elusive. To elucidate this aspect, we investigated the characteristics of macrophages in the presence of VAMP5 overexpression or knockdown and found that VAMP5 participates in Fcγ receptor-mediated phagosome formation, although not directly in phagosome maturation. Overexpressed VAMP5 was localized to the early phagosomal membrane but no longer localized to the lysosomal-associated membrane protein 1-positive maturing phagosomal membrane. Analyses using compound-based selective inhibitors demonstrated that VAMP5 dissociation from early phagosomes occurs in a clathrin- and dynamin-dependent manner and is indispensable for SNAP23 function in subsequent membrane fusion during phagosome maturation. Accordingly, to the best of our knowledge, we demonstrate, for the first time, that VAMP5 exerts an immunologically critical function during phagosome formation and maturation via SNARE-based membrane trafficking in macrophages.

Regulatory Mechanism of SNAP23 in Phagosome Formation and Maturation.

Synaptosomal associated protein of 23 kDa (SNAP23), a plasma membrane-localized soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE), is a ubiquitously expressed protein that is generally involved in fusion of the plasma membrane and secretory or endosomal recycling vesicles during several types of exocytosis. SNAP23 is expressed in phagocytes, such as neutrophils, macrophages, and dendritic cells, and functions in both exocytosis and phagocytosis. This review focuses on the function of SNAP23 in immunoglobulin G Fc receptor-mediated phagocytosis by macrophages. SNAP23 and its partner SNAREs mediate fusion of the plasma membrane with intracellular organelles or vesicles to form phagosomes as well as the fusion of phagosomes with endosomes or lysosomes to induce phagosome maturation, characterized by reactive oxygen species production and acidification. During these processes, SNAP23 function is regulated by phosphorylation. In addition, microtubule-associated protein 1A/1B light chain 3 (LC3)-associated phagocytosis, which tightly promotes or suppresses phagosome maturation depending on the foreign target, requires SNAP23 function. SNAP23 that is enriched on the phagosome membrane during LC3-associated phagocytosis may be phosphorylated or dephosphorylated, thereby enhancing or inhibiting subsequent phagosome maturation, respectively. These findings have increased our understanding of the SNAP23-associated membrane trafficking mechanism in phagocytes, which has important implications for microbial pathogenesis and innate and adaptive immune responses.

Characterization of MORN2 stability and regulatory function in LC3-associated phagocytosis in macrophages.

Microtubule-associated protein A1/B1-light chain 3 (LC3)-associated phagocytosis (LAP) is a type of non-canonical autophagy that regulates phagosome maturation in macrophages. However, the role and regulatory mechanism of LAP remain largely unknown. Recently, the membrane occupation and recognition nexus repeat-containing-2 (MORN2) was identified as a key component of LAP for the efficient formation of LC3-recruiting phagosomes. To characterize MORN2 and elucidate its function in LAP, we established a MORN2-overexpressing macrophage line. At a steady state, MORN2 was partially cleaved by the ubiquitin-proteasome system. MORN2 overexpression promoted not only LC3-II production but also LAP phagosome (LAPosome) acidification during Escherichia coli uptake. Furthermore, the formation of LAPosomes containing the yeast cell wall component zymosan was enhanced in MORN2-overexpressing cells and depended on reactive oxygen species (ROS). Finally, MORN2-mediated LAP was regulated by plasma membrane-localized soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) such as SNAP-23 and syntaxin 11. Taken together, these findings demonstrate that MORN2, whose expression is downregulated via proteasomal digestion, is a limiting factor for LAP, and that membrane trafficking by SNARE proteins is involved in MORN2-mediated LAP.

OCT3/4-binding sequence-dependent maintenance of the unmethylated state of CTCF-binding sequences with DNA demethylation and suppression of de novo DNA methylation in the H19 imprinted control region.

DNA demethylation and suppression of de novo DNA methylation are activities that maintain an unmethylated state. However, the strength of these two activities at the same locus has not been estimated separately. Furthermore, the association between these two activities and the unmethylated state remains unclear. Octamer-binding transcription factor-binding sequences (OBSs) and CCCTC-binding factor-binding sequences (CBSs) within the mouse H19-imprinted control region (ICR) are involved in the induction of DNA demethylation and maintenance of the unmethylated state in mouse undifferentiated embryonic cell lines. To reveal the association between the two cis-elements and the two unmethylated state maintenance activities in maintaining the unmethylated state of the ICR, we evaluated the altered DNA methylation levels at sites that were initially methylated or unmethylated using a stable transfection-based assay, and estimated the strength of the two unmethylated state maintenance activities separately via a Poisson process model that described the DNA methylation state regulatory process. Although DNA demethylation depending on OBSs affected almost the entire ICR, DNA demethylation depending on CBSs occurred near CBSs, resulting in redundant demethylation of CBS regions. Detailed analysis of the CBS4 region suggested that OBSs were required to induce unmethylated state maintenance activities, and that CBSs-dependent activities contributed, but diminished, during incubation when protection of the CBS4 region by OBSs-dependent activities was absent. Analysis via the Poisson process model indicated that the unmethylated state at the CBS4 region was maintained by OBSs-dependent suppression of de novo DNA methylation rather than DNA demethylation. We propose that the hierarchical regulation of redundant protection of the CBS region via cooperation between the two unmethylated state maintenance activities is a potential function of the ICR that effectively maintains allele-specific methylation status in the same DNA sequence.

Increased PD-1-positive macrophages in the tissue of gastric cancer are closely associated with poor prognosis in gastric cancer patients.

BACKGROUND: Programmed cell death 1 (PD-1) is one of the immune checkpoint molecules that negatively regulate the function of T cells. Although recent studies indicate that PD-1 is also expressed on other immune cells besides T cells, its role remains unclear. This study aims to evaluate PD-1 expression on macrophages and examine its effect on anti-tumor immunity in gastric cancer (GC) patients. METHODS: The frequency of PD-1+ macrophages obtained from GC tissue was determined by multicolor flow cytometry (n = 15). Double immunohistochemistry staining of PD-1 and CD68 was also performed to evaluate the correlations among the frequency of PD-1+ macrophages, clinicopathological characteristics, and prognosis in GC patients (n = 102). RESULTS: The frequency of PD-1+ macrophages was significantly higher in GC tissue than in non-tumor gastric tissue. The phagocytotic activity of PD-1+ macrophages was severely impaired compared with that of PD-1- macrophages. The 5-year disease-specific survival rates in patients with PD-1+ macrophageLow (the frequency of PD-1+ macrophages; < 0.85%) and those with PD-1+ macrophageHigh (the frequency of PD-1+ macrophages; ≥ 0.85%) were 85.9 and 65.8%, respectively (P = 0.008). Finally, multivariate analysis showed the frequency of PD-1+ macrophage to be an independent prognostic factor. CONCLUSIONS: The function of PD-1+ macrophage was severely impaired and increased frequency of PD-1+ macrophage worsened the prognosis of GC patients. PD-1-PD-L1 therapies may function through a direct effect on macrophages in GC.

Oct motif variants in Beckwith-Wiedemann syndrome patients disrupt maintenance of the hypomethylated state of the H19/IGF2 imprinting control region.

The methylation status of imprinting control center 1 (IC1) regulates the monoallelic transcription of H19 and Igf2 in mammalian cells. Several single nucleotide variants in Oct motifs within IC1 occur in patients with Beckwith-Wiedemann syndrome (BWS) who have hypermethylated maternal IC1. However, the importance of Oct motifs in the regulation of IC1 methylation status remains unclear. Here, we demonstrate that three variants found in BWS (BWS variants) suppress intensive induction of DNA demethylation, whereas consensus disruption of motifs unrelated to BWS only slightly affects the induction of demethylation. BWS variants reduce DNA demethylation levels and trigger the accumulation of DNA methylation downstream of the IC1 transgenes. Thus, the risk of IC1 hypermethylation is associated with inhibitory levels of Oct motif-dependent hypomethylation maintenance activities.

Syntaxin 11 regulates the stimulus-dependent transport of Toll-like receptor 4 to the plasma membrane by cooperating with SNAP-23 in macrophages.

Syntaxin 11 (stx11) is a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) that is selectively expressed in immune cells; however, its precise role in macrophages is unclear. We showed that stx11 knockdown reduces the phagocytosis of Escherichia coli in interferon-γ-activated macrophages. stx11 knockdown decreased Toll-like receptor 4 (TLR4) localization on the plasma membrane without affecting total expression. Plasma membrane-localized TLR4 was primarily endocytosed within 1 h by lipopolysaccharide (LPS) stimulation and gradually relocalized 4 h after removal of LPS. This relocalization was significantly impaired by stx11 knockdown. The lack of TLR4 transport to the plasma membrane is presumably related to TLR4 degradation in acidic endosomal organelles. Additionally, an immunoprecipitation experiment suggested that stx11 interacts with SNAP-23, a plasma membrane-localized SNARE protein, whose depletion also inhibits TLR4 replenishment in LPS-stimulated cells. Using an intramolecular Förster resonance energy transfer (FRET) probe for SNAP-23, we showed that the high FRET efficiency caused by LPS stimulation is reduced by stx11 knockdown. These findings suggest that stx11 regulates the stimulus-dependent transport of TLR4 to the plasma membrane by cooperating with SNAP-23 in macrophages. Our results clarify the regulatory mechanisms underlying intracellular transport of TLR4 and have implications for microbial pathogenesis and immune responses.

IL-13 attenuates early local CXCL2-dependent neutrophil recruitment for Candida albicans clearance during a severe murine systemic infection.

To investigate the role of IL-13 during a severe systemic Candida albicans infection, BALB/c control and IL-13-/- mice were examined for colony forming units (CFU) in the kidneys and survival days after intravenous infection. Proinflammatory mediators and cell recruitment into the tissue were measured by quantitative real-time PCR, a multiple ELISA system, and morphological cell differentiation. The IL-13-/- group exhibited a lower CFU number in the kidneys at 4 days and survived longer than the control mice, which was accompanied by significantly higher expression of C-X-C motif ligand 2 (CXCL2), IFN-γ, and polymorphonuclear neutrophils (PMNs) in the infected kidneys. By contrast, the expression of transforming growth factor ß (TGF-ß) and IL-17 A on day 10 were significantly higher in the control mice than in the IL-13-/- group. When using an intratracheal infection model, the IL-13-/- group recruited a greater number of PMNs in 6 h, with rapidly increased CXCL2 in the alveolar space. In vitro testing with cultured bone-marrow-derived cells demonstrated rapid CXCL2 mRNA upregulation at 3 h after contact with C. albicans, which decreased with recombinant IL-13 pretreatment, whereas rIL-13 retained TGF-ß upregulation. In a murine model of Candida systemic infection, preexistent IL-13 limits both the rapid CXCL2 elevation and PMN aggregation in the target organ to suppress inflammatory mediators, which also attenuates local pathogen clearance within four days.

Phosphorylation of SNAP-23 at Ser95 causes a structural alteration and negatively regulates Fc receptor-mediated phagosome formation and maturation in macrophages.

SNAP-23 is a plasma membrane-localized soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) involved in Fc receptor (FcR)-mediated phagocytosis. However, the regulatory mechanism underlying its function remains elusive. Using phosphorylation-specific antibodies, SNAP-23 was found to be phosphorylated at Ser95 in macrophages. To understand the role of this phosphorylation, we established macrophage lines overexpressing the nonphosphorylatable S95A or the phosphomimicking S95D mutation. The efficiency of phagosome formation and maturation was severely reduced in SNAP-23-S95D-overexpressing cells. To examine whether phosphorylation at Ser95 affected SNAP-23 structure, we constructed intramolecular Förster resonance energy transfer (FRET) probes of SNAP-23 designed to evaluate the approximation of the N termini of the two SNARE motifs. Interestingly, a high FRET efficiency was detected on the membrane when the S95D probe was used, indicating that phosphorylation at Ser95 caused a dynamic structural shift to the closed form. Coexpression of IκB kinase (IKK) 2 enhanced the FRET efficiency of the wild-type probe on the phagosome membrane. Furthermore, the enhanced phagosomal FRET signal in interferon-γ-activated macrophages was largely dependent on IKK2, and this kinase mediated a delay in phagosome-lysosome fusion. These results suggested that SNAP-23 phosphorylation at Ser95 played an important role in the regulation of SNARE-dependent membrane fusion during FcR-mediated phagocytosis.

Fate of methylated/unmethylated H19 imprinting control region after paternal and maternal pronuclear injection.

The paternal-allele-specific methylation of the Igf2/H19 imprinting control region (ICR) is established during gametogenesis and maintained throughout development. To elucidate the requirement of the germline passage in the maintenance of the imprinting methylation, we established a system introducing a methylated or unmethylated ICR-containing DNA fragment (ICR-F) into the paternal or maternal genome by microinjecting into the paternal or maternal pronucleus of fertilized eggs, and traced the methylation pattern in the ICR-F. When the ICR-F was injected in a methylated form, it was demethylated approximately to half degree at blastocyst stage but was almost completely remethylated at 3 weeks of age. In the case of the unmethylated form, the ICR-F remained unmethylated at the blastocyst stage, but was almost half-methylated at 3 weeks of age. Interestingly, the paternally injected ICR-F was highly methylated compared with maternally injected ICR-F at 3 weeks of age, partially mimicking the endogenous methylation pattern. Moreover, introduction of mutations in the CTCF (CCCTC binding factor) binding sites of the ICR-F, which are known to be important for the maintenance of hypomethylated maternal ICR, induced hypermethylation of the mutated ICR-F in both paternal and maternal pronuclear injected 3-week-old mice. Our results suggest the presence of a protection-against-methylation activity of the CTCF binding site in establishing the preferential paternal methylation during post-fertilization development and the importance of germline passage in the maintenance of the parental specific methylation at H19 ICR.

Quantitative analysis of phagosome formation and maturation using an Escherichia coli probe expressing a tandem fluorescent protein.

Phagosome formation and maturation are essential innate immune mechanisms to engulf and digest foreign particles. To analyze these processes quantitatively, we established a specific Escherichia coli probe expressing a tandem fluorescent protein, comprising glutathione S-transferase fused with monomeric Cherry (mCherry) and monomeric Venus (mVenus). We demonstrated that mVenus was more susceptible to bleaching in an acidic environment than mCherry, and that the mVenus:mCherry fluorescence intensity ratio can be used to monitor phagosomal pH changes during maturation. Using this probe, we revealed that synaptosomal-associated protein of 23 kDa, a plasma membrane soluble N-ethylmaleimide-sensitive factor attachment protein receptor protein, actively regulated phagocytosis of E. coli and subsequent phagosome maturation in macrophages. Our results indicated that this probe has the potential to be a powerful tool in understanding the molecular mechanisms of phagosome formation and maturation.

Histone macroH2A1.2 promotes metabolic health and leanness by inhibiting adipogenesis.

BACKGROUND: Obesity has tremendous impact on the health systems. Its epigenetic bases are unclear. MacroH2A1 is a variant of histone H2A, present in two alternatively exon-spliced isoforms macroH2A1.1 and macroH2A1.2, regulating cell plasticity and proliferation, during pluripotency and tumorigenesis. Their role in adipose tissue plasticity is unknown. RESULTS: Here, we show evidence that macroH2A1.1 protein levels in the visceral adipose tissue of obese humans positively correlate with BMI, while macroH2A1.2 is nearly absent. We thus introduced a constitutive GFP-tagged transgene for macroH2A1.2 in mice, and we characterized their metabolic health upon being fed a standard chow diet or a high fat diet. Despite unchanged food intake, these mice exhibit lower adipose mass and improved glucose metabolism both under a chow and an obesogenic diet. In the latter regimen, transgenic mice display smaller pancreatic islets and significantly less inflammation. MacroH2A1.2 overexpression in the mouse adipose tissue induced dramatic changes in the transcript levels of key adipogenic genes; genomic analyses comparing pre-adipocytes to mature adipocytes uncovered only minor changes in macroH2A1.2 genomic distribution upon adipogenic differentiation and suggested differential cooperation with transcription factors. MacroH2A1.2 overexpression markedly inhibited adipogenesis, while overexpression of macroH2A1.1 had opposite effects. CONCLUSIONS: MacroH2A1.2 is an unprecedented chromatin component powerfully promoting metabolic health by modulating anti-adipogenic transcriptional networks in the differentiating adipose tissue. Strategies aiming at enhancing macroH2A1.2 expression might counteract excessive adiposity in humans.

SNAP23/25 and VAMP2 mediate exocytic event of transferrin receptor-containing recycling vesicles.

We recently showed that Rab11 is involved not only in formation of recycling vesicles containing the transferrin (Tfn)-transferrin receptor (TfnR) complex at perinuclear recycling endosomes but also in tethering of recycling vesicles to the plasma membrane (PM) in concert with the exocyst tethering complex. We here aimed at identifying SNARE proteins responsible for fusion of Tfn-TfnR-containing recycling vesicles with the PM, downstream of the exocyst. We showed that exocyst subunits, Sec6 and Sec8, can interact with SNAP23 and SNAP25, both of which are PM-localizing Qbc-SNAREs, and that depletion of SNAP23 and/or SNAP25 in HeLa cells suppresses fusion of Tfn-TfnR-containing vesicles with the PM, leading to accumulation of the vesicles at the cell periphery. We also found that VAMP2, an R-SNARE, is colocalized with endocytosed Tfn on punctate endosomal structures, and that its depletion in HeLa cells suppresses recycling vesicle exocytosis. These observations indicate that fusion of recycling vesicles with the PM downstream of the exocyst is mediated by SNAP23/25 and VAMP2, and provide novel insight into non-neuronal roles of VAMP2 and SNAP25.

SNAP-23 regulates phagosome formation and maturation in macrophages.

Synaptosomal associated protein of 23 kDa (SNAP-23), a plasma membrane-localized soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE), has been implicated in phagocytosis by macrophages. For elucidation of its precise role in this process, a macrophage line overexpressing monomeric Venus-tagged SNAP-23 was established. These cells showed enhanced Fc receptor-mediated phagocytosis. Detailed analyses of each process of phagocytosis revealed a marked increase in the production of reactive oxygen species within phagosomes. Also, enhanced accumulation of a lysotropic dye, as well as augmented quenching of a pH-sensitive fluorophore were observed. Analyses of isolated phagosomes indicated the critical role of SNAP-23 in the functional recruitment of the NADPH oxidase complex and vacuolar-type H(+)-ATPase to phagosomes. The data from the overexpression experiments were confirmed by SNAP-23 knockdown, which demonstrated a significant delay in phagosome maturation and a reduction in uptake activity. Finally, for analyzing whether phagosomal SNAP-23 entails a structural change in the protein, an intramolecular Förster resonance energy transfer (FRET) probe was constructed, in which the distance within a TagGFP2-TagRFP was altered upon close approximation of the N-termini of its two SNARE motifs. FRET efficiency on phagosomes was markedly enhanced only when VAMP7, a lysosomal SNARE, was coexpressed. Taken together, our results strongly suggest the involvement of SNAP-23 in both phagosome formation and maturation in macrophages, presumably by mediating SNARE-based membrane traffic.

Development of cysteine-free fluorescent proteins for the oxidative environment.

Molecular imaging employing fluorescent proteins has been widely used to highlight specific reactions or processes in various fields of the life sciences. Despite extensive improvements of the fluorescent tag, this technology is still limited in the study of molecular events in the extracellular milieu. This is partly due to the presence of cysteine in the fluorescent proteins. These proteins almost cotranslationally form disulfide bonded oligomers when expressed in the endoplasmic reticulum (ER). Although single molecule photobleaching analysis showed that these oligomers were not fluorescent, the fluorescent monomer form often showed aberrant behavior in folding and motion, particularly when fused to cysteine-containing cargo. Therefore we investigated whether it was possible to eliminate the cysteine without losing the brightness. By site-saturated mutagenesis, we found that the cysteine residues in fluorescent proteins could be replaced with specific alternatives while still retaining their brightness. cf(cysteine-free)SGFP2 showed significantly reduced restriction of free diffusion in the ER and marked improvement of maturation when fused to the prion protein. We further applied this approach to TagRFP family proteins and found a set of mutations that obtains the same level of brightness as the cysteine-containing proteins. The approach used in this study to generate new cysteine-free fluorescent tags should expand the application of molecular imaging to the extracellular milieu and facilitate its usage in medicine and biotechnology.

Disruption of the fusion of Leishmania parasitophorous vacuoles with ER vesicles results in the control of the infection.

Parasitophorous vacuoles (PV) that harbour Leishmania parasites acquire some characteristics from fusion with host cell vesicles. Recent studies have shown that PVs acquire and display resident endoplasmic reticulum (ER) molecules. We investigated the importance of ER molecules to PV biology by assessing the consequence of blocking the fusion of PVs with vesicles that originate from the early secretory pathway. This was achieved by targeting the N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) that mediate the fusion of early secretory vesicles. In the presence of dominant negative variants of sec22b or some of its known cognate partners, D12 and syntaxin 18, PVs failed to distend and harboured fewer parasites. These observations were confirmed in studies in which each of the SNAREs listed above including the intermediate compartment ER/Golgi SNARE, syntaxin 5, was knocked down. The knock-down of these SNARES had little or no measurable effect on the morphology of the ER or on activated secretion even though they resulted in a more significant reduction of PV size. Moreover, the knock-down of the ER/Golgi SNAREs resulted in significant reduction in parasite replication. Taken together, these studies provide further evidence that PVs acquire ER components by fusing with vesicles derived from the early secretory pathway; disruption of this interaction results in inhibition of the development of PVs as well as the limitation of parasite replication within infected cells.

Leishmania parasitophorous vacuoles interact continuously with the host cell's endoplasmic reticulum; parasitophorous vacuoles are hybrid compartments.

Macrophages that express representative endoplasmic reticulum (ER) molecules tagged with green fluorescence protein were generated to assess the recruitment of ER molecules to Leishmania parasitophorous vacuoles (PVs). More than 90% of PVs harbouring Leishmania pifanoi or Leishmania donovani parasites recruited calnexin, to their PV membrane. An equivalent proportion of PVs also recruited the membrane-associated soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), Sec22b. Both ER molecules appeared to be recruited very early in the formation of nascent PVs. Electron microscopy analysis of infected Sec22b/YFP expressing cells confirmed that Sec22b was recruited to Leishmania PVs. In contrast to PVs, it was found that no more than 20% of phagosomes that harboured Zymosan particles recruited calnexin or Sec22b to their limiting phagosomal membrane. The retrograde pathway that ricin employs to access the cell cytosol was exploited to gain further insight into ER-PV interactions. Ricin was delivered to PVs in infected cells incubated with ricin. Incubation of cells with brefeldin A blocked the transfer of ricin to PVs. This implied that molecules that traffic to the ER are transferred to PVs. Moreover the results show that PVs are hybrid compartments that are composed of both host ER and endocytic pathway components.

Sec22b is a negative regulator of phagocytosis in macrophages.

The endoplasmic reticulum (ER) is proposed to be a membrane donor for phagosome formation. In support of this, we have previously shown that the expression level of syntaxin 18, an ER-localized SNARE protein, correlates with phagocytosis activity. To obtain further insights into the involvement of the ER in phagocytosis we focused on Sec22b, another ER-localized SNARE protein that is also found on phagosomal membranes. In marked contrast to the effects of syntaxin 18, we report here that phagocytosis was nearly abolished in J774 macrophages stably expressing mVenus-tagged Sec22b, without affecting the cell surface expression of the Fc receptor or other membrane proteins related to phagocytosis. Conversely, the capacity of the parental J774 cells for phagocytosis was increased when endogenous Sec22b expression was suppressed. Domain analyses of Sec22b revealed that the R-SNARE motif, a selective domain for forming a SNARE complex with syntaxin18 and/or D12, was responsible for the inhibition of phagocytosis. These results strongly support the ER-mediated phagocytosis model and indicate that Sec22b is a negative regulator of phagocytosis in macrophages, most likely by regulating the level of free syntaxin 18 and/or D12 at the site of phagocytosis.

Regulated motion of glycoproteins revealed by direct visualization of a single cargo in the endoplasmic reticulum.

The quality of cargo proteins in the endoplasmic reticulum (ER) is affected by their motion during folding. To understand how the diffusion of secretory cargo proteins is regulated in the ER, we directly analyze the motion of a single cargo molecule using fluorescence imaging/fluctuation analyses. We find that the addition of two N-glycans onto the cargo dramatically alters their diffusion by transient binding to membrane components that are confined by hyperosmolarity. Via simultaneous observation of a single cargo and ER exit sites (ERESs), we could exclude ERESs as the binding sites. Remarkably, actin cytoskeleton was required for the transient binding. These results provide a molecular basis for hypertonicity-induced immobilization of cargo, which is dependent on glycosylation at multiple sites but not the completion of proper folding. We propose that diffusion of secretory glycoproteins in the ER lumen is controlled from the cytoplasm to reduce the chances of aggregation.