Cell Contact Sites Research Articles

Cell-to-Cell Spread of Dengue Viral RNA in Mosquito Cells

Dengue virus (DENV) is an important mosquito-borne arbovirus that is particularly prevalent in tropical and subtropical areas of the world. The virus is generally ingested with a blood meal, replicates in host tissues, and disseminates into salivary glands for transmission to the next host. Membrane-bound vacuoles carrying DENV particles have been documented in mosquito cells and play a role in the cell-to-cell transmission of DENV2. C189 is one member of the tetraspanin family and generally increases its expression as one component of the vacuoles (C189-VCs) within C6/36 cells infected with DENV2. In the present study, we have further demonstrated via sucrose gradient centrifugation as well as magnetic immune isolation (MI) that the RNA of DENV2 was eventually carried by C189-VCs. In addition, viral RNA was shown to spread from donor to recipient cells in a coculture assay even when 20 mM NH4Cl was added to inhibit virus replication in the culture. In an alternate assay using the transwell system, viral RNA was only detected in recipient cells in the absence of 40 mM NH4Cl, suggesting that cell-cell contact is required for the intercellular spread of DENV2. In turn, the formation of viral synapse (VS) derived from aggregates of viral particles was frequently observed at sites of cell contact. Taken together, the formation of C189-VCs in C6/36 cells is induced by DENV2 infection, which may serve as a vehicle for transferring virions and also viral RNA to neighboring cells by cell-to-cell transmission after cell-cell contact. This finding provides insight into the understanding of viral spread between mosquito cells. It may also elucidate the benign persistent infection in mosquito cells and efficient dissemination of DENV infection within a mosquito vector.

Cbl-Associated Protein CAP contributes to correct formation and robust function of the Drosophila heart tube.

The formation of a tube-like structure is a basic step in the making of functional hearts in vertebrates and invertebrates and therefore, its understanding provides important information on heart development and function. In Drosophila, the cardiac tube originates from two bilateral rows of dorsally migrating cells. On meeting at the dorsal midline, coordinated changes in cell shape and adhesive properties transform the two sheets of cells into a linear tube. ECM and transmembrane proteins linked to the cytoskeleton play an important role during these dynamic processes. Here we characterize the requirement of Cbl-Associated Protein (CAP) in Drosophila heart formation. In embryos, CAP is expressed in late migrating cardioblasts and is located preferentially at their luminal and abluminal periphery. CAP mutations result in irregular cardioblast alignment and imprecisely controlled cardioblast numbers. Furthermore, CAP mutant embryos show a strongly reduced heart lumen and an aberrant shape of lumen forming cardioblasts. Analysis of double heterozygous animals reveals a genetic interaction of CAP with Integrin- and Talin-encoding genes. In post-embryonic stages, CAP closely colocalizes with Integrin near Z-bands and at cell–cell contact sites. CAP mutants exhibit a reduced contractility in larval hearts and show a locally disrupted morphology, which correlates with a reduced pumping efficiency. Our observations imply a function of CAP in linking Integrin signaling with the actin cytoskeleton. As a modulator of the cytoskeleton, CAP is involved in the establishment of proper cell shapes during cardioblast alignment and cardiac lumen formation in the Drosophila embryo. Furthermore, CAP is required for correct heart function throughout development.

Contact-ID, a tool for profiling organelle contact sites, reveals regulatory proteins of mitochondrial-associated membrane formation.

The mitochondria-associated membrane (MAM) has emerged as a cellular signaling hub regulating various cellular processes. However, its molecular components remain unclear owing to lack of reliable methods to purify the intact MAM proteome in a physiological context. Here, we introduce Contact-ID, a split-pair system of BioID with strong activity, for identification of the MAM proteome in live cells. Contact-ID specifically labeled proteins proximal to the contact sites of the endoplasmic reticulum (ER) and mitochondria, and thereby identified 115 MAM-specific proteins. The identified MAM proteins were largely annotated with the outer mitochondrial membrane (OMM) and ER membrane proteins with MAM-related functions: e.g., FKBP8, an OMM protein, facilitated MAM formation and local calcium transport at the MAM. Furthermore, the definitive identification of biotinylation sites revealed membrane topologies of 85 integral membrane proteins. Contact-ID revealed regulatory proteins for MAM formation and could be reliably utilized to profile the proteome at any organelle-membrane contact sites in live cells.

HIV Reactivation in Latently Infected Cells With Virological Synapse-Like Cell Contact.

HIV reactivation from latency is induced by cytokines but also by cell contact with other cells. To better understand this, J1.1 cells, a latent HIV-1-infected Jurkat derivative, were cocultured with its parental Jurkat. J1.1 cells became p17MA-positive and produced a high level of HIV p24CA antigen, only when they were cocultured with stimulated Jurkat with cell-to-cell contact. In contrast, very little p24CA was produced when they were cocultured without cell contact. Similar results were obtained when latent ACH-2 and its parental A3.01 cells were cocultured. Confocal microscopy revealed that not only HIV-1 p17MA and gp120Env but also LFA-1, CD81, CD59, and TCR CD3 accumulated at the cell contact site, suggesting formation of the virological synapse-like structure. LFA-1–ICAM-1 interaction was involved in the cell-to-cell contact. When J1.1 was cocultured with TCR-deficient Jurkat, the p17MA-positive rate was significantly lower, although the cell-to-cell contact was not impaired. Quantitative proteomics identified 54 membrane molecules, one of which was MHC class I, that accumulated at the cell contact site. Reactivation from latency was also influenced by the presence of stromal cells. Our study indicated that latent HIV-1 in J1.1/ACH-2 cells was efficiently reactivated by cell-to-cell contact with stimulated parental cells, accompanying the virological synapse-like structure.

The Rho-guanine nucleotide exchange factor Solo decelerates collective cell migration by modulating the Rho-ROCK pathway and keratin networks.

Collective cell migration plays crucial roles in tissue remodeling, wound healing, and cancer cell invasion. However, its underlying mechanism remains unknown. Previously, we showed that the RhoA-targeting guanine nucleotide exchange factor Solo (ARHGEF40) is required for tensile force–induced RhoA activation and proper organization of keratin-8/keratin-18 (K8/K18) networks. Here, we demonstrate that Solo knockdown significantly increases the rate at which Madin-Darby canine kidney cells collectively migrate on collagen gels. However, it has no apparent effect on the migratory speed of solitary cultured cells. Therefore, Solo decelerates collective cell migration. Moreover, Solo localized to the anteroposterior regions of cell–cell contact sites in collectively migrating cells and was required for the local accumulation of K8/K18 filaments in the forward areas of the cells. Partial Rho-associated protein kinase (ROCK) inhibition or K18 or plakoglobin knockdown also increased collective cell migration velocity. These results suggest that Solo acts as a brake for collective cell migration by generating pullback force at cell–cell contact sites via the RhoA-ROCK pathway. It may also promote the formation of desmosomal cell–cell junctions related to K8/K18 filaments and plakoglobin.

Innate Antifungal Immune Receptor, Dectin-1, Undergoes Ligand-Induced Oligomerization with Highly Structured β-glucans and at Fungal Cell Contact Sites

Dectin-1A is a C-type Lectin innate immunoreceptor that recognizes β-(1,3)-glucan, a structural component of Candida species cell walls. The higher order structure of β-glucans ranges from random coil to insoluble fiber due to varying degrees of tertiary (helical) and quaternary structure. Model Saccharomyces cerevisiae β-glucans of medium and high molecular weight (MMW and HMW, respectively) are highly structured. In contrast, low MW glucan (LMW) are much less structured. Despite similar affinity for Dectin-1, the ability of glucans to induce Dectin-1A mediated calcium influx and Syk phosphorylation positively correlates with their degree of higher order structure. Chemical denaturation and renaturation of MMW glucan showed that glucan structure determines agonistic potential, but not binding affinity, for Dectin-1A. We explored the role of glucan structure on Dectin-1A oligomerization, which is thought to be required for Dectin-1 signaling. Decreased Dectin-1A diffusion coefficient inversely proportional to glucan structural content was consistent with Dectin-1A aggregation. Förster Resonance Energy Transfer (FRET) measurements revealed that molecular aggregation of Dectin-1 occurs in a manner dependent upon glucan higher order structure. Numbers and Brightness analysis specifically confirmed an increase in the Dectin-1A dimer and oligomer populations that is correlated with glucan structure content. Receptor modeling data confirms that in resting cells, Dectin-1A is predominantly in a monomeric state. Super Resolution Microscopy revealed that glucan-stimulated Dectin-1 aggregates are small (<15 nm) collections of engaged receptor. Finally, FRET measurements confirmed increased molecular aggregation of Dectin-1A at fungal particle contact sites in a manner that positively correlated with the degree of exposed glucan on the particle surface. These results indicate that Dectin-1A senses the solution conformation of β-glucans through their varying ability to drive receptor dimer/oligomer formation and activation of membrane proximal signaling events.

Restoration of calcium-induced differentiation potential and tight junction formation in HaCaT keratinocytes by functional attenuation of overexpressed highmobility group box-1 protein.

HaCaT cells have been widely used as undifferentiated epidermal keratinocytes, since these non-tumorigenic cells can be readily maintained in conventional medium and partly retain epidermal differentiation potential upon stimulation with high concentration of calcium. In contrast to primary epidermal keratinocytes, however, these cells never form tight junction (TJ), a specific structure in highly differentiated keratinocytes, solely by the differentiation stimulation. Here, we show that HaCaT cells secrete a considerable amount of highmobility group box-1 protein (HMGB1), one of major inflammatory mediator, which appeared to be responsible, at least in part, for such aberrant differentiation response. So far, inhibition of c-Jun N-terminal kinase (JNK) in high calcium medium has been supposed to be the only way to induce TJ formations in HaCaT cells; however, SP600125, a potent inhibitor of JNK showed cytostatic effects and clearly attenuated epidermal differentiation and stratification. In contrast, dipotassium glycyrrhizate (GK2), a soluble analogue of HMGB1-blocker Glycyrrhizin, down-regulated interferon-β, a typical inflammatory cytokine induced by secreted HMGB1, and accelerated differentiation responses to the calcium treatment in these cells. In addition, GK2-treatmenrt resulted in the formation of double cell layers in cultured HaCaT cells, where the stratified upper cells transiently accumulated TJ proteins at the cell-cell contact sites. These results highlight the importance of attenuation of secreted HMGB1-signals in cultured HaCaT cells for studies of functional keratinocytes.

CD162 Is a Key E-Selectin Receptor Promoting Acute Myeloid Leukemia Chemo-Resistance in the Bone Marrow Niche

We have recently found that the vascular adhesion molecule E(endothelial)-selectin is a critical bone marrow niche component mediating acute myeloid leukemia (AML) chemo-resistance. Clinical trials involving the use of E-selectin mimetics to improve efficacy of conventional AML therapy are in progress. In this study we investigate the identity of the AML cell surface receptors mediating vascular E-selectin-induced chemo-resistance. E-selectin has two well-characterised receptors, CD162 also known as P-Selectin Glycoprotein Ligand-1 (PSGL-1), and CD44 isoform HCELL (Hematopoietic Cell E-selectin/L-selectin Ligand) with other cell surface glycoproteins (such as ESL-1) and glycolipids also binding E-selectin after Sialyl Lewisx/a glycosylation. To investigate which of these AML cell surface receptors are responsible for mediating vascular E-selectin survival signalling, we first investigated if each co-localized with E-selectin on AML cell surface by confocal imaging. Human CD34+ AML KG1a cells were labelled ± adhesion to fluorescently E-selectin-IgM. Confocal imaging revealed that although both canonical CD44 and CD162 receptors co-localized with E-selectin at the site of cell contact, only CD162 became strongly polarized at E-selectin binding site, while CD44 remained widely distributed across the cell surface. To dissect a functional role for each of these canonical receptors in human AML, CRISPR-Cas9 gene editing was used to selectively delete CD44 and/or CD162 from human AML KG1a cells. We found that although deletion of both CD44 and CD162 receptors reduced KG1a E-selectin-IgM binding potential (3-fold), deletion of either receptor alone did not. Next, we investigated whether deletion of either receptor reversed E-selectin-mediated chemo-resistance in an in vitro chemosensitivity assay. KG1a cells were seeded in wells pre-coated with a range of vascular adhesion molecules commonly expressed in the bone marrow niche, then monitored for cell survival after 48hr treatment ± cytarabine. In this in vitro assay, we found that adhesion to E-selectin significantly increased parental KG1a survival to chemotherapy (p=0.0035). No similar increase in survival was observed following adhesion to P-selectin, or with integrin ligands ICAM-1 and PE-CAM-1. When we repeated the assay using Crispr CD44 deleted KG1a AML we found significant E-selectin-mediated chemo-resistance was still observed (p=0.027) even in the absence of CD44. These results suggest CD44 is not the receptor mediating AML chemo-resistance. In contrast E-selectin-mediated chemo-resistance was abrogated in the CD162 Crispr deleted human KG1a AMLs. Together these data suggest CD162/PSGL-1 expressed on the surface of human AML KG1a appears to be the receptor mediating vascular E-selectin chemo-resistance. This would be a completely novel role described for CD162 which is conventionally known as a homing molecule. To confirm this new role for CD162 in mediating AML chemo-resistance can be replicated in pre-clinical models in vivo, we next generated (11q23-rearranged) AML from CD44-/- and/or CD162-/- gene-deleted mice by retroviral transduction of murine hematopoietic stem cells with MLL-AF9 which then were transplanted into wildtype mice. Cohorts of leukemic mice (n=8/gp) were administered induction therapy (cytarabine/doxorubicin) to monitor impact on disease-free survival. In contrast to AMLs from wildtype, we found absence of CD162 in murine AMLs lead to a pronounced chemo-sensitisation in vivo resulting in a significant (6-fold, p=0.0004) extension in overall disease-free survival duration, compared to either no chemotherapy gene-deleted AML controls, or to treated wildtype AML controls. These in vivo murine data confirm the identification of an exciting new role for CD162 as an important cell surface receptor mediating therapy resistance in AML. In conclusion, we describe a novel form of niche-mediated chemo-resistance and identify CD162 as a key AML cell surface receptor involved in both human and mouse AML therapy resistance. CD162/PSGL-1 expression has not previously been implicated in direct therapy resistance. Together these findings help extend our knowledge on the potential mechanisms by which therapeutic blockade of vascular E-selectin can significantly improves therapy outcomes. Disclosures Levesque: GlycoMimetics: Equity Ownership. Magnani:GlycoMimetics Inc: Employment, Equity Ownership. Winkler:GlycoMimetics: Patents &amp; Royalties.

Cytotoxic Granule Exocytosis From Human Cytotoxic T Lymphocytes Is Mediated by VAMP7.

Cytotoxic T lymphocytes kill infected or malignant cells through the directed release of cytotoxic substances at the site of target cell contact, the immunological synapse. While genetic association studies of genes predisposing to early-onset life-threatening hemophagocytic lymphohistiocytosis has identified components of the plasma membrane fusion machinery, the identity of the vesicular components remain enigmatic. Here, we identify VAMP7 as an essential component of the vesicular fusion machinery of primary, human T cells. VAMP7 co-localizes with granule markers throughout all stages of T cell maturation and simultaneously fuses with granule markers at the IS. Knock-down of VAMP7 expression significantly decreased the killing efficiency of T cells, without diminishing early T cell receptor signaling. VAMP7 exerts its function in a SNARE complex with Syntaxin11 and SNAP-23 on the plasma membrane. The identification of the minimal fusion machinery in T cells provides a starting point for the development of potential drugs in immunotherapy.

C-FLIP and CD95 signaling are essential for survival of renal cell carcinoma

Clear cell renal cell carcinoma (ccRCC) is the most-prominent tumor type of kidney cancers. Resistance of renal cell carcinoma (RCC) against tumor therapy is often owing to apoptosis resistance, e.g., by overexpression of anti-apoptotic proteins. However, little is known about the role of the apoptosis inhibitor c-FLIP and its potential impact on death receptor-induced apoptosis in ccRCC cells. In this study, we demonstrate that c-FLIP is crucial for resistance against CD95L-induced apoptosis in four ccRCC cell lines. Strikingly, downregulation of c-FLIP expression by short hairpin RNA (shRNA)interference led to spontaneous caspase activation and apoptotic cell death. Of note, knockdown of all c-FLIP splice variants was required to induce apoptosis. Stimulation of ccRCC cells with CD95L induced NF-κB and MAP kinase survival pathways as revealed by phosphorylation of RelA/p65 and Erk1/2. Interestingly, CD95L surface expression was high in all cell lines analyzed, and CD95 but not TNF-R1 clustered at cell contact sites. Downstream of CD95, inhibition of the NF-κB pathway led to spontaneous cell death. Surprisingly, knockdown experiments revealed that c-FLIP inhibits NF-κB activation in the context of CD95 signaling. Thus, c-FLIP inhibits apoptosis and dampens NF-κB downstream of CD95 but allows NF-κB activation to a level sufficient for ccRCC cell survival. In summary, we demonstrate a complex CD95-FLIP-NF-κB-signaling circuit, in which CD95-CD95L interactions mediate a paracrine survival signal in ccRCC cells with c-FLIP and NF-κB both being required for inhibiting cell death and ensuring survival. Our findings might lead to novel therapeutic approaches of RCC by circumventing apoptosis resistance.

Diacylglycerol kinase control of protein kinase C.

The diacylglycerol kinases (DGK) are lipid kinases that transform diacylglycerol (DAG) into phosphatidic acid (PA) in a reaction that terminates DAG-based signals. DGK provide negative regulation to conventional and novel protein kinase C (PKC) enzymes, limiting local DAG availability in a tissue- and subcellular-restricted manner. Defects in the expression/activity of certain DGK isoforms contribute substantially to cognitive impairment and mental disorders. Abnormal DGK overexpression in tumors facilitates invasion and resistance to chemotherapy preventing tumor immune destruction by tumor-infiltrating lymphocytes. Effective translation of these findings into therapeutic approaches demands a better knowledge of the physical and functional interactions between the DGK and PKC families. DGKζ is abundantly expressed in the nervous and immune system, where physically and functionally interacts with PKCα. The latest discoveries suggest that PDZ-mediated interaction facilitates spatial restriction of PKCα by DGKζ at the cell-cell contact sites in a mechanism where the two enzymes regulate each other. In T lymphocytes, DGKζ interaction with Sorting Nexin 27 (SNX27) guarantees the basal control of PKCα activation. SNX27 is a trafficking component required for normal brain function whose deficit has been linked to Alzheimer's disease (AD) pathogenesis. The enhanced PKCα activation as the result of SNX27 silencing in T lymphocytes aligns with the recent correlation found between gain-of-function PKCα mutations and AD and suggests that disruption of the mechanisms that provides a correct spatial organization of DGKζ and PKCα may lie at the basis of immune and neuronal synapse impairment.

Pannexin 2 Localizes at ER-Mitochondria Contact Sites.

Endomembrane specialization allows functional compartmentalization but imposes physical constraints to information flow within the cell. However, the evolution of an endomembrane system was associated with the emergence of contact sites facilitating communication between membrane-bound organelles. Contact sites between the endoplasmic reticulum (ER) and mitochondria are highly conserved in terms of their morphological features but show surprising molecular diversity within and across eukaryote species. ER-mitochondria contact sites are thought to regulate key processes in oncogenesis but their molecular composition remains poorly characterized in mammalian cells. In this study, we investigate the localization of pannexin 2 (Panx2), a membrane channel protein showing tumor-suppressing properties in cancer cells. Using a combination of subcellular fractionation, particle tracking in live-cell, and immunogold electron microscopy, we show that Panx2 localizes at ER-mitochondria contact sites in mammalian cells and sensitizes cells to apoptotic stimuli.

UBTD1 is a mechano-regulator controlling cancer aggressiveness.

Ubiquitin domain-containing protein 1 (UBTD1) is highly evolutionary conserved and has been described to interact with E2 enzymes of the ubiquitin-proteasome system. However, its biological role and the functional significance of this interaction remain largely unknown. Here, we demonstrate that depletion of UBTD1 drastically affects the mechanical properties of epithelial cancer cells via RhoA activation and strongly promotes their aggressiveness. On a stiff matrix, UBTD1 expression is regulated by cell-cell contacts, and the protein is associated with β-catenin at cell junctions. Yes-associated protein (YAP) is a major cell mechano-transducer, and we show that UBTD1 is associated with components of the YAP degradation complex. Interestingly, UBTD1 promotes the interaction of YAP with its E3 ubiquitin ligase β-TrCP Consequently, in cancer cells, UBTD1 depletion decreases YAP ubiquitylation and triggers robust ROCK2-dependent YAP activation and downstream signaling. Data from lung and prostate cancer patients further corroborate the in cellulo results, confirming that low levels of UBTD1 are associated with poor patient survival, suggesting that biological functions of UBTD1 could be beneficial in limiting cancer progression.

Natural ligand-nonmimetic inhibitors of the lipid-transfer protein CERT

Lipid transfer proteins mediate inter-organelle transport of membrane lipids at organelle contact sites in cells, playing fundamental roles in the lipidome and membrane biogenesis in eukaryotes. We previously developed a ceramide-mimetic compound as a potent inhibitor of the ceramide transport protein CERT. Here we develop CERT inhibitors with structures unrelated to ceramide. To this aim, we identify a seed compound with no ceramide-like structure but with the capability of forming a hydrogen-bonding network in the ceramide-binding START domain, by virtual screening of ~3 × 106 compounds. We also establish a surface plasmon resonance-based system to directly determine the affinity of compounds for the START domain. Then, we subject the seed compound to a series of in silico docking simulations, efficient chemical synthesis, affinity analysis, protein-ligand co-crystallography, and various in vivo assays. This strategy allows us to obtain ceramide-unrelated compounds that potently inhibited the function of CERT in human cultured cells.

Sensory neuropathy-causing mutations in ATL3 affect ER-mitochondria contact sites and impair axonal mitochondrial distribution.

Axonopathies are neurodegenerative disorders caused by axonal degeneration, affecting predominantly the longest neurons. Several of these axonopathies are caused by genetic defects in proteins involved in the shaping and dynamics of the endoplasmic reticulum (ER); however, it is unclear how these defects impinge on neuronal survival. Given its central and widespread position within a cell, the ER is a pivotal player in inter-organelle communication. Here, we demonstrate that defects in the ER fusion protein ATL3, which were identified in patients suffering from hereditary sensory and autonomic neuropathy, result in an increased number of ER–mitochondria contact sites both in HeLa cells and in patient-derived fibroblasts. This increased contact is reflected in higher phospholipid metabolism, upregulated autophagy and augmented Ca2+ crosstalk between both organelles. Moreover, the mitochondria in these cells display lowered motility, and the number of axonal mitochondria in neurons expressing disease-causing mutations in ATL3 is strongly decreased. These results underscore the functional interdependence of subcellular organelles in health and disease and show that disorders caused by ER-shaping defects are more complex than previously assumed.

Andrographolide prevented toluene diisocyanate-induced occupational asthma and aberrant airway E-cadherin distribution via p38 MAPK-dependent Nrf2 induction

Toluene diisocyanate (TDI) is a major cause of chemical-induced occupational asthma, which contributes about 15% of global asthma burden. Resistance and compounded side effects associated with the use of corticosteroid in asthma necessitate the search for alternative drugs. Andrographolide (AGP), a naturally occurring diterpene lactone is known to exhibit various bioactivities. Its ability to ameliorate cardinal features of allergic asthma was previously suggested in an eosinophilic asthma endotype. However, its potential antiasthma activity and mechanism of action in a neutrophilic occupational asthma model, as well as its effect on epithelial dysfunction remain unknown. BALB/c mice were dermally sensitised with 0.3% TDI or acetone olive oil (AOO) vehicle on day 1 and 8, followed by 0.1% TDI intranasal challenge on days 15, 18 and 21. Endpoints were evaluated via bronchoalveolar lavage fluid (BALF) cell analysis, 2′,7′-dichlorofluorescein diacetate (DCFDA) assays, immunoblotting, immunohistochemistry and methacholine challenge test. Decreases in total and differential leukocyte counts of BALF were recorded in AGP-treated animals. The compound dose-dependently reduced intracellular de-esterification of DCFDA, thus suggesting AGP's potential to inhibit intracellular reactive oxygen species (ROS). Mechanistically, the treatment prevented TDI-induced aberrant E-cadherin distribution and restored airway epithelial β-catenin at cell to cell contact site. Furthermore, AGP ameliorated TDI induced pulmonary collagen deposition. In addition, the treatment significantly upregulated pulmonary HO-1, Nrf2 and phospho-p38 levels. Airway hyperresponsiveness was markedly suppressed among AGP-treated animals. Collectively, these findings suggest AGP's protective function against TDI-induced airway epithelial barrier dysfunction and oxidative lung damage possibly through the upregulation of adherence junction proteins and the activation of p38/Nrf2 signalling. This study elucidates the therapeutic potential of AGP in the control and management of chemical-induced allergic asthma. To the best of our knowledge, the potential anti-asthma activity of AGP in TDI-induced occupational asthma has not been reported previously.

Cadherins Interact With Synaptic Organizers to Promote Synaptic Differentiation.

Classical cadherins, a set of ~20 related recognition and signaling molecules, have been implicated in many aspects of neural development, including the formation and remodeling of synapses. Mechanisms underlying some of these steps have been studied by expressing N-cadherin (cdh2), a Type 1 cadherin, in heterologous cells, but analysis is complicated because widely used lines express cdh2 endogenously. We used CRISPR-mediated gene editing to generate a Human embryonic kidney (HEK)293 variant lacking Cdh2, then compared the behavior of rodent cortical and hippocampal neurons co-cultured with parental, cdh2 mutant and cdh2-rescued 293 lines. The comparison demonstrated that Cdh2 promotes neurite branching and that it is required for three synaptic organizers, neurologin1 (NLGL1), leucine-rich repeat transmembrane protein 2 (LRRtm2), and Cell Adhesion Molecule 1 (Cadm1/SynCAM) to stimulate presynaptic differentiation, assayed by clustering of synaptic vesicles at sites of neurite-293 cell contact. Similarly, Cdh2 is required for a presynaptic organizing molecule, Neurexin1β, to promote postsynaptic differentiation in dendrites. We also show that another Type I cadherin, Cdh4, and a Type II cadherin, Cdh6, can substitute for Cdh2 in these assays. Finally, we provide evidence that the effects of cadherins require homophilic interactions between neurites and the heterologous cells. Together, these results indicate that classical cadherins act together with synaptic organizers to promote synaptic differentiation, perhaps in part by strengthening the intracellular adhesion required for the organizers to act efficiently. We propose that cadherins promote high affinity contacts between appropriate partners, which then enable synaptic differentiation.

Visualizing multiple inter-organelle contact sites using the organelle-targeted split-GFP system

Functional integrity of eukaryotic organelles relies on direct physical contacts between distinct organelles. However, the entity of organelle-tethering factors is not well understood due to lack of means to analyze inter-organelle interactions in living cells. Here we evaluate the split-GFP system for visualizing organelle contact sites in vivo and show its advantages and disadvantages. We observed punctate GFP signals from the split-GFP fragments targeted to any pairs of organelles among the ER, mitochondria, peroxisomes, vacuole and lipid droplets in yeast cells, which suggests that these organelles form contact sites with multiple organelles simultaneously although it is difficult to rule out the possibilities that these organelle contacts sites are artificially formed by the irreversible associations of the split-GFP probes. Importantly, split-GFP signals in the overlapped regions of the ER and mitochondria were mainly co-localized with ERMES, an authentic ER-mitochondria tethering structure, suggesting that split-GFP assembly depends on the preexisting inter-organelle contact sites. We also confirmed that the split-GFP system can be applied to detection of the ER-mitochondria contact sites in HeLa cells. We thus propose that the split-GFP system is a potential tool to observe and analyze inter-organelle contact sites in living yeast and mammalian cells.

GRAM marks the spot for STIM. Commentary on “GRAM domain proteins specialize functionally distinct ER-PM contact sites in human cells”

GRAM domain proteins were reported as novel ER-PM tethers defining specific membrane contact sites (MCS) subdomains. GRAMD2a pre-marks the sites occupied by STIM1 at MCS and its ablation impairs STIM1 translocation, but not store-operated Ca2+ entry. We discuss these apparently counterintuitive findings in the context of STIM/ORAI signaling at MCS.

Calcium and lipid exchange machinery in Sertoli cells

Sperm production (spermatogenesis) takes place in the seminiferous tubules of the testis. Specialized junction complexes termed ‘ectoplasmic specializations' (ESs) in the seminiferous epithelium have several key functions. ESs between Sertoli cells disassemble during the movement of early spermatogenic cells towards the apex of the epithelium and those ESs between a Sertoli cell and late stage spermatids disassemble during sperm release. There is evidence that structures called ‘Tubulobulbar Complexes' (TBCs) are endocytic structures responsible for the internalization and eventual degradation of junctions associated with ESs, allowing for early spermatocyte translocation from the basal epithelium and release of late spermatids from the apical epithelium. One key feature of both ESs and TBCs is their extensive association with peripheral ER. ESs are tripartite in structure consisting of Sertoli cell plasma membrane (PM), a layer of actin bundles, and an overlying leaflet of ER. TBCs are double membrane invaginations that appear as ESs are beginning to break down. They are long, endocytic in‐growths, with a swelled bulb region that contains an extensive ER‐PM contact site. TBC bulbs eventually ‘bud’, fuse, and become endosomes. Little is understood about the function of the ER at both ESs and TBCs, though it is well established that ER contact sites in other cells function in both calcium signalling and lipid exchange. If ER in ESs and ER‐TBC contacts function in calcium and lipid exchange, then known calcium signalling machinery and lipid exchange proteins should be localized to these structures. Using confocal imaging, we localized the CACNA1H voltage gated calcium channel to the PM of apical ESs, and the VAPA integral ER protein, which is associated with lipid synthesis and exchange machinery, to the apical ER as well as the ER in basal regions known to contain ESs. This data adds further weight to the hypothesis that calcium exchange is integral to regulation of ES and TBC structure, as well as begins our exploration into the possible role of lipid exchange at these sites.Support or Funding InformationThis work was supported by the Natural Sciences and Engineering Research Council (NSERC) of Canada (155397‐2013 to A. Wayne Vogl)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.