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.     

Antibodies to MHC Class II Molecules Induce Autoimmunity: Critical Role for Macrophages in the Immunopathogenesis of Obliterative Airway Disease

Previous studies have shown that intrabronchial administration of antibodies (Abs) to MHC class I resulted in development of obliterative airway disease (OAD), a correlate of chronic human lung allograft rejection. Since development of Abs specific to mismatched donor HLA class II have also been associated with chronic human lung allograft rejection, we analyzed the role of Abs to MHC class II in inducing OAD. Administration of MHC class II Abs (M5/114) to C57BL/6 mice induced the classical features of OAD even though MHC class II expression is absent de novo on murine lung epithelial and endothelial cells. The induction of OAD was accompanied by enhanced cellular and humoral immune responses to self-antigens (Collagen V and K- α1Tubulin). Further, lung-infiltrating macrophages demonstrated a switch in their phenotype predominance from MΦ1 (F4/80⁺CD11c⁺) to MΦ2 (F4/80⁺CD206⁺) following administration of Abs and prior to development of OAD. Passive administration of macrophages harvested from animals with OAD but not from naïve animals induced OAD lesions. We conclude that MHC class II Abs induces a phenotype switch of lung infiltrating macrophages from MΦ1 (F4/80⁺CD11c⁺) to MΦ2 (F4/80⁺CD206⁺) resulting in the breakdown of self-tolerance along with an increase in autoimmune Th17 response leading to OAD.     

Expression of calmodulin and myosin light chain kinase during larval settlement of the Barnacle Balanus amphitrite

Barnacles are one of the most common organisms in intertidal areas. Their life cycle includes seven free-swimming larval stages and sessile juvenile and adult stages. The transition from the swimming to the sessile stages, referred to as larval settlement, is crucial for their survivor success and subsequent population distribution. In this study, we focused on the involvement of calmodulin (CaM) and its binding proteins in the larval settlement of the barnacle, Balanus ( = Amphibalanus) amphitrite. The full length of CaM gene was cloned from stage II nauplii of B. amphitrite (referred to as Ba-CaM), encoding 149 amino acid residues that share a high similarity with published CaMs in other organisms. Quantitative real-time PCR showed that Ba-CaM was highly expressed in cyprids, the stage at which swimming larvae are competent to attach and undergo metamorphosis. In situ hybridization revealed that the expressed Ba-CaM gene was localized in compound eyes, posterior ganglion and cement glands, all of which may have essential functions during larval settlement. Larval settlement assays showed that both the CaM inhibitor compound 48/80 and the CaM-dependent myosin light chain kinase (MLCK) inhibitor ML-7 effectively blocked barnacle larval settlement, whereas Ca(2+)/CaM-dependent kinase II (CaMKII) inhibitors did not show any clear effects. The subsequent real-time PCR assay showed a higher expression level of Ba-MLCK gene in larval stages than in adults, suggesting an important role of Ba-MLCK gene in larval development and competency. Overall, the results suggest that CaM and CaM-dependent MLCK function during larval settlement of B. amphitrite.     

Mapping of the phosphorylation sites on the phototropic signal transducer, NPH3

The phototropic response in Arabidopsis thaliana is initiated by the blue-light photoreceptors, phototropin (phot)1 and phot2. A recent study has shown that one of the phototropic signal transducers, NPH3, is phosphorylated under dark conditions and dephosphorylated by blue-light irradiation. To further understand the function of phosphorylation and dephosphorylation of NPH3 during this phototropic response, we have mapped the phosphorylation sites of NPH3 in our current study. The NPH3 protein has at least three phosphorylation sites at serine residues, Ser212, Ser222, and Ser236, and these sites are dephosphorylated by blue-light irradiation. By immunoblotting analysis, these phosphorylation sites in phot1 mutants are not dephosphorylated following blue-light irradiation at both low and high fluence rates, even though such irradiations induce the phot2-dependent phototropic response in phot1. These results suggest that the dephosphorylated NPH3 is involved in the phot1-dependent phototropic response and is not essential for the phot2-dependent phototropic response. We generated two types of transgenic nph3 plants expressing a NPH3^{S212A/S222A/S232A/S236A} protein and a NPH3^Δ212–238 protein in which the phosphorylation region is deleted, and assessed the phototropic phenotype of these. Based upon our present findings, we discuss the role of dephosphorylated and phosphorylated NPH3 in mediating the phototropic response.     

Mechanism of asymmetric ovarian development in chick embryos

In most animals, the gonads develop symmetrically, but most birds develop only a left ovary. A possible role for estrogen in this asymmetric ovarian development has been proposed in the chick, but the mechanism underlying this process is largely unknown. Here, we identify the molecular mechanism responsible for this ovarian asymmetry. Asymmetric PITX2 expression in the left presumptive gonad leads to the asymmetric expression of the retinoic-acid (RA)-synthesizing enzyme, RALDH2, in the right presumptive gonad. Subsequently, RA suppresses expression of the nuclear receptors Ad4BP/SF-1 and estrogen receptor alpha in the right ovarian primordium. Ad4BP/SF-1 expressed in the left ovarian primordium asymmetrically upregulates cyclin D1 to stimulate cell proliferation. These data suggest that early asymmetric expression of PITX2 leads to asymmetric ovarian development through up- or downregulation of RALDH2, Ad4BP/SF-1, estrogen receptor alpha and cyclin D1.     

Arabidopsis CAPRICE-LIKE MYB 3 (CPL3) controls endoreduplication and flowering development in addition to trichome and root hair formation

CAPRICE (CPC) encodes a small protein with an R3 MYB motif and promotes root hair cell differentiation in Arabidopsis thaliana. Three additional CPC-like MYB genes, TRY (TRIPTYCHON), ETC1 (ENHANCER OF TRY AND CPC 1) and ETC2 (ENHANCER OF TRY AND CPC 2) act in a redundant manner with CPC in trichome and root hair patterning. In this study, we identified an additional homolog, CPC-LIKE MYB 3 (CPL3), which has high sequence similarity to CPC, TRY, ETC1 and ETC2. Overexpression of CPL3 results in the suppression of trichomes and overproduction of root hairs, as has been observed for CPC, TRY, ETC1 and ETC2. Morphological studies with double, triple and quadruple homolog mutants indicate that the CPL3 gene cooperatively regulates epidermal cell differentiation with other CPC homologs. Promoter-GUS analyses indicate that CPL3 is specifically expressed in leaf epidermal cells, including stomate guard cells. Notably, the CPL3 gene has pleiotropic effects on flowering development, epidermal cell size and trichome branching through the regulation of endoreduplication.     

The DEFECTIVE IN ANTHER DEHISCENCE1 Gene Encodes a Novel Phospholipase A1 Catalyzing the Initial Step of Jasmonic Acid Biosynthesis, Which Synchronizes Pollen Maturation, Anther Dehiscence, and Flower Opening in Arabidopsis

The Arabidopsis mutant defective in anther dehiscence1 (dad1) shows defects in anther dehiscence, pollen maturation, and flower opening. The defects were rescued by the exogenous application of jasmonic acid (JA) or linolenic acid, which is consistent with the reduced accumulation of JA in the dad1 flower buds. We identified the DAD1 gene by T-DNA tagging, which is characteristic to a putative N-terminal transit peptide and a conserved motif found in lipase active sites. DAD1 protein expressed in Escherichia coli hydrolyzed phospholipids in an sn-1–specific manner, and DAD1–green fluorescent protein fusion protein expressed in leaf epidermal cells localized predominantly in chloroplasts. These results indicate that the DAD1 protein is a chloroplastic phospholipase A1 that catalyzes the initial step of JA biosynthesis. DAD1 promoter::β-glucuronidase analysis revealed that the expression of DAD1 is restricted in the stamen filaments. A model is presented in which JA synthesized in the filaments regulates the water transport in stamens and petals.