We determined the organization of target (t) SNARE proteins on the basolateral endothelial plasma membrane (PM) and their role in the mechanism of caveolar fusion. Studies were performed in a cellfree system involving endothelial PM sheets and isolated biotinlabeled caveolae. We monitored the fusion of caveolae with the PM by the detection of biotin-streptavidin complexes using correlative high resolution fluorescence microscopy and gold labeling electron microscopy on ultrathin sections of PM sheets. Imaging of PM sheets demonstrated and biochemical findings showed that the t-SNARE proteins present in endothelial cells (SNAP-23 and syntaxin-4) formed cholesterol-dependent clusters in discrete areas of the PM. Upon fusion of caveolae with the target PM, 50% of the caveolae co-localized with the t-SNARE clusters, indicating that these caveolae were at the peak of the fusion reaction. Fluorescent streptavidin staining of PM sheets correlated with the ultrastructure in the same area. These findings demonstrate that t-SNARE clusters in the endothelial target PM serve as the fusion sites for caveolae during exocytosis. The endothelial cells (ECs)3 lining the blood vessel walls are differentiated to mediate the rapid exchange of substances between the plasma and interstitial fluid. The process involving the fission of caveolae from the apical plasma membrane (PM) and fusion with the basolateral PM is termed transcytosis. Caveolae "pinch off " from the apical PM through a process requiring the recruitment of the GTPase dynamin to the caveolar necks as regulated by another protein intersectin (1, 2). Upon fission, caveolae form vesicular carriers that shuttle through the cytosol and deliver their cargo to the subendothelium by fusion with the basolateral PM (3). The basis of caveolar fusion may involve the same machinery as other vesicular carriers, i.e. the SNARE proteins of the syntaxin, synaptobrevin/cellubrevin, and SNAP-23/25 families (4 -7). However, the organization of SNARE proteins and their function in the fusion of caveolae in ECs are incompletely understood. It is known that syntaxin, cellubrevin, SNAP-23, and the cytosolic factors N-ethylmaleimide-sensitive factor (NSF) and ␣-and ␥-SNAP are key components of the endothelial multimolecular transcytotic complex, the assembly of which depends on the membrane fusion ATPase, NSF, which can be inhibited by alkylation of NSF by N-ethylmaleimide (7). Studies have also shown that N-ethylmaleimide interferes with transcytosis in ECs (4, 8). Based on the SNARE hypothesis, membrane fusion occurs when SNARE proteins on opposing membranes form four helix bundles, bringing the membranes in close apposition, thus providing the driving force necessary for fusion (9, 10). Ultrastructural studies have shown several states of association between caveolae and their target PM, varying in proximity, stability, and readiness for fusion (11). However, the pre-fusion states of caveolae have not been experimentally clarified, and the sequence of events responsible for caveolar fusion...

Key Points• Distinct T-cell patterns characterize the acute and chronic forms of cutaneous GVHD.• Increased TSLP expression is an indicator of acute cutaneous GVHD development.Graft-versus-host disease (GVHD) is a major complication of allogeneic hematopoietic stem cell transplantation (HCT) and can present in an acute (aGVHD), a chronic lichenoid (clGVHD), and a chronic sclerotic form (csGVHD). It is unclear whether similar or different pathomechanisms lead to these distinct clinical presentations. To address this issue, we collected lesional skin biopsies from aGVHD (n 5 25), clGVHD (n 5 17), and csGVHD (n 5 7) patients as well as serial nonlesional biopsies from HCT recipients (prior to or post-HCT) (n 5 14) and subjected them to phenotypic and functional analyses. Our results revealed striking differences between aGVHD and clGVHD. In aGVHD, we found a clear predominance of T helper (Th)2 cytokines/chemokines and, surprisingly, of interleukin (IL)-22 messenger RNA as well as an increase of IL-22-producing CD4 1 T cells.Thymic stromal lymphopoietin, a cytokine skewing the immune response toward a Th2 direction, was elevated at day 20 to 30 post-HCT in the skin of patients who later developed aGVHD. In sharp contrast to aGVHD, the immune response occurring in clGVHD showed a mixed Th1/Th17 signature with upregulated Th1/Th17 cytokine/chemokine transcripts and elevated numbers of interferon-g-and IL-17-producing CD8

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Intersectins (ITSNs) are multidomain adaptor proteins implicated in endocytosis, regulation of actin polymerization, and Ras/MAPK signaling. We have previously shown that ITSN-1s is required for caveolae fission and internalization in endothelial cells (ECs). In the present study, using small interfering RNA to knock down ITSN-1s protein expression, we demonstrate a novel role of ITSN-1s as a key antiapoptotic protein. Knockdown of ITSN-1s in ECs activated the mitochondrial pathway of apoptosis as determined by genomic DNA fragmentation, extensive mitochondrial fission, activation of the proapoptotic proteins BAK and BAX, and cytochrome c efflux from mitochondria. ITSN-1 knockdown acts as a proapoptotic signal that causes mitochondrial outer membrane permeabilization, dissipation of the mitochondrial membrane potential, and generation of reactive oxygen species. These effects were secondary to decreased activation of Erk1/2 and its direct activator MEK. Bcl-X L overexpression prevented BAX activation and the apoptotic ECs death induced by suppression of ITSN-1s. Our findings demonstrate a novel role of ITSN-1s as a negative regulator of the mitochondrial pathway-dependent apoptosis secondary to activation of the Erk1/2 survival signaling pathway.ITSN-1 is a multidomain adaptor protein, which binds to and scaffolds the endocytic machinery of clathrin-and caveolaemediated endocytic pathways (1-3). Two major ITSN-1 transcripts have been described in mammals, the ubiquitously expressed ITSN-1s and the neuron specific long isoform, 4,5). A highly similar human gene, ITSN-2, generates by alternative splicing two ITSN-2 isoforms widely expressed in human tissues (lung endothelium included) and showing a high degree of similarity to ITSN-1 proteins (2, 6). ITSN-1s, the only ITSN-1 isoform present in the ECs lining the blood vessels wall (3), comprises two Eps15 homology domains, a central coiled-coil domain, and five consecutive SH3 2 domains (SH3A to -E) (1, 7). Although ITSN-1 is best known for its role in endocytosis, recent studies also implicate ITSNs as mediators of the MAPK signaling pathway (8). ITSNs interact via the SH3A domain with the mammalian Son-of-sevenless, a guanine-nucleotide exchange factor for Ras (9). The association of ITSN with mammalian Son-of-sevenless leads to increased levels of RasGTP with functional consequences in signaling Erk1/2 activation (9). Activated Ras initiates the phosphorylation cascade, leading to phosphorylation and activation of p42/ p44 MAPKs (Erk1 and Erk2). In both neurons and nonneuronal cell lines, overexpression of the SH3A to -E, presumed to function in a dominant negative manner (11), attenuated the activation of EGF-mediated Ras/MAPK pathway (8). The concept of ITSNs as mediators of MAPK signaling is further supported by studies showing that full-length ITSN overexpression in HEK cells generated increased Ras-GTP levels associated with cytoplasmic vesicles, resulting in the c-Jun NH 2 -terminal kinase (JNK) activation (12). More recent work (13) in NIH 3T3 fibroblast...