A Membrane Fusion Protein αSNAP Is a Novel Regulator of Epithelial Apical Junctions

Abstract

Tight junctions (TJs) and adherens junctions (AJs) are key determinants of the structure and permeability of epithelial barriers. Although exocytic delivery to the cell surface is crucial for junctional assembly, little is known about the mechanisms controlling TJ and AJ exocytosis. This study was aimed at investigating whether a key mediator of exocytosis, soluble N-ethylmaleimide sensitive factor (NSF) attachment protein alpha (αSNAP), regulates epithelial junctions. αSNAP was enriched at apical junctions in SK-CO15 and T84 colonic epithelial cells and in normal human intestinal mucosa. siRNA-mediated knockdown of αSNAP inhibited AJ/TJ assembly and establishment of the paracellular barrier in SK-CO15 cells, which was accompanied by a significant down-regulation of p120-catenin and E-cadherin expression. A selective depletion of p120 catenin effectively disrupted AJ and TJ structure and compromised the epithelial barrier. However, overexpression of p120 catenin did not rescue the defects of junctional structure and permeability caused by αSNAP knockdown thereby suggesting the involvement of additional mechanisms. Such mechanisms did not depend on NSF functions or induction of cell death, but were associated with disruption of the Golgi complex and down-regulation of a Golgi-associated guanidine nucleotide exchange factor, GBF1. These findings suggest novel roles for αSNAP in promoting the formation of epithelial AJs and TJs by controlling Golgi-dependent expression and trafficking of junctional proteins.

Loss of a membrane trafficking protein αSNAP induces non-canonical autophagy in human epithelia

“…30,31 Since several recent studies suggested that the Golgi can supply membranes to the autophagic pathway, 12,14,37-39 we investigated if disintegration of this organelle can contribute to the increased autophagic flux in αSNAP-deficient cells. Using HeLa-GFP-LC3 cells, we observed that formation of autophagosomes after αSNAP knockdown occurred in parallel to fragmentation of both cys and trans-Golgi compartments as visualized by giantin and TGN46 labeling, respectively (Fig.…”

“…Surprisingly, do not understand how loss of αSNAP results in the decreased expression of mTOR or 4E-BP1 as well as some other proteins (Bcl-2 and p120-catenin) described in our previous publications. 30,31 Future studies are required to address this important question.…”

“…SK-CO15 cells with stable overexpression of bovine αSNAP and their appropriate controls were generated as described elsewhere. 30 For biochemical and immunolabeling experiments, the cells were thiol groups of many cellular proteins and, as such, is a far less selective tool than the siRNA-mediated NSF knockdown used in the present study. Our results strongly suggest that NSF is not an obligate regulator of mammalian autophagy.…”

“…30,31 Although a key role of NSF in regulating yeast autophagy has been recently reported, 32 the involvement of αSNAP in autophagosomal biogenesis remains unexplored. The present study examines the role of αSNAP in regulating autophagy in model human epithelia.…”

“…41 On the other hand, we recently found that knockdown of epithelial αSNAP downregulated expression of all BFA-sensitive GEFs. 30 Therefore, we asked if loss of Golgi-resident GEFs can be responsible for the increased autophagy following αSNAP depletion. First, we confirmed decreased expression of GBF1, BIG1 and BIG2 in αSNAP-deficient SK-CO15 cells (Fig.…”