Abstract
The formation and maintenance of cell-cell junctions, both under physiological and pathological conditions, requires the targeting and trafficking of junctional proteins. Proteins of the syntaxin (Stx)-family localize to a variety of subcellular membranes and contribute to intracellular transport of cargo by regulating vesicle fusion events at these sites. Unlike plasma membrane localized Stxs, the roles of endosome- and Golgi-localized stx proteins in epithelial morphogenesis are less understood. Here we show that Stx16– an endosome- and Golgi-localized target-membrane soluble N-ethylmaleimide attachment protein receptor (t-SNARE) that plays a role in membrane trafficking between these compartments – is essential for lumen development. In cultured Madin Darby Canine Kidney (MDCK) cells, Stx16 was selectively upregulated as sparsely plated cells attained confluency. Stx16-depleted confluent monolayers consistently showed lower transepithelial resistance than control monolayers, and failed to maintain endogenous and ectopically expressed E-cadherin at the adherens junctions due to decreased recycling. We further found that whereas cysts formed by MDCK cells cultured in Matrigel have a single hollow lumen, those formed by stx16-depleted counterparts had multiple lumens, due to abnormal orientiation of the mitotic spindle. Finally, a similar role for stx16 function in vivo is indicated by our analysis of pronephric-duct development in zebrafish expressing the claudinB:lynGFP transgene; lack of stx16 function in this structure (in stx16-morphant embryos) led to the development of enlarged, torturous pronephric ducts with more than one lumen. Taken together, our in vitro and in vivo studies establish a role for Stx16 in maintaining the integrity of cell-cell junctions, and thereby in morphogenesis of the kidney epithelial lumen.
Highlights
The formation of polarized epithelia requires a functional apical junctional complex, major components of which are adherens junctions (AJs) and tight junctions (TJs)
An AJ component that is key to these functions is E-cadherin, a calcium-dependent, homophilic, cell-to-cell adhesion receptor located in the basolateral domain
As proof of concept that Stx16 is required for kidney morphogenesis in vivo, we show that zebrafish stx16morphants show aberrant pronephric duct morphogenesis
Summary
The formation of polarized epithelia requires a functional apical junctional complex, major components of which are adherens junctions (AJs) and tight junctions (TJs). The AJs promote cell-cell adhesion and coordinate the changes in cell shape that are necessary for morphogenesis and organogenesis. An AJ component that is key to these functions is E-cadherin, a calcium-dependent, homophilic, cell-to-cell adhesion receptor located in the basolateral domain. AJ-localized E-cadherin is linked to the actin cytoskeleton by scaffolding proteins such as the catenins. Given that it contributes to AJ formation as well as to the maintenance of epithelial integrity during tissue homeostasis and remodeling, its activities must be precisely regulated. E-cadherin regulation is achieved in part by the transport of cadherin- and catenin-containing vesicles to and from the plasma membrane (PM) via precisely tuned exocytic and endocytic events [1]
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