The proteins and functional protein networks of the tight junction remain incompletely defined. This impedes better understanding of how the barrier is formed, regulated and interacts with the action cytoskeleton. Among the currently known proteins are barrier-forming proteins like occludin and the claudin family;scaffolding proteins like ZO-1;and some cytoskeletal, signaling, and cell polarity proteins. To define a more complete list of proteins and infer their functional implications, we have identified the proteins that are within molecular dimensions of ZO-1, cadherin, occludin and claudin-2 and -4 by fusing these proteins to biotin ligase (BL), expressing these fusion proteins in Madin-Darby canine kidney epithelial cells, and purifying and identifying the resulting biotinylated proteins by mass spectrometry. Results for tagging by ZO-1 were published in 2013. In 2014 we characterized the unexpected finding that found ZO-1 is near several BAR-domain membrane curvature/sensing proteins including TOCA-1, TUBA and FBP17, BIN3 and Endophilin-A2 . Using Caco-2 epithelial cells, we showed an alternative splice of TOCA-1 adds a PDZ-binding motif which binds ZO-1, targeting TOCA-1 to barrier contacts. ShRNA-mediated knockdown of TOCA-1 levels leads to delayed recovery after Latrunculin A washout and in calcium switch assays and increases paracellular flux. Restoring TOCA-1 to knockdown cells results in recruitment of N-WASP and WIPF2 and accumulation of junctional actin. The ability to recruit N-WASP and induce actin accumulation is dependent on the SH3 domain of TOCA-1 but not the F-BAR or Cdc42-binding domains. Recognition of this complex provides insight into how the barrier proteins are coupled to the actin cytoskeleton and raises new questions about roles for membrane bending/sensing BAR-domains proteins under the tight junction strands. We are developing freeze-fracture EM protocols to test whether TOCA-1 bends the membrane at junctions with functional consequences. We found cadherin is near the LIM domain protein, lipoma preferred partner (LPP) and validated its role in junctional actin organization. Similarly, we found occludin is close to a large number of trafficking proteins and are sorting through the implications of proximal proteins for claudins. The prevalence of tagging for all constructs was high for a subset of proteins but fell exponentially suggesting that the first 50-100 most heavily tagged proteins may be most relevant. Consistent with this, the most heavily tagged protein was always the fusion protein itself and the next few were always known proximal proteins. This finding as well as the high spatial resolution suggests this is a valid method for identifying new proximal proteins. We are currently finishing the comparison of proteins near claudin- andoccludin with ZO-1 and cadherin. Among these proteins we decided to characterize SORBS2 (Sorbin and SH3 domain-containing protein 2) which is a complex actin-binding scaffold also present at focal contacts. We have defined the splice forms of SORBS2 in several cell lines in preparation for making and characterizing shRNA studies. We expect this protein will have a significant role in regulating perijunctional action filaments and thus paracellular permeability, adhesiveness and/or cell motility. Overall, these results provide a rich inventory of proteins and potential novel insights into functions and protein networks that should catalyze further understanding of tight junction biology. Additionally, the technique demonstrates unexpectedly high spatial resolution, which could be generally applied to defining other subcellular protein compartmentalization.
