SUMMARY Differentiating enterocytes build ~3000 microvilli on their apical surface and organize these protrusions into an array referred to as the brush border (BB). BB microvilli exhibit perfect tight packing and this unique morphology is critical for maximizing the number of protrusions, the holding capacity for membrane associated transporters and channels, and in turn, the functional capacity of the cell. The physiological importance of the BB is underscored by the fact that numerous intestinal diseases that are linked to the destruction and/or malformation of microvilli. Our group has made fundamental discoveries on mechanisms that enterocytes use to organize microvilli into functional, ordered BB arrays. Groundbreaking studies from our group identified two BB-specific protocadherins, CDHR2 and CDHR5, which form a heterophilic intermicrovillar adhesion complex (IMAC) that links the tips of adjacent microvilli and promotes ordered packing on mature villus enterocytes. Importantly, our work with CDHR2 KO mice established that loss of IMAC function leads to ~35% fewer microvilli on the apical surface and a corresponding growth rate reduction at the whole animal level. How IMACs drive the accumulation of thousands of microvilli over time during enterocyte differentiation remains the critical open question that we will tackle in this proposal. In exciting preliminary ultrastructural studies, we discovered that crypt microvilli initially exhibit robust accumulation at cell margins, which implies the existence of a mechanism for anchoring nascent protrusions at these sites. We also observed similar marginal accumulation of microvilli on the surface of differentiating intestinal and kidney epithelial cell lines. In all models examined, microvilli extending from one cell span intercellular space to make physical contact with microvilli on a neighboring cell. Remarkably, super- resolution microscopy of native tissue and epithelial culture models showed that microvilli in these contacts contain both CDHR2 and CDHR5, suggesting they represent transjunctional IMACs, a novel form of epithelial cell-cell contact. Using a cell mixing approach to drive the formation of transjunctional IMACs, photobleaching measurements revealed that transjunctional IMACs are much longer lived vs. medial IMACs. Finally, we learned that CDHR2 loss-of-function models, which are unable to form IMACs, exhibit defects in tight junctions. These initial findings lead us to propose the following hypothesis: transjunctional IMACs drive the accumulation of nascent microvilli into a mature BB while promoting the integrity of canonical cell junctions. Using state-of-the- art microscopy and novel epithelial model systems, we will: (Aim 1) define the subcellular mechanism of IMAC formation, (Aim 2) determine if transjunctional IMACs promote microvillus accumulation, and (Aim 3) determine if transjunctional IMACs promote the integrity of canonical cell junctions. These studies will reveal h...