Role of AJC in umbrella cell function and dysfunction

Abstract: A critical component of the umbrella cell barrier is the apical junctional complex (AJC), a
multipartite, belt-like structure comprised of the tight junction, the adherens junction, desmosomes, and an
associated cytoskeleton. Functions of the AJC include regulation of paracellular flux, cell-cell adhesion, and
mechanotransduction. Despite evidence that the umbrella cell AJC is integral to urothelial function and
disrupted in several lower urinary tract disorders, we have limited understanding of key aspects of umbrella cell
AJC biology and pathobiology including: (i) how the AJC maintains its continuity in the face of cyclical bladder
filling and voiding; (ii) how the AJC is organized to undergo these transitions and the function of the
cytoskeleton in these events; and (iii) how the umbrella cell AJC senses tension and whether pathologically
high intravesical pressures stimulate AJC-associated mechanotransduction pathways. Our preliminary studies
include the novel findings that during bladder filling the AJC perimeter expands dramatically, a process that
depends on changes in the actin cytoskeleton and vesicular traffic, likely directed toward the AJC. In contrast,
the AJC contracts soon after bladder voiding, events driven by the non-muscle myosin II-triggered contraction
of the actin cytoskeleton, RhoA, as well as endocytosis. Based on available data, we hypothesize that critical
functions of the umbrella cell AJC are to maintain urothelial barrier function by undergoing dynamic expansion
and contraction and to serve as a site of mechanotransduction under normal and pathological conditions. To
test this global hypothesis, we propose the following experiments. In Aim 1, we will use a newly developed
biaxial stretching device, coupled with live-cell image analysis, to determine if increased strain triggers
exocytosis of junction-associated proteins directed toward the AJC, and if release of strain stimulates their
endocytosis. We will also assess if blocking AJC expansion perturbs urothelial barrier function. In Aim 2, we
will focus on deciphering the function and organization of the umbrella cell AJC-associated cytoskeleton. We
will use super-resolution confocal imaging, as well as electron microscopy to reconstruct the umbrella cell AJC
in 3D. In addition, we will determine if formins drive actin polymerization in response to filling. In Aim 3, we will
use tension sensors to determine if transmembrane proteins associated with the umbrella cell AJC sense
force, and assess whether junction-associated signaling pathways are activated in response to partial bladder
outlet obstruction (PBOO). Upon completion of these studies we will have new insights into how umbrella cell
AJC dynamics contribute to urothelial barrier function, the organization of the AJC and the function of its
associated cytoskeleton, and important new information about how the AJC senses and responds to
perturbations in its mechanical milieu, including in response to PBOO.

Grant Number: 5R01DK129473-05
NIH Institute/Center: NIH
Principal Investigator: Gerard Apodaca