Quantitative apical membrane proteomics reveals vasopressin-induced actin dynamics in collecting duct cells

Abstract

In kidney collecting duct cells, filamentous actin (F-actin) depolymerization is a critical step in vasopressin-induced trafficking of aquaporin-2 to the apical plasma membrane. However, the molecular components of this response are largely unknown. Using stable isotope-based quantitative protein mass spectrometry and surface biotinylation, we identified 100 proteins that showed significant abundance changes in the apical plasma membrane of mouse cortical collecting duct cells in response to vasopressin. Fourteen of these proteins are involved in actin cytoskeleton regulation, including actin itself, 10 actin-associated proteins, and 3 regulatory proteins. Identified were two integral membrane proteins (Clmn, Nckap1) and one actin-binding protein (Mpp5) that link F-actin to the plasma membrane, five F-actin end-binding proteins (Arpc2, Arpc4, Gsn, Scin, and Capzb) involved in F-actin reorganization, and two actin adaptor proteins (Dbn1, Lasp1) that regulate actin cytoskeleton organization. There were also protease (Capn1), protein kinase (Cdc42bpb), and Rho guanine nucleotide exchange factor 2 (Arhgef2) that mediate signal-induced F-actin changes. Based on these findings, we devised a live-cell imaging method to observe vasopressininduced F-actin dynamics in polarized mouse cortical collecting duct cells. In response to vasopressin, F-actin gradually disappeared near the center of the apical plasma membrane while consolidating laterally near the tight junction. This F-actin peripheralization was blocked by calcium ion chelation. Vasopressin-induced apical aquaporin-2 trafficking and forskolin-induced water permeability increase were blocked by F-actin disruption. In conclusion, we identified a vasopressin-regulated actin network potentially responsible for vasopressin-induced apical F-actin dynamics that could explain regulation of apical aquaporin-2 trafficking and water permeability increase.V asopressin regulates aquaporin-2 (AQP2) and osmotic water transport in the collecting duct by regulating total AQP2 protein abundance and AQP2 trafficking to and from the apical plasma membrane of principal cells (1). Understanding these regulatory mechanisms at the molecular level is important to the ultimate understanding of the pathophysiology of multiple water balance disorders (2). This goal is abetted by the methods of molecular systems biology (proteomics and transcriptomics), which not only reveal "parts lists" for collecting duct cells but also can identify what biological and molecular processes are regulated by vasopressin. These proteomic and transcriptomic data have been made publicly available in databases (3). Many of these studies have focused on whole cells (4-6), whereas several analyzed subcellular fractions (7-12). Analysis of subcellular fractions is technically demanding, but it has the benefit of identifying low abundance proteins with important functions. To identify proteins potentially involved in apical AQP2 trafficking, we previously used NHS-based surface biotinylation coupled to streptavidin...

AKAP220 manages apical actin networks that coordinate aquaporin-2 location and renal water reabsorption

“…This actin barrier physically prevents the fusion of AQP2-containing vesicles with the apical plasma membrane. Conversely, upon dehydration and the release of the hormone vasopressin, the actin barrier is depolymerized (4,33). Thus, water homeostasis requires equilibrium between AQP2 vesicles that are sequestered in the cytoplasm by the actin barrier and active water pores fused to the apical membranes.…”

“…The next step was to explore the role of AKAP220 signaling elements in the modulation of actin barrier dynamics and kidney function. Because the actin barrier forms in the absence of vasopressin stimulation (13,33), we overhydrated the animals to block vasopressin release. Animals were water-loaded by oral gavage (3% total body weight) as described previously (38).…”

Ankyrin G Expression Regulates Apical Delivery of the Epithelial Sodium Channel (ENaC)

“…Additionally, TEM images of kidney tubules from this paper show AnkG closely associated with some but not all vesicles near the apical surface, whereas AnkG on the lateral side appears more tightly associated with the plasma membrane itself (34). The 190-kDa AnkG isoform was also identified as an apically associated protein in an apical membrane proteomics screen of cultured mouse cortical collecting duct (mpkCCD-clone 11) cells (64). It is possible therefore, that AnkG serves a structural role along the basolateral membrane, such as its anchoring effect on E-cadherin (33,63), whereas near the apical surface, its association with vesicles plays a role in recycling.…”

Functional roles of MMP14 and MMP15 in early postnatal mammary gland development

“…However, given the number of genes associated with epithelial cell adhesion complexes and cytoskeletal organization [e.g. calmin, plakophilin 2, folliculin (Arimoto et al, 2014;Khabibullin et al, 2014;Loo et al, 2013)], we examined mammary ducts recovered from P10 Mmp14 +/+ and Mmp14 −/− mice by transmission electron microscopy (TEM). Interestingly, Mmp14 −/− ducts form more intimate cell-cell junctions, with a marked increase in the number of apical microvilli (Fig.…”