Fig. 3

Hypothesized mechanism of membrane proximal NAD+ action in muscle-ECM adhesion. (A–B) Model of membrane proximal NAD+ regulation of subcellular protein localization, post-translational modification, laminin-binding affinity, and ECM organization in muscle. (A) Damage scenario. NRK enzymes localize to cytoplasmic tails of Integrin receptors for laminin and generate an intracellular membrane proximal pool of NAD+. In response to damage, intracellular membrane proximal NAD+ generated by NRK enzymes is actively transported or leaks across the sarcolemma into the extracellular environment. Extracellular membrane proximal NAD+ is consumed by ART enzymes as ADP-ribose moieties are added to Integrin receptors for laminin in mono-ADP-ribosylation reactions. Intracellular localization of Paxillin to cell-matrix adhesion complexes is disrupted. Extracellular organization of laminin is disrupted. (B) NAD+-mediated damage response scenario. Due to the movement of intracellular membrane proximal NAD+ to the extracellular environment, ADP-ribosylation of Integrin receptors for laminin change to a high-affinity binding conformation. Laminin organization is increased, Paxillin localization to cell-matrix adhesion complexes is restored, and Integrin-laminin binding is augmented. (C–E) Reprinted with permission from [33]. c Model of genetic mosaic experiment to determine if wild-type Nrk2b is sufficient in a cell autonomous manner for subcellular localization of beta-Dystroglycan and/or Paxillin to cell-matrix adhesions. Fluorescent dextran labeled wild-type cells were transplanted into a Nrk2b-deficient background and subcellular localization of beta-Dystroglycan and Paxillin were determined by immunohistochemistry. (D–E) Side mount, anterior left, 26 hpf nrk2b morphant hosts with transplanted wild-type cells (red) and beta-Dystroglycan (blue) or Paxillin antibody staining (green). (D-D1) Beta-Dystroglycan robustly localizes to MTJs in nrk2b morphants in the presence and the absence of transplanted wild-type cells. (E-E1) Robust Paxillin localization to cell-matrix adhesion complexes in nrk2b morphants is rescued in the transplanted wild-type cells, but not in the Nrk2b-deficient cells surrounding them. Therefore, Nrk2b is cell autonomously sufficient for subcellular localization of Paxillin, likely through generation of membrane proximal NAD+