The N-cadherin interactome in primary cardiomyocytes as defined by quantitative proximity proteomics

Abstract

Statement: Proximity proteomics reveals a specific and specialized Ncadherin (CDH2) interactome along the cell-cell contacts of primary cardiomyocytes. AbstractThe junctional complexes that couple cardiomyocytes must transmit the mechanical forces of contraction while maintaining adhesive homeostasis. The adherens junction (AJ) connects the actomyosin networks of neighboring cardiomyocytes and is required for proper heart function. Yet little is known about the molecular composition of the cardiomyocyte AJ or how it is organized to function under mechanical load. Here we define the architecture, dynamics and proteome of the cardiomyocyte AJ. Mouse neonatal cardiomyocytes assemble stable AJs along intercellular contacts with organizational and structural hallmarks similar to mature contacts. We combine quantitative mass spectrometry with proximity labeling to identify the N-cadherin (CDH2) interactome. We define over 350 proteins in this interactome, nearly 200 of which are unique to CDH2 and not part of the E-cadherin (CDH1) interactome. CDH2-specific interactors are comprised primarily of adaptor and adhesion proteins that promote junction specialization. Finally, we find evidence of dynamic interplay between AJ and Z-disc proteins. Together, our results provide novel insight into the cardiomyocyte AJ and provide a proteomic atlas for defining the molecular complexes that regulate cardiomyocyte intercellular adhesion. . et al. (2016). The alternatively-included 11a sequence modifies the effects of Mena on actin cytoskeletal organization and cell behavior. Sci Rep 6, 35298.Barton, L. J., Soshnev, A. A. and Geyer, P. K. (2015). Networking in the nucleus: a spotlight on LEM-domain proteins.

Vinculin anchors contractile actin to the cardiomyocyte adherens junction

“…Fluorescence recovery over 15 minutes was quantified, plotted and fit to a double exponential curve. The Ncad-GFP recovery prolife was similar to previously published data from our group (Figure 5, A and E;Li, 2019), consistent with the ability of Ncad-GFP to restore AJs in an Ncadherin-null background. The mobile fraction of Ncad-GFP and all fusion constructs was ~30%, similar to those observed for AJ components in cardiomyocytes and epithelial cells ( Figure 5I; Li, 2019.…”

“…Lastly, Ncad-GFP-ABD formed poor contacts that failed to recruit vinculin but did display limited afadin recruitment (Supplemental Figure S2J). We speculate that this weak afadin recruitment to cardiomyocyte cell-cell contacts may be mediated by nectins (Satomi-Kobayashi et al, 2009;Li et al, 2019). Thus, we were able to specifically recruit E-catenin ligands to nascent cardiomyocyte contacts and observed morphological changes as a function of the selective association of AJ components.…”

“…Vinculin and afadin are recruited to epithelial AJs in a force-dependent manner (le Duc et al, 2010;Yonemura et al, 2010;Kale et al, 2018;Matsuzawa et al, 2018). Vinculin and afadin localize to the ICD in adult heart and proximity proteomics revealed that both are enriched at the AJ in cultured neonatal cardiomyocytes (Li et al, 2019). We sought to determine if vinculin and afadin recruitment to cardiomyocyte AJs is tensiondependent.…”

“…Cardiomyocytes have at least two distinct actin networks at cell-cell contactsmyofibrils and the cortical cytoskeleton (Li et al, 2019)that must be integrated at AJs. Many actin-binding ligands interact with E-catenin, including vinculin, afadin, ZO-1 and Eplin (Yonemura, 2017).…”

“…Likewise, epithelial afadin can also bind E-catenin in a force-dependent manner (Matsuzawa et al, 2018), where it functions to strengthen the AJ under tension (Choi et al, 2016). Both vinculin and afadin localize to the ICD (Geiger et al, 1985;Borrmann et al, 2006) and are recruited to cardiomyocyte AJs (Li et al, 2019). Vinculin is required for proper heart development and functions in cardiomyocyte adhesion and contraction (Shiraishi et al, 1997;Xu et al, 1998).…”