Cytosolic Interactome In article number 2300105, Alireza Mashaghi and co-workers conduct single-molecule mechanical interrogation of proteins in a cytosolic environment, providing insights into the folding and unfolding of a single protein molecule in its native setting. The cytosolic interactome, including molecular chaperones, collectively shapes the folding landscape of a protein, necessitating single-molecule analysis within the cytosol.