Morphological control enables nanometer-scale dissection of cell-cell signaling complexes

• Published in: Nature communications 2022-12, Vol.13 (1), p.7831-7831, Article 7831
• Main Authors: Dow, Liam P., Gaietta, Guido, Kaufman, Yair, Swift, Mark F., Lemos, Moara, Lane, Kerry, Hopcroft, Matthew, Bezault, Armel, Sauvanet, Cécile, Volkmann, Niels, Pruitt, Beth L., Hanein, Dorit
• Format: Article
• Language: English
• Subjects: 13; 14; 147/143; 147/3; 38/1; 38/109; 38/35; 631/136; 631/45/535/1262; 631/535/1258/1260; 631/57/2272; 631/80/86; Adherens junctions; Atomic force microscopy; Boundaries; Carbon - chemistry; Cell signaling; Cryoelectron Microscopy - methods; Cytology; Electron microscopy; Humanities and Social Sciences; Image Processing, Computer-Assisted - methods; Life Sciences; Microenvironments; Micropatterning; Microscopy; Morphology; multidisciplinary; Proteins; Robust control; Scanning electron microscopy; Science; Science (multidisciplinary); Signal Transduction; Signaling; Smoothness; Spring constant; Stiffness; Substrates; Workflow
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-022-35409-9

Protein micropatterning enables robust control of cell positioning on electron-microscopy substrates for cryogenic electron tomography (cryo-ET). However, the combination of regulated cell boundaries and the underlying electron-microscopy substrate (EM-grids) provides a poorly understood microenvironment for cell biology. Because substrate stiffness and morphology affect cellular behavior, we devised protocols to characterize the nanometer-scale details of the protein micropatterns on EM-grids by combining cryo-ET, atomic force microscopy, and scanning electron microscopy. Measuring force displacement characteristics of holey carbon EM-grids, we found that their effective spring constant is similar to physiological values expected from skin tissues. Despite their apparent smoothness at light-microscopy resolution, spatial boundaries of the protein micropatterns are irregular at nanometer scale. Our protein micropatterning workflow provides the means to steer both positioning and morphology of cell doublets to determine nanometer details of punctate adherens junctions. Our workflow serves as the foundation for studying the fundamental structural changes governing cell-cell signaling. Micropatterning on electron microscopy grids enables control of cell positioning and arrangement. Here, the authors describe detailed nanoscale characterization of a micropatterning workflow for reproducible molecular characterization through cryo-ET