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

Liam P Dow; Guido Gaietta; Yair Kaufman; Mark F Swift; Moara Lemos; Kerry Lane; Matthew Hopcroft; Armel Bezault; Cécile Sauvanet; Niels Volkmann; Beth L Pruitt; Dorit Hanein

doi:10.1038/s41467-022-35409-9

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

Nat Commun. 2022 Dec 20;13(1):7831. doi: 10.1038/s41467-022-35409-9.

Authors

Liam P Dow¹, Guido Gaietta², Yair Kaufman¹, Mark F Swift², Moara Lemos³, Kerry Lane¹, Matthew Hopcroft¹, Armel Bezault³, Cécile Sauvanet³, Niels Volkmann^{4

5}, Beth L Pruitt⁶, Dorit Hanein^{7

8

9}

Affiliations

¹ Mechanical Engineering and Biomolecular Science and Engineering, University of California, Santa Barbara, CA, USA.
² Scintillon Institute, San Diego, CA, USA.
³ Institut Pasteur, CNRS UMR3528, Structural Studies of Macromolecular Machines in Cellulo Unit, F-75015, Paris, France.
⁴ Scintillon Institute, San Diego, CA, USA. niels.volkmann@pasteur.fr.
⁵ Institut Pasteur, Université de Paris, CNRS UMR3528, Structural Image Analysis Unit, Paris, France. niels.volkmann@pasteur.fr.
⁶ Mechanical Engineering and Biomolecular Science and Engineering, University of California, Santa Barbara, CA, USA. blp@ucsb.edu.
⁷ Scintillon Institute, San Diego, CA, USA. dorit@ucsb.edu.
⁸ Institut Pasteur, CNRS UMR3528, Structural Studies of Macromolecular Machines in Cellulo Unit, F-75015, Paris, France. dorit@ucsb.edu.
⁹ Department of Chemistry and Biochemistry, and of Biomedical Engineering, University of California, Santa Barbara, CA, USA. dorit@ucsb.edu.

Abstract

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.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

Carbon / chemistry
Cryoelectron Microscopy / methods
Image Processing, Computer-Assisted* / methods
Proteins*
Signal Transduction

Substances

Proteins
Carbon

Abstract

Publication types

MeSH terms

Substances

Grants and funding