Analysis of the H-Ras mobility pattern in vivo shows cellular heterogeneity inside epidermal tissue.

Developments in single-molecule microscopy (SMM) have enabled imaging individual proteins in biological systems, focusing on the analysis of protein mobility patterns inside cultured cells. In the present study, SMM was applied in vivo, using the zebrafish embryo model. We studied dynamics of the membrane protein H-Ras, its membrane-anchoring domain, C10H-Ras, and mutants, using total internal reflection fluorescence microscopy. Our results consistently confirm the presence of fast- and slow-diffusing subpopulations of molecules, which confine to microdomains within the plasma membrane. The active mutant H-RasV12 exhibits higher diffusion rates and is confined to larger domains than the wild-type H-Ras and its inactive mutant H-RasN17. Subsequently, we demonstrate that the structure and composition of the plasma membrane have an imperative role in modulating H-Ras mobility patterns. Ultimately, we establish that differences between cells within the same embryo largely contribute to the overall data variability. Our findings agree with a model in which the cell architecture and the protein activation state determine protein mobility, underlining the importance of SMM imaging for studying factors influencing protein dynamics in an intact living organism. This article has an associated First Person interview with the first author of the paper.

Analysis of intracellular protein dynamics in living zebrafish embryos using light-sheet fluorescence single-molecule microscopy.

Single-molecule microscopy techniques have emerged as useful tools to image individual molecules and analyze their dynamics inside cells, but their application has mostly been restricted to cell cultures. Here, a light-sheet fluorescence microscopy setup is presented for imaging individual proteins inside living zebrafish embryos. The optical configuration makes this design accessible to many laboratories and a dedicated sample-mounting system ensures sample viability and mounting flexibility. Using this setup, we have analyzed the dynamics of individual glucocorticoid receptors, which demonstrates that this approach creates multiple possibilities for the analysis of intracellular protein dynamics in intact living organisms.