Staff member

The invasion of human embryos in the uterus overcoming the maternal tissue barrier is a crucial step in embryo implantation and subsequent development. Although tissue invasion is fundamentally a mechanical process, most studies have focused on the biochemical and genetic aspects of implantation. Here, we fill the gap by using a deformable ex vivo platform to visualize traction during human embryo implantation. We demonstrate that embryos apply forces remodeling the matrix with species-specific displacement amplitudes and distinct radial patterns: principal displacement directions for mouse embryos, expanding on the surface while human embryos insert in the matrix generating multiple traction foci. Implantation-impaired human embryos showed reduced displacement, as well as mouse embryos with inhibited integrin-mediated force transmission. External mechanical cues induced a mechanosensitive response, human embryos recruited myosin, and directed cell protrusions, while mouse embryos oriented their implantation or body axis toward the external cue. These findings underscore the role of mechanical forces in driving species-specific invasion patterns during embryo implantation.

JTD Keywords: Animals, Anterior-posterior axis, Biomechanical phenomena, Cells, Collagen, Differentiatio, Embryo implantation, Embryo, mammalian, Female, Humans, Mechanotransduction, cellular, Mice, Morphogenesis, Pregnancy, Range, Self-organization, Species specificity, Trophoblast invasion, Uterine contractions

Protein dimerization and oligomerization are essential to most cellular functions, yet measurement of the size of these oligomers in live cells, especially when their size changes over time and space, remains a challenge. A commonly used approach for studying protein aggregates in cells is number and brightness (N&B), a fluorescence microscopy method that is capable of measuring the apparent average number of molecules and their oligomerization (brightness) in each pixel from a series of fluorescence microscopy images. We have recently expanded this approach in order to allow resampling of the raw data to resolve the statistical weighting of coexisting species within each pixel. This feature makes enhanced N&B (eN&B) optimal for capturing the temporal aspects of protein oligomerization when a distribution of oligomers shifts toward a larger central size over time. In this protocol, we demonstrate the application of eN&B by quantifying receptor clustering dynamics using electron-multiplying charge-coupled device (EMCCD)-based total internal reflection microscopy (TIRF) imaging. TIRF provides a superior signal-to-noise ratio, but we also provide guidelines for implementing eN&B in confocal microscopes. For each time point, eN&B requires the acquisition of 200 frames, and it takes a few seconds up to 2 min to complete a single time point. We provide an eN&B (and standard N&B) MATLAB software package amenable to any standard confocal or TIRF microscope. The software requires a high-RAM computer (64 Gb) to run and includes a photobleaching detrending algorithm, which allows extension of the live imaging for more than an hour.

JTD