Effect of osmotic stress on live cell plasma membranes, probed via Laurdan general polarization measurements.

Here we seek to gain insight into changes in the plasma membrane of live cells upon the application of osmotic stress using Laurdan, a fluorescent probe that reports on membrane organization, hydration, and dynamics. It is known that the application of osmotic stress to lipid vesicles causes a decrease in Laurdan's generalized polarization (GP), which has been interpreted as an indication of membrane stretching. In cells, we see the opposite effects, as GP increases when the osmolarity of the solution is decreased. This increase in GP is associated with the presence of caveolae, which are known to disassemble and flatten in response to osmotic stress.

Pre-complexation of talin and vinculin without tension is required for efficient nascent adhesion maturation.

Talin and vinculin are mechanosensitive proteins that are recruited early to integrin-based nascent adhesions (NAs). In two epithelial cell systems with well-delineated NA formation, we find these molecules concurrently recruited to the subclass of NAs maturing to focal adhesions. After the initial recruitment under minimal load, vinculin accumulates in maturing NAs at a ~ fivefold higher rate than in non-maturing NAs, and is accompanied by a faster traction force increase. We identify the R8 domain in talin, which exposes a vinculin-binding-site (VBS) in the absence of load, as required for NA maturation. Disruption of R8 domain function reduces load-free vinculin binding to talin, and reduces the rate of additional vinculin recruitment. Taken together, these data show that the concurrent recruitment of talin and vinculin prior to mechanical engagement with integrins is essential for the traction-mediated unfolding of talin, exposure of additional VBSs, further recruitment of vinculin, and ultimately, NA maturation.

A versatile oblique plane microscope for large-scale and high-resolution imaging of subcellular dynamics.

We present an oblique plane microscope (OPM) that uses a bespoke glass-tipped tertiary objective to improve the resolution, field of view, and usability over previous variants. Owing to its high numerical aperture optics, this microscope achieves lateral and axial resolutions that are comparable to the square illumination mode of lattice light-sheet microscopy, but in a user friendly and versatile format. Given this performance, we demonstrate high-resolution imaging of clathrin-mediated endocytosis, vimentin, the endoplasmic reticulum, membrane dynamics, and Natural Killer-mediated cytotoxicity. Furthermore, we image biological phenomena that would be otherwise challenging or impossible to perform in a traditional light-sheet microscope geometry, including cell migration through confined spaces within a microfluidic device, subcellular photoactivation of Rac1, diffusion of cytoplasmic rheological tracers at a volumetric rate of 14 Hz, and large field of view imaging of neurons, developing embryos, and centimeter-scale tissue sections.