Visualizing Spatiotemporal Dynamics of Intercellular Mechanotransmission upon Wounding

Abstract

During cell-to-cell communications, the interplay between physical and biochemical cues is essential for informational exchange and functional coordination, especially in multicellular organisms. However, it remains a challenge to visualize intercellular signaling dynamics in single live cells. Here, we report a photonic approach, based on laser microscissors and Förster resonance energy transfer (FRET) microscopy, to study intercellular signaling transmission. First, using our high-throughput screening platform, we developed a highly sensitive FRET-based biosensor (SCAGE) for Src kinase, a key regulator of intercellular interactions and signaling cascades. Notably, SCAGE showed a more than 40-fold sensitivity enhancement than the original biosensor in live mammalian cells. Next, upon local severance of physical intercellular connections by femtosecond laser pulses, SCAGE enabled the visualization of a transient Src activation across neighboring cells. Lastly, we found that this observed transient Src activation following the loss of cell-cell contacts depends on the passive structural support of cytoskeleton but not on the active actomyosin contractility. Hence, by precisely introducing local physical perturbations and directly visualizing spatiotemporal transmission of ensuing signaling events, our integrated approach could be broadly applied to mimic and investigate the wounding process at single-cell resolutions. This integrated approach with highly sensitive FRET-based biosensors provides a unique system to advance our in-depth understanding of molecular mechanisms underlying the physical-biochemical basis of intercellular coupling and wounding processes.

Src kinases relax adherens junctions between the neighbors of apoptotic cells to permit apical extrusion

“…In our experiments, this appears to be mediated by SFK signaling. Thus, SFK activity increased at the orthogonal junctions, consistent with the recent demonstration that Src is activated in response to laser-mediated cell injury, as revealed using a newly designed Src biosensor (Wang et al, 2018). Importantly, blocking SFK with either inhibitors or kinase RNAi prevented junctions from relaxing and also interrupted their elongation.…”

Integration of FRET and sequencing to engineer kinase biosensors from mammalian cell libraries

“…Third, the counter-sorting strategy incorporating a kinase-active or kinase-dead domain in biosensor variants promotes the biosensor specificity during the screening process. While the focus of this work is on the substrate sequence of the biosensor, optimizations of linkers 54 , SH2 domains 9 , and FPs 3 may further enhance the performance of the biosensor and our FRET-Seq platform can be extended to screen more diverse libraries to optimize these important components of the FRET biosensors in the future. The integration with in silico simulation to identify the hot spots for mutagenesis should further increase the success rate of library screening 55 .…”

“…Several restriction sites were introduced: two Esp3I sites at each end of LacZ to be replaced by the sensing domain (including SH2 domain, a flexible linker (15 amino acids), and substrate peptide), and XbaI/EcoRI flanking the kinase domain to be replaced by different domain versions. To construct the biosensor, the cDNA of the sensing domain, which was amplified by PCR from the mutated c-Src SH2 domain (C185A) 9 with a sense primer containing an Esp3I and a reverse primer containing the cDNA of a flexible linker, a substrate peptide, and an Esp3I site, was used to replace the LacZ domain via the Golden Gate assembly (NEB). To vary the sequence of the substrate peptide, the PCR products of a common forward primer and a NNK degenerate primer that target the substrate were inserted into the ELYK template by Golden Gate assembly.…”

“…At present, optimization of FRET biosensors is rather empirical and labor-intensive, and the design of these biosensors is often limited by the availability of accurate protein structures 5 . To address these problems, several methods have been proposed that utilize evolutionary strategies in bacteria and yeasts 3 , 6 – 9 . While these methods are very well-designed, they generally need additional selection steps to identify the optimized FRET biosensors since results from purified proteins or bacteria/yeasts cannot be translated directly to biosensor responses in mammalian cells.…”

Quantifying Tensile Forces at Cell–Cell Junctions with a DNA-based Fluorescent Probe

“…In another strategy, genetically encoded protein-based tension probes have been developed to measure intercellular forces mediated by cadherins or platelet endothelial cell adhesion molecule 8,[24][25][26] . However, the routine use of these sensors is still limited due to their labor-intensive design and validation.…”