Automatic Multi-functional Integration Program (AMFIP) towards all-optical mechano-electrophysiology interrogation

Abstract

Automatic operations of multi-functional and time-lapse live-cell imaging are necessary for the biomedical science community to study active, multi-faceted, and long-term biological phenomena. To achieve automatic control, most existing solutions often require the purchase of extra software programs and hardware that rely on the manufacturers’ own specifications. However, these software programs are usually non-user-programmable and unaffordable for many laboratories. To address this unmet need, we have developed a novel open-source software program, titled Automatic Multi-functional Integration Program (AMFIP), as a new Java-based and hardware-independent system that provides proven advantages over existing alternatives to the scientific community. Without extra hardware, AMFIP enables the functional synchronization of the μManager software platform, the Nikon NIS-Elements platform, and other 3rd party software to achieve automatic operations of most commercially available microscopy systems, including but not limited to those from Nikon. AMFIP provides a user-friendly and programmable graphical user interface (GUI), opening the door to expanding the customizability for myriad hardware and software systems according to user-specific experimental requirements and environments. To validate the intended purposes of developing AMFIP, we applied it to elucidate the question whether single cells, prior to their full spreading, can sense and respond to a soft solid substrate, and if so, how does the interaction depend on the cell spreading time and the stiffness of the substrate. Using a CRISPR/Cas9-engineered human epithelial Beas2B (B2B) cell line that expresses mNeonGreen2-tagged mechanosensitive Yes-associated protein (YAP), we show that single B2B cells develop distinct substrate-stiffness-dependent YAP expressions within 10 hours at most on the substrate, suggesting that cells are able to sense, distinguish, and respond to mechanical cues prior to the establishment of full cell spreading. In summary, AMFIP provides a reliable, open-source, and cost-free solution that has the validated long-term utility to satisfy the need of automatic imaging operations in the scientific community.

YAP at the Crossroads of Biomechanics and Drug Resistance in Human Cancer

“…In general, ECM stiffness (Young's modulus) is sensed by transmembrane integrins on the cell surface, which sequentially induce activation of Src, FAK at focal adhesion sites and intracellular Rho-ROCK machinery to increase cytoskeleton contractility, which is mechanical tension in cells generated by the sliding of actin and myosin filaments along each other [58][59][60][61]. As demonstrated by our own CRISPR/Cas9 imaging of real-time YAP dynamics and by other independent research groups, increased cytoskeleton contractility triggers nuclear translocation of YAP, while reduced contractility induces cytoplasmic retention of YAP [19,22,34,57,[62][63][64]. For example, in human mammary epithelial cells, 80% of the cells cultured on stiff plastic (~10 MPa; 1 Pa = 1 Newton/m 2 ) substrate show nuclear YAP translocation, while the rest 20% of the cells show homogenous intracellular YAP distribution.…”