All-optical Mechanobiology Interrogation of Yes-associated Protein in Human Cancer and Normal Cells using a Multi-functional System

Abstract

Long-term multi-functional imaging and analysis of live cells require streamlined, functional coordination of various hardware and software platforms. However, manual control of various equipment produced by different manufacturers is labor-intensive and time-consuming, potentially decreasing the accuracy, reproducibility, and quality of acquired data. Therefore, an all-in-one and user-programmable system that enables automatic, multi-functional, and long-term image acquisition and is compatible with most fluorescent microscopy platforms can benefit the scientific community. This paper introduces the complete operating protocols of utilizing a novel integrated software system that consists of (1) a home-built software program, titled "Automatic Multi-functional Integration Program (AMFIP)," which enables automatic multi-channel imaging acquisition, and (2) a suite of quantitative imaging analysis and cell traction computation packages.This integrated system is applied to reveal the previously unknown relationship between the spatial-temporal distribution of mechano-sensitive Yes-associated protein (YAP) and the cell mechanics, including cell spreading and traction, in CRISPR/Cas9-engineered human normal cells (B2B) and lung cancer cells (PC9).Leveraging this system's capability of multi-channel control and readout, the result shows: (1) B2B normal cells and PC9 cancer cells show a distinct relationship between YAP expression, traction, and cell dynamics during cell spreading and migration processes; and (2) PC9 cancer cells apply noticeable peri-nuclear forces on substrates. In summary, this paper presents a detailed stepwise protocol on how to utilize an integrated user-programmable system that enables automatic multi-

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.…”

Towards an integrative understanding of cancer mechanobiology: calcium, YAP, and microRNA under biophysical forces

“…Most importantly, further mechanobiological studies, leveraging the in vivo imaging, 395–399 CRISPR/Cas9 genome editing, 400–402 and data science, 29,403–405 could facilitate elucidating the roles and mechanisms of Ca 2+ /YAP/miRNA within mechanotranduction in vivo and empower the development of mechano-medicine for combinatorial cancer therapeutics.…”

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

“…Setting up the programming environment. The following steps show how to set up the software environment to program and implement AMFIP [ 72 ].…”

“…The ratio of fluorescence intensity between nucleus and cytoplasm (N/C ratio) is widely used in live-cell experiments for the analysis of protein dynamics and cell functions. To examine the relationship between YAP N/C ratio and cell-area/nucleus-area ratio, we analyzed time-lapse images of single spreading cells and non-spreading cells on different substrates ( Figs 3 – 6 ) by using Fiji ImageJ to process the acquired confocal and bright-field images ( S1 Protocol ) [ 72 , 73 ].…”