Microscopy-based cellular contractility assay for adult, neonatal, and hiPSC cardiomyocytes.

This protocol provides instructions to acquire high-quality cellular contractility data from adult, neonatal, and human induced pluripotent stem cell-derived cardiomyocytes. Contractility parameters are key to unravel mechanisms underlying cardiac pathologies, yet difficulties in acquiring data can compromise measurement accuracy and reproducibility. We provide optimized steps for microscope and camera setup, as well as cellular selection criteria for different cardiomyocyte cell types, aiming to obtain robust and reliable data. Moreover, we use CONTRACTIONWAVE software to analyze and show the optimized results. For complete details on the use and execution of this profile, please refer to Scalzo et al. (2021).

Dense optical flow software to quantify cellular contractility.

Cell membrane deformation is an important feature that occurs during many physiological processes, and its study has been put to good use to investigate cardiomyocyte function. Several methods have been developed to extract information on cardiomyocyte contractility. However, no existing computational framework has provided, in a single platform, a straightforward approach to acquire, process, and quantify this type of cellular dynamics. For this reason, we develop CONTRACTIONWAVE, high-performance software written in Python programming language that allows the user to process large data image files and obtain contractility parameters by analyzing optical flow from images obtained with videomicroscopy. The software was validated by using neonatal, adult-, and human-induced pluripotent stem-cell-derived cardiomyocytes, treated or not with drugs known to affect contractility. Results presented indicate that CONTRACTIONWAVE is an excellent tool for examining changes to cardiac cellular contractility in animal models of disease and for pharmacological and toxicology screening during drug discovery.

Investigating the Effect of Chain Connectivity on the Folding of a Beta-Sheet Protein On and Off the Ribosome.

Determining the relationship between protein folding pathways on and off the ribosome remains an important area of investigation in biology. Studies on isolated domains have shown that alteration of the separation of residues in a polypeptide chain, while maintaining their spatial contacts, may affect protein stability and folding pathway. Due to the vectorial emergence of the polypeptide chain from the ribosome, chain connectivity may have an important influence upon cotranslational folding. Using MATH, an all ß-sandwich domain, we investigate whether the connectivity of residues and secondary structure elements is a key determinant of when cotranslational folding can occur on the ribosome. From Φ-value analysis, we show that the most structured region of the transition state for folding in MATH includes the N and C terminal strands, which are located adjacent to each other in the structure. However, arrest peptide force-profile assays show that wild-type MATH is able to fold cotranslationally, while some C-terminal residues remain sequestered in the ribosome, even when destabilized by 2-3 kcal mol-1. We show that, while this pattern of Φ-values is retained in two circular permutants in our studies of the isolated domains, one of these permutants can fold only when fully emerged from the ribosome. We propose that in the case of MATH, onset of cotranslational folding is determined by the ability to form a sufficiently stable folding nucleus involving both ß-sheets, rather than by the location of the terminal strands in the ribosome tunnel.

Folding and binding pathways of BH3-only proteins are encoded within their intrinsically disordered sequence, not templated by partner proteins.

Intrinsically disordered regions are present in one-third of eukaryotic proteins and are overrepresented in cellular processes such as signaling, suggesting that intrinsically disordered proteins (IDPs) may have a functional advantage over folded proteins. Upon interacting with a partner macromolecule, a subset of IDPs can fold and bind to form a well-defined three-dimensional conformation. For example, disordered BH3-only proteins bind promiscuously to a large number of homologous BCL-2 family proteins, where they fold to a helical structure in a groove on the BCL-2-like protein surface. As two protein chains are involved in the folding reaction, and the structure is only formed in the presence of the partner macromolecule, this raises the question of where the folding information is encoded. Here, we examine these coupled folding and binding reactions to determine which component determines the folding and binding pathway. Using Φ value analysis to compare transition state interactions between the disordered BH3-only proteins PUMA and BID and the folded BCL-2-like proteins A1 and MCL-1, we found that, even though the BH3-only protein is disordered in isolation and requires a stabilizing partner to fold, its folding and binding pathway is encoded in the IDP itself; the reaction is not templated by the folded partner. We suggest that, by encoding both its transition state and level of residual structure, an IDP can evolve a specific kinetic profile, which could be a crucial functional advantage of disorder.

Disorder drives cooperative folding in a multidomain protein.

Many human proteins contain intrinsically disordered regions, and disorder in these proteins can be fundamental to their function-for example, facilitating transient but specific binding, promoting allostery, or allowing efficient posttranslational modification. SasG, a multidomain protein implicated in host colonization and biofilm formation in Staphylococcus aureus, provides another example of how disorder can play an important role. Approximately one-half of the domains in the extracellular repetitive region of SasG are intrinsically unfolded in isolation, but these E domains fold in the context of their neighboring folded G5 domains. We have previously shown that the intrinsic disorder of the E domains mediates long-range cooperativity between nonneighboring G5 domains, allowing SasG to form a long, rod-like, mechanically strong structure. Here, we show that the disorder of the E domains coupled with the remarkable stability of the interdomain interface result in cooperative folding kinetics across long distances. Formation of a small structural nucleus at one end of the molecule results in rapid structure formation over a distance of 10 nm, which is likely to be important for the maintenance of the structural integrity of SasG. Moreover, if this normal folding nucleus is disrupted by mutation, the interdomain interface is sufficiently stable to drive the folding of adjacent E and G5 domains along a parallel folding pathway, thus maintaining cooperative folding.