The shift from quiescent to effector T cells (TC) is controlled at the translational level. Post-translational modifications (PTMs) are key factors in the diversification of protein function. Advancements in mass spectrometry-based proteomics enable proteome-wide, hypothesis-free quantitative PTM analysis. Current research highlights the centrosome role in TC activation. Here the diversity of PTMs in the TC centrosome is studied by analyzing centrosome-enriched fractions from human resting and activated T lymphoblasts. Our results show that oxidative modifications predominate in this organelle, with tryptophan as the most frequently oxidized residue. These PTMs are enriched in proteins involved in translation, vesicular trafficking, cytoskeleton organization, among others. We also demonstrate the existence of PTM changes on specific protein regions during TC activation in Myh9 (hyperoxidized), and Gzma (hypoxidized). These hyper- or hypoxidized proteins display distinct functional distributions. Our study provides the first comprehensive PTM mapping of the TC centrosome, underscoring the PTM regulatory role in TC activation.

The limited regenerative capacity in adult mammalian heart is mainly attributed to the low cell cycle activity of cardiomyocytes. Achieving cardiomyocyte division is a challenging undertaking, as the contractile function, a major characteristic of cardiomyocytes, is not overall compatible with cell cycle progression. To dissect the relationship between sarcomeric contraction and proliferation in cardiomyocytes, we utilized Blebbistatin (Blebb), a Myosin II inhibitor commonly used for cardiomyocyte culture ex vivo, and revealed that Myosin inhibition by Blebb in cardiomyocytes resulted in enhanced cell cycle entry with cytokinesis failure, thus increasing polyploidy. Elevated H3K9me3 modification was found to be required for the increased cell cycle entry induced by Blebb, and the increased H3K9me3 modifications were enriched on genes that encode negative regulators of cell cycle and controlled by NFAT transcription factors. Furthermore, SIRT1 was identified to be a nucleocytoplasmic shuttling protein that dissociates from Z lines of sarcomeres and translocates into the nucleus upon Myosin inhibition, directly interacts with NFATc3, and is required for the Blebb-induced elevation of H3K9me3 modification and cell cycle activity. Our results identified a signaling pathway transducing sarcomeric signals to epigenetic modifications modulating the cardiomyocyte cell cycle, which may facilitate the understanding of the complex regulatory network controlling cardiomyocyte proliferation and provide therapeutic targets for regenerative medicine.

Holotomograhic microscopy (HTM) has emerged as a non-invasive imaging technique that offers high-resolution, quantitative 3D imaging of biological samples. This study explores the application of HTM in examining endothelial cells (ECs). HTM overcomes the limitations of traditional microscopy methods in capturing the real-time dynamics of ECs by leveraging the refractive index (RI) to map 3D distributions label-free. This work demonstrates the utility of HTM in visualizing key cellular processes during endothelialization, wherein ECs anchor, adhere, migrate, and proliferate. Leveraging the high resolution and quantitative power of HTM, we show that lipid droplets and mitochondria are readily visualized, enabling more comprehensive studies on their respective roles during endothelialization. The study highlights how HTM on a commercial instrument can uncover novel insights into HUVEC cell behavior, offering potential applications in medical diagnostics and research, particularly in developing treatments for cardiovascular diseases. This advanced imaging technique not only enhances our understanding of EC biology but also presents a significant step forward in the study of cardiovascular diseases, providing a robust platform for future research and therapeutic development.