ADVANCED MICROSCOPY TECHNIQUES REVEAL THE ROLE OF BCLAF1-PTK2 NUCLEAR CONDENSATES IN CARDIOMYOCYTE SURVIVAL DURING OXIDATIVE STRESS

Protein tyrosine kinase 2 (PTK2) is a non-receptor tyrosine kinase essential for regulating distinct cellular functions, including migration, growth, and maintenance of survival under stress conditions. Stabilization of PTK2 has been linked to its involvement in cell survival mechanisms, particularly under stress conditions, where it plays a crucial role in preventing apoptosis and maintaining cellular integrity. This study aimed to characterize the molecular function of the PTK2-Bclaf1 interaction in H9c2 cardiomyocytes under stress induced by chemotherapy drug doxorubicin (dox). Proximity ligation assays confirmed the direct interaction between PTK2 and Bclaf1, while Super-resolution Structured Illumination Microscopy (SR-SIM) revealed the formation of nuclear biomolecular condensates of Bclaf1 containing PTK2 in cardiomyocytes under stress. In silico analyses using AlphaFold and PSpredictor confirmed the unstructured nature of Bclaf1 and its high propensity for liquid-liquid phase separation (LLPS). Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Lifetime Imaging Microscopy (FLIM) were crucial to assess the biophysical properties of these condensates, revealing their high fluidity and dynamic nature, characteristic of LLPS. In dox-treated cardiomyocytes, PTK2 localized into the Bclaf1 condensate was shown to be protected from ubiquitination and degradation induced by dox-generated OS. Moreover, the ubiquitination site on PTK2, K926, was identified using mass spectrometry. These findings indicate that Bclaf1 biomolecular condensates are generated by LLPS and function sequestering and stabilizing PTK2, allowing cardiomyocytes to resist cytotoxicity caused by OS. This interaction plays a critical role in protein quality control during oxidative stress, when protein destabilization can determine cell fate. The elucidation of this mechanism provides insights into potential therapeutic strategies for enhancing cardioprotection in conditions associated with oxidative damage, such as chemotherapy-induced cardiotoxicity.