BCLAF1 PHASE SEPARATION AND ITS REGULATION IN CARDIOMYOCYTES DURING STRESS

Cells organize their intracellular environment not only through membrane-bound organelles but also through membraneless structures called biomolecular condensates, which compartmentalize proteins and RNAs to regulate diverse cellular functions, including signaling in response to changes in cellular state. BCLAF1 (Bcl-2-associated transcription factor 1) is a multifunctional protein that undergoes phase separation to form nuclear biomolecular condensates under doxorubicin-induced oxidative stress. In our previous studies, we showed that these assemblies sequester the pro-survival kinase PTK2, shielding it from ubiquitination and degradation—a mechanism critical for cardiomyocyte survival. Understanding the formation, stability and dissolution of these condensates is essential to elucidate how cells organize their nuclear environment to activate survival pathways under stress. Therefore, this study aims to delineate the BCLAF1 sequence responsible for phase separation and evaluate how the chaperones Hsp70 and VCP, key components of the protein quality control system, modulate condensate dynamics. Initially, sequence analysis predicted a propensity for liquid-liquid phase separation across BCLAF1, guiding the design of truncated constructs to map phase-separation-competent domains. BCLAF1 distribution was assessed via Super-Resolution Structured Illumination Microscopy (SR-SIM), showing that doxorubicin induced BCLAF1 condensate formation with transient, shell-like architectures over time. Once the stressor was removed, a gradual decrease in condensate number was observed. Under cisplatin treatment, condensates displayed different dynamics; although this drug also induced BCLAF1 condensate formation, no significant changes in morphology or number were observed over time. A Proximity Ligation Assay confirmed the interaction of BCLAF1 with the chaperones Hsp70 and VCP in the nucleus after stress induction, while SR-SIM validated their recruitment to the condensate region. Fluorescence Recovery After Photobleaching experiments revealed that Hsp70 inhibition significantly alters BCLAF1 mobility, indicating that Hsp70 activity is a key regulator of condensate dynamics. In conclusion, BCLAF1 undergoes phase separation in response to distinct genotoxic stressors, such as doxorubicin and cisplatin. Hsp70 and VCP are recruited to these condensates, with Hsp70 specifically modulating their dynamics, highlighting the essential role of protein quality control in governing BCLAF1 condensate behavior during the cellular stress response.