MECHANISMS OF HSPA1A EXPORT TO THE EXTRACELLULAR MEDIUM IN HEAT-SHOCKED CELLS

Heat shock proteins (HSPs) are essential molecular chaperones that regulate key stress-response pathways. Hsp70 family member HspA1A, localizes to the plasma membrane (mHspA1A) and is actively secreted into the extracellular space (eHspA1A) during cellular stress and in tumor cells. Distinctly, mHspA1A contributes to protein transport and lysosomal degradation, while eHspA1A functions as an immunogenic “danger” signal that activates immune responses, reflecting divergent cellular responses under stress and tumor progression. Previous studies suggest that mHspA1A binds to the plasma membrane via lipid interactions, namely phosphatidylserine (PS) and phosphatidylinositol 4-phosphate (PI4P). In this thesis, I tested the hypothesis that eHspA1A is independently regulated from mHspA1A. To evaluate this, I implemented lipid biosensors, pharmacological inhibitors, membrane fractionation, extracellular vesicle (EV) isolation, and endocytosis assays. My results contrast membrane localization, critically dependent on PS and PI4P, with eHspA1A, notably lipid independent. Kinetic analyses revealed that mHspA1A and eHspA1A accumulation differs after stress. Endocytosis assays further revealed that HspA1A internalizes within minutes of stress recovery and recycles through the endosomal pathway, a model where mHspA1A and eHspA1A are governed by distinct regulatory mechanisms. While mHspA1A relies on specific lipid interactions for membrane association, eHspA1A secretion is driven by vesicular pathways. Although largely lipid-independent, eHspA1A release is likely supported by compensatory mechanisms including exosomes, membrane shedding, and endocytic recycling, advancing our understanding of HspA1A transport.