Research Projects

Macrophages, the frontline immune cells, operate in dynamic tissue environments encountering mechanical stress during migration, adhesion, and phagocytosis. These physiological forces strain the actin cytoskeleton and its associated focal adhesion (FA) proteins such as Filamin A, α Actinin, and Vinculin, which are mechanosensitive scaffolds that reversibly unfold and refold under tension. While this structural plasticity is essential for function, chronic mechanical stress may lead to irreversible misfolding and aggregation, contributing to immune dysfunction and fibrosis. Yet, how macrophages maintain cytoskeletal proteostasis under such conditions remains poorly understood.Molecular chaperones like Hsp70 and its co chaperone BAG3 are essential regulators of proteostasis under thermal and oxidative stress. However, their role in preserving the integrity of force-sensitive proteins in immune cells remains largely unexplored. Hypothesis: Hsp70 and BAG3 mitigate force-induced misfolding of focal adhesion proteins in macrophages, thus preserving cytoskeletal proteostasis. Aim 1: FA assembly occurs under physiological forces (1 to 20 pN), yet how these forces modulate the conformational stability of FA proteins in macrophages is unclear. We will use single molecule magnetic tweezers (MT) to apply defined piconewton-scale pulses to recombinant Filamin A, α Actinin, and Vinculin domains. These assays will characterize unfolding thresholds and refolding kinetics under physiological constraints. Aim 2: Chaperones are known to influence protein folding, but their function under force remains unresolved. This aim will assess whether Hsp70 and BAG3 can facilitate folding or prevent misfolding of mechanically strained FA proteins. MT-based folding assays will be performed in the presence of individual or combined chaperones, including ATPase-deficient mutants, to evaluate their energy-coupled activity. Aim 3: Mechanical proteotoxicity has been implicated in immune cell dysfunction, but direct validation in macrophages is lacking. We will test chaperone-mediated rescue in RAW264.7 and THP 1 cells expressing HaloTagged FA proteins and cultured on stiffness-tuned substrates. Mechanical and oxidative stress will be applied, and chaperone activity modulated via siRNA, inhibitors, or overexpression. Readouts include aggregate formation, p62 or ubiquitin markers and NF-κB activation. This interdisciplinary study integrates single molecule biophysics with cell biology to uncover how chaperones respond to mechanical destabilization of cytoskeletal proteins in immune cells. It will generate real-time kinetic benchmarks for chaperone activity under force, map domains of mechanical vulnerability, and define molecular rescue pathways in stressed macrophages. These insights will not only advance our fundamental understanding of immune cell mechanoproteostasis but may also identify novel chaperone-based therapeutic strategies for inflammatory and fibrotic diseases.