Student Theses and Dissertations
Date of Award
2025
Document Type
Thesis
Degree Name
Doctor of Philosophy (PhD)
Thesis Advisor
Gregory M. Alushin
Keywords
mechanotransduction, LIM proteins, actin cytoskeleton, force-activated binding, stress fiber repair, cytoskeleton-to-nucleus signaling
Abstract
For tissues to develop and maintain mechanical homeostasis, cells must be able to perceive and respond to mechanical cues in their local environments (mechanosense). While there has been significant progress in understanding the physiological significance of mechanosensation, the mechanisms by which proteins convert mechanical stimuli into biochemical signals (mechanotransduction) are poorly understood. The actin cytoskeleton serves as a nexus for cellular mechanotransduction, generating and transducing forces into intracellular signals that coordinate cell migration, cellular contractility, organelle dynamics, and gene expression. However, our understanding of the molecular mechanisms by which cytoskeletal mechanotransduction is achieved is limited. LIM domain proteins are a superfamily of cytoskeleton-associated proteins involved in cytoskeletal remodeling and transcriptional regulation, but how mechanical forces modulate their functions is unclear. Here, I employed an interdisciplinary toolkit of structural, biophysical, and cell biology methods to investigate mechanisms of cytoskeletal mechanotransduction through LIM proteins. In a collaborative project, we characterized the LIM domain superfamily and tested the hypothesis that LIM proteins are mechanosensitive actin binding proteins. Using an imaging-based screen of mechanically-stretched fibroblasts, I identified the zyxin, FHL, and paxillin families of tandem LIM domain proteins to be mechanoresponsive cytoskeletal proteins. A single conserved phenylalanine in each LIM domain was identified to be necessary for mechanosensitivity. Through development of an in vitro force reconstitution system, we discovered that LIM proteins are the first class of actin binding proteins to only bind actin filaments when they are under force. We show that strained actin binding acts as a cytoplasmic sink to retain FHL2 from shuttling into the nucleus, potentially inhibiting its activity as a transcriptional co-regulator. These studies unearthed a novel force-activated actin-binding mechanism, shedding light on how LIM proteins can associate with the cytoskeleton despite lacking canonical actin-binding domains, and established a pathway that could mediate cytoskeleton-to-nucleus mechanotransduction by FHL2. These studies set the stage for investigating how force-activated binding by LIM proteins could coordinate downstream mechanotransduction. Thus, I next focused on investigating how these force-activated binding events could mediate stress fiber repair: a paradigmatic example of cytoskeletal mechanotransduction mediated by the LIM protein zyxin. While cellular studies have uncovered the molecular components involved in stress fiber repair, the precise molecular mechanism by which zyxin coordinates the recognition of tensed actin filaments with downstream effector functions of repair proteins is unknown. Through reconstitution of the stress fiber repair reaction with purified proteins, I found that zyxin and other mechanosensitive LIM proteins form force-activated assemblies that bridge actin fragments, acting as a molecular bandage and sustaining the stress across broken filaments. We hypothesize that these force-activated assemblies are polymers of LIM proteins, representing a new class of force-evoked biomolecular assembly. I found that these force-activated zyxin assemblies also serve as hubs for the binding and recruitment of stress fiber repair factors. Through a minimal reconstitution of a contractile stress fiber-like network, I found that force-activated zyxin assemblies coordinate actin nucleation and crosslinking by repair factors VASP and α-actinin, respectively, to orchestrate stress fiber repair at the filament network scale. Recent studies have shown that zyxin localization is a strong molecular marker of high traction stresses in cells. Based on our findings that zyxin only binds actin filaments in the presence of myosin-evoked forces in vitro, we hypothesized that zyxin-enriched regions of the cytoskeleton would feature myosin-evoked actin filament (F-actin) conformations. Notably, studies applying our in vitro force reconstitution assays on EM grids revealed striking force-evoked superhelical actin filament segments. Thus, I investigated whether these F-actin structures are present at zyxin enriched cytoskeletal structures, such as focal adhesions and stress fibers, using cryo-electron tomography. In a collaborative project, we found oscillatory F-actin structures enriched at adhesions and stress fibers high in zyxin, reminiscent of myosin-evoked superhelical F-actin. These results suggest that myosin-evoked forces can generate superhelical F-actin structures in cells that may be sensed by mechanosensitive actin binding proteins. Most recently, I investigated the cytoskeleton-to-nucleus mechanotransduction pathway of FHL2, a mechanosensitive LIM protein reported to be a transcriptional co-regulator. Using multi-omics, cell biology, and biochemical assays, I characterized the cytoskeletal and nuclear functions of FHL2. While the nuclear functions of FHL2 remain elusive, I found that FHL2 is negative regulator of cytoskeletal mechanics, modulating focal adhesion morphology and cellular contractility. Collectively, the research presented in this thesis sheds new insights to the molecular mechanisms of LIM protein mechanotransduction through strained actin binding. Moreover, these studies reveal how mechanical homeostasis is coordinated by the LIM protein zyxin while providing new tools for studying the mechanisms of cytoskeletal mechanotransduction and the assembly of contractile networks. Lastly, my cellular studies of FHL2 provide a molecular atlas of its potential functions as a signal relay between the cytoskeleton and the nucleus.
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This work is licensed under a Creative Commons Attribution-NonCommercial-Share Alike 4.0 International License.
Recommended Citation
Phua, Donovan Yong Zhi, "Molecular and Cellular Mechanisms of LIM Protein Mechanotransduction" (2025). Student Theses and Dissertations. 822.
https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/822
Comments
A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy