Biology subjects

Gardner, M. K.

Publications and source records attributed to Gardner, M. K..

6 recordsLinked to original sources

bioRxiv2025

17β-Estradiol Counteracts Pathological Microtubule Remodeling To Enhance Cardiac Function

The female-predominate sex hormone 17{beta}-estradiol exerts cardioprotective effects via multiple mechanisms. Available data demonstrate 17{beta}-estradiol modulates microtubule dynamics in vitro, but its effects on pathogenic microtubule remodeling in pressure-overloaded cardiomyocytes are unexplored. Here, we show 17{beta}-estradiol directly blunts microtubule polymerization in vitro, counteracts endothelin-mediated microtubule remodeling in iPSC-cardiomyocytes, and mitigates microtubule stabilization in pulmonary artery banded right ventricular cardiomyocytes. 17{beta}-estradiol treatment blunts cardiomyocyte and nuclear hypertrophy, restores t-tubule architecture, and prevents mislocalization of connexin-43 in RV cardiomyocytes of pulmonary artery banded rats. These cellular phenotypes are paired with significant improvements in RV function. Thus, we propose 17{beta}-estradiol exerts cardioprotective effects via direct modulation of microtubules in addition to its well ascribed signaling functions.

physiology↗

bioRxiv2022

Rapid binding to protofilament edge sites facilitates tip tracking of EB1 at growing microtubule plus-ends

EB1 is a key cellular protein that delivers critical regulatory molecules throughout the cell via the tip-tracking of growing microtubule plus-ends. Thus, it is important to understand the mechanism for how EB1 efficiently tracks growing microtubule plus-ends. It is widely accepted that EB1 binds with higher affinity to GTP-tubulin subunits at the growing microtubule tip, relative to GDP-tubulin along the microtubule length. However, it is unclear whether this difference in affinity alone is sufficient to explain the tip-tracking of EB1 at growing microtubule tips. Previously, we found that EB1 binds to exposed microtubule protofilament-edge sites at a [~]70-fold faster rate than to closed-lattice sites, due to diffusional steric hindrance to binding. Thus, we asked whether rapid protofilament-edge binding could contribute to efficient EB1 tip tracking. A computational simulation with differential EB1 on-rates based on closed-lattice or protofilament-edge binding, and with EB1 off-rates that were dependent on tubulin hydrolysis state, recapitulated experimental EB1 tip tracking. To test this model, we used cell-free biophysical assays, as well as live-cell imaging, in combination with the chemotherapy drug Eribulin. We found that Eribulin blocked EB1 protofilament-edge binding, which led to a dramatic decrease in EB1 tip tracking on dynamic microtubules. We conclude that rapid EB1 binding to microtubule protofilament-edge sites increases the efficiency of EB1 tip tracking at the growing microtubule plus end.

cell biology↗

bioRxiv2020

Oxidative stress pathogenically remodels the cardiac myocyte cytoskeleton via structural alterations to the microtubule lattice

In the failing heart, the cardiac myocyte microtubule network is remodeled, which increases cellular stiffness and disrupts contractility, contributing to heart failure and death. However, the origins of this deleterious cytoskeletal reorganization are unknown. We now find that oxidative stress, a condition characteristic of failing heart cells, leads to cysteine oxidation of microtubules. Further, our electron and fluorescence microscopy experiments revealed regions of structural damage within the oxidized microtubule lattice. These damaged regions led to the lengthening, realignment, and acetylation of dynamic microtubules within cardiac myocytes. Thus, we found that oxidative stress acts inside of cardiac myocytes to facilitate a dramatic, pathogenic shift from a dynamic, multifaceted microtubule network into a highly acetylated, longitudinally aligned, and static microtubule network. Our results demonstrate how a disease condition characterized by oxidative stress can trigger a molecular oxidation event, which propagates a toxic cellular-scale transformation of the cardiac myocyte microtubule network.Competing Interest StatementThe authors have declared no competing interest.View Full Text

cell biology↗

bioRxiv2020

UNC-45A Destabilizes MTs Independent of its Effect on Non-Muscle Myosin II

In invertebrates, UNC-45 regulates myosin stability and functions. Vertebrates have two distinct isoforms of the protein: UNC-45B, expressed in muscle cells only and UNC-45A, expressed in all cells and implicated in regulating both Non-Muscle Myosin II (NMII)- and microtubule (MT)-associated functions. Here we show for the first time that: a) in vitro UNC-45A binds to the MT lattice and weakens its integrity leading to MT bending, breakage and depolymerization, b) in cells, UNC-45A overexpression causes loss of MT mass and increase in MT breakages, c) both in vitro and in cells, UNC-45A destabilizes MTs independent of its NMII C-terminal binding domain and destabilization occurs even in presence of the NMII inhibitor blebbistatin. These findings are consistent with a not mutually exclusive but rather dual role of UNC-45A in regulating NMII activity and MT stability. Because many human diseases, from cancer to neurodegenerative diseases, are caused by or associated with deregulation of MT stability our findings have profound implications in both, the biology of MTs as well as the biology of human diseases and possible therapeutic implications for their treatment.

cell biology↗

bioRxiv2019

Stutter: a Transient Dynamic Instability Phase that is Strongly Associated with Catastrophe

Microtubules (MTs) are cytoskeletal fibers that undergo dynamic instability (DI), a remarkable process involving phases of growth and shortening separated by stochastic transitions called catastrophe and rescue. Dissecting dynamic instability mechanism(s) requires first characterizing and quantifying these dynamics, a subjective process that often ignores complexity in MT behavior. We present a Statistical Tool for Automated Dynamic Instability Analysis (STADIA), which identifies and quantifies not only growth and shortening, but also a category of intermediate behaviors that we term stutters. During stutters, the rate of MT length change tends to be smaller in magnitude than during typical growth or shortening phases. Quantifying stutters and other behaviors with STADIA demonstrates that stutters precede most catastrophes in our dimer-scale MT simulations and in vitro experiments, suggesting that stutters are mechanistically involved in catastrophes. Related to this idea, we show that the anti-catastrophe factor CLASP2{gamma} works by promoting the return of stuttering MTs to growth. STADIA enables more comprehensive and data-driven analysis of MT dynamics compared to previous methods. The treatment of stutters as distinct and quantifiable DI behaviors provides new opportunities for analyzing mechanisms of MT dynamics and their regulation by binding proteins.

cell biology↗

bioRxiv2019

Structural state recognition facilitates tip tracking of EB1 at growing microtubule ends in cells

The microtubule binding protein EB1 specifically targets the growing ends of microtubules in cells, where EB1 facilitates the interactions of cellular proteins with microtubule plus-ends. Microtubule end targeting of EB1 has been attributed to high affinity binding of EB1 to GTP-tubulin that is present at growing microtubule ends. However, our 3D single-molecule diffusion simulations predicted a [~]6000% increase in EB1 arrivals to open, tapered microtubule tip structures relative to closed lattice conformations. Using quantitative fluorescence, single-molecule, and electron microscopy experiments, we found that the binding of EB1 onto opened, structurally disrupted microtubules was dramatically increased relative to closed, intact microtubules, regardless of hydrolysis state. Correspondingly, in cells, the conversion of growing microtubule ends from a tapered into a blunt configuration resulted in reduced EB1 targeting. Together, our results suggest that microtubule structural recognition, based on a fundamental diffusion-limited binding model, facilitates the tip tracking of EB1 at growing microtubule ends.

cell biology↗