Oxidative Stress Restructures the Cellular Microtubule Cytoskeleton via Repair-Mediated Rescue Events

Abstract

Microtubules are cylindrical cytoskeletal polymers composed of a/β-tubulin heterodimers that make up an ordered tubulin lattice. Microtubules display dynamic length changes, termed “dynamic instability”, in which slow elongation phases are followed by rapid shortening. In cells, microtubules form a network that is a key component of the cellular cytoskeleton. Under pathological conditions of oxidative stress, we and others have found that cardiomyocytes display a denser microtubule cytoskeleton, which may lead to the progressive structural and functional cellular changes associated with myocardial ischemia and systolic dysfunction. This reorganization of the microtubule network occurs despite only small increases in tubulin expression, suggesting that alterations to microtubule length regulation are involved. Using biophysical reconstitution experiments and live-cell imaging, we found that oxidative stress directly provokes microtubule ‘rescue,’ the transition from rapid microtubule shortening to slow elongation, while it has little effect on other microtubule dynamic instability parameters or the microtubule nucleation rate. To explore a mechanism for this observation, we used electron microscopy, and observed structural damage, consisting of holes and sheet-like structures, under conditions of moderate oxidative stress. Further, using a quantitative two-color tubulin “repair” assay (Reid et al, 2017), we found that repair of structural defects within the microtubule lattice via the incorporation of free tubulin was 133% higher under conditions of oxidative stress compared to controls. Such repaired regions have been termed ‘rescue islands’ because they facilitate rescue events, and thus promote net microtubule elongation (Dimitrov et al., 2008). We conclude that microtubule structural damage may explain our observations of oxidative stress-mediated increases in microtubule density in cardiomyocytes, potentially providing insight into the progressive myocardial changes that accompany Ischemic Heart Disease.