Abstract P1072: Hypoxia Inhibits Cellular Contractility Via Allosteric Regulation Of Microtubule Acetylation

Abstract

Hypoxia is implicated in pathogenesis of many cardiovascular diseases (CVDs), including loss of cardiac contractility; but its effects on cytoskeletal dynamics are not well understood. The microtubule (MT) network provides mechanical strength to cardiomyocytes and its dysregulation is observed in many CVDs. Acetylated MTs provide structural flexibility to cardiomyocytes and protect against proteinopathy-induced cardiac failure. MT acetylation is exclusively catalyzed by α-TAT1, but little is known about how α-TAT1 is regulated. Here we report that hypoxia inhibits MT acetylation and promotes nuclear accumulation of α-TAT1. We show that cytosolic localization of α-TAT1, disrupted in hypoxia, is critical for MT acetylation. Using computational, live cell microscopy and biochemical approaches, we identified a conserved localization motif in the intrinsically disordered C-terminus of α-TAT1, consisting of three competing regulatory elements - nuclear export, nuclear import, and cytosolic retention. Inhibiting Exportin 1 induced nuclear accumulation of α-TAT1. We also found that α-TAT1 cytosolic localization is mediated by CDK1, CK2 and PKA kinases, and inhibited by PP2A phosphatase, through a critical residue: T 322 that binds to 14-3-3 proteins. α-TAT1 knock-out (KO) fibroblasts show defects in actomyosin contractility and focal adhesions. We developed an optogenetic tool, named optoATAT1, which rapidly and reversibly shuttled from nucleus to cytosol on blue light stimulation. optoATAT1 stimulation in HeLa cells increased MT acetylation unlike those kept in dark, validating the tool. optoATAT1 stimulation also led to increase in cell contractility indicated by myosin accumulation and focal adhesion maturation, thus confirming a causal relationship between MT acetylation and cell contractility. Also, α-TAT1 KO fibroblasts are highly proliferative, which may pertain to cardiac hypertrophy. In summary, we have identified a novel role of α-TAT1 C-terminus in regulating MT acetylation and cell contractility under hypoxia. Along with this novel regulatory mechanism of MT acetylation, multiple pharmacological agents identified in this study to modulate spatial regulation of α-TAT1 may offer new insights into the treatment of CVDs.