Date of Award

2023

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

Abstract

Vascular smooth muscle cells (SMCs) surround many blood vessels throughout the body,where they dynamically alter vessel diameter to regulate blood pressure, provide structural integrity, and absorb shock on a beat-to-beat timescale. As smooth muscle function fails, profound vascular disease ensues, often with tragic results- even death. Smooth muscle myosin 2 (SM2) is the dominant motor protein that actuates contractility and allows SMCs to perform these vital functions. To function, SM2 monomers dynamically assemble into filaments, which upon SMC activation, associate with filamentous actin to drive contractility. Despite the critical contribution of SM2 to SMC function, foundational aspects of SM2 assembly and dynamics remain unexplored. To remedy this, we expressed EGFP-tagged SM2 in rat aortic smooth muscle cells (A7R5), which retained a cytosolic calcium and contractile response to the acetylcholine agonist carbachol. Using fluorescence recovery after photobleaching (FRAP), we observed rapid polymer exchange kinetics for SM2, more similar to non-muscle myosin 2 (NM2) than striated myosin 2s. Consistently, super-resolution imaging of SM2 and NM2 suggests they form filamentous co-polymers. Using a single cell filament assembly assay, we observed that the majority of SM2 is assembled in filaments at steady state, but that SMC activation with carbachol rapidly increases SM2 assembly levels. Carbachol also reduced polymer exchange kinetics, suggesting stabilization of filaments during SMC activation. This carbachol-induced increase in SM2 assembly and decrease in exchange kinetics closely parallels the cytosolic calcium and contractility kinetics. Furthermore, both dynamics and assembly of smooth muscle myosin were altered in pathological mutations associated with thoracic aortic aneurysm and dissection, suggesting regulation of dynamics and assembly are vital for smooth muscle function. Collectively, our data supports an updated model in which highly dynamic SM2 filaments assemble, are stabilized, and are activated to produce cell-scale contractile forces during SMC activation.

Creative Commons License

Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License.

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