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bioRxiv · 10.1101/2022.10.10.511341

Smooth Muscle Myosin 2 Filaments Dynamically Assemble and Stabilize During Induced Contractility

Bennett, M. A.·Demeulenaere, S. G.·Wu, H.·Patel, H.·Sala, S.·Longtine, E. R.·Oakes, P. W.·Beach, J. R.
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Abstract

BackgroundVascular smooth muscle cells (SMCs) dynamically tune blood vessel diameter to regulate blood pressure, provide vessel wall structural integrity, and absorb shock on a beat-to-beat timescale. Smooth muscle myosin 2 (SMII) is the dominant motor protein driving SMC contraction. To function, SMII monomers dynamically assemble into filaments, which associate with the actin cytoskeleton to drive contractility. Precisely how SMII filaments assemble and exchange in living SMCs, however, both at steady-state and during induced contractility, remains poorly defined. MethodsWe used a single-cell filament assembly assay to determine SMII assembly into filaments at steady-state and upon induced contractility in rat aortic SMCs (A7R5) transiently-expressing EGFP-tagged SM1A isoform of SMII. We then used fluorescence recovery after photobleaching (FRAP) to characterize SMII exchange kinetics at steady-state and upon induced contractility, and measured changes in force production using traction force microscopy. Finally, we developed a CRISPR knock-in EGFP-SMII murine model to quantify SMII dynamics at endogenous expression in primary SMCs and intact arterioles. ResultsWhile predominantly filamentous at baseline, induced contraction rapidly increased SMII filament assembly. FRAP revealed rapid SMII exchange kinetics, more similar to non-muscle myosin II than striated myosin II, and induced contractility consistently stabilized SMII filaments. Super-resolution imaging revealed SMII and non-muscle myosin II filament structures consistent with co-assembly. Endogenous EGFP-SMII in primary SMCs and intact arterioles paralleled cell culture studies with similar baseline exchange kinetics and activation-dependent stabilization. ConclusionsTogether, these data support a model in which SMII is surprisingly dynamic and co-assembles with non-muscle myosin II. Vascular SMC activation further increases SMII filament assembly while reducing filament exchange, consistent with stabilization of a dynamic SMII pool during force generation, which allows cells to dynamically adapt their overall contractility in response to environmental conditions. CLINICAL IMPLICATIONSSmooth muscle myosin II (SMII) is the principal contractile motor protein of vascular smooth muscle tissue, but its molecular behavior in living differentiated SMCs remains poorly defined. Here, we show that SMII is not a static contractile scaffold but highly dynamic. SMII exhibits dynamic exchange between the filament and monomeric forms at baseline, but becomes acutely stabilized during agonist-induced contraction, including in primary SMCs and intact arterioles at endogenous Myh11 expression. These findings suggest that vascular tone is tuned not only by activating pre-existing myosin filaments, but also by rapidly shifting SMII filament assembly levels and exchange rates. This framework is relevant to diseases in which SMC contractility and arterial wall mechanics are abnormal, including MYH11-associated thoracic aortic disease and related disorders of arterial stiffness or maladaptive vasomotor regulation. Although this study is mechanistic, it identifies SMII filament dynamics as a measurable layer of vascular regulation and a potential readout for future studies of pathogenic MYH11 variants, disease modeling, and therapies that alter myosin activation and/or cytoskeletal stability. O_FIG O_LINKSMALLFIG WIDTH=192 HEIGHT=200 SRC="FIGDIR/small/511341v2_ufig1.gif" ALT="Figure 1"> View larger version (78K): org.highwire.dtl.DTLVardef@d64a8forg.highwire.dtl.DTLVardef@b9000corg.highwire.dtl.DTLVardef@1b24a91org.highwire.dtl.DTLVardef@3d81be_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Bennett, M. A., Demeulenaere, S. G., Wu, H., Patel, H., Sala, S., Longtine, E. R., Oakes, P. W., Beach, J. R.. 2022-10-11. Smooth Muscle Myosin 2 Filaments Dynamically Assemble and Stabilize During Induced Contractility. https://doi.org/10.1101/2022.10.10.511341

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