Biology subjects

Beach, J. R.

Publications and source records attributed to Beach, J. R..

4 recordsLinked to original sources

bioRxiv2023

Local Monomer Levels and Established Filaments Potentiate Non-Muscle Myosin 2 Assembly

The ability to dynamically assemble contractile networks is required throughout cell physiology, yet the biophysical mechanisms regulating non-muscle myosin 2 filament assembly in living cells are lacking. Here we use a suite of dynamic, quantitative imaging approaches to identify deterministic factors that drive myosin filament appearance and amplification. We find that actin dynamics regulate myosin assembly, but that the actin architecture plays a minimal direct role. Instead, remodeling of actin networks modulates the local myosin monomer levels and facilitates assembly through myosin:myosin driven interactions. Using optogenetically controlled myosin, we demonstrate that locally concentrating myosin is sufficient to both form filaments and jump-start filament amplification and partitioning. By counting myosin monomers within filaments, we demonstrate a myosin-facilitated assembly process that establishes sub-resolution filament stacks prior to partitioning into clusters that feed higher-order networks. Together these findings establish the biophysical mechanisms regulating the assembly of non-muscle contractile structures that are ubiquitous throughout cell biology.

cell biology↗

bioRxiv2023

Non-muscle myosin 2 filaments are processive in cells

Directed transport of cellular components is often dependent on the processive movements of cytoskeletal motors. Myosin 2 motors predominantly engage actin filaments of opposing orientation to drive contractile events, and are therefore not traditionally viewed as processive. However, recent in vitro experiments with purified non-muscle myosin 2 (NM2) demonstrated myosin 2 filaments could move processively. Here, we establish processivity as a cellular property of NM2. Processive runs in central nervous system-derived CAD cells are most apparent as processive movements on bundled actin in protrusions that terminate at the leading edge. We find that processive velocities in vivo are consistent with in vitro measurements. NM2 makes these processive runs in its filamentous form against lamellipodia retrograde flow, though anterograde movement can still occur in the absence of actin dynamics. Comparing the processivity of NM2 isoforms, we find that NM2A moves slightly faster than NM2B. Finally, we demonstrate that this is not a cell-specific property, as we observe processive-like movements of NM2 in the lamella and subnuclear stress fibers of fibroblasts. Collectively, these observations further broaden NM2 functionality and the biological processes in which the already ubiquitous motor can contribute.

cell biology↗

bioRxiv2022

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

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

cell biology↗

bioRxiv2021

Inhibitory and Stimulatory Micropeptides Preferentially Bind to Different Conformations of the Cardiac Calcium Pump

The ATP-dependent ion pump SERCA sequesters Ca2+ in the endoplasmic reticulum to establish a reservoir for cell signaling. Because of its central importance in physiology, this transporter is tightly controlled by physical interactions with tissue-specific regulatory micropeptides that tune SERCA function to match changing physiological conditions. In the heart, phospholamban (PLB) inhibits SERCA, while dwarf open reading frame (DWORF) stimulates SERCA. These competing interactions determine cardiac performance by modulating the amplitude of Ca2+ signals that drive the contraction/relaxation cycle. The distinct functions of these peptides may relate to their reciprocal preferences for SERCA binding. While SERCA binds PLB more avidly at low cytoplasmic Ca2+, it binds DWORF better at high Ca2+. In the present study, we determined that this opposing Ca2+ sensitivity is due to preferential binding of DWORF and PLB to different intermediate conformations that the pump samples during the Ca2+ transport cycle. The results suggest a mechanistic basis for inhibitory and stimulatory micropeptide function. In addition, fluorescence resonance energy transfer (FRET) measurements revealed dynamic shifts in SERCA-micropeptide binding equilibria during cellular Ca2+ elevations. The data suggest Ca2+-dependent dynamic exchange of inhibitory and stimulatory micropeptides from SERCA during the cardiac cycle. Together, these mechanisms provide beat-to-beat modulation of cardiac Ca2+ handling and contribute to the hearts adaptation to the increased physiological demands of exercise. O_TEXTBOXSignificance Interactions between SERCA and its regulatory micropeptides modulate cardiac performance. A previous study demonstrated that while the SERCA inhibitor, phospholamban, loses affinity during intracellular Ca2+ elevations, its competitor DWORF stimulates SERCA and binds better at elevated Ca2+. Here, we found this Ca2+-dependent difference in affinity is driven by preferential micropeptide binding to different SERCA conformations sampled during its enzymatic cycle. Tight binding to different conformations of the pump may underly the distinct mechanistic functions of these regulators. Lower affinity during alternating phases of the cardiac cycle may drive dynamic exchange of inhibitory and stimulatory micropeptides during contraction and relaxation. Our results reveal mechanisms that modulate cardiac Ca2+ handling on a beat-to-beat basis and help the heart adapt to exercise. C_TEXTBOX

biophysics↗