Biology subjects

Geeves, M.

Publications and source records attributed to Geeves, M..

3 recordsLinked to original sources

bioRxiv2025

Direct measurement of mavacamten and deoxyATP perturbation of the SRX/DRX ratio in porcine cardiac myofibrils using a simple, accessible and multiplexed approach

Cardiac muscle adapts to varying physiological demands by modulating the number of active myosin II motors available for contraction. These motors are organized into thick filaments in the sarcomere and generate force through an ATP-dependent interaction with thin filaments that contain actin. To conserve energy, when demand is low myosin can occupy the super-relaxed (SRX) state, which acts as a reserve. Here, we build upon the earlier studies from the Cooke lab to quantify the size of this cardiac reserve using fluorescence imaging of Cy3-ATP directly in myofibrils. This approach employs a pulse-chase method and exploits the high permeability of isolated myofibrils to monitor nucleotide release in situ. By preserving sarcomeric architecture while enabling rapid reagent exchange, this method bridges the gap between complex single-molecule imaging and traditional stopped-flow bulk assays using MANT-ATP. Using this approach we have studied biochemical perturbation of the SRX reserve with deoxyATP and mavacamten. DeoxyATP caused large depletion of the cardiac reserve, and mavacamten increased its size, consistent with its clinical application. Our results demonstrate the utility of this technique and the potential for further enhancement using multiplexing, holding promise for future applications in health and disease.

biophysics↗

bioRxiv2024

Spatially resolving how phosphorylation affects β-cardiac myosin activity in porcine myofibril sarcomeres with single molecule resolution

Cardiac muscle contraction is mediated by myosin binding from the thick filament of the sarcomere to the thin filament in an ATP powered reaction. This process is highly regulated on a beat-to-beat basis by calcium interactions with the thin filament. Additionally, the number of heads available for participation in contraction is also regulated, resulting in a dynamically variable reserve of heads for controlling contractile force. We aimed to discover the size of this reserve and how it is modulated by phosphorylation. Using single molecule imaging of fluorescently labelled ATP molecules binding and releasing myosins within porcine cardiac sarcomeres, we could determine myosin activity with high spatial resolution. We find three kinetic species when examining the myosin ATPase. The fastest is consistent with non-specific ATP binding to myosins surface, and the slower two species are consistent with the previously identified DRX and SRX states. The former is thought to represent myosins in an ON state, ready to interact with the thin filament and the latter an OFF state with slowed ATPase that constitutes the cardiac reserve. We find that the cardiac reserve is 50% in the sarcomere and this can be sub-divided into the P-, C- and D-zones, with the D-zone having the least population of OFF heads (44%). Treatment with PKA phosphorylates cardiac myosin binding protein-C (cMyBP-C) leading to a 16% reduction in reserve in the C-zone (where cMyBP-C is found), a 10% reduction in the P-zone, and an unexpected 8% increase in the D-zone. By contrast, myosin regulatory light chain (RLC) phosphorylation with myosin light chain kinase (MLCK) resulted in a large 24% decrease in reserve myosins, interestingly the least affected area of the sarcomere was the C-zone. Altogether these data suggest that cMyBP-Cs interaction with RLC governs the degree of activation due to RLC phosphorylation.

biophysics↗

bioRxiv2017

Ultrafast glutamate sensors resolve synaptic short-term plasticity

Glutamatergic synapses display a rich repertoire of plasticity mechanisms on many different time scales, involving dynamic changes in the efficacy of transmitter release as well as changes in the number and function of postsynaptic glutamate receptors. The genetically encoded glutamate sensor iGluSnFR enables visualization of glutamate release from presynaptic terminals at frequencies up to [~]10 Hz. However, to resolve glutamate dynamics during high frequency bursts, faster indicators are required. Here we report the development of fast (iGluf) and ultrafast (iGluu) variants with comparable brightness, but increased Kd for glutamate (137 M and 600 M, respectively). Compared to iGluSnFR, iGluu has a 6-fold faster dissociation rate in vitro and 5-fold faster kinetics in synapses. Fitting a three-state model to kinetic data, we identify the large conformational change after glutamate binding as the rate-limiting step. In rat hippocampal slice culture stimulated at 100 Hz, we find that iGluu is sufficiently fast to resolve individual glutamate release events, revealing that glutamate is rapidly cleared from the synaptic cleft. Depression of iGluu responses during 100 Hz trains correlates with depression of postsynaptic EPSPs, indicating that depression during high frequency stimulation is purely presynaptic in origin. At individual boutons, the recovery from depression could be predicted from the amount of glutamate released on the second pulse (paired pulse facilitation/depression), demonstrating differential frequency-dependent filtering of spike trains at Schaffer collateral boutons.\n\nSignificance StatementExcitatory synapses convert presynaptic action potentials into chemical signals that are sensed by postsynaptic glutamate receptors. To eavesdrop on synaptic transmission, genetically encoded fluorescent sensors for glutamate have been developed. However, even the best available sensors lag behind the very fast glutamate dynamics in the synaptic cleft. Here we report the development of an ultrafast genetically encoded glutamate sensor, iGluu, which allowed us to image glutamate clearance and synaptic depression during 100 Hz spike trains. We found that only boutons showing paired-pulse facilitation were able to rapidly recover from depression. Thus, presynaptic boutons act as frequency-specific filters to transmit select features of the spike train to specific postsynaptic cells.

neuroscience↗