Significance Diverse demands on cardiac muscle require the fine-tuning of contraction. Cardiac myosin binding protein-C (cMyBP-C) is involved in this regulation; however, its precise molecular mechanism of action remains uncertain. By imaging the interactions of single myosin and cMyBP-C molecules interacting with suspended thin filaments in vitro we observe cMyBP-C N-terminal fragments assist activation and modulate contraction velocity by affecting myosin binding to the thin filament. Fluorescent imaging of Cy3-labeled cMyBP-C revealed that it diffusively scans the thin filament and then strongly binds to displace tropomyosin and activate at low calcium. At high calcium, cMyBP-C decorates the filament more extensively, reducing myosin binding through competing with binding sites. Understanding the mechanism of MyBP-C action has important implications for heart disease.
Regulated thin filaments (RTFs) tightly control striated muscle contraction through calcium binding to troponin, which enables tropomyosin to expose myosin-binding sites on actin. Myosin binding holds tropomyosin in an open position, exposing more myosin-binding sites on actin, leading to cooperative activation. At lower calcium levels, troponin and tropomyosin turn off the thin filament; however, this is antagonised by the high local concentration of myosin, questioning how the thin filament relaxes. To provide molecular details of deactivation, we used single-molecule imaging of green fluorescent protein (GFP)-tagged myosin-S1 (S1-GFP) to follow the activation of RTF tightropes. In sub-maximal activation conditions, RTFs are not fully active, enabling direct observation of deactivation in real time. We observed that myosin binding occurs in a stochastic step-wise fashion; however, an unexpectedly large probability of multiple contemporaneous detachments is observed. This suggests that deactivation of the thin filament is a coordinated active process.
Mitosis is a complex self-organising process that achieves high fidelity separation of duplicated chromosomes into two daughter cells through capture and alignment of chromosomes to the spindle mid-plane. Chromosome movements are driven by kinetochores, multi-protein machines that attach chromosomes to microtubules (MTs), both controlling and generating directional forces. Using lattice light sheet microscopy imaging and automated near-complete tracking of kinetochores at fine spatio-temporal resolution, we produce a detailed atlas of kinetochore metaphase-anaphase dynamics in untransformed human cells (RPE1). We fitted 18 biophysical models of kinetochore metaphase-anaphase dynamics to experimental data using Bayesian inference, and determined support for the models with model selection methods, demonstrating substantial sister force asymmetry and time dependence of the mechanical parameters. Our analysis shows that K-fiber pulling and pushing strengths are inversely correlated and that there is substantial spatial organisation of KT dynamic properties both within, and transverse to the metaphase plate. Further, K-fiber forces are tuned over the last 5 mins of metaphase towards a set point, which we refer to as the anaphase ready state.
In this paper we describe the design, fabrication and characterization of gold nano-patches, deposited on gallium nitride substrate, acting as optical nanoantennas able to efficiently localize the electric field at the metal–dielectric interface. We analyse the performance of the proposed device, evaluating the transmission and the electric field localization by means of a three-dimensional finite difference time domain (FDTD) method. We detail the fabrication protocol and show the morphological characterization. We also investigate the near-field optical transmission by means of scanning near-field optical microscope measurements, which reveal the excitation of a localized surface plasmon resonance at a wavelength of 633 nm, as expected by the FDTD calculations. Such results highlight how the final device can pave the way for the realization of a single optical platform where the active material and the metal nanostructures are integrated together on the same chip.
Abstract Cardiac muscle contraction is activated by calcium binding to troponin and the consequent motion of tropomyosin on actin within the sarcomere. These movements permit myosin binding, filament sliding and motion generation. One potential mechanism by which the N-terminal domains of cardiac myosin-binding protein C (cMyBP-C) play a modulatory role in this activation process is by cMyBP-C binding directly to the actin-thin filament at low calcium levels to enhance the movement of tropomyosin. To determine the molecular mechanisms by which cMyBP-C enhances myosin recruitment to the actin-thin filament, we directly visualized fluorescently-labelled cMyBP-C N-terminal fragments and GFP-labelled myosin molecules binding to suspended actin-thin filaments in a fluorescence-based single molecule microscopy assay. Binding of the C0C3 N-terminal cMyBP-C fragment to the thin filament enhanced myosin association at low calcium levels. However, at high calcium levels, C0C3 bound cooperatively, blocking myosin binding. Dynamic imaging of thin filament-bound Cy3-C0C3 molecules demonstrated that these fragments diffuse along the thin filament before statically binding, suggesting a mechanism that utilizes a weak-binding mode to search for access to the thin filament and a tight-binding mode to sensitize the thin filament to calcium and thus, enhance myosin binding. Although shorter N-terminal fragments (Cy3-C0C1 and Cy3-C0C1f) bound to the thin filaments and displayed modes of motion on the thin filament similar to that of the Cy3-C0C3 fragment, the shorter fragments were unable to sensitize the thin filament. Therefore, the longer N-terminal fragment (C0C3) must possess the requisite domains needed to bind specifically to the thin filament in order for the cMyBP-C N terminus to modulate cardiac contractility.
Muscle contraction is a finely tuned mechanism involving cyclical interactions between
actin and myosin, regulated by calcium through troponin and tropomyosin and
modulated by myosin binding protein-C. Genetic mutations of the proteins involved in
such complex mechanism can thus lead to potential life threatening diseases, such as
Hypertrophic Cardiomyopathy (HCM). Although being mostly asymptomatic, HCM
affects 1 in 500 people, ultimately leading to poor prognosis and sudden death,
thought to occur through the impairment of relaxation during diastole.
In this thesis I present the experiments conducted to improve our current
understanding of the molecular mechanism behind HCM, specifically on the role of
tropomyosin and myosin binding protein-C in modulating thin filament activation and
relaxation. Using a single molecule approach, we first visualised fluorescent myosin
binding to reconstituted thin filaments and examined their dynamics in the presence
of the tropomyosin HCM causing E180G mutation, demonstrating a shift of the thin
filament activation state towards the closed state, facilitating myosin binding at low
calcium, and a reduction of the thin filament regulatory unit.
We then looked at the dynamics of very highly concentrated clusters of myosin,
showing how the sudden collapse of these active regions cannot be explained by
normal relaxation mechanisms, thus suggesting an alternative mechanistic role for
tropomyosin and how its mutations could lead to impaired relaxation in HCM.
Finally, we turned our focus on N-terminal fragments of cardiac myosin binding
protein-C (cMyBP-C) and study their role in thin filament activation, by looking at how
they affect acto-myosin interactions. We found that only the presence of the whole
cMyBP-C N-terminus was able to promote acto-myosin interactions at low Ca2+ or
repressing them at high Ca2+. Moreover, by looking at the dynamics of the fragments,
we were able to determine that cMyBP-C possesses a two steps binding mechanism to
actin, leading us to define its mechanism by which it activates the thin filament.
Abstract Current models infer that the microtubule-based mitotic spindle is built from GDP-tubulin with small GTP caps at microtubule plus-ends, including those that attach to kinetochores (K-fibres). Here we reveal that K-fibres additionally contain a dynamic mixed-nucleotide zone that reaches several microns in length. This zone becomes visible in cells expressing fluorescently labelled EBs, a known marker for GTP-tubulin, and endogenously-labelled HURP - a protein which we show to preferentially bind the GDP microtubule lattice in vitro . In living cells HURP accumulates on the ends of depolymerising K-fibres, whilst avoiding recruitment to nascent polymerising K-fibres. This gives rise to a growing “HURP-gap” which we can recapitulate in a minimal computational simulation. We therefore postulate that the K-fibre lattice contains a dynamic, micron-sized mixed-nucleotide zone. One Sentence Summary We reveal that the microtubules of the mitotic spindle contain a third, uncharacterized domain, a mixed nucleotide zone that resides between the GTP-cap and the GDP-tubulin lattice.
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