Boglárka H. Várkuti

para-Nitroblebbistatin, the non-cytotoxic and photostable myosin II inhibitor.

Blebbistatin, the best characterized myosin II-inhibitor, is commonly used to study the biological roles of various myosin II isoforms. Despite its popularity, the use of blebbistatin is greatly hindered by its blue-light sensitivity, resulting in phototoxicity and photoconversion of the molecule. Additionally, blebbistatin has serious cytotoxic side effects even in the absence of irradiation, which may easily lead to the misinterpretation of experimental results since the cytotoxicity-derived phenotype could be attributed to the inhibition of the myosin II function. Here we report the synthesis as well as the in vitro and in vivo characterization of a photostable, C15 nitro derivative of blebbistatin with unaffected myosin II inhibitory properties. Importantly, para-nitroblebbistatin is neither phototoxic nor cytotoxic, as shown by cellular and animal tests; therefore it can serve as an unrestricted and complete replacement of blebbistatin both in vitro and in vivo.

Azidoblebbistatin, a photoreactive myosin inhibitor

Photoreactive compounds are important tools in life sciences that allow precisely timed covalent crosslinking of ligands and targets. Using a unique technique we have synthesized azidoblebbistatin, which is a derivative of blebbistatin, the most widely used myosin inhibitor. Without UV irradiation azidoblebbistatin exhibits identical inhibitory properties to those of blebbistatin. Using UV irradiation, azidoblebbistatin can be covalently crosslinked to myosin, which greatly enhances its in vitro and in vivo effectiveness. Photo-crosslinking also eliminates limitations associated with the relatively low myosin affinity and water solubility of blebbistatin. The wavelength used for photo-crosslinking is not toxic for cells and tissues, which confers a great advantage in in vivo tests. Because the crosslink results in an irreversible association of the inhibitor to myosin and the irradiation eliminates the residual activity of unbound inhibitor molecules, azidoblebbistatin has a great potential to become a highly effective tool in both structural studies of actomyosin contractility and the investigation of cellular and physiological functions of myosin II. We used azidoblebbistatin to identify previously unknown low-affinity targets of the inhibitor (EC50 ≥ 50 μM) in Dictyostelium discoideum, while the strongest interactant was found to be myosin II (EC50 = 5 μM). Our results demonstrate that azidoblebbistatin, and potentially other azidated drugs, can become highly useful tools for the identification of strong- and weak-binding cellular targets and the determination of the apparent binding affinities in in vivo conditions.

A highly soluble, non-phototoxic, non-fluorescent blebbistatin derivative

Blebbistatin is a commonly used molecular tool for the specific inhibition of various myosin II isoforms both in vitro and in vivo. Despite its popularity, the use of blebbistatin is hindered by its poor water-solubility (below 10 micromolar in aqueous buffer) and blue-light sensitivity, resulting in the photoconversion of the molecule, causing severe cellular phototoxicity in addition to its cytotoxicity. Furthermore, blebbistatin forms insoluble aggregates in water-based media above 10 micromolar with extremely high fluorescence and also high adherence to different types of surfaces, which biases its experimental usage. Here, we report a highly soluble (440 micromolar in aqueous buffer), non-fluorescent and photostable C15 amino-substituted derivative of blebbistatin, called para-aminoblebbistatin. Importantly, it is neither photo- nor cytotoxic, as demonstrated on HeLa cells and zebrafish embryos. Additionally, para-aminoblebbistatin bears similar myosin II inhibitory properties to blebbistatin or para-nitroblebbistatin (not to be confused with the C7 substituted nitroblebbistatin), tested on rabbit skeletal muscle myosin S1 and on M2 and HeLa cells. Due to its drastically improved solubility and photochemical feature, as well as lack of photo- or cytotoxicity, para-aminoblebbistatin may become a feasible replacement for blebbistatin, especially at applications when high concentrations of the inhibitor or blue light irradiation is required.

Structural Model of Weak Binding Actomyosin in the Prepowerstroke State

Background: Actomyosin generates mechanical force in all eukaryotic cells including muscle. Results: By dynamic computational simulations we revealed structural rearrangements in myosin upon actin binding, leading to the initial state of force generation. Conclusion: The actin binding-induced structural rearrangements in myosin are transmitted specifically through the activation loop of the myosin. Significance: The first actomyosin atomic structural model of the initial state of force generation. We present the first in silico model of the weak binding actomyosin in the initial powerstroke state, representing the actin binding-induced major structural changes in myosin. First, we docked an actin trimer to prepowerstroke myosin then relaxed the complex by a 100-ns long unrestrained molecular dynamics. In the first few nanoseconds, actin binding induced an extra primed myosin state, i.e. the further priming of the myosin lever by 18° coupled to a further closure of switch 2 loop. We demonstrated that actin induces the extra primed state of myosin specifically through the actin N terminus-activation loop interaction. The applied in silico methodology was validated by forming rigor structures that perfectly fitted into an experimentally determined EM map of the rigor actomyosin. Our results unveiled the role of actin in the powerstroke by presenting that actin moves the myosin lever to the extra primed state that leads to the effective lever swing.

High Content, Phenotypic Assays and Screens for Compounds Modulating Cellular Processes in Primary Neurons

High content, phenotypic screens offer a powerful approach to systems biology at the cellular level. The approach employs cells carrying fluorescently labeled molecules or organelles in 384- or 1536-well microplates, and an automated confocal screening microscope for capturing images from each well. Although some specifics vary according to the assay type, each will apply some degree of image processing and feature extraction followed by a data analysis pipeline to identify the perturbations (small molecules, etc.) of interest. We describe and discuss the advantages and limitations of high content assays and screens using the specific example of assaying mitochondrial dynamics in primary neurons. We provide a detailed description of our culturing methods, imaging and data analysis techniques and provide an open source, ready to use CellProfiler pipeline for high-throughput image segmentation and quantification tool for mitochondrial parameters.

A Novel Actin Binding Site of Myosin is Responsible for Effective Muscle Contraction

F-actin is a track protein for myosin motors as well as an activator of the myosin ATPase activity. Actin activation provides effective contraction by increasing the ratio of productive myosin heads over futile heads by several orders of magnitudes. Despite the functional significance of actin activation, its structural mechanism was still unrevealed. We demonstrate a new, conserved actin binding region, called activation loop. It is located at the relay region which swings the lever of myosin upon the powerstroke. We prove that activation loop interacts with the N-terminal segment of actin. We found that this interaction specifically activates myosin ATPase. Biochemical (steady state and transient kinetic fluorescent measurements) and in vivo experiments using transgenic C. elegans strains proved that activation loop is responsible for force production but not essential for motility per se. We conclude that actin binding to activation loop directly accelerates the lever movement. This process increases the ratio of working myosin heads and produce effective muscle contraction.View Large Image | View Hi-Res Image | Download PowerPoint Slide