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July 12-15, 2017 Our lab participated: EMBL Symposia, Mechanical Forces in Biology, Heidelberg, Germany

On behalf of Ádám Horváth (PhD student), Máté Pénzes (PhD student), Demeter Túrós (MSc student) and Dániel Batora (MSc student) our laboratory also participated with 3 substantial topics at the conference.

EMBO I EMBL Symposia

Mechanical Forces in Biology

EMBL Heidelberg, Germany

12-15 July 2017

Taking into account the facts the field of mechanical forces in biology is expanding rapidly therefore the aims of symposium was to unite the field of mechanobiology by bringing together world-leading experts in the generation and sensing of forces from the molecular scale to the organismal scale.An important emphasis was the combination of physics and computer modelling with molecular genetics and live-imaging in vitro or in vivo.

Ádám Horváth presented a poster about subcellular spatial control of non-muscle myosin 2 redistribution and stress fiber strain by Molecular Tattoo, Demeter Túrós and Máté Pénzes presented a poster about Neurelaxin, a novel blebbistatin derivative that can cause rapid neurite outgrowth and last but not least Dániel Batora presented also a poster about Molecular Tattoo which is a novel optopharmacological tool to permanently modulate neuronal functions even in subcellular level. Below one can view and read both the posters and the abstracts. All three topics were supported by The National Research, Development and Innovation Office (VKSZ_14-1-2015-0052 and NVKP_16-1-1016_0051).

Subcellular spatial control of non-muscle myosin II redistribution and stress fiber strain by Molecular Tattoo

The cellular distribution of the motor protein non-muscle myosin 2 (NM2) leads to different  forms of intracellular strain driving cell motility, cytokinesis and morphogenetic processes including axonal growth and retraction. However, it remains elusive how these cellular processess are governed by the dynamic changes in NM2 localization and supramolecular assembly. To address this problem, we determined the effect of different types of NM2 inhibition on the dynamics of load-bearing stress fibers and unloaded inner cytoplasmatic NM2 structures in live HeLa cells. We followed NM2 redistribution via FRAP, applied also in combination with our recently developed optopharmacological tool, Molecular Tattoo, which allows subcellular confinement of drug effects via 2-photon induced photocrosslinking to targets. We found that the Rho-kinase inhibitor, Y-27632, dramatically accelerates NM2 redistribution and induces stress fiber dissolution, due to NM2 filament disassembly resulting from myosin light chain dephosphorylation. When NM2 was inhibited by para-nitroblebbistatin  (pNBleb) or locally by tattooed azidoblebbistatin, in the stress fibers a significant acceleration and suprression of NM2 redistribution was detected at moderate and high inhibitor concentrations, respectively. The observed effects were local and specific for load-bearing peripheral stress fibers, implying the roal of mechanical load in NM2 redistribution. Furthermore, in these tests stress fibers remained intact, contrary to that seen upon Rho-kinase inhibition. These results highlight that variations in the localization and/or pharmacological mechnism of NM2 inhibition produce distinct effects in intracellular strain and cellular morphogenesis.

Neurelaxin, a novel blebbistatin derivative causes rapid neurite outgrowth in vivo

Blebbistatin, a myosin 2 specific inhibitor, has been implicated in vitro in having effects on neurons by promoting dendritic- and axonal cone growth as well as resulting in dynamic filopodia-like spineprecursors. Due to the low solubility, chemical instability and cytotoxicity of blebbistatin, its in vivo effect on the nervous system could not been studied. Recently, a novel blebbistatin derivative, Neurelaxin has been developed, which has the same target specificity as blebbistatin but without causing significant adverse effects. Thus, Neurelaxin enables us to investigate the role of NMII in neuronal functions in vivo. Our experiments were conducted on transgenic zebrafish embryos, expressing pan-neural GFP signal. Two-photon microscopy and 3D visualization and analysis software Imaris was used to investigate the drug induced effects on the brain. Upon Neurelaxin treatment the volume of the brain was significantly increased. We also examined the total dendrite number and length in tectum opticum and found that Neurelaxin caused 15% and 10% increases, respectively. By labeling single neurons of tectum opticum we were also able to investigate the structural changes of axonal tree in response to Neurelaxin. Our results suggesting that Neurelaxin by inhibiting the function of NMII also causes significant in vivo architectural changes in neurons.

Molecular Tattoo: A novel pharmacological tool to permanently modulate neuronal functions even in subcellular level

Although optogenetic actuators are widely used selectively modulate the overall activity of neurons, manipulatin other neuronal functions such as a specific receptor’s activity or synaptic plasticity remains a great challenge in modern neuroscience. After our finding that two-photon excitation can initiate crosslinking between photoreactive drug compounds and their target proteins we have developed a novel optopharmacological technique named Molecular Tattooing which can hopefully overcome the limitations of optogenetic methods. The technique enables us to confine drug effects into even subcellular regions without causing any systemic effects outside the targeted area. We have developed series of photoreactive bioactive compounds targeting different neuronal and cell signaling receptors, ion channels and cytoskeletal enzymes. One such compund, azidoblebbistatin, a myosin 2 inhibitor was successfully utilized to induce neurite outgrowth in a single growth cone of an embryonic hippocampal neuron. Other aims of our group was to develop a photoreactive NMDA receptor antagonist which can be used to selectively manipulate the basics of learning, memory formation and other NMDA receptor induced neuronal functions. Monitoring a simple behvaiour, the habituation of the escape response in zebrafish, we identified a series of drugs affecting the fish’s memory. A photoreactive quinoxaline derivative, N3BFQX induced robust alterations in the fish’s behavior, similar to the non-competitive NMDA receptor antagonist, MK-801. Both UV and two-photon irradiation caused changes in the compunds structure confirming it’s photoreactivitym however further in vivo and in vitro experiments are still required to spprove the compunds’s exact effects.

 

 

 

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