Maksim M. Khalisov

Mechanosensitive ion channels of the Piezo family, discovered in 2010, are crucial participants of intracellular mechanotransduction. The Piezo1 and Piezo2 channels are found in various cell types, including primary sensory neurons, although their precise roles remain poorly understood. Although Jedi2, a pharmacological activator of Piezo1 channels, has been shown to modulate Piezo1 activity, its effects on neurons have not been studied yet. In this work, we investigate the impact of Jedi2 on primary sensory neurons. Using organotypic tissue culture, we demonstrate that Jedi2 regulates neurite growth of sensory neurons of dorsal root ganglia in a dose-dependent manner. Atomic force microscopy revealed that a 10 μM concentration of Jedi2 — while having no effect on neurite growth — induces a significant increase in the stiffness of primary sensory neurons. This response can be explained by the triggering of intracellular Ca2+-dependent signaling pathways mediated by Piezo1 channel activation. Our findings suggest that Jedi2 at 10 μM concentration can serve as a useful tool for exploring the molecular mechanisms of Piezo1- mediated mechanotransduction in primary sensory neurons under physiologically relevant conditions. Одними из важнейших участников механизмов внутриклеточной механотрансдукции являются открытые в 2010 году механочувствительные ионные каналы семейства Piezo. Эти каналы (Piezo1 и Piezo2) обнаружены во многих типах клеток, включая первичные сенсорные нейроны, причем их роль здесь пока неясна. Изучению механизмов функционирования каналов Piezo1 может способствовать применение специфических фармакологических активаторов этих каналов, например, Jedi2. В литературе отсутствуют данные о влиянии Jedi2 на нейроны. В настоящей работе исследованы эффекты Jedi2 на первичные сенсорные нейроны. С помощью метода органотипической культуры ткани было установлено, что Jedi2 дозозависимо регулирует рост нейритов сенсорных нейронов спинномозговых ганглиев. Методом атомно-силовой микроскопии было показано, что указанный агент в концентрации 10 мкМ, которая не влияет на рост нейритов сенсорных нейронов, приводит к увеличению жесткости первичных сенсорных нейронов. Полученный результат можно объяснить опосредованным каналами Piezo1 запуском внутриклеточных Са2+-активируемых сигнальных путей, что указывает на возможность эффективного применения Jedi2 в концентрации 10 мкМ для выяснения молекулярных механизмов Piezo1-опосредованной механотрансдукции в первичных сенсорных нейронах при физиологически адекватных условиях.

Jedi2, an activator of mechanosensitive Piezo1 channels, was found to affect the growth of embryonic heart tissue explants. The dependence of the change in the area index on the Jedi2 concentration is described by the Hill equation (Kd ≈ 20 μM, Hill coefficient — 1.6). A concentration of 10 μM was chosen to chemically activate mechanosensitive Piezo1 channels in the atomic force microscopy (AFM) study since it did not impact the growth of heart explants. Based on the obtained stimulus–response relationship, a force of 3 nN was chosen for mechanical action from the AFM probe when studying the effect of Jedi2 on fibroblasts, which does not lead to a change in cell stiffness in response to mechanical stimulation. In contrast to low forces (1–5 nN), high stimulation forces (6–7 nN) were associated with a sharp increase in the Young’s modulus of fibroblasts. An AFM study showed that Jedi2 augments fibroblast stiffness: the Young’s modulus of cells after exposure to the test substance (68 ± 7 kPa, n = 33) increases compared to the control (37 ± 4 kPa, n = 29). The effect of Jedi2 becomes stronger with time, with the maximum effect on fibroblast mechanical characteristics achieved after more than two hours of exposure to the substance. We assume that the increase in fibroblast stiffness observed under the influence of Jedi2 and at a stimulation force of 3 nN is associated with a shift in the threshold for triggering the cell response towards lower forces caused by the modulation of the Piezo1 channels. Установлено, что Jedi2, активатор механочувствительных каналов Piezo1, влияет на рост эксплантатов сердца эмбриональной ткани. Зависимость изменения индекса площади от концентрации действующего агента описывается уравнением Хилла (Кd ≈ 20 мкМ, коэффициент Хилла — 1,6). Концентрация Jedi2, равная 10 мкМ, была выбрана для химической активации механочувствительных каналов Piezo1 в исследовании с помощью метода атомно-силовой микроскопии, поскольку она не влияла на рост эксплантатов сердца. На основании полученной зависимости стимул–ответ для механического воздействия со стороны зонда атомно-силового микроскопа при исследовании влияния Jedi2 на фибробласты была выбрана сила 3 нН, не приводящая к изменению жесткости клеток в ответ на механическую стимуляцию. В отличие от малых сил (1–5 нН), при больших силах стимуляции (6–7 нН) наблюдалось резкое увеличение модуля Юнга фибробластов. Исследование с помощью атомно-силовой микроскопии показало, что Jedi2 вызывает увеличение жесткости фибробластов — модуль Юнга клеток после воздействия исследуемого вещества (68 ± 7 кПа, n = 33) растет по сравнению с контролем (37 ± 4 кПа, n = 29). Эффект воздействия Jedi2 усиливается со временем: в рамках рассмотренного периода максимальное влияние на механические характеристики фибробластов достигается спустя более двух часов воздействия вещества. Мы предполагаем, что наблюдаемый при воздействии Jedi2 и силе стимуляции 3 нН рост жесткости фибробластов связан с вызванным модуляцией работы каналов Piezo1 сдвигом порога запуска ответа клеток в сторону меньших сил.

A group of phyllosilicate nanoscrolls conjoins several hydrosilicate layered compounds with a size mismatch between octahedral and tetrahedral sheets. Among them, synthetic Mg₃Si₂O₅(OH)₄ chrysotile nanoscrolls (obtained via the hydrothermal method) possess high thermal stability and mechanical properties, making them prospective composite materials fillers. However, accurate determination of these nano-objects with Young’s modulus remains challenging. Here, we report on a study of the mechanical properties evolution of individual synthetic phyllosilicate nanoscrolls after a series of heat treatments, observed with an atomic force microscopy and calculated using the density functional theory. It appears that the Young’s modulus, as well as shear deformation’s contribution to the nanoscrolls mechanical behavior, can be controlled by heat treatment. The main reason for this is the heat-induced formation of covalent bonding between the adjacent layers, which complicate the shear deformation.

Consoles and bridges of MgNi₂Si₂O₅(OH)₄ nanoscrolls were tested for bending in atomic force microscope. Using test data, we analyze how the consoles or bridges were fixed, and took this information into account when calculating the Young's modulus of the nanoscrolls. The results on the consoles are in good agreement with the results on the bridges when modeling the latter as three-span beams, and the former as beams on an elastic foundation with a suspended console. Keywords: AFM, nanoscroll, bending, Young's modulus, Krylov's functions.

Some phyllosilicate compounds have the ability of spontaneous scrolling because of the size mismatch between the covalently bounded metal oxide and silica sheets. Their unique structure and high theoretically predicted Young's modulus (around 210–230 GPa) induce phyllosilicates’ application as reinforcing fillers. However, previous nanomechanical experiments with individual phyllosilicate nanoscrolls are in poor agreement with theory. The main reason for this is the low accuracy of experiments, which leads to large measurement errors compared to measured average values. Here, the study of the mechanical properties of synthetic (Mg_1–xNi_x)₃Si₂O₅(OH)₄ phyllosilicates is reported by testing a suspended nanoobject (a nanobridge) with an atomic force microscope (AFM). The Young's modulus of corresponding phyllosilicate model layers is also calculated by means of the density functional theory (DFT). The original AFM approach makes it possible to account for the probe slipping off the nanobridge and determine its boundary conditions. The measured Young's modulus values are considered within the models of surface tension and shear strain contributions. The shear strain appears to have a decisive impact on the measured Young's modulus (from 150 ± 70 GPa to 200 ± 210 GPa) and its spread.

The effects of ouabain on the mechanical characteristics of primary sensory neurons and fibroblasts of 10- to 12-day-old chicken embryos were investigated by atomic force microscopy under physiologically relevant conditions. Fibroblasts express only the α1 isoform of Na,K-ATPase, while sensory neurons express the α1 and α3 isoforms. It was found that exposure to ouabain in the concentration corresponding to its endogenous level led to an increase in membrane rigidity of sensory neurons, which was apparently due to activation of the transducer rather than the pumping function of Na,K-ATPase. The mechanical parameters of fibroblasts were not affected by exposure to endogenous concentrations of ouabain. These results suggest that endogenous ouabain specifically modulates the transducer function of the α3 Na,K-ATPase isoform in the sensory neuron membrane. Thus, atomic force microscopy was efficiently applied to perform a comparative study of intracellular signaling cascades in living cells.