Mechanical stretch regulates macropinocytosis in Hydra vulgaris

Cells rely on a diverse array of engulfment processes to sense, exploit, and adapt to their environments. Macropinocytosis is a versatile example of such a process, allowing for the indiscriminate and rapid uptake of large volumes of fluid and membrane. Much of the molecular machinery essential for macropinocytosis has been well established. However, most of these studies relied on tissue culture models, leaving the regulation of this process within the context of organs and organisms unresolved. Here, we report that large-scale macropinocytosis occurs in the outer epithelial layer of the cnidarian Hydra vulgaris. Exploiting Hydra’s relatively simple body plan, we developed approaches to visualize macropinocytosis over extended periods of time in living tissue, revealing constitutive engulfment across the entire body axis. Using pharmacological perturbations, we establish a role for stretch-activated channels, including Piezo, and downstream calcium influx in inhibiting this process. Finally, we show that the direct application of planar stretch leads to calcium influx and a corresponding inhibition of macropinocytosis. Together, our approaches provide a platform for the mechanistic dissection of constitutive macropinocytosis in physiological contexts and reveal a role for macropinocytosis in responding to membrane tension.

Stretch-induced endogenous electric fields drive neural crest directed collective cell migration in vivo

Directed collective cell migration (dCCM) is essential for morphogenesis. Cell clusters migrate in inherently complex in vivo environments composed of chemical, electrical, mechanical as well as topological features. While these environmental factors have been shown to allow dCCM in vitro, our understanding of dCCM in vivo is mostly limited to chemical guidance. Thus, despite its wide biological relevance, the mechanisms that guide dCCM in vivo remain unclear. To address this, we study endogenous electric fields in relation to the migratory environment of the Xenopus laevis cephalic neural crest, an embryonic cell population that collectively and directionally migrates in vivo. Combining bioelectrical, biomechanical and molecular tools, we show that endogenous electric fields drive neural crest dCCM via electrotaxis in vivo. Moreover, we identify the voltage-sensitive phosphatase 1 (Vsp1) as a key component of the molecular mechanism used by neural crest cells to transduce electric fields into a directional cue. Furthermore, Vsp1 function is specifically required for electrotaxis, being dispensable for cell motility and chemotaxis. Finally, we reveal that endogenous electric fields are mechanoelectrically established. Mechanistically, convergent extension movements of the neural fold generate membrane tension, which in turn opens stretch-activated channels to mobilise the ions required to fuel electric fields. Overall, our results reveal a mechanism of cell guidance, where electrotaxis emerges from the mechanoelectrical and molecular interplay between neighbouring tissues. More broadly, our data contribute to validate the, otherwise understudied, functions of endogenous bioelectrical stimuli in morphogenetic processes.

Muscle stretching induces twitch contractions without activation of stretch-activated channels in intact rat trabeculae

Introduction Mechano-electric coupling (MEC) means that muscle stretching can induce action potentials. Stretch-activated channels (SACs) have been believed to play important roles in their induction. Purpose To investigate what degree of muscle stretching can induce MEC-mediated action potentials and what roles SACs play in their induction. Methods Trabeculae were obtained from right ventricles of rat hearts. Force was measured with a strain gauge, sarcomere length (SL) with a laser diffraction technique, and [Ca2+]i with fura-2 (24°C). The SL was set at 2.0 μm at the resting condition. Trabeculae were stimulated electrically at 400-ms intervals for 7.5 s. Various degrees of muscle stretching were applied at 500 ms after the last stimulus of the electrical train to determine the minimal SL (SL-AP) at which an action potential or a twitch contraction was induced by the stretching (0.7 mM [Ca2+]o). Results The SL-AP was 2.34±0.02 μm (n=8) when trabeculae were stretched rapidly from a SL of 2.0 μm (400-ms stimulation intervals, 0.7 mM [Ca2+]o). The SL-AP was not changed by increasing the stimulation intervals from 400 to 2000 ms (n=7), by increasing [Ca2+]o from 0.7 to 2 mM (n=8), and by adding 1 μM isoproterenol (n=8), suggesting that Ca2+ loading within the myocardium has no effect on the SL-AP. Surprisingly, the SL-AP was not changed by adding 5 μM GsMTx4 (n=8), 10 mM Gd3+ (n=9), 100 μM (n=8) and 200 μM streptomycin (n=11), revealing that SACs play no roles in the determination of SL-AP. The SL-AP was not changed by adding 1 μM ryanodine (n=5) and 30 μM cyclopiazonic acid and was not changed by adding 3 μM diphenyleneiodonium chloride (n=8) and 10 μM colchicine, suggesting that Ca2+ leak from the SR and activation of NADPH oxidase has no effect on the SL-AP. In contrast, elevation of temperature from 23 to 36°C decreased the SL-AP from 2.35±0.01 to 2.34±0.02 μm (p<0.05, n=7). Elevation of extracellular K+ ([K+]o) from 5 to 10 mM increased the SL-AP from 2.35±0.01 to 2.38±0.01 μm (p<0.01, n=7), while reduction of [K+]o to 5 mM decreased it to 2.36±0.01 μm (p<0.05, n=7), suggesting that depolarization of membrane potential suppresses MEC-mediated twitch contractions. The SL-AP was increased from 2.34±0.01 to 2.36±0.01 μm (p<0.01, n=7) when stretching was applied at a shorter interval after the last stimulus, i.e., 200 ms. After electrical stimulation at 300-ms stimulation intervals for 30 s, arrhythmias were induced by a MEC-mediated twitch contraction in 6 out of 9 trabeculae when stretching was applied at 500 ms after the last stimulus, while they were induced only in 2 out of 9 trabeculae without the stretching (4 mM [Ca2+]o, 1 μM isoproterenol). Conclusions These results suggest that muscle stretching causes membrane excitation, which potentially induces arrhythmias and that activation of SACs, Ca2+ release from the SR, and activation of NADPH oxidase by muscle stretching are not involved in the excitation. FUNDunding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Grant-in-Aid for Scientific Research (C) from Japan Society for the Promotion of Science.

Mechanosensitivity in Pulmonary Circulation: Pathophysiological Relevance of Stretch-Activated Channels in Pulmonary Hypertension

A variety of cell types in pulmonary arteries (endothelial cells, fibroblasts, and smooth muscle cells) are continuously exposed to mechanical stimulations such as shear stress and pulsatile blood pressure, which are altered under conditions of pulmonary hypertension (PH). Most functions of such vascular cells (e.g., contraction, migration, proliferation, production of extracellular matrix proteins, etc.) depend on a key event, i.e., the increase in intracellular calcium concentration ([Ca2+]i) which results from an influx of extracellular Ca2+ and/or a release of intracellular stored Ca2+. Calcium entry from the extracellular space is a major step in the elevation of [Ca2+]i, involving a variety of plasmalemmal Ca2+ channels including the superfamily of stretch-activated channels (SAC). A common characteristic of SAC is that their gating depends on membrane stretch. In general, SAC are non-selective Ca2+-permeable cation channels, including proteins of the TRP (Transient Receptor Potential) and Piezo channel superfamily. As membrane mechano-transducers, SAC convert physical forces into biological signals and hence into a cell response. Consequently, SAC play a major role in pulmonary arterial calcium homeostasis and, thus, appear as potential novel drug targets for a better management of PH.

Abstract 14409: TRPA1 Channels Are a Source of Calcium-driven Cellular Mechano-arrhythmogenicity

Introduction: Pathologic changes in myocardial mechanics and hemodynamic load result in arrhythmias via mechanically-induced changes in electrophysiology or intracellular Ca 2+ (‘mechano-arrhythmogenicity’). While molecular mechanisms driving mechano-arrhythmogenicity are poorly defined, they are associated with disease-related alterations in voltage-Ca 2+ dynamics. Objective: Define mechanisms of mechano-arrhythmogenicity during alterations in voltage-Ca 2+ dynamics in rabbit ventricular myocytes. Methods: Rabbit (♀, NZW) LV myocytes were transiently stretched (8-16% change in sarcomere length, 100ms) during diastole or late repolarisation in control or during K ATP channel activation (pinacidil). Drugs were used to buffer Ca 2+ (BAPTA), stabilise RyR (dantrolene), non-selectively block stretch-activated channels (streptomycin), or specifically block (HC-030031) or activate (AITC) mechano-sensitive TRPA1 channels. Voltage-Ca 2+ dynamics were simultaneously monitored with fluorescent dyes (di-4-ANBDQPQ, Fluo-5F) and a single camera-optical splitter system and diastolic Ca 2+ was measured using Fura Red. Results: Pinacidil caused greater shortening of the AP than Ca 2+ transient (-144±17 vs -74±11ms; n =24 cells, N =7 rabbits; p <0.001) with no change in cell stiffness or contractility. Stretch during pinacidil application caused arrhythmias in both diastole and late repolarisation (8 and 10% of stretches; n =46, N =5), which voltage-Ca 2+ imaging revealed were Ca 2+ -driven. Arrhythmias were reduced with BAPTA (3 and 0% of stretches; p <0.05), streptomycin (4 and 2%; p <0.05), and HC-030031(2 and 1%; p <0.01), while dantrolene had no effect ( n =40, N =5 for each). Stretch in diastole during AITC application also caused arrhythmias (15%; p <0.001), which were blocked by HC-030031 (4%; p <0.001) or BAPTA (3%; p <0.001; n =40, N =5 each). Both AITC and pinacidil caused an increase in diastolic Ca 2+ (112±29 and 78±29% of control; p<0.05), which was reduced by HC-030031 with AITC (26±24%; p <0.05), but not with pinacidil ( n =25, N =5 each). Conclusions: TRPA1 activation increases mechano-arrhythmogenicity via a Ca 2+ -driven mechanism and may represent a novel anti-arrhythmic target in pathologies involving altered cardiac mechanics.

Cell cycle progression in confining microenvironments is regulated by a growth-responsive TRPV4-PI3K/Akt-p27Kip1 signaling axis

In tissues, cells reside in confining microenvironments, which may mechanically restrict the ability of a cell to double in size as it prepares to divide. How confinement affects cell cycle progression remains unclear. We show that cells progressed through the cell cycle and proliferated when cultured in hydrogels exhibiting fast stress relaxation but were mostly arrested in the G0/G1 phase of the cell cycle when cultured in hydrogels that exhibit slow stress relaxation. In fast-relaxing gels, activity of stretch-activated channels (SACs), including TRPV4, promotes activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, which in turn drives cytoplasmic localization of the cell cycle inhibitor p27Kip1, thereby allowing S phase entry and proliferation. Cell growth during G1 activated the TRPV4-PI3K/Akt-p27Kip1 signaling axis, but growth is inhibited in the confining slow-relaxing hydrogels. Thus, in confining microenvironments, cells sense when growth is sufficient for division to proceed through a growth-responsive signaling axis mediated by SACs.

Loss of stretch-activated channels, PIEZOs, accelerates non-small cell lung cancer progression and cell migration

PIEZO channels are stretch-activated channels involved in wound sealing and cell proliferation in many cell types. A recent study focussing on lung cancer (LC), using next-generation sequencing analysis, has indicated that PIEZO functions were implicated in LC development. However, the expression and role of PIEZO channels in non-small cell LC (NSCLC) progression require elucidation. In the current study, we investigated the gene expression and alteration frequency in human NSCLC tissue, accessed the prognostic roles of PIEZO channels in NSCLC patients, and further studied the effect of PIEZOs in NSCLC cell proliferation and tumor growth in vivo. The mRNA expression of PIEZO1 and 2 was clearly decreased in NSCLC tumor tissue compared with that in matched adjacent non-tumor tissue. In human NSCLC tissues, PIEZO1 gene expression exhibits a highly deep deletion rate, and PIEZO2 mainly exhibits mutation in gene expression. High mRNA expression of PIEZO channels was found to correlate with better overall survival (OS) for NSCLC patients, especially for patients with lung adenocarcinoma (LUAD), but not for patients with lung squamous cell carcinoma (LUSC). The prognostic role of PIEZO channels was more sensitive in female patients than male patients, and more sensitive in patients at earlier stages than patients at latter stages. Knockdown of PIEZO1 or PIEZO2 in NSCLC cells significantly promoted cell migration in vitro and tumor growth in vivo. These results indicate the critical prognostic values of the PIEZO channels in NSCLC. This information will be beneficial to understand the pathological mechanism of NSCLC and to generate effective therapeutic approaches for NSCLC patients.