Calcium Signaling in Live Cells on Elastic Gels under Mechanical Vibration at Subcellular Levels

Wagner Shin Nishitani,Taher A Saif,Yingxiao Wang,Fan Yuan

doi:10.1371/journal.pone.0026181

Wagner Shin Nishitani, Taher A Saif + Show 2 more

Open Access

https://doi.org/10.1371/journal.pone.0026181

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Abstract

A new device was designed to generate a localized mechanical vibration of flexible gels where human umbilical vein endothelial cells (HUVECs) were cultured to mechanically stimulate these cells at subcellular locations. A Fluorescence Resonance Energy Transfer (FRET)-based calcium biosensor (an improved Cameleon) was used to monitor the spatiotemporal distribution of intracellular calcium concentrations in the cells upon this mechanical stimulation. A clear increase in intracellular calcium concentrations over the whole cell body (global) can be observed in the majority of cells under mechanical stimulation. The chelation of extracellular calcium with EGTA or the blockage of stretch-activated calcium channels on the plasma membrane with streptomycin or gadolinium chloride significantly inhibited the calcium responses upon mechanical stimulation. Thapsigargin, an endoplasmic reticulum (ER) calcium pump inhibitor, or U73122, a phospholipase C (PLC) inhibitor, resulted in mainly local calcium responses occurring at regions close to the stimulation site. The disruption of actin filaments with cytochalasin D or inhibition of actomyosin contractility with ML-7 also inhibited the global calcium responses. Therefore, the global calcium response in HUVEC depends on the influx of calcium through membrane stretch-activated channels, followed by the release of inositol trisphosphate (IP3) via PLC activation to trigger the ER calcium release. Our newly developed mechanical stimulation device can also provide a powerful tool for the study of molecular mechanism by which cells perceive the mechanical cues at subcellular levels.

Highlights

Mechanical cues, such as substrate properties and mechanical forces, can affect a wide variety of cell behaviors and diseases [1]
The characterization of mechanical stimulation The vibration frequency and magnitude of the probe tip generated by the mechanical stimulation equipment (Figure 1A) were determined while inserted inside the gel substrate or positioned out of the gel
Both displacement and strain were larger at regions closer to the stimulation site and became negligible at the farthest cell edge away from the probe (Figure 1C, Video S3 and Video S4)

Summary

Introduction

Mechanical cues, such as substrate properties and mechanical forces, can affect a wide variety of cell behaviors and diseases [1]. Different patterns of fluid shear stress may be related to the development of atherosclerosis [4] It remains unclear how cells perceive mechanical forces and correspondingly coordinate intracellular molecular signals. Force can be applied to cells through fluid shear stress in a flow chamber [4,6] or by stretching a flexible substrate where the cells are plated on, such as a silicone membrane [7]. Another way to deliver mechanical forces is through laser tweezers [8], where a laser trap system is able to apply and measure forces on a cell-bound bead in the order of pN. Glass probes were shown to touch the cells and evoke calcium signaling [12]

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