U. ILLINOIS (US) — Using live cell imaging, researchers have gotten a clearer view of how mechanical force regulates calcium signaling in the body.
Reported in the journal PLoS One, the work was led by Peter Wang and Taher Saif of the Beckman Institute at the University of Illinois. Wagner Shin Nishitani, a graduate student in Wang’s laboratory is the study’s lead author.
A main focus of research in Wang’s Molecular Engineering and Live Cell Imaging laboratory is on manipulating large proteins in live cells toward the development of technologies like biosensors for detecting disease. Wang’s approach is to get the body’s own cells to do the sensing work.
Left image shows the probe and gel substrate with 1 µm beads embedded. The right images show a typical displacement (in µm). (Credit: Peter Wang.)
The method combines mechanical stimulation with genetically encoded biosensors that enable live cell imaging. The genetically encoded biosensors are based on a molecular interaction—called fluorescence resonance energy transfer (FRET)—between dye molecules that enables live cell imaging for the visualization and quantification of crucial molecular signals at subcellular levels.
For this research, they created a novel probe device for mechanical stimulation of the cells and used the FRET biosensors to measure the results.
In order to stimulate the cells, Wang and his collaborators developed a probe that uses the body’s own mechanical cues such as, “substrate properties and mechanical forces that can affect a wide variety of cell behaviors and diseases” for stimulation of cells on gels.
This method reveals how cells perceive mechanical forces and correspondingly coordinate intracellular molecular signals. The researchers focused on intracellular signaling of calcium ions because their concentration has been shown to “play crucial roles in a variety of physiological consequences and is sensitive to mechanical cues.”
The probe consisted of a glass capillary on an aluminum rod; the probe tip is inserted into the substrate gel where cells are seeded and the vibration mechanism vibrates the substrate gel, stimulating the cell.
Flexible gels were used to culture human umbilical vein endothelial cells (HUVECs) used in the study. The mechanical stimulation delivers a localized and subcellular vibrational stretch on the HUVECs with a high temporal frequency.
This method of mechanical stimulation “has no interference on the cell membrane and intracellular structure, different from other methods involving probes or beads attached to the cell surface directly prior to the stimulation which may alter the cell membrane structures and cytoskeletal network,” Wang says.
Using this method, the researchers were able to “monitor the spatiotemporal distribution of intracellular calcium concentrations in the cells upon this mechanical stimulation,” revealing 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 results have implications for those working in fields such as vascular biology and those seeking to apply an advanced molecular imaging technique toward areas such as mechanotransduction, the cellular process that converts mechanical signals into biochemical responses. Wang says the technology also has potential diagnostic applications, such as a biomarker that is used to indicate the state of a cell toward disease detection.
“Abnormal calcium responses of diseased cells under this mechanical stimulation could be utilized a signature/biomarker to identify and diagnose the diseased cells.”
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