title: Rapid and Localized Mechanical Stimulation and Adhesion Assay: TRPM7 Involvement in Calcium Signaling and Cell Adhesion.
abstract: A cell mechanical stimulation equipment, based on cell substrate deformation, and a more sensitive method for measuring adhesion of cells were developed. A probe, precisely positioned close to the cell, was capable of a vertical localized mechanical stimulation with a temporal frequency of 207 Hz, and strain magnitude of 50%. This setup was characterized and used to probe the response of Human Umbilical Endothelial Vein Cells (HUVECs) in terms of calcium signaling. The intracellular calcium ion concentration was measured by the genetically encoded Cameleon biosensor, with the Transient Receptor Potential cation channel, subfamily M, member 7 (TRPM7) expression inhibited. As TRPM7 expression also regulates adhesion, a relatively simple method for measuring adhesion of cells was also developed, tested and used to study the effect of adhesion alone. Three adhesion conditions of HUVECs on polyacrylamide gel dishes were compared. In the first condition, the substrate is fully treated with Sulfo-SANPAH crosslinking and fibronectin. The other two conditions had increasingly reduced adhesion: partially treated (only coated with fibronectin, with no use of Sulfo-SANPAH, at 5% of the normal amount) and non-treated polyacrylamide gels. The cells showed adhesion and calcium response to the mechanical stimulation correlated to the degree of gel treatment: highest for fully treated gels and lowest for non-treated ones. TRPM7 inhibition by siRNA on HUVECs caused an increase in adhesion relative to control (no siRNA treatment) and non-targeting siRNA, but a decrease to 80% of calcium response relative to non-targeting siRNA which confirms the important role of TRPM7 in mechanotransduction despite the increase in adhesion.
authors.affiliation: Department of Bioengineering & Beckman Institute for Advanced Science and Technology, Center for Biophysics and Computational Biology, Institute for Genomic Biology, University of Illinois, Urbana-Champaign, Urbana, Illinois, 61801, United States of America; Instituto de Física, Universidade de São Paulo, São Paulo, São Paulo, Brazil.
authors.forename: Wagner Shin
authors.initials: WS
authors.lastname: Nishitani
authors.affiliation: Instituto de Física, Universidade de São Paulo, São Paulo, São Paulo, Brazil.
authors.forename: Adriano Mesquita
authors.initials: AM
authors.lastname: Alencar
authors.affiliation: Department of Bioengineering & Beckman Institute for Advanced Science and Technology, Center for Biophysics and Computational Biology, Institute for Genomic Biology, University of Illinois, Urbana-Champaign, Urbana, Illinois, 61801, United States of America; Department of Mechanical Science & Engineering, Integrative and Molecular Physiology, Center for Biophysics and Computational Biology, Institute for Genomic Biology, University of Illinois, Urbana-Champaign, Urbana, Illinois, 61801, United States of America; Department of Bioengineering & Institute of Engineering in Medicine, University of California, San Diego, La Jolla, California, 92093, United States of America.
authors.forename: Yingxiao
authors.initials: Y
authors.lastname: Wang
mesh_terms: D015374:Biosensing Techniques; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000235:genetics / Q000502:physiology*; D002448:Cell Adhesion / Q000235:genetics / Q000502:physiology*; D002478:Cells, Cultured; D061307:Human Umbilical Vein Endothelial Cells / Q000378:metabolism; D006801:Humans; D055595:Mechanical Phenomena; D040542:Mechanotransduction, Cellular / Q000502:physiology*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering; D050053:TRPM Cation Channels / Q000235:genetics* / Q000378:metabolism
chemical_list: D034741:RNA, Small Interfering; D050053:TRPM Cation Channels; D017346:Protein Serine-Threonine Kinases; C486583:TRPM7 protein, human; D002118:Calcium