Substrate rigidity regulates Ca2+ oscillation via RhoA pathway in stem cells.
Substrate rigidity plays crucial roles in regulating cellular functions, such as cell spreading, traction forces, and stem cell differentiation. However, it is not clear how substrate rigidity influences early cell signaling events such as calcium in living cells. Using highly sensitive Ca(2+) biosensors based on fluorescence resonance energy transfer (FRET), we investigated the molecular mechanism by which substrate rigidity affects calcium signaling in human mesenchymal stem cells (HMSCs). Spontaneous Ca(2+) oscillations were observed inside the cytoplasm and the endoplasmic reticulum (ER) using the FRET biosensors targeted at subcellular locations in cells plated on rigid dishes. Lowering the substrate stiffness to 1 kPa significantly inhibited both the magnitudes and frequencies of the cytoplasmic Ca(2+) oscillation in comparison to stiffer or rigid substrate. This Ca(2+) oscillation was shown to be dependent on ROCK, a downstream effector molecule of RhoA, but independent of actin filaments, microtubules, myosin light chain kinase, or myosin activity. Lysophosphatidic acid, which activates RhoA, also inhibited the frequency of the Ca(2+) oscillation. Consistently, either a constitutive active mutant of RhoA (RhoA-V14) or a dominant negative mutant of RhoA (RhoA-N19) inhibited the Ca(2+) oscillation. Further experiments revealed that HMSCs cultured on gels with low elastic moduli displayed low RhoA activities. Therefore, our results demonstrate that RhoA and its downstream molecule ROCK may mediate the substrate rigidity-regulated Ca(2+) oscillation, which determines the physiological functions of HMSCs.
Neuroscience Program, Beckman Institute for Advanced Science and Technology, University of Illinois, Urbana-Champaign, Urbana, Illinois, USA.
Tae-Jin
TJ
Kim
Jihye
J
Seong
Mingxing
M
Ouyang
Jie
J
Sun
Shaoying
S
Lu
Jun Pyu
JP
Hong
Ning
N
Wang
Yingxiao
Y
Wang
D015374:Biosensing Techniques; D020013:Calcium Signaling; D002462:Cell Membrane / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005110:Extracellular Space / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D008164:Luminescent Proteins / Q000378:metabolism; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000201:enzymology*; D013379:Substrate Specificity; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*
D008164:Luminescent Proteins; D020742:rhoA GTP-Binding Protein