Pingping Han1, 2, Jessica E. Frith3, Guillermo A. Gomez4,5, Alpha Yap4, Geraldine O’Neill6,7,8, Justin J. Cooper-White*,1,2,9,10

1The UQ Centre in Stem Cell Ageing and Regenerative Engineering (StemCARE), University of Queensland, QLD, Australia.
2Australian Institute for Bioengineering and Nanotechnology (AIBN), University of Queensland, QLD, Australia.
3Materials Science and Engineering, Monash University, VIC, Australia.
4Institute for Molecular Bioscience, Division of Cell Biology and Molecular Medicine, The University of Queensland, St. Lucia, Brisbane, Queensland, Australia 4072.
5Centre for Cancer Biology, SA Pathology and the University of South Australia, Adelaide, SA, Australia.
6The Institute for Molecular Bioscience (IMB), University of Queensland, QLD, Australia.
7Kids Research Institute, Children’s Hospital at Westmead, NSW, Australia,
8Discipline of Child and Adolescent Health, University of Sydney, NSW, Australia. 9Commonwealth Scientific and Industrial Research Organization (CSIRO), VIC, Australia. 10School of Chemical Engineering, University Of Queensland, QLD, Australia

The ability of mesenchymal stem cells to sense changes in extracellular matrix (ECM) composition in their local microenvironment is crucial to their survival. Our previous data showed that increased lateral nanospacing (30 to 60 nm) of RGD peptides led to less mature FA formation in human mesenchymal stem cells (hMSCs). However, the exact underlying mechanism driving these substantial differences remains unclear. In this work, we created tailored surfaces of self-assembled, azide-functionalized polystyrene-block-poly (ethylene oxide) copolymers (PS-PEO-N3) with controlled lateral spacing of RGD peptides (~30 and 60nm) on PEO-N3 nanodomains. Fluorescence resonance energy transfer (FRET)-based reporters were utilized in hMSCs to investigate the molecular effectors and regulators involved in mechanotransductive signaling. We observed that smaller (30nm) nanospacing resulted in lower vinculin tension sensor (VinTS) FRET activity and longer fluorescence life times of up to τ=2.78ns, indicating the development of higher levels of tension at the site of focal adhesions compared to larger (60nm) nanodomain spacings. Higher FAK-GFP expression was colocalized at FA sites with longer FA lifetimes on smaller nanodomain spacings, when compared to larger nanospacings, suggesting the formation of more mature FAs. Higher RhoA and Src FRET activity and lower Rac FRET activity were noted when the lateral spacing of peptides was decreased from 60nm to 30nm, confirming modulation of intracellular signaling pathways associated with mechanotransduction. Smaller lateral nanospacing of adhesion peptides enables the recruitment of mature FA proteins, enabling the transmission and development of higher levels of tension into the cytoskeleton, and the biased upregulation of critical mechanosensitive RhoGTPases.

Biographic Details

Name: Pingping Han
Title: Dr
Affiliation, Country: The UQ Centre in Stem Cell Ageing and Regenerative Engineering (StemCARE), Australia
Phone: +61 4 68 608 359
Research interests: musco-skeletal ageing, nanomaterials, mechanosensing