title: Matrix rigidity regulates spatiotemporal dynamics of Cdc42 activity and vacuole formation kinetics of endothelial colony forming cells.
abstract: Recent evidence has shown that endothelial colony forming cells (ECFCs) may serve as a cell therapy for improving blood vessel formation in subjects with vascular injury, largely due to their robust vasculogenic potential. The Rho family GTPase Cdc42 is known to play a primary role in this vasculogenesis process, but little is known about how extracellular matrix (ECM) rigidity affects Cdc42 activity during the process. In this study, we addressed two questions: Does matrix rigidity affect Cdc42 activity in ECFC undergoing early vacuole formation? How is the spatiotemporal activation of Cdc42 related to ECFC vacuole formation? A fluorescence resonance energy transfer (FRET)-based Cdc42 biosensor was used to examine the effects of the rigidity of three-dimensional (3D) collagen matrices on spatiotemporal activity of Cdc42 in ECFCs. Collagen matrix stiffness was modulated by varying the collagen concentration and therefore fibril density. The results showed that soft (150 Pa) matrices induced an increased level of Cdc42 activity compared to stiff (1 kPa) matrices. Time-course imaging and colocalization analysis of Cdc42 activity and vacuole formation revealed that Cdc42 activity was colocalized to the periphery of cytoplasmic vacuoles. Moreover, soft matrices generated faster and larger vacuoles than stiff matrices. The matrix-driven vacuole formation was enhanced by a constitutively active Cdc42 mutant, but significantly inhibited by a dominant-negative Cdc42 mutant. Collectively, the results suggest that matrix rigidity is a strong regulator of Cdc42 activity and vacuole formation kinetics, and that enhanced activity of Cdc42 is an important step in early vacuole formation in ECFCs.
authors.affiliation: Department of Biomedical Engineering, Indiana University-Purdue University Indianapolis, Indianapolis, IN 46202, USA.
authors.forename: Seung Joon
authors.initials: SJ
authors.lastname: Kim
authors.affiliation: Department of Biomedical Engineering, Indiana University-Purdue University Indianapolis, Indianapolis, IN 46202, USA; Weldon School of Biomedical Engineering, Purdue University, West Lafayette, IN 47907, USA.
authors.forename: Qiaoqiao
authors.initials: Q
authors.lastname: Wan
authors.affiliation: Department of Biomedical Engineering, Indiana University-Purdue University Indianapolis, Indianapolis, IN 46202, USA.
authors.forename: Eunhye
authors.initials: E
authors.lastname: Cho
authors.affiliation: School of Mechanical Engineering, Purdue University, West Lafayette, IN 47907, USA; Weldon School of Biomedical Engineering, Purdue University, West Lafayette, IN 47907, USA.
authors.forename: Bumsoo
authors.initials: B
authors.lastname: Han
authors.affiliation: Department of Pediatrics, Indiana University School of Medicine, Indianapolis, IN 46202, USA; Herman B. Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202, USA; Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN 46202, USA.
authors.forename: Mervin C
authors.initials: MC
authors.lastname: Yoder
authors.affiliation: Weldon School of Biomedical Engineering, Purdue University, West Lafayette, IN 47907, USA; Department of Basic Medical Sciences, Purdue University, West Lafayette, IN 47907, USA.
authors.forename: Sherry L
authors.initials: SL
authors.lastname: Voytik-Harbin
authors.affiliation: Department of Biomedical Engineering, Indiana University-Purdue University Indianapolis, Indianapolis, IN 46202, USA. Electronic address: sungna@iupui.edu.
authors.forename: Sungsoo
authors.initials: S
authors.lastname: Na
mesh_terms: D055592:Biophysical Phenomena; D015374:Biosensing Techniques; D002478:Cells, Cultured; D003094:Collagen / Q000737:chemistry / Q000378:metabolism; D003114:Colony-Forming Units Assay; D042783:Endothelial Cells / Q000166:cytology* / Q000378:metabolism*; D005109:Extracellular Matrix / Q000737:chemistry / Q000378:metabolism*; D005312:Fetal Blood / Q000166:cytology; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D007700:Kinetics; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D018919:Neovascularization, Physiologic; D014617:Vacuoles / Q000378:metabolism*; D020764:cdc42 GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*
chemical_list: D050505:Mutant Proteins; D003094:Collagen; D020764:cdc42 GTP-Binding Protein
keywords: Endothelial colony forming cells (ECFCs); Fluorescence resonance energy transfer (FRET); Live cell imaging; Matrix stiffness; Mechanotransduction; Rho family GTPases