Nanofiber Curvature Sensing Through an Arfaptin and Rac1 Dependent Mechanism

Open Access
Higgins, Andrew Michael
Area of Honors:
Bachelor of Science
Document Type:
Thesis Supervisors:
  • Justin Lee Brown, Thesis Supervisor
  • William O Hancock, Honors Advisor
  • Peter J Butler, Faculty Reader
  • nanofibers
  • Rac1
  • arfaptin
  • curvature sensing
More than one million bone grafting procedures occur each year at costs reaching into the millions of dollars. Although synthetic biomaterials have great potential, 80% of bone grafts are dependent upon donor material in the form of autografts or allografts. Should a synthetic bone graft be developed whose grafting efficacy matches that of autografts, the current gold standard, then the dependence on donor materials can be eliminated. Current synthetic grafts lack a crucial property of an ideal bone graft: osteoinduction, or the ability to induce stem cell differentiation down an osteoblastic lineage. Through the use of nanofiber surface modifications, it is hoped that synthetic grafts can become innately osteoinductive and replace other grafting technologies. An empirical approach was used to develop nanofibers with diameters ranging from <100 nm to >1 μm. This range of diameters would permit the testing of a theory first proposed by Vogel et al.: that the curvature induced by the cell membrane bending around a small nanofiber will promote the adhesion of arfaptin, a GTPase binding vesicle trafficking protein, which will then release Rac1, a GTPase associated with focal adhesion turnover, lamellipodial extension, and proliferation. Electrospinning resulted in the formation of three distinct nanofiber groups 80±40 nm, 270±53 nm, and 1,048±353 nm. Immunostaining of MC3T3 cells adherent to these fibers and a control surface revealed that active Rac1 localizes along the smallest nanofibers but not the control or larger fibers. Quantitative G-LISA results on POR1 siRNA treated cells showed that small nanofibers do lead to elevated levels of active Rac1, but that these levels were enabled by POR1, not inhibited by it. The results suggest that POR1 binds active Rac1 and acts as a competitive inhibitor of GTPase-activating protein (GAP), thus decreasing the rate at which Rac1 is inactivated.