Fibronectin Assembly Dynamics in Osteoblasts is Integrated With Cell Motility

Objectives: Fibronectin (FN) is an extracellular matrix (ECM) glycoprotein important for osteoblast function. We have previously shown that FN is a key orchestrator for assembly of other bone ECM proteins and that the assembly of FN by osteoblasts is a highly dynamic process that appears to be driven by cell motility. The goal of this study was to further define the dynamic mechanisms by which fibronectin is assembled in osteoblasts and the role of cell motility in assembly and reorganization of fibronectin fibrils. Methods: Dynamic time lapse imaging was performed in living cultures of primary osteoblasts and the 2T3 osteoblast-like cell line using alexafluor488 or 555-labeled fibronectin as a probe. Fibronectin assembly dynamics were monitored in the presence and absence of the cell motility inhibitor Cytochalasin D. To determine the kinetics of fibronectin incorporation/assembly as a function of time, image stacks were thresholded and quantified. Cell and fibril motions were quantified using the MTrackJ plugin within the ImageJ software. Results: Time-lapse imaging showed that FN assembly in living osteoblasts was a highly dynamic process in which short fibrils were first assembled on the cell surface. As cells moved, apparently randomly, larger fibrils formed through coalescence of surface-assembled material, with multiple cells contributing to the formation of fibronectin fibrils. Even after assembly, the fibril network was continually stretched and reorganized by the motile cell layer. Cytochalasin D (0.1-1µM) inhibited motility of the osteoblasts dose-dependently, with maximal inhibition at 1µM. Addition of 1µM cytochalasin D also inhibited assembly of fibronectin by more than 90% compared to controls. When added after cells had incorporated FN on their surface, cytochalasin D also prevented the progression to form larger fibrillar networks. Conclusions: The data suggest that cell motility is key for the assembly of extracellular fibronectin fibril networks. Funded by: UMKC-SOD Rinehart Foundation, NIH RO1AR051517