Associate Professor Hoxworth Center, United States
Introduction: Filopodia are hair-like cell membrane extensions formed in various cell types including cancer cells, immune cells, platelets and neurons. As adhesive cellular structures, filopodia recruit integrins to facilitate adhesion to the underlying matrix. These integrins likely transmit forces which are important for cell adhesion, migration, mechanotransduction, etc. However, these forces (termed integrin tensions) remain poorly investigated within filopodia. The challenge originates from the ultrathin structure of filopodia, which makes integrin tensions exceedingly weak to visualize or calibrate.
Materials and
Methods: To study integrin tensions in filopodia, we developed a powerful platform enabling the direct imaging of single integrin tensions in live cells. We functionalized glass surfaces with PLL-PEG, a composite polymer with a poly-L-lysine (PLL) decorated with multiple polyethylene glycol (PEG). The brush-like PEG structure offers a non-fouling surface required for single molecule imaging, whereas the positively charged PLL supports cell adhesion through electrostatic absorption (Figure 1A). On top of the PLL-PEG, DNA-based integrative tension sensor (ITS) [1] was grafted to report integrin tensions with single molecule sensitivity. On this platform, we studied filopodia integrin tensions (FIT) and force-protein interplay in filopodia.
Results, Conclusions, and Discussions: Results show that FIT is generated in discrete foci (force nodes) along each filopodium with a spacing of ~1 μm (Fig. 1B-1E). Inhibitions of actin polymerization or myosin II activity markedly reduced FIT signals in force nodes at filopodia tips and at filopodia bases, respectively, suggesting differential force sources of FIT in the distal force nodes and proximal ones in filopodia. We further showed that the molecular force level of FIT is greater at filopodia bases than that at filopodia tips. By co-imaging FIT and structural proteins, we showed that vinculin is dispensable for the formation of filopodia and the transmission of low-level FIT. However, vinculin is indeed required for the transmission of strong FIT (>50 pN). We also demonstrated that myosin X does not participate in FIT generation, despite its prominent role in filopodium formation and elongation.
In summary, integrin tensions in filopodia were imaged and calibrated at the single molecule level. The results revealed a remarkable fact that FIT is generated at discrete and periodic patterns. This work shed new insights to the mechanobiology of filopodia which are fundamental adhesion complexes in many cell types, and also presented a platform feasible for the study of single molecule forces in live cells.
Acknowledgements (Optional): This research was sponsored by National Institute of General Medical Sciences (1R35GM128747)
.jpg "Xuefeng Wang, PhD photo")