UNC-CH Physics and Astronomy Masters Presentation

Megan Kern

“Correlating Engulfment Forces to Actin Distribution and GEFH1 Activation to Study the Mechanisms of Force Generation and Mechanosensing in Phagocytosis”

Phagocytosis is the process by which immune cells engulf harmful bacteria, foreign pathogens, and other cell debris. It is well documented that the actin driven engulfment process depends on upstream GTPase activation, but it is still unclear how phagocytosis relies on the activation of GEFs. Active GEF-H1 has recently been associated with regulating force response and leading-edge dynamics during migration. Using a single-chain FRET biosensor we are able to correlate relative changes in the activation state of GEF-H1. The GEF-H1 biosensor consists of a yPet and mCerulean3 FRET pair inserted into the hinge of the autoinhibitory domain to signal the change in conformation of the protein upon activation. We have made a RAW 264.7 murine macrophage cell line stably expressing this biosensor as well as Ftractin-Halo in order to study the dynamics of GEF-H1 activation and actin during phagocytosis. Using a vertical light sheet system coupled to an AFM, we have recorded the XZ-plane and volumetric actin distribution of individual macrophages during early stages of engulfment and correlated them to engulfment forces. In addition, we have captured the actin distribution and GEF-H1 activity during frustrated phagocytic spreading of cells on IgG functionalized substrates as well as in side-view. During these protrusions, GEF-H1 activation is highest at the leading edge of the phagocytic cup. This suggests that GEF-H1 activation plays a role in the mechanosensatory process of phagocytosis.