Forces of Phagocytosis with Two-Channel Live Cell Bessel Light Sheet 3D Imaging

Abstract

A critical function of the immune system is the search and elimination of foreign pathogens by macrophages, neutrophils and other cells. These cells eliminate large particles (>0.5μm) by consuming them through a process called phagocytosis. There are several receptor-ligand pairs and associated signaling pathways that initiate phagocytosis. The best understood is the Fcϒ receptor (FcϒR)-immunoglobulin G (IgG) pair. Recognition by FcϒR of an IgG coated particle triggers the cell's cytoskeleton to form a pseudopod that reaches around and engulfs the particle. Evaluating the mechanics of macrophage target engulfment requires careful monitoring of forces and high quality florescence imaging of the membrane and cytoskeleton. We combined an atomic force microscope (AFM) with a versatile optics system, a first of its kind, to monitor piconewton scale forces while imaging the phagocytosis engulfment from the side using Pathway Rotated Imaging for Sideways Microscopy with vertical light sheet (PRISM-LS). The macrophage produces a dynamic response a few nanonewtons during its envelopment of an IgG covered bead attached to an AFM cantilever. We observe the macrophages pull on the bead with downward forces in the range of 300pN-8nN. The macrophage exerts these downward forces before the pseudopod envelops past the midpoint of the bead. These downward forces can last from 5 seconds to 2 minutes. The phagocytic cup formation is associated with punctate brightening of actin underneath the bead. In addition, the versatile optics system can be used to record two-channel, three dimensional images of cells while engaging with the AFM. Bessel beam light sheet imaging with controlled force data will inform mechanical models of phagocytosis which will improve understanding of this important immunological process and inform mammalian disease progression.