Investigating Actin Mechanics during Phagocytic Uptake and Transport

Abstract

Phagocytosis, the internalization and consecutive digestion of biological material by macrophages is a major part of the innate mammalian immune response. Fc gamma receptor-mediated uptake is driven by actin recruitment to the internalization site and consecutive formation of membrane protrusions around the target object. Molecular motors and cytoskeletal elements contribute to the subsequent transport of phagosomes inside the cells. Although a large number of molecules that are involved in the phagocytic uptake and the phagosomal transport are identified by now, the mechanics of these processes are largely unknown. We therefore investigated the mechanics of phagocytic uptake and phagosomal transport by using live cell microscopy in combination with optical and magnetic tweezers with a focus on the role of actin filaments. We hypothesize that the actin recruitment during phagocytic uptake leads to a transient and localized increase of the stiffness of the uptake region.To test this hypothesis we induced Fc gamma receptor-mediated phagocytosis in J774A.1 mouse macrophages by offering the cells target microbeads coated with immunoglobulin-G. During the uptake we measured local cell stiffness changes with a ‘blinking optical traps’ technique executed via periodic intensity modulation of the optical force. Preliminary data indicates that that the stiffness of the uptake region increases temporarily during the uptake. In addition, we investigated the influence of actin filaments on the phagosomal transport by magnetic tweezers-based bead displacement assays and cytochalasin treatment of the cells.Our measurements are expected to identify characteristic length- and time-scales for the variation of cell mechanical properties during phagocytosis and to contribute to a more comprehensive understanding of this medically relevant process.