Abstract
Phagocytosis requires locally coordinated cytoskeletal rearrangements driven by actin polymerization and myosin motor activity. How this actomyosin dynamics is dependent upon systems that provide access to ATP at phagosome microdomains has not been determined. We analyzed the role of brain-type creatine kinase (CK-B), an enzyme involved in high-energy phosphoryl transfer. We demonstrate that endogenous CK-B in macrophages is mobilized from the cytosolic pool and coaccumulates with F-actin at nascent phagosomes. Live cell imaging with XFP-tagged CK-B and β-actin revealed the transient and specific nature of this partitioning process. Overexpression of a catalytic dead CK-B or CK-specific cyclocreatine inhibition caused a significant reduction of actin accumulation in the phagocytic cup area, and reduced complement receptor–mediated, but not Fc-γR–mediated, ingestion capacity of macrophages. Finally, we found that inhibition of CK-B affected phagocytosis already at the stage of particle adhesion, most likely via effects on actin polymerization behavior. We propose that CK-B activity in macrophages contributes to complement-induced F-actin assembly events in early phagocytosis by providing local ATP supply.
Highlights
Dynamic reorganization and stabilization of the actin cytoskeleton and membrane-shape alterations of cells are intimately and reciprocally coupled events that are essential for a variety of distinct cell functions such as adhesion, motility, cytokinesis, and endocytosis [1]
One view proposes that ATP is heterogeneously distributed, but the cytoskeletal proteins actin and myosin receive regional and preferential access to ATP. This model raises another question: how is ATP funneled to these proteins from distant sources? To address some of these questions, we studied the highly localized molecular events controlling actin dynamics around phagocytic activity of macrophages
We demonstrate that actin and creatine kinase-B (CK-B), a long-known enzyme involved in ATP supply, are simultaneously recruited into the sites of action during the early phases of particle ingestion
Summary
Dynamic reorganization and stabilization of the actin cytoskeleton and membrane-shape alterations of cells are intimately and reciprocally coupled events that are essential for a variety of distinct cell functions such as adhesion, motility, cytokinesis, and endocytosis [1]. One of the processes that is critically dependent on proper regulation of actin polymerization is phagocytosis, essential for food intake in lower eukaryotes or the elimination of invading microbial pathogens and scavenging of dead cells in higher multicellular eukaryotes [2]. In Fc-cR–mediated phagocytosis, signals are mediated through Rac and Cdc, whereas CR3-regulated phagocytosis of complement-opsonized targets requires only RhoA activation [7]. These pathways converge and lead to the induction of Arp2/3mediated actin polymerization, which is considered the main driving force for the formation of circular pseudopod protrusions (i.e., a ‘‘phagocytic cup’’) around the target [8]. Once the wrapping in cellular membrane protrusions is complete, a contractile force is generated to engulf the particle or dead cell completely and guide the contents of the vesicle into the endocytotic pathway for degradation [9]
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