The Balance in the Delivery of ER Components and the Vacuolar Proton Pump to the Phagosome Depends on Myosin IK in Dictyostelium

Abstract

In Dictyostelium, the cytoskeletal proteins Actin binding protein 1 (Abp1) and the class I myosin MyoK directly interact and couple actin dynamics to membrane deformation during phagocytosis. Together with the kinase PakB, they build a regulatory switch that controls the efficiency of uptake of large particles. As a basis for further functional dissection, exhaustive phagosome proteomics was performed and established that about 1300 proteins participate in phagosome biogenesis. Then, quantitative and comparative proteomic analysis of phagosome maturation was performed to investigate the impact of the absence of MyoK or Abp1. Immunoblots and two-dimensional differential gel electrophoresis of phagosomes isolated from myoK-null and abp1-null cells were used to determine the relative abundance of proteins during the course of maturation. Immunoblot profiling showed that absence of Abp1 alters the maturation profile of its direct binding partners such as actin and the Arp2/3 complex, suggesting that Abp1 directly regulates actin dynamics at the phagosome. Comparative twodimensional differential gel electrophoresis analysis resulted in the quantification of mutant-to-wild type abundance ratios at all stages of maturation for over one hundred identified proteins. Coordinated temporal changes in these ratio profiles determined the classification of identified proteins into functional groups. Ratio profiling revealed that the early delivery of ER proteins to the phagosome was affected by the absence of MyoK and was coupled to a reciprocal imbalance in the delivery of the vacuolar proton pump and Rab11 GTPases. As direct functional consequences, a delayed acidification and a reduced intraphagosomal proteolysis were demonstrated in vivo in myoK-null cells. In conclusion, the absence of MyoK alters the balance of the contributions of the ER and an endo-lysosomal compartment, and slows down phagosome acidification as well as the speed and efficiency of particle degradation inside the phagosome. Molecular & Cellular Proteomics 11: 10.1074/mcp.M112.017608, 886 -900, 2012.Professional phagocytes, ranging from phagotrophic protozoa to specialized cells of the innate immune system such as macrophages, neutrophils, or dendritic cells, ingest and digest large particles (Ͼ 250 nm). Indeed, the core machineries acting in phagocytosis have been conserved as its basic purpose evolved from predation and feeding to antigen presentation (1). Particle recognition by the phagocytes initiate the internalization process and triggers remodeling of the plasma membrane and its underlying cytoskeleton, to project a circular cup-shaped lamella around the particle. The phagocytic cup finally encloses the particle into a de novo membranebound vacuole, the phagosome. Endomembrane compartments, such as early and late endosomes (2-4) and the endoplasmic reticulum (5), are recruited to provide membrane to form the phagosome. Modifications of the environment within the closed phagosome generates a microbicidal milieu and leads to particle degradation. M...

WASH is required for lysosomal recycling and efficient autophagic and phagocytic digestion

“…To establish the mechanism underlying the defects in degradation, we used quantitative comparative proteomics to identify differentially represented proteins in the isolated phagosomes of Ax2, WASH , and FAM21 mutants (Dieckmann et al ., 2012; Gotthardt et al ., 2006a). To confirm this approach, we first looked at the levels of v-ATPase.…”

Proteomic characterization of theMycobacterium marinum-containing vacuole inDictyostelium discoideum

“…They have also been functionally clustered with the help of the DAVID functional annotation clustering tool ( Figure 4C and Table S2). Comparison of the MCV proteome functional classification with the previously published phagosome proteome classification (Dieckmann, Gueho et al 2012) indicated that overall, these compartments are very similar (Figure 4B). This again confirms that the MCV conserves some of its phagosomal identity.…”

“…In order to study the MCV proteome, the first step of this study has been to establish an experimental strategy to isolate pure fractions of those compartments from infected D. discoideum cells. We decided to adapt the efficient method to isolate pure fractions of latex beads-containing phagosomes (LPCBs) described previously (Dieckmann, Gueho et al 2012). This method is based on the light buoyant density and flotation properties of latex beads.…”

The Dictyostelium discoideum GPHR Ortholog Is an Endoplasmic Reticulum and Golgi Protein with Roles during Development

“…The regulation of ER dynamics in D. discoideum has not been under extensive investigation. Involvement of the class I myosin MyoK in the delivery of ER membranes to the early phagosome has been reported, and recruitment of calnexin-positive ER to Legionella pneumophila-containing vacuoles (45,46). From the published data, it is reasonable to assume that the ER associates with the actin and the microtubule cytoskeletons in D. discoideum.…”