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1.
Traffic ; 13(1): 120-30, 2012 Jan.
Article in English | MEDLINE | ID: mdl-22008230

ABSTRACT

The role of actin, class I myosins and dynamin in endocytic uptake processes is well characterized, but their role during endo-phagosomal membrane trafficking and maturation is less clear. In Dictyostelium, knockout of myosin IB (myoB) leads to a defect in membrane protein recycling from endosomes back to the plasma membrane. Here, we show that actin plays a central role in the morphology and function of the endocytic pathway. Indeed, latrunculin B (LatB) induces endosome tubulation, a phenotype also observed in dynamin A (dymA)-null cells. Knockout of dymA impairs phagosome acidification, whereas knockout of myoB delays reneutralization, a phenotype mimicked by a low dose of LatB. As a read out for actin-dependent processes during maturation, we monitored the capacity of purified phagosomes to bind F-actin in vitro, and correlated this with the presence of actin-binding and membrane-trafficking proteins. Phagosomes isolated from myoB-null cells showed an increased binding to F-actin, especially late phagosomes. In contrast, early phagosomes from dymA-null cells showed reduced binding to F-actin while late phagosomes were unaffected. We provide evidence that Abp1 is the main F-actin-binding protein in this assay and is central for the interplay between DymA and MyoB during phagosome maturation.


Subject(s)
Actins/metabolism , Dynamins/metabolism , Endosomes/metabolism , Microfilament Proteins/metabolism , Myosin Type I/metabolism , Phagosomes/metabolism , Protozoan Proteins/metabolism , Blotting, Western , Dictyostelium/metabolism , Dictyostelium/ultrastructure , Dynamins/genetics , Endosomes/ultrastructure , Gene Knockout Techniques , Models, Biological , Myosin Type I/genetics , Phagocytosis , Phagosomes/ultrastructure , Protein Transport , Protozoan Proteins/genetics
2.
Mol Biol Cell ; 21(9): 1505-18, 2010 May 01.
Article in English | MEDLINE | ID: mdl-20200225

ABSTRACT

Actin dynamics and myosin (Myo) contractile forces are necessary for formation and closure of the phagocytic cup. In Dictyostelium, the actin-binding protein Abp1 and myosin IK are enriched in the closing cup and especially at an actin-dense constriction furrow formed around the neck of engulfed budded yeasts. This phagocytic furrow consists of concentric overlapping rings of MyoK, Abp1, Arp3, coronin, and myosin II, following an order strikingly reminiscent of the overall organization of the lamellipodium of migrating cells. Mutation analyses of MyoK revealed that both a C-terminal farnesylation membrane anchor and a Gly-Pro-Arg domain that interacts with profilin and Abp1 were necessary for proper localization in the furrow and efficient phagocytosis. Consequently, we measured the binding affinities of these interactions and unraveled further interactions with profilins, dynamin A, and PakB. Due to the redundancy of the interaction network, we hypothesize that MyoK and Abp1 are restricted to regulatory roles and might affect the dynamic of cup progression. Indeed, phagocytic uptake was regulated antagonistically by MyoK and Abp1. MyoK is phosphorylated by PakB and positively regulates phagocytosis, whereas binding of Abp1 negatively regulates PakB and MyoK. We conclude that a MyoK-Abp1-PakB circuit acts as a switch regulating phagocytosis efficiency of large particles.


Subject(s)
Microfilament Proteins/metabolism , Myosin Type I/metabolism , Phagocytosis/physiology , Protein Kinases/metabolism , Protozoan Proteins/metabolism , Animals , Dictyostelium/genetics , Dictyostelium/metabolism , Dictyostelium/ultrastructure , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Immunoblotting , Microfilament Proteins/genetics , Microscopy, Electron , Microscopy, Fluorescence , Mutation , Myosin Type I/genetics , Phosphorylation , Protein Binding , Protein Kinases/genetics , Protein Prenylation , Protozoan Proteins/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Surface Plasmon Resonance , Yeasts/physiology , Yeasts/ultrastructure
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