Dictyostelium MEGAPs: F-BAR domain proteins that regulate motility and membrane tubulation in contractile vacuoles

Abstract

PCH family proteins are fundamentally important proteins, linking membrane curvature events with cytoskeletal reorganisation. One group, the MEGAPs (also called srGAPs and WRPs) contain RhoGAP domains in addition to the F-BAR domain. We disrupted MEGAP1 and MEGAP2 in Dictyostelium both singly and in combination. We found a strong cytoskeletal phenotype in MEGAP1– cells and a subtle phototaxis defect in MEGAP2– slugs. MEGAP1–/2– cells have an overabundance of filopodia and slug motility and function are affected. The most dramatic changes, however, are on contractile vacuoles. MEGAP1–/2– cells empty their contractile vacuoles less efficiently than normal and consequently have three times the usual number. GFP-tagged MEGAP1 localises to tubules of the contractile vacuole network and when vacuoles start to empty they recruit cytosolic GFP-MEGAP1. Mutants in the Saccharomyces homologues RGD1 and RGD2 also show abnormal vacuoles, implying that this role is conserved. Thus, MEGAP is an important regulator of the contractile vacuole network, and we propose that tubulation of the contractile vacuole by MEGAP1 represents a novel mechanism for driving vacuole emptying.

The inverse BAR-domain protein IBARa drives membrane remodelling to control osmoregulation, phagocytosis and cytokinesis

“…Interestingly, MEGAP1 2 /2 2 double mutants empty their CVs less efficiently and have three times more CVs as compared to wild type. As assessed by time-lapse imaging, these proteins localize transiently to the CV membrane and subsequently fragment into smaller vesicles as the vacuole empties (Heath and Insall, 2008). Provided that IBARa, MEGAP1 and MEGAP2 (and possibly other F-BAR proteins) act on the same side of the CV membrane, we surmise that I-BAR and F-BAR proteins might synergize in CV discharge because the appearance of negative curvature is accompanied by positive curvature and vice versa.…”

“…How could IBARa operate in this process? Of note, the F-BAR proteins MEGAP1 and MEGAP2 have also been previously shown to localize at the CV membrane (Heath and Insall, 2008). Interestingly, MEGAP1 2 /2 2 double mutants empty their CVs less efficiently and have three times more CVs as compared to wild type.…”

“…3A), next we quantified the numbers of this organelle in WT and mutant cells using the live-cell dye FM2-10 ( Heath and Insall, 2008). Analysis by confocal microscopy revealed that, in the absence of IBARa, the cells displayed a significantly increased number of CVs as compared to control (Fig.…”

“…The Dictyostelium genome encodes six F-BAR proteins, including four MEGAPs and one syndapinlike protein, of which only MEGAP1 and MEGAP2 have been characterized. These two proteins have been shown to be implicated in cell motility and tubulation of the CV system (Eichinger et al, 2005;Heath and Insall, 2008). Thus, it remains to be seen whether some of the remaining F-BAR proteins contribute to filopodium formation.…”

“…The CVs of live cells were visualized with the lipophilic dye FM2-10 (Molecular Probes) as described previously (Heath and Insall, 2008), and were imaged using an Olympus Fluoview FV1000 confocal microscope (Olympus) at a rate of 1 frame every 5 s. The topography of the ventral cell surface, including filopodia and cell-to-substratum contacts, was imaged by RICM using a LSM 510 confocal microscope (Zeiss) and the 633-nm line of the He-Ne laser. Immunolabeling of fixed cells with polyclonal anti-IBARa antibodies or monoclonal anti-cortexillin antibody (241-71-3) was essentially performed as described previously (Faix et al, 2001).…”