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First published online 11 March 2008
doi: 10.1242/jcs.021113

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

¹ School of Biosciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK
² The Beatson Institute for Cancer Research, Glasgow, G61 1BD, UK

View larger version (39K): [in this window] [in a new window]   Fig. 1. (A) Domains of Dictyostelium MEGAPs as predicted by SMART db. (B) Alignment of MEGAP RhoGAP domains with known human GAP proteins. (C) Growth of MEGAP mutants in shaken culture. Data are mean ± s.e.m. of three experiments.

View larger version (103K): [in this window] [in a new window]   Fig. 2. MEGAP mutant morphology and chemotaxis. (A) Vegetative morphology of mutant and wild-type (AX3) cell lines during axenic growth. Axenically growing cells were observed using Nomarski differential interference contrast (DIC). (B) Developed morphology of cell lines during under-agar chemotaxis. Pulse developed cells were observed during under-agar chemotaxis to 100 nM cAMP. (C) Chemotaxis of respective cell lines to a point source of 1 µM cAMP. cAMP-pulsed AX3 and mgp– cells were resuspended in KK2 medium and allowed to adhere to glass-bottomed dishes. Cells were then filmed for 10 minutes by DIC time-lapse microscopy, responding to a point source of cAMP delivered by micropipette. Images are representative of three experiments. Scale bars: 10 µm.

View larger version (134K): [in this window] [in a new window]   Fig. 3. MEGAP mutant phototaxis and development. (A) Terminal fruiting bodies of cell lines developed on nitrocellulose membranes for 48 hours. Scale bar: 2 mm. (B) Aggregation territories of cell lines on nutrient-free agar. A large quantity of nonstreaming cells can be observed in mgp1 mutants. mgp2 mutants often have larger aggregation centres than the wild type. Scale bar: 500 µm. (C) Phototaxis of slugs to a point light source. Light-directed migration of slugs after 48 hours on 0.5% charcoal agar shows that mgp– slugs are defective in phototaxis. Asterisks indicate the direction of the light source. Scale bar: 2 mm. (D) Representative examples of wild-type and mgp mutant slugs when cells are developed on nutrient-free agar. mgp1– slugs are shorter than the wild type and large clumps of cells remain on the substratum. mgp2– mutants form a few very large slugs and numerous shorter slugs. mgp1–/2– slugs are also longer than wild-type slugs. Scale bar: 250 µm. Images are representative of three experiments.

View larger version (11K): [in this window] [in a new window]   Fig. 4. MEGAP mutant slugs are significantly varied in length. Wild-type AX3 and mgp mutant cells were allowed to develop on nutrient-free agar plates. The length of at least 150 slugs from three experiments was measured and plotted as a box and whisker plot. mgp1– slugs (n=194) are significantly shorter than wild-type slugs (n=192; P0.001). mgp2– (n=151) and mgp1–/2– (n=170) slugs are significantly longer than wild-type slugs (P0.0001 in both instances), but not significantly different from each other.

View larger version (49K): [in this window] [in a new window]   Fig. 5. Mutant MEGAP cells have more contractile vacuoles. (A) Contractile vacuole network of cells visualised with the lipophilic styryl dye FM2-10. Both mgp1– and mgp1–/2– show many more contractile vacuoles than the wild type. Pictures are representative of at least three experiments. Scale bar: 10 µm. (B) Distribution of vacuole number in wild-type AX3 and mgp– cells visualised with FM2-10. Average number of vacuoles per cell ± s.e.m. is shown in inset. The number of vacuoles in each cell was counted for 30 minutes. The distribution in B reflects the frequency of cells containing (n) vacuoles for all cells over this time. mgp1– and mgp1–/2– have, on average, significantly more contractile vacuoles than the wild type (P=0.0012 and P=0.0052, respectively). GFP-MEGAP1 expressed in the mgp1– background significantly rescues the contractile vacuole phenotype (P=0.0042).

View larger version (19K): [in this window] [in a new window]   Fig. 6. The rate of contractile vacuole expulsion is unaffected in mgp1–/2– cells. (A) Average contractile vacuole (CV) expulsions rate of wild-type AX3 (squares) and mgp1–/2– cells (circles). (B) Average contractile vacuole expulsions rate of AX3 (squares) and mgp1–/2– (circles) when stimulated with 25 µM folate (arrow). Each point of the graph represents the cumulative average number of vacuole expulsions in ten cells (from three experiments) over time. The slope therefore reflects the average rate of vacuole expulsion events.

View larger version (148K): [in this window] [in a new window]   Fig. 7. GFP-MEGAP1 labels the contractile vacuole network. (A) GFP-MEGAP1 localises to tubules associated with the contractile vacuole membrane in an iso-osmotic environment (arrowheads). (B) Live imaging of GFP-MEGAP1-expressing cells in a hypo-osmotic environment reveals transient localisation to contractile vacuoles at the point of discharge, suggesting that MEGAP1 is important in contractile vacuole emptying. Contractile vacuole activity was stimulated by incubating cells for 30 minutes in Lo Flo. Cells were imaged using an agar-overlay technique (Yumura et al., 1984) by confocal microscopy. Scale bars: 5 µm.

View larger version (42K): [in this window] [in a new window]   Fig. 8. Macropinocytosis is unaffected in MEGAP mutants. (A) Quantification of fluid-phase uptake. The rate of fluid-phase uptake is unaltered in MEGAP mutants. Mean ± s.e.m. of three experiments. (B) Visualisation of fluid-phase uptake. Confocal images showing normal TRITC-dextran internalisation in MEGAP mutant cell lines. Scale bar: 10 µm.

View larger version (90K): [in this window] [in a new window]   Fig. 9. S. cerevisiae Rgd1 and Rgd2 cells have numerous small vacuoles. Cells were labelled with FM4-64 dye and visualised by confocal microscopy. Both Rgd1 and Rgd2 lines have a greater number of vacuoles (arrowheads), which are smaller than those in the wild type. Scale bar: 5 µm.

View larger version (8K): [in this window] [in a new window]   Fig. 10. Model of MEGAP1 function. (A) The contractile vacuole network is mostly tubular when MEGAP1 is bound. (B) As water enters the cell, MEGAP1 moves to the cytosol, allowing a vacuole to form. (C) The bladder grows until the vacuole has reached full size and requires emptying. (D) The contractile vacuole contacts the plasma membrane, forming a discrete pore (contained by a ring of F-actin) and begins to empty. MEGAP1 detects the change in curvature. (E) As the contractile vacuole empties, MEGAP1 binds, tubulating the bladder, adding force to the expulsion. (F) The bladder continues emptying and tubulating, and the cycle begins anew.

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