RacB regulates cytoskeletal function in Dictyostelium spp

Abstract

Thus far, 14 homologues of mammalian Rac proteins have been identified in Dictyostelium. It is unclear whether each of these genes has a unique function or to what extent they play redundant roles in actin cytoskeletal organization. To investigate the specific function of RacB, we have conditionally expressed wild-type (WT-RacB), dominant negative (N17-RacB), and constitutively activated (V12-RacB) versions of the protein. On induction, cells expressing V12-RacB stopped growing, detached from the surface, and formed numerous spherical surface protrusions while cells overexpressing WT-RacB became flattened on the surface. In contrast, cells overexpressing N17-RacB did not show any significant morphological abnormalities. The surface protrusions seen in V12-RacB cells appear to be actin-driven protrusions because they were enriched in F-actin and were inhibitable by cytochalasin A treatment. The protrusions in V12-RacB cells did not require myosin II activity, which distinguishes them from blebs formed by wild-type cells under stress. Finally, we examined the functional consequences of expression of wild-type and mutant RacB. Phagocytosis, endocytosis, and fluid phase efflux rates were reduced in all cell lines expressing RacB proteins but the greatest decrease was observed for cells expressing V12-RacB. From these results, we conclude that like other members of the Rho family, RacB induces polymerization of actin but the consequences of activation appear to be different from other Dictyostelium Rac proteins so far investigated, resulting in different morphological and functional changes in cells.

FIG. 1.

Cell morphology changes induced by expression of RacB. (A to E) Cells were grown for 36 h in the presence or absence of folic acid, and phase-contrast images were captured. Wild-type Ax4 cells in the absence of folic acid (A), V12-RacB cells in fresh medium containing 1 mM folic acid (B), and WT-RacB cells (C), V12-RacB cells (D), and WT-RacC cells (E) in the absence of folic acid are shown. The morphology of WT-RacB, V12-RacB, and WT-RacC cells in the presence of folic acid is almost identical to that of parental cells but is dramatically changed on induction. WT-RacB cells became flattened, V12-RacB cells became detached with numerous protrusions on the cell surface, and WT-RacC cells became relatively flat with irregular contours on the surface. Bar, 20 μm. (F) Expression of WT-RacB and V12-RacB. Extracts of 2 × 10⁵ parental, V12-RacB, and WT-RacB cells were harvested and run on an SDS-10% polyacrylamide gel. Expression of RacB was detected by rabbit polyclonal antibody against RacB (right panel) or by anti-HA antibody (left panel). In the presence of folate at low cell density (2 × 10⁵ cells/ml), the recombinant protein was barely detectable (− lanes); however, it was induced twofold over the endogenous RacB level at high cell density in the absence of folic acid (+ lanes).

FIG. 2.

Growth of parental, WT-RacB, and V12-RacB cells. Equal numbers of cells from each cell line were added to wells of a microtiter plate. Cells from two wells were taken every 10 to 14 h to determine the average cell density. Parental Ax4 cells have an 11-h generation time, while V12-RacB cells stopped growing 36 h after folic acid removal. WT-RacB cells had a slightly lower growth rate.

FIG. 3.

Nuclear staining of parental and V12-RacB cells. (A) Cells were grown for 3 days under induced conditions and stained with 1 μM propidium iodide to visualize nuclei. Left panels are DIC images, and right panels show the nuclear staining. The upper panels are wild-type cells, and the lower panels V12-RacB cells. The arrowhead indicates heterogeneous sizes of nuclei in V12-RacB cells. (B) The number of nuclei was counted from 157 parental and 97 V12-RacB-expressing cells. In parental cells, 87% contain one or two nuclei, as opposed to 26% in V12-RacB population. The number of nuclei in cells expressing V12-RacB ranged from 1 to 15, compared to 1 to 5 in wild-type cells.

FIG. 4.

Scanning electron micrographs of parental and V12-RacB cells. Cells were grown on glass coverslips and prepared for SEM after 2 days of growth in the absence of folic acid. (A) Parental cells have a thick center with protrusions at the cell edge. (B) V12-RacB cells have numerous irregular protrusions on the cell surface. (C) Some V12-RacB cells remain flattened on the surface and contain numerous protrusions at the cell edges. Bar, 5 μm.

FIG. 5.

Comparison of V12-RacB protrusions to stress-induced blebs. (A and E) Cells were sealed between two coverslips in a Rose chamber and imaged at 1-min intervals. The cells stopped moving after 5 to 10 min in the sealed chamber and began to form blebs. Control cells before sealing (A) and after 20 min in the chamber (E) are shown. (B and F) V12-RacB (Ax4 parental) cells contained surface protrusions (B), but after treatment with 10 μM cytochalasin A for 5 min, the protrusions were lost from the surface (F). (C and G) Myosin null cells (mhcA) before sealing (C) and after a 20-min (G) or 1-h incubation. The cells did not show any blebbing after incubation (our unpublished results). (D and H) Expression of V12-RacB was induced for 3 days in JH10, a parental cell line of mhcA cells (D), and in mhcA cells (H). Surface protrusions were induced in both cell lines. Bar, 5 μm.

FIG. 6.

V12-RacB increases the amount of F-actin in cells. (A and B) The forward scatter (FSC) data from flow cytometry of parental (A) and V12-RacB (B) cells shows that the average size was increased about twofold in V12-RacB-expressing cells. F-actin was quantified by measuring the TRITC-phalloidin fluorescence of cells of similar size. Two areas, marked R3 and R5, indicate two different size ranges of cells. (C and D) The fluorescence intensity of cells in each size range was plotted (R3 and R5, respectively). The solid gray line represents the fluorescence intensity of the parental Ax4 cells, and the black line represents the V12RacB cells. (E) The amount of actin was also quantified by SDS-polyacrylamide gel electrophoresis by loading whole-cell extracts and cytoskeletal fractions on a gel. The total amount of actin was not changed significantly; however, the amount of cytoskeleton-associated F-actin increased about twofold in V12-RacB-expressing cells (see Table 1).

FIG. 6.

V12-RacB increases the amount of F-actin in cells. (A and B) The forward scatter (FSC) data from flow cytometry of parental (A) and V12-RacB (B) cells shows that the average size was increased about twofold in V12-RacB-expressing cells. F-actin was quantified by measuring the TRITC-phalloidin fluorescence of cells of similar size. Two areas, marked R3 and R5, indicate two different size ranges of cells. (C and D) The fluorescence intensity of cells in each size range was plotted (R3 and R5, respectively). The solid gray line represents the fluorescence intensity of the parental Ax4 cells, and the black line represents the V12RacB cells. (E) The amount of actin was also quantified by SDS-polyacrylamide gel electrophoresis by loading whole-cell extracts and cytoskeletal fractions on a gel. The total amount of actin was not changed significantly; however, the amount of cytoskeleton-associated F-actin increased about twofold in V12-RacB-expressing cells (see Table 1).

FIG. 7.

Three-dimensional localization of F-actin. Cells were fixed and stained with TRITC-phalloidin, and confocal z sections were acquired. (A) Wild-type cells. Images are shown at 1.4-μm z intervals. (B) V12-RacB cells. Images are shown at 3-μm z intervals. Note that the protrusions on the cell surface are enriched in F-actin. (C) WT-RacC cells. Images are shown at 1.5-μm z intervals. Note that these deformations of the cortex are not actin filled. Bar, 5 μm.

FIG.8.

Visualization of actin dynamics in live cells, using GFP-ABD. (A) Parental Ax4 cells expressing GFP-ABD were observed by confocal microscopy at 20-s intervals. GFP-ABD localizes to the actin cortex and new protrusions as they form at the periphery of the cell (arrow). (B) Cells coexpressing GFP-ABD and V12-RacB were induced for 2 days, and time-lapse images were acquired at 20-s intervals. Cells became detached and actively formed actin-containing spherical protrusions (arrow). (C) Ax4 cells expressing GFP-ABD were electroporated, and the process of bleb formation was visualized by confocal microscopy at 7-s intervals. Note the lack of staining of the bleb. (D) Cells coexpressing GFP-ABD and WT-RacC were induced for 2 days, and time-lapse images were acquired at 20-s intervals. Cells deform the actin cortex both inwardly (arrowhead) and outwardly (arrow). Bar, 5 μm.

FIG. 9.

Quantitation of phagocytosis, macropinocytosis, and recycling rates. (A) Uptake of fluorescent beads by phagocytosis. (B) Uptake of fluorescent dextran by endocytosis. (C) Efflux of the endocytic probe. Efflux was measured by feeding cells with fluorescent dextran for 3 h and then measuring the fluorescence remaining at each time point.