Regulation of cell diameter, For3p localization, and cell symmetry by fission yeast Rho-GAP Rga4p

Abstract

Control of cellular dimensions and cell symmetry are critical for development and differentiation. Here we provide evidence that the putative Rho-GAP Rga4p of Schizosaccharomyces pombe controls cellular dimensions. rga4 Delta cells are wider in diameter and shorter in length, whereas Rga4p overexpression leads to reduced diameter of the growing cell tip. Consistent with a negative role in cell growth control, Rga4p protein localizes to the cell sides in a "corset" pattern, and to the nongrowing cell tips. Additionally, rga4 Delta cells show an altered growth pattern similar to that observed in mutants of the formin homology protein For3p. Consistent with these observations, Rga4p is required for normal localization of For3p and for normal distribution of the actin cytoskeleton. We show that different domains of the Rga4p protein mediate diverse morphological functions. The C-terminal GAP domain mediates For3p localization to the cell tips and maintains cell diameter. Conversely, overexpression of the N-terminal LIM homology domain of Rga4p promotes actin cable formation in a For3p-dependent manner. Our studies indicate that Rga4p functionally interacts with For3p and has a novel function in the control of cell diameter and cell growth.

Figure 1.

Loss of Rga4p alters cellular dimensions. (A) a, wild-type 972. b, rga4Δ (FV513). c, rga4Δ strain expressing HA-Rga4p under the control of the nmt1 promoter (FV531), grown in the absence of thiamine for 16 h at 32°C. Cells were stained with calcofluor. Bar, 10 μm. (B) Effect of rga4 deletion on cell length and cell diameter at division expressed as average ± SD. Twenty-five cells were measured for each condition. (C) Localization of chromosomally tagged Rga4-GFP (FV781) and Rga4-HA (FV532). a and b, Rga4-GFP; d and e, corresponding DIC images. c, Rga4-HA; f, corresponding DAPI visualization. Note that in d and e overall cell dimensions are altered by methanol fixation. Bar, 10 μm.

Figure 2.

rga4 deletion results in disorganized cytoplasmic actin cables. a and c, phalloidin staining of actin; e and g, Myo52-GFP visualization in live cells; i and k, staining of microtubules. b, d, j, and l, DAPI staining to visualize the nucleus; f and h, DIC images; a, b, i, and j, wild-type 972; c, d, k, and l, rga4Δ (FV513); e and f, control Myo52-GFP (DM3415); g and h, rga4Δ Myo52-GFP (FV797). Cells were grown exponentially at 32°C for 24 h and then stained with phalloidin-conjugated Alexa Fluor 488 for actin and immunofluorescent anti-TAT1 staining for microtubules. Bar, 10 μm.

Figure 3.

Pattern of polarized cell growth in rga4Δ cells. (A) Growth patterns of wild-type 972 and rga4Δ (FV513) cells and percentage of cell in each class. Arrows indicate the direction of growth in daughter cell pairs. (B) Time-lapse images of wild-type and rga4Δ daughter cell pairs. Cells were recorded every 10 min for 6 h at 25°C. (C) Example of measurement of growth at cell tips of daughter cell pairs from wild-type and rga4Δ. (Time 0 is at the time of cell separation.) (D) Total growth for each cell tip during the cell cycle for wild-type (n = 15) and in rga4Δ (n = 24) daughter cell pairs (average ± SD). Tip a is defined as the tip showing the most growth, which in rga4Δ strains is always found in the cell that does not activate the new end. Cells were grown on YE at 23–25°C (room temperature).

Figure 4.

Intracellular localization of For3p-YFP is altered in rga4 deletion cells. a, b, e, f, i, j, m, n, q, and r, control rga4+ cells; c, d, g, h, k, l, o, p, s, and t, rga4Δ cells. For3p-YFP visualization in live cells: a, BFY81; c, FV572. Tea4-GFP visualization in live cells: e, CA2301; g, FV796. DIC visualization of cells: b, d, f, and h. Tea1p visualization with a polyclonal anti-Tea1p antibody: i, wild-type, 972; k, FV513 (Mata and Nurse, 1997). Orb2p-HA visualization with an anti-HA antibody: m, FV607; o, FV610. Orb6p-HAp visualization with an anti- HA antibody: q, FV542; s, FV527. DAPI visualization of the nucleus: j, l, n, p, r, and t. Note that panels i–t are merged images of different microscopic fields. Bar, 10 μm.

Figure 5.

The GAP domain of Rga4p is required for For3p-YFP localization and for regulation of cell diameter. (A) LIM domains, coiled-coil domain and GAP domain in the Rga4p protein and the different constructs used in this study. (B) Calcofluor staining of rga4Δ cells expressing different domains of Rga4p; a, rga4Δ cells with pREP3X (FV516); b, rga4Δ cells expressing HA-Rga4p (FV531); c, rga4Δ cells expressing HA-Rga4p-ΔGAP (FV575); d, rga4Δ cells expressing HA-Rga4p-ΔN (FV574). Cells were grown for eight generations in the presence of thiamine. (C) For3p-YFP localization in rga4Δ cells expressing different domains of Rga4p. a, rga4Δ cells carrying empty pREP3X (FV516); b, rga4Δ cells expressing full-length HA-Rga4p (FV516); c, rga4Δ cells expressing HA-Rga4p-ΔGAP (FV575); d, rga4Δ cells expressing HA-Rga4p-ΔN (FV574). Cells were grown for 20 h at 25°C in the absence of thiamine. Bar, 10 μm.

Figure 6.

Overexpression of the N-terminus of Rga4p induces actin cable formation in a For3p-dependent manner. (a) Wild-type cells carrying an empty Rep3X plasmid (FV800). (b) Wild-type cells expressing full length HA-Rga4p (FV801). (c) Wild-type cells expressing HA-Rga4p-ΔGAP (FV804). (d) Wild-type cells expressing HA-Rga4p- ΔN (FV802). (e) rga4Δ cells carrying an empty Rep3X plasmid (FV416). (f) rga4Δ cells expressing full length HA-Rga4p (FV531). (g) rga4Δ cells expressing HA-Rga4p-ΔGAP (FV575). (h) rga4Δ cells expressing HA-Rga4p- ΔN (FV574). (i) for3Δ cells carrying an empty Rep3X plasmid (FV605). (j) for3Δ cells expressing HA-Rga4p (FV606). (k) for3Δ cells expressing HARga4p- ΔGAP (FV604). (l) for3Δ cells expressing HA-Rga4p-ΔN (FV603). (m) tea4Δ cells carrying an empty Rep3X plasmid (FV759). (n) tea4Δ cells expressing HA-Rga4p (FV760). (o) tea4Δ cells expressing HA-Rga4p-ΔGAP (FV761). (p) tea4Δ cells expressing HA-Rga4p-ΔN (FV762). Cells were grown for 16 h at 32°C in the absence of thiamine. Bar, 10 μm.

Figure 7.

Rga4p localization during cell growth. (A) a and b, control cells. c and d, tea4Δ; a and c, Rga4-GFP visualization in live cells; b and d, corresponding Calcofluor staining. (B) Localization of Rga4-GFP in cells at different stages of cell cycle. a–e, Rga4-GFP; f–j, Calcofluor staining. a and f, cell in G2 phase; b and g, cell in early mitosis, c and h, cell in mitosis during septum formation, with arrows showing the initial localization of Rga4p-GFP to the site of septation, d and i, cell during mitosis after completion of septation; e and j, cells immediately after cell separation. (C) Effect of latrunculin A on Rga4-GFP localization; a and b, DMSO-treated cells; c and d, cells treated with 200 μM latrunculin A for 20 min; a and c, Rga4-GFP visualization in live cells; b and d, DIC visualization.