Regulation of a formin complex by the microtubule plus end protein tea1p

Abstract

The plus ends of microtubules have been speculated to regulate the actin cytoskeleton for the proper positioning of sites of cell polarization and cytokinesis. In the fission yeast Schizosaccharomyces pombe, interphase microtubules and the kelch repeat protein tea1p regulate polarized cell growth. Here, we show that tea1p is directly deposited at cell tips by microtubule plus ends. Tea1p associates in large "polarisome" complexes with bud6p and for3p, a formin that assembles actin cables. Tea1p also interacts in a separate complex with the CLIP-170 protein tip1p, a microtubule plus end-binding protein that anchors tea1p to the microtubule plus end. Localization experiments suggest that tea1p and bud6p regulate formin distribution and actin cable assembly. Although single mutants still polarize, for3Deltabud6Deltatea1Delta triple-mutant cells lack polarity, indicating that these proteins contribute overlapping functions in cell polarization. Thus, these experiments begin to elucidate how microtubules contribute to the proper spatial regulation of actin assembly and polarized cell growth.

Figure 1.

Microtubule plus ends deposit tea1p on the cell surface. (A) Time-lapse images of a wild-type cell expressing tea1p-YFP (green) and CFP-atb2p (α-tubulin, red). Times in seconds are listed. Arrowheads show examples of growing microtubules with tea1p at the plus end; empty arrowheads show a shrinking microtubule without tea1p. (B) Time-lapse images of an rsp1-1 cell expressing tea1p-YFP (green) and CFP-atb2p (red) grown in liquid minimal media to late log phase at 30°C. Arrowheads show an example of a tea1p dot that is retained at the cell surface after microtubule shrinks. Additional examples are shown in Videos 1–3 (available at http://www.jcb.org/cgi/content/full/jcb.200403090/DC1). Bar, 2.5 μm.

Figure 2.

For3p, bud6p, tea1p, and tip1p coimmunoprecipitate. (a) For3p-myc and bud6p-HA coimmunoprecipitate. Extracts expressing (1) for3p-myc (BFY19), (2) for3p-myc bud6p-HA (BFY168), or (3) bud6p-HA (FC592) were immunoprecipitated using protein A–Sepharose beads complexed with either α-HA or α-myc antibodies and were Western blotted. (b) For3p-TAP immunoprecipitates tea1p. Extracts expressing (1) for3-myc (BFY19) or (2) for3p-TAP (BFY198) were immunoprecipitated with mouse α-rabbit Dynal magnetic beads complexed with rabbit IgG, and were Western blotted with α-myc antibodies (the HRP anti–rabbit secondary recognizes both the α-myc antibody and the TAP tag) or anti-tea1p antibodies. (c) Tea1p-HA immunoprecipitates for3p-myc and tip1p. Extracts expressing tea1p-HA for3p-myc (BFY190, lanes 1 and 2) or for3p-myc (BFY19, lanes 3 and 4) were immunoprecipitated with sheep α-mouse Dynal magnetic beads complexed with mouse anti-HA antibodies, washed in buffer containing either 250 mM NaCl (lanes 1 and 3) or 150 mM NaCl (lanes 2 and 4; see Materials and methods), and immunoblotted. White lines indicate that intervening lanes have been spliced out.

Figure 3.

Tea1p, for3p, bud6p, and tip1p reside in multiple complexes. (a) Soluble yeast extracts were fractionated on velocity sucrose gradients, and fractions were immunoblotted with appropriate antibodies. Fractions were loaded so the fractions from the top of the gradient (containing smaller complexes) are on the left. Colored boxes mark peaks of proteins in complexes. Four complexes of ∼12, 20, 45, and 75S are labeled A, B, C, and D, respectively. Labels on the right denote the genotype of the yeast strain analyzed. a and b represent two representative experiments in which multiple sucrose gradients were prepared and centrifuged in parallel, so that gradients can be directly compared. (a) Fractionation of for3p-myc bud6p-HA (wild-type, BFY168), for3Δbud6-HA (BFY186), and tea1Δfor3-myc (BFY184) extracts. (b) Fractionation of for3p-myc bud6-HA and bud6Δfor3-myc (BFY192) extracts.

Figure 4.

Localization dependency relationships between for3p, bud6p, and tea1p. Localization of YFP- or CFP-tagged proteins were imaged by fluorescence microscopy in living wild-type and mutant cells. (a) Localization of for3p-YFP (green) and tea1p-CFP (red; BFY125) in interphase cells. (b) Localization of for3p-YFP (green) and bud6p-CFP (red; BFY112) in interphase cells. (c) Localization of for3p-YFP in wild-type cells (BFY81). (d) Localization of for3p-YFP in a tea1Δ cell (BFY122). Calcofluor preferentially stains the active site of cell growth. (e) Localization of for3p-YFP in a T-shaped tea1Δ cell. (f) Localization of for3p-YFP in bud6Δ cells (BFY120). Arrows show for3p-YFP dots on the sides of cells. (g) Localization of tea1p-YFP in wild-type (FC871) and for3Δ cells (BFY191). (h) Localization of bud6p-CFP in wild-type (BFY104) and for3Δ cells (BFY148). Bar, 5 μm.

Figure 5.

Tea1p and bud6p affect actin cable organization. (a) Wild-type (FC421), bud6Δ (FC592), and tea1Δ (FC691) cells were fixed and stained for F-actin using Alexa Fluor^® phalloidin. Single focal planes in the middle of the cell of deconvolved confocal images are shown. Actin cables are the filamentous structures. (b) Fluorescence intensity of Alexa Fluor^® phalloidin–stained cables relative to Alexa Fluor^® phalloidin–stained actin patches in the same cell. (c) Number of actin cables. In each cell, the number of actin cables in all focal planes was counted at three points corresponding to 25 (a), 50 (b), and 75% (c) of the cell length. In tea1Δ cells, (a) was assigned to the growing cell tip and (c) to the nongrowing cell tip. In bipolar wild-type cells, no significant difference was seen at the two tips, and the assignment of (a) and (c) was random. Bars, 5 μm.

Figure 6.

Synthetic genetic interactions between for3 ⁺ , bud6 ⁺ , and tea1 ⁺ reveal functions in general cell polarity. (a) Growth rates. 10-fold serial dilutions of cells with the indicated genotype were spotted onto rich media and grown at 30, 32, and 36°C on agar plates for 4 d. (b) Differential interference contrast images of representative cells with the indicated genotype. (c) Quantitation of shapes of cells of the indicated genotype grown in liquid culture (n > 150 cells). Bar, 5 μm.

Figure 7.

Model of how microtubule plus ends regulate actin assembly. Step 1: tea1p is bound to the microtubule plus ends by interaction with the CLIP-170 tip1p, and the growth of microtubules (green) delivers tea1p to the cell tip. Step 2: tea1p leaves the microtubule and docks at the cell surface when the microtubule shrinks back. Step 3: at certain cell tips, tea1p may recruit, stabilize, or otherwise regulate the polarisome complex, which includes bud6p and the formin for3p. Step 4: the complex promotes polarized cell growth, with for3p functioning to assemble actin cables (red) at the cell tip.