Roles of a fimbrin and an alpha-actinin-like protein in fission yeast cell polarization and cytokinesis

Abstract

Eukaryotic cells contain many actin-interacting proteins, including the alpha-actinins and the fimbrins, both of which have actin cross-linking activity in vitro. We report here the identification and characterization of both an alpha-actinin-like protein (Ain1p) and a fimbrin (Fim1p) in the fission yeast Schizosaccharomyces pombe. Ain1p localizes to the actomyosin-containing medial ring in an F-actin-dependent manner, and the Ain1p ring contracts during cytokinesis. ain1 deletion cells have no obvious defects under normal growth conditions but display severe cytokinesis defects, associated with defects in medial-ring and septum formation, under certain stress conditions. Overexpression of Ain1p also causes cytokinesis defects, and the ain1 deletion shows synthetic effects with other mutations known to affect medial-ring positioning and/or organization. Fim1p localizes both to the cortical actin patches and to the medial ring in an F-actin-dependent manner, and several lines of evidence suggest that Fim1p is involved in polarization of the actin cytoskeleton. Although a fim1 deletion strain has no detectable defect in cytokinesis, overexpression of Fim1p causes a lethal cytokinesis defect associated with a failure to form the medial ring and concentrate actin patches at the cell middle. Moreover, an ain1 fim1 double mutant has a synthetical-lethal defect in medial-ring assembly and cell division. Thus, Ain1p and Fim1p appear to have an overlapping and essential function in fission yeast cytokinesis. In addition, protein-localization and mutant-phenotype data suggest that Fim1p, but not Ain1p, plays important roles in mating and in spore formation.

Figure 1

Identification of the fim1 start codon (see text for details). The putative start codon is underlined and in boldface; an upstream ATG (−125 to −123) is in boldface; the possible intron donor and acceptor sequences are underlined; and the 5′ ends of the three cDNAs are underscored by *.

Figure 2

Sequences of S. pombe Ain1p and Fim1p. (A) Alignment of Ain1p (GenBank/EMBL/DDBJ accession number Z97208) and Drosophila muscle α-actinin (DmActinin; accession number X51753). Proteins were aligned using the GCG GAP program with some modifications according to the results of a BLAST analysis (http://www.ncbi.nlm.nih.gov/BLAST/). Identical and similar (V/I/L, D/E, K/R, N/Q, S/T) amino acids are shaded in black and gray, respectively. Dots indicate gaps introduced to optimize the alignment; underscoring by ∼ indicates the presumed actin-binding motif; and –//– indicates amino acids 370 to 599 of DmActinin, omitted here to optimize alignment of the NH₂- and COOH-terminal regions of the proteins (see text). (B) Alignment of Fim1p (GenBank/EMBL/DDBJ accession number AF053722), S. cerevisiae Sac6p (SWISS-PROT accession number P32599), and the human fimbrin L-plastin (SWISS-PROT accession number P13796). Proteins were aligned using the GCG PileUp program. Amino acids identical or similar (see above) to those in Fim1p are shaded in black and gray, respectively. Two putative actin-binding motifs are underscored with ∼. The amino acids of Sac6p and L-plastin underscored with * were used to design the degenerate PCR primers that allowed the cloning of fim1 (see MATERIALS AND METHODS).

Figure 3

Localization of Ain1p and Fim1p during the cell cycle. In A and B, upper and lower panels show the same cells, and individual cells are numbered for reference in the text; in C, the two panels show the same cells. (A) Localization of Ain1p to the medial ring. Cells expressing Ain1p-GFP (strain JW46) were grown in EMM medium at 25°C and stained with Calcofluor and bisBenzimide to allow visualization of septa and DNA. (B and C) Localization of Fim1p to actin patches and the medial ring. Cells expressing Fim1p-3HA (strain JW106) were grown in EMM at 30°C, fixed, and double-stained either with HA-specific antibody and bisBenzimide (B) or with HA-specific antibody and rhodamine-phalloidin (C). Bar, 10 μm.

Figure 4

Actin dependence of Ain1p and Fim1p localization. Cells expressing Ain1p-GFP (strain JW46; A-D) or Fim1p-GFP (strain JW109; E and F) were treated with 100 μM Lat-A (added from a stock solution in DMSO; see MATERIALS AND METHODS) (A and C–F) or an equal volume of DMSO alone (B) at 25°C for 20 min, and then stained for actin (A and B) or examined for the GFP-fusion proteins (C and E) after staining with Calcofluor and bisBenzimide to allow visualization of septa and DNA in the same cells (D and F). Bar, 10 μm.

Figure 5

Morphological abnormalities of ain1 deletion and fim1 deletion cells. (A–C) Cells were double-stained with Calcofluor and bisBenzimide to visualize septa and DNA. (D–F) Cells were observed directly in culture medium by DIC microscopy. (A) ain1-Δ1 cells (strain JW45) grown in EMM medium at 25°C. (B and C) Wild-type strain 972 (B) and strain JW45 (C) grown in EMM + 1 M KCl medium at 18°C for 40 h. (D) fim1-Δ1 cells (strain JW142) grown in EMM at 25°C. (E and F) Strains 972 (E) and JW142 (F) grown for 5 h (20 h for the cells in the inset) after a shift from 25 to 36°C in EMM. Bar, 10 μm.

Figure 6

Abnormal actin organization in ain1 deletion and fim1 deletion cells. (A) Failure of actin-ring formation in ain1-Δ1 cells under stress conditions. Strains 972 (left panels) and JW45 (right panels) growing in YES medium at 30°C were shifted to EMM + 1 M KCl medium at 18°C for 20 h, fixed, and double-stained with rhodamine-phalloidin (upper panels) and bisBenzimide (lower panels). (B) Mislocalization and disorganization of actin patches in fim1-Δ1 cells. Strains 972 (left panels) and JW142 (right panels) growing in EMM medium at 25°C were shifted to 36°C for 5 h, fixed, and double-stained with rhodamine-phalloidin (upper panels) and bisBenzimide (lower panels). Bar, 10 μm.

Figure 7

Defects in cytokinesis, septation, and actin organization resulting from overexpression of Ain1p or Fim1p. Cells in which the expression of Ain1p or Fim1p was under the control of the 3nmt1 promoter were grown under repressing conditions and then shifted to inducing conditions (see MATERIALS AND METHODS) at 30°C. (A) Cells of strains JW49 (left panels) and JW110 (right panels) were double-stained with Calcofluor and bisBenzimide at 16 h (upper panels) or 19 h (lower panels). (B) Cells of strains JW49 (left panels) and JW110 (right panels) were fixed and double-stained with rhodamine-phalloidin (upper panels) and bisBenzimide (lower panels) at 16 h. The arrowhead indicates an interphase cell (see text). Bars, 10 μm.

Figure 8

Cell division defect of an ain1 fim1 double mutant. ain1-Δ1 41nmt1-fim1⁺ cells (strain JW164) growing in EMM medium without thiamine (inducing conditions) at 25°C (A) or 16 h after a shift to YES medium (repressing conditions, leading to depletion of Fim1p) at 25°C (B–E) were double-stained for septa and DNA without fixation (A and B), stained for septa alone without fixation (C), or double-stained for F-actin (D) and DNA (E) after fixation (see MATERIALS AND METHODS). Bar, 10 μm.

Figure 9

(A–F) Localization of Fim1p-GFP during mating (A), sporulation (B and D), and spore germination (F). (C and E) DIC images of the cells shown in B and D, respectively. fim1⁺-GFP strains JW109 and JW117 were crossed on an SPA plate at 25°C, and samples taken after 12 h (A), 24 h (B and C), and 50 h (D and E) were examined after resuspension in liquid SPA medium. After 60 h, the mature asci were digested with Glusulase (NEN, Boston, MA) to release spores, which were then incubated in liquid EMM medium at 25°C and examined after 12 h (F). (G) Defective sporulation in fim1 deletion cells. fim1-Δ1 strains JW144 and JW142 were crossed on an SPA plate at 25°C and examined by DIC after 50 h. Bar, 10 μm.