Pxl1p is a paxillin-related protein in fission yeast. (A) Sequence alignment between Pxl1p and its related proteins. ClustalW (1.82) and Boxshade (3.21) programs were used for multiple sequence alignment and shading, respectively. Identical residues are shaded in black and conservative substitutions are shaded in gray. Sp, Schizosaccharomyces pombe; Sc, S. cerevisiae; Gg, Gallus gallus. (B) Schematic representation of Pxl1p showing three C-terminal LIM domains. The location and amino acid sequences of these LIM domains are shown at the bottom. X represents any amino acid.

Localization of Pxl1p depends on F-actin and essential components of the acytomyosin ring. (A) F-actin is required for the medial localization of Pxl1p. cdc25-22 cells expressing pxl1-GFP were grown at 24°C and shifted to 36°C for 3.5 h to block cells at G2 and then were released to 24°C for 1 h in the presence of latrunculin A (LatA) or DMSO. (B) Mutations in several components of the actomyosin ring disrupt Pxl1p distribution in the division site. Wild-type and mutant cells such as cdc3-124, cdc12-112, cdc15-140, rng2-D5, and myo2-E1 expressing pxl1-GFP were grown at 24°C and shifted to 36°C for 3–4 h. Images were taken both at 24 and 36°C. (C) Localization of Pxl1p is independent of vesicle trafficking and microtubules. Left panels, cdc25-22 cells expressing pxl1-GFP were treated similarly as in A except for release in the presence of brefeldin A (BFA) or ethanol. Right panels, nda3-KM311 cells expressing pxl1-GFP were grown at 32°C and shifted to 19°C for 6.5 h. Scale bar, 5 μm.

pxl1Δ mutant cells have a defect in the organization of the actomyosin ring. (A) pxl1Δ mutant cells have a double ring structure. Wild-type and pxl1Δ mutant cells expressing rlc1-GFP were grown at 24°C and observed by confocal microscopy. Z-stack images were taken and 3D reconstruction images with maximal projection are shown. (B) pxl1Δ mutant cells have a single unconstricted ring at early mitosis and a double ring at late stages. Individual cells from A were observed by confocal microscopy, Z-stack images were taken, and 3D reconstruction images are shown at different angles. Cells a and c, early stage. Cells b and d, late stage. Arrow indicates the primary ring and star indicates the secondary ring. (C) Cdc4p and F-actin are present in the double ring structure. pxl1Δ cells were grown in YES medium at 24°C. Samples were fixed and stained with DAPI, α-cdc4 antibodies, and phalloidin to visualize nuclei, Cdc4p, and F-actin, respectively. (D) Formation of the secondary ring structure in pxl1Δ mutant cells. Wild-type cells expressing rlc1-GFP were grown at 24°C and observed by time-lapse confocal microscopy. Z-stack images were taken at different time points, and 3D reconstruction images with maximal projection are shown panel a. pxl1Δ cells expressing rlc1-GFP were treated and observed as above (b and c). Arrow indicates the appearance of the secondary ring structure. (E) Formation of metaphase actomyosin ring is normal in pxl1Δ mutant cells. nda3-KM31 and nda3-KM311 pxl1Δ cells expressing rlc1-GFP were grown at 32°C to log-phase and shifted to 19°C for 6.5 h to arrest cells at metaphase. Images were taken by fluorescence microscopy. (F) Quantification of the number of metaphase actomyosin rings in wild-type and pxl1Δ mutant cells. Cells from E were quantified for the presence of actomyosin rings (n = 200). Time is indicated in min::sec. BF, bright field. Scale bar, 5 μm.

Rate of actomyosin ring constriction slows down in pxl1Δ mutant cells. (A) Percentage of cells with Rlc1p ring is higher in pxl1Δ mutant. Wild-type and pxl1Δ mutant cells expressing rlc1-GFP were grown at 24°C and observed by fluorescence microscopy. The number of cells with Rlc1p ring was quantified (n = 500). (B) Rate of ring constriction is constant in both wild-type and pxl1Δ mutant cells. Wild-type cells expressing rlc1-GFP were grown at 24°C and observed by time-lapse confocal microscopy. Z-stack images were taken at different time points. 3D-reconstruction images are shown at a 45° angle. The diameter of the Rlc1p ring was measured and plotted over time. Two examples from wild-type and pxl1Δ mutant cells are shown, and the rate of ring constriction was calculated using the stated formula. d_n represents the diameter of the actomyosin ring at the time t_n. (C) Rate of ring constriction is slower in pxl1Δ mutant cells. Wild-type and pxl1Δ mutant cells expressing rlc1-GFP were treated and observed as in B, and the calculation of the rate of ring constriction was based on the formula in B (n = 10).

Pxl1p localizes to the division site in cells undergoing cytokinesis. (A) Localization of Pxl1p in wild-type cells. Cells expressing pxl1-GFP were grown in YES medium at 24°C and visualized by confocal microscopy. (B) Pxl1p-GFP forms a ring structure. Z-stack images were taken for two individual cells from A and processed for 3D reconstruction, and the obtained images are shown at different angles. (C) The Pxlp ring constricts during cytokinesis. Cells expressing pxl1-GFP sid4-GFP were grown on a YES agar pad at 24°C and visualized by time-lapse confocal microscopy, and Z-stack images were taken at different time points. 3D reconstruction images with maximal projection are shown. (D) Colocalization of Pxl1p and Cdc4p at late stages of cytokinesis. Cells expressing pxl1-GFP were grown in YES medium at 24°C, and samples were fixed and stained with DAPI, α-GFP antibodies, and α-cdc4 antibodies to visualize nuclei, Pxl1p and Cdc4p, respectively. Time is indicated in min::sec. BF, bright field. Scale bar, 5 μm.

pxl1Δ cells have cell separation defects and display genetic interaction with clp1Δ. (A) Cell separation defects in pxl1Δ mutant. Wild-type and pxl1Δ mutant cells were grown in YES medium at 24°C and observed by light microscopy. (B) Quantification of the percentage of cells with septa as well as the number(s) of septa in wild-type and pxl1Δ mutant cells (n = 500). (C) Examples of abnormal cells in the pxl1Δ mutant. pxl1Δ cells were grown at 24°C, and samples were fixed and stained with aniline blue and DAPI to visualize septa and nuclei, respectively. (D) pxl1Δ clp1Δ double mutant cells accumulate multiple nuclei in the same compartment. Cells of indicated genotypes were grown in YES medium (or YES plus 1.2 M sorbitol medium) at 24°C. Samples were fixed and stained with aniline blue and DAPI to visualize septa and nuclei, respectively. (E) ace2Δ clp1Δ double mutant cells do not accumulate multiple nuclei in the same compartment. Cells of indicated genotypes were treated as D. Scale bar, 5 μm.

Assessment of localization of various cytokinetic proteins to the secondary rings in pxl1Δ mutant cells. (A) Mid1p localizes to the nucleus during the primary ring constriction in pxl1Δ mutant cells. pxl1Δ cells expressing rlc1-mcherry mid1-4GFP were grown at 24°C to log-phase. Samples were taken and observed by fluorescence microscopy. (B) Cdc7p was distributed randomly with respect to the primary rings in pxl1Δ mutant cells. pxl1Δ cells expressing rlc1-GFP cdc7-GFP were grown at 24°C to log phase. Samples were taken and observed by confocal laser microscopy. Z-stack images were taken and 3D reconstruction images with maximal projection are shown. (C) Spn1p localizes to the primary ring in pxl1Δ mutant cells. pxl1Δ cells expressing rlc1-mcherry spn1-GFP were treated as A. (D) Agn1p localizes to the primary ring in pxl1Δ mutant cells. pxl1Δ cells expressing rlc1-mcherry agn1-GFP were treated as A. Scale bar, 5 μm.

LIM domains are required for Pxl1p function. (A) pxl1-limΔ cells show a cell separation phenotype as in pxl1Δ. Wild-type, pxl1Δ, and pxl1-limΔ cells were grown at 24°C and observed by light microscopy. (B) LIM domains can rescue the pxl1Δ phenotype. pxl1Δ cells bearing the plasmids pREP81, pREP81-pxl1, pREP81-pxl1-N, and pREP81-pxl1-C were grown at 24°C. Samples were taken and stained with aniline blue to visualize septa. (C) Quantification of the number of septa. Cells from B were quantified for the number of septa (n = 500). (D) Localization of N- and C-terminal parts of Pxl1p. pxl1Δ cells bearing the plasmids pREP81-GFP-pxl1, pREP81-GFP-pxl1-N, and pREP81-GFP-pxl1-C were grown at 24°C and observed by fluorescence microscopy. Images with Calcoflour white staining of the cell wall are shown at the bottom. Arrow indicates the nucleus localization of GFP-Pxl1p-C. Scale bar, 5 μm.