Figure 1.
Loss of the cytokinesis protein Fic1 causes defects in S. pombe growth polarity.
(A) Live-cell images of calcofluor-stained wild-type and fic1Δ cells. Birth scars remain unstained and appear as dark bands across cells. Arrowheads indicate monopolar cells, i.e. cells that have only grown at one end, with birth scars abutting cell ends. (B) Quantification of (A), with three trials per genotype and n>300 for each trial. Data are presented as mean ± SEM for each category. (C) Quantification of septated cells in (A) and (B), with three trials per genotype and n>200 for each trial. Data are presented as mean ± SEM for each category. (D) Live-cell GFP images of crn1-GFP and fic1Δ crn1-GFP cells. (E) Quantification of (D), with three trials per genotype and n>200 for each trial. Data are presented as mean ± SEM for each category. (F) Live-cell GFP (in green) and RFP (in magenta) merged images of rgf1-GFP sid4-RFP and fic1Δ rgf1-GFP sid4-RFP cells. (G) Quantification of (F), with three trials per genotype and n>200 for each trial. Data are presented as mean ± SEM for each category (Bars = 5 µm).
Figure 2.
fic1Δ cells fail at new end growth independently of known cell cycle controls on NETO.
(A–B) Live-cell DIC movies of wild-type or fic1Δ cells. Solid arrows denote old end growth, whereas dashed arrows indicate new end growth. Birth scars are marked by asterisks. Time points are noted. (C) Quantification of growth patterns for cells imaged in (A) and (B), with sample size (n) provided. (D) Quantification of growth patterns for tea1Δ and tea1Δ fic1Δ cells, with sample size (n) provided. (E) Live-cell DIC movie of a tea1Δ fic1Δ cell that gives rise to a T-shaped daughter cell. The solid arrow denotes old end growth, whereas the dashed arrow indicates non-tip growth. Birth scars are marked by asterisks. Time points are noted. (F) Quantification of T-shaped cells in tea1Δ and tea1Δ fic1Δ strains grown at 25°C, with three trials per genotype and n>300 for each trial. Data are presented as mean ± SEM for each genotype. (G) Live-cell images of calcofluor-stained tea1Δ and tea1Δ fic1Δ cells grown at 25°C. Arrows indicate T-shaped cells. (H) Quantification of times from septum splitting to initiation of new end growth in cells that undergo NETO prior to the next septation in (A–C). Data are presented in box-and-whisker plots showing the median (line in the box), 25th–75th percentiles (box), and 5th–95th percentiles (whiskers) for each genotype. (I) Live-cell images of calcofluor-stained cdc25-22 and fic1Δ cdc25-22 cells that had been arrested in G2. Arrowheads indicate monopolar cells. (J) Quantification of (I), with three trials per genotype and n>300 for each trial. Data are presented as mean ± SEM for each category. (K) Quantification of cell lengths at cell division, with n>200 for each genotype. Data are presented as box-and-whisker plots showing the median (line in the box), 25th–75th percentiles (box), and 5th–95th percentiles (whiskers) for each genotype. The dashed line represents the minimum length required for NETO [5] (Bars = 5 µm).
Figure 3.
Fic1's C terminus is necessary and sufficient for Fic1's polarity function at the division site.
(A) Live-cell bright field (BF) and GFP images of fic1-GFP cells. Localization to cell tips (*), the cytokinetic ring (>), and the division site (#) are marked. (B) Schematic of Fic1 protein domain organization. Residues and fragments of interest are marked. (C) Live-cell BF, GFP (in green), mCherry (mCh) (in magenta), and GFP/mCherry merged images of fic1-GFP sid4-GFP cdc15-mCherry, fic1N-GFP sid4-GFP cdc15-mCherry, and fic1C-GFP sid4-GFP cdc15-mCherry cells. (D) Live-cell images of calcofluor-stained fic1N and fic1C cells. Arrowheads indicate monopolar cells. (E) Quantification of (D), with three trials per genotype and n>300 for each trial. Data are presented as mean ± SEM for each category. (F) Quantification of septated cells in (D) and (E), with three trials per genotype and n>200 for each trial. Data are presented as mean ± SEM for each category. (G) Live-cell BF and GFP images of interphase cell tips of fic1-GFP and fic1C-GFP cells (Bars = 5 µm, except for 3G where Bar = 1 µm).
Figure 4.
Fic1 participates in protein–protein interactions at the CR that guide subsequent growth polarity.
(A) Live-cell images of calcofluor-stained cdc15ΔSH3, fic1-P257A, imp2Δ, and cyk3Δ cells. Arrowheads indicate monopolar cells. (B) Quantification of (A), with three trials per genotype and n>300 for each trial. Data are presented as mean ± SEM for each category. (C) Quantification of septated cells in (A) and (B), with three trials per genotype and n>300 for each trial. Data are presented as mean ± SEM for each category. (D) Anti-Cdc15 and anti-FLAG immunoprecipitates from cells of the indicated genotypes were blotted with anti-Cdc15 and/or anti-FLAG antibodies. cdc25-22 cells and nda3-KM311 cells were arrested in G2 and prometaphase, respectively, prior to pelleting and lysis. (E) Anti-FLAG and anti-V5 immunoprecipitates from prometaphase-arrested cells of the indicated genotypes were blotted with anti-FLAG and/or anti-V5 antibodies. (F) Live-cell GFP movie of a cyk3-GFP sid4-GFP cell, with images every 3 min (Bars = 5 µm).
Figure 5.
Loss of Fic1 impairs CR disassembly and leads to persistence of division site factors.
(A) Fixed-cell DIC and DAPI/methyl blue images of asynchronous and G2-arrested cells of the indicated genotypes. Arrowheads indicate cells that are still joined following ingression. (B) Quantification of (A), with four trials per genotype and n>300 for each trial. Percentages are presented as mean ± SEM. (C) Live-cell GFP movies of rlc1-GFP sid4-GFP and fic1Δ rlc1-GFP sid4-GFP cells. Images were acquired every 2 min, and representative images are given for 10 min intervals. (D) Quantification of times from spindle pole body (SPB) separation to the completion of CR constriction in (C). n>20 for each genotype. Data are presented in box-and-whisker plots showing the median (line in the box), 25th–75th percentiles (box), and 5th–95th percentiles (whiskers) for each genotype. (E) Quantification of times from septum closure to disappearance of the CR at the division site for GFP-cps1 rlc1-mCherry3 and fic1Δ GFP-cps1 rlc1-mCherry3 cells. n>30 for each genotype. Data are presented in box-and-whisker plots showing the median (line in the box), 25th–75th percentiles (box), and 5th–95th percentiles (whiskers) for each genotype. (F) Live-cell GFP (colored green) and mCherry (mCh) (colored magenta) movies of GFP-cps1 rlc1-mCherry3 and fic1Δ GFP-cps1 rlc1-mCherry3 cells, with time intervals indicated and GFP/mCherry images merged. White arrows in GFP images mark the septa's leading edges. The time point with only one arrow drawn marks septum closure. In the mCh images, “C” marks the point of CR closure, and arrowheads denote CR remnants persisting after this point. (G) Fixed-cell images of actin stained with Alexa Fluor 488 Phalloidin. Single z planes as well as maximum projections of multiple z planes are given. Red arrows indicate division planes, whereas yellow arrows indicate unusual actin masses lining the division plane (Bars = 5 µm).
Figure 6.
Late cytokinesis mutants phenocopy the new end-growth polarity defects of fic1Δ cells.
(A) Quantification of growth patterns for cells of the indicated genotypes. Sample size (n) is provided for each genotype. The percentage of the most prevalent faulty growth pattern is boxed for each genotype. (B) Live-cell DIC movies of cells of the indicated genotypes scored in (A). The most prevalent faulty growth pattern for each genotype is pictured. Solid arrows denote old end growth, whereas dashed arrows indicate new end growth. Birth scars are marked by asterisks. Time points are noted. (C) Quantification of polarity phenotypes of calcofluor-stained cells of the indicated genotypes, with three trials per genotype and n>300 for each trial. Data are presented as mean ± SEM for each category. All cells were grown at 25°C unless otherwise noted. (D) Quantification of septated cells in (C), with three trials per genotype and n>200 for each trial. Data are presented as mean ± SEM for each category. A dashed gray line marks 20% on the y-axis (Bar = 5 µm).
Figure 7.
An endogenous Tea1-For3 fusion protein is functional but impinges on the cell division machinery.
(A) Schematic of Tea1, For3, and Tea1-For3 protein domains and organization. (B) Anti-V5 immunoprecipitates from asynchronous tea1-V53, for3-V53, and tea1-for3-V53 cells were blotted with anti-V5 antibodies. Arrows indicate full-length proteins. Lysates were blotted with anti-Cdc2 as a loading control. (C) Fixed-cell DAPI/methyl blue images of stained wild-type, tea1Δ, for3Δ, tea1Δ for3Δ, and tea1-for3 cells. (D) Quantification of phenotypes of cells in (C), with three trials per genotype and n>300 for each trial. Data are presented as mean ± SEM for each category. (E) Quantification of cell lengths at cell division, with n>200 for each genotype. Data are presented as box-and-whisker plots showing the median (line in the box), 25th–75th percentiles (box), and 5th–95th percentiles (whiskers) for each genotype (Bar = 5 µm).
Figure 8.
Constitutive NETO signaling does not fully rescue cytokinesis-based growth polarity defects.
(A) Quantification of growth patterns for wild-type and tea1-for3 cells. Sample size (n) is provided for each genotype. (B) Quantification of times from septum splitting to initiation of tip growth at previous division sites for tea1-for3 cells. Times were carried into the next cell cycle where applicable. Data are presented in box-and-whisker plots showing the median (line in the box), 25th–75th percentiles (box), and 5th–95th percentiles (whiskers). n>200. (C) Data for tea1-for3 cells in (B) grouped according to the amount of time needed for the mother cell to complete septation. Data are presented in box-and-whisker plots showing the median (line in the box), 25th–75th percentiles (box), and 5th–95th percentiles (whiskers) for each category. (D) Live-cell DIC movies of tea1-for3 cells with different times needed to complete septation. The time of septum splitting of the mother cell is marked as point zero. The initiation of tip growth at previous division sites is denoted by yellow arrows. Tip growth at these sites was also scored for cells that did not initiate such growth until the subsequent cell cycle. (E) Live-cell images of calcofluor-stained tea1-for3 and tea1-for3 fic1Δ cells. Arrowheads indicate monopolar cells. (F) Quantification of (E), with three trials per genotype and n>300 for each trial. Data are presented as mean ± SEM for each category. (G) Quantification of septated cells in (E) and (F), with three trials per genotype and n>200 for each trial. Data are presented as mean ± SEM for each category (Bars = 5 µm).
Figure 9.
Cytokinesis mutants with growth polarity defects exhibit enhanced invasiveness.
(A) Invasive growth assays for strains of the indicated genotypes on 2% agar. Cells were spotted on rich medium and incubated for 20 days at 29°C (top panel). Colonies were then rinsed under a stream of water and rubbed off (bottom panel). (B) Quantification of pseudohyphae in (A), with n≥3 for each genotype. Data are presented as mean ± SEM for each genotype. (C) Image of fic1Δ pseudohyphae in 2% agar, with enlarged images on the right. (D) Invasive growth assays for tea1-for3 and tea1-for3 fic1Δ strains on 2% agar. Cells were spotted on rich medium and incubated for 20 days at 29°C (top panel). Colonies were then rinsed under a stream of water and rubbed off (bottom panel). (E) Quantification of pseudohyphae in (D), with n≥3 for each genotype. Data are presented as mean ± SEM for each genotype. (F) Colony growth of strains of the indicated genotypes on rich medium containing 2% (top panel) or 0.3% agar (middle panel). Cells were spotted and incubated for 12 days at 29°C. Schematics of colony growth on 0.3% agar are also given (bottom panel), with white areas representing growth on the agar surface and black areas representing growth into the agar (Bars = 5 µm).
Figure 10.
Model of Fic1's involvement in cytokinesis and the establishment of bipolar growth in S.
pombe. During cytokinesis, Fic1 serves as a scaffold for SH3 proteins, including Cdc15, at the cytokinetic ring. In the absence of Fic1, its interactions, or a parallel pathway, the completion of cell division is perturbed, and the cell division machinery persists at the previous division site. Failure to robustly complete cytokinesis impedes new end growth, even if NETO signaling is prematurely activated. Cytokinesis-based constraints on new end growth polarity aid in the transition into invasive fungal growth.