PP1-Dependent Formin Bnr1 Dephosphorylation and Delocalization from a Cell Division Site.

Cell cycle ends with cytokinesis that is the physical separation of a cell into two daughter cells. For faithful cytokinesis, cells integrate multiple processes, such as actomyosin ring formation, contraction and plasma membrane closure, into coherent responses. Linear actin assembly by formins is essential for formation and maintenance of actomyosin ring. Although budding yeast's two formins, Bni1 and Bnr1, are known to switch their subcellular localization at the division site prior to cytokinesis, the underlying mechanisms were not completely understood. Here, we provide evidence showing that Bnr1 is dephosphorylated concomitant with its release from the division site. Impaired PP1/Glc7 activity delayed Bnr1 release and dephosphorylation, Bni1 recruitment and actomyosin ring formation at the division site. These results suggest the involvement of Glc7 in this regulation. Further, we identified Ref2 as the PP1 regulatory subunit responsible for this regulation. Taken together, Glc7 and Ref2 may have a role in actomyosin ring formation by modulating the localization of formins during cytokinesis

Glc7 is involved in Bnr1 release and dephosphorylation.

<p>A, <i>glc7-129</i> cells and isogenic control cells expressing Bnr1-13myc from own promoter were synchronized, released and analyzed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146941#pone.0146941.g002" target="_blank">Fig 2A</a>. B, <i>glc7-129</i> cells and isogenic control cells expressing <i>BNI1-3GFP</i> or <i>3GFP-BNR1</i> from own promoters were synchronized, released as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146941#pone.0146941.g002" target="_blank">Fig 2A</a>. Cells were fixed with formaldehyde at 25°C for 5 min, and then GFP signal forming a single ring at the division site was counted under the fluorescent microscopy. (Left) Representative results of three independent experiments were shown. (Right) % cells with single ring Bni1-3GFP signal (Bni1-3GFP) and % cells with 3GFP-Bnr1 signal (3GFP-Bnr1) at 60 min after release were shown. N>200 for each experiment. Means of three independent experiments were shown. Error bars, s.e.m. *; p<0.05 by student’s t-test. C, Cells expressing <i>GLC7-mCherry</i> and <i>3GFP-BNR1</i> from own promoters were observed under the fluorescent microscopy. D, Bnr1-13myc was immunoprecipitated from cell lysate prepared from asynchronous yeast culture using anti-myc antibody. PP1 treatment was performed at 30°C for 30 min in the presence or absence of phosphatase inhibitors Na₃VO₄ and Okadaic Acid (OA).</p

Genetic evidence supporting <i>REF2</i>’s involvement in cytokinesis.

<p>Indicated strains with a plasmid (pRS316-<i>CDC10</i>) were serially diluted (4X), spotted onto FOA (right) or SD-URA (left) plates and incubated at 25°C for 4 days.</p

Bnr1 is dephosphorylated during cytokinesis.

<p>A, in mitosis by nocodazole and released into fresh media. Samples were collected at indicated time points. Cell lysates were prepared and were subjected to western blotting using anti-myc antibody and anti-Tub1 antibody (control). % cells with large buds in the bright-field microscopic images were counted after formaldehyde fixation to monitor cytokinesis. N>200 for each experiment. Means of three independent experiments are shown. Error bars indicate SD of three independent experiments. B, Bnr1-13myc was immunoprecipitated from cell lysate prepared from asynchronous yeast culture using anti-myc antibody. Beads-bound Bnr1-13myc was subjected to CIP treatment at 37°C in the presence or absence of phosphatase inhibitor Na₃VO₄. Samples were subjected to western blotting. Bnr1-13myc signals were detected using anti-myc antibody. C, Cells lacking Bnr1 kinases were synchronized and released as in A, and were subjected to western blotting using anti-myc antibody and anti-Tub1 antibody (control).</p

Live-cell imaging for 3GFP-Bnr1 and Bni1-mCherry.

<p>Cells expressing 3GFP-Bnr1 and Bni1-mCherry from own promoter were cultured to log phase, spotted on SD medium containing 1.2% agarose on glass slides. Fluorescent signals were monitored under the microscope. (A) Mean relative signal intensities of 3GFP-Bnr1 and Bni1-mCherry at the bud neck were shown. N = 11. Error bars indicate SD. (B) Left, a representative kymograph of (A) is shown. Right, a rectangle indicates an area shown in left panels.</p

Ref2 is required for formin switching and actomyosin ring formation.

<p>A, <i>ref2Δ</i> cells and isogenic control cells were synchronized, released and analyzed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146941#pone.0146941.g003" target="_blank">Fig 3</a>. Note that in this and subsequent figures, time points for the observation were not identical between <i>ref2Δ</i> and control cells, due to slow growth phenotype of <i>ref2Δ</i> cells. N>100 for each experiment. Means of three independent experiments were shown. Error bars, s.e.m. B, <i>ref2Δ</i> cells and isogenic control cells were synchronized, released and analyzed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146941#pone.0146941.g003" target="_blank">Fig 3</a>. N>100 for each experiment. Means of three independent experiments were shown. Error bars, s.e.m. C, <i>ref2Δ</i> cells expressing <i>BNI1-3GFP</i> or <i>3GFP-BNR1</i> from own promoters were synchronized, released and analyzed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146941#pone.0146941.g003" target="_blank">Fig 3</a>. N>100 for each experiment. Means of three independent experiments were shown. Error bars, s.e.m. D, <i>ref2Δ</i> cells expressing Bnr1-13myc were synchronized, released and analyzed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146941#pone.0146941.g002" target="_blank">Fig 2A</a>.</p

In silico screening identifies PP1 regulatory subunit Ref2 as a potential cytokinesis regulator.

<p>Saccharomyces cerevisiae morphological database [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146941#pone.0146941.ref021" target="_blank">21</a>] was used for in silico screening. We focused on two parameters, dispersed actin with divided nuclei (A109_C) and directed actin at the bud neck with divided nuclei (A110_C). Median of all mutant strains analyzed in the database were shown as a control value. Deletion mutants for Glc7 regulatory subunits (from <i>afr1Δ</i> to <i>ypi1Δ</i>), formins (<i>bni1Δ</i> and <i>bnr1Δ</i>) septin related kinases (<i>gin4Δ</i> and <i>elm1Δ</i>) and a septin (<i>cdc10Δ</i>) were analyzed. N.A., not assigned.</p

Glc7 is involved in cytokinesis.

<p>A, <i>glc7-129</i> cells and isogenic control cells were synchronized and released at 25°C (permissive temperature) as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146941#pone.0146941.g002" target="_blank">Fig 2</a>. Mitosis and cytokinesis were monitored after formaldehyde fixation by DAPI staining and cell separation, respectively. (Left and center) Representative results of three independent experiments were shown. (Right) % cells after nuclear division (2 nuclei) and % cells after cytokinesis (After Cytokinesis) at 60 min after release were shown. N>150 for each experiment. Means of three independent experiments. Error bars, s.e.m. n.s. (not significant), statistically insignificant by student’s t-test. B, <i>glc7-129</i> cells and isogenic control cells were synchronized and released at 25°C (permissive temperature) as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146941#pone.0146941.g002" target="_blank">Fig 2</a>. Large budded cells with actin ring was counted after formaldehyde fixation and Allexa-phalloidin staining. (Upper left) Representative results of three independent experiments were shown. (Upper right) % cells with actin ring at 60 min after release were shown. N>150 for each experiment. Means of three independent experiments. Error bars, s.e.m. **; p<0.01 by student’s t-test. (Lower) Arrows indicate actin ring (<i>GLC7</i>), arrow heads indicate absence of actin ring (<i>glc7-129</i>). A scale bar, 2μm.</p