<i>An-vps23</i> and <i>An-vps25</i> are essential when <i>nimA</i> is partially inhibited.

<p>(A and B) At 35°C, a semi-permissive temperature for <i>nimA7</i>, absence of <i>An-vps23</i> or <i>An-vps25</i> is poorly tolerated in combination with the temperature sensitive <i>nimA7</i> allele, resulting in the absence of any colony growth even after 96 hours of incubation in the double mutant <i>nimA7</i> + <i>ΔAn-vps23</i> and <i>nimA7</i> + <i>ΔAn-vps25</i> strains. Black arrowheads mark Δ<i>An-vps23</i> and Δ<i>An-vps25</i> colonies that exhibit the presence of suppressor mutations at 96 hours but not at 72 hours. (C) The inset in (A) has been magnified to show the emergence of colonies carrying suppressor mutations from one colony and not from the adjacent one. Strains: WT  =  R153, <i>nimA7</i>  =  MG44, <i>ΔAn-vps23</i>  =  MGH21, <i>ΔAn-vps25</i>  =  MGH26, <i>nimA7</i> + <i>ΔAn-vps23</i>  =  MGH17, <i>nimA7</i> + <i>ΔAn-vps25</i>  =  MGH14.</p

Ectopic NIMA-GFP locates to the plus ends of microtubules in an EB1 dependent manner.

<p>(A) When <i>alcA</i> driven NIMA expression is induced by germinating cells in the presence of glycerol, ectopic NIMA-GFP comets locate to microtubule plus ends defined by their colocalizaton with EB1-CR (arrows, strain: MG385). (B) The location of ectopic NIMA-GFP to microtubule plus ends is abolished in strains deleted for EB1. Strains: WT  =  MG409, ΔEB1  =  MG410. (C) Ectopic NIMA-GFP comets colocalize with EB1 through time (strain: MG385). See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004248#pgen.1004248.s008" target="_blank">Movie 3 in supporting material</a>. (D) Expression of ectopic NIMA-GFP promotes EB1 comet movement to and then around cell tips (tracked by the yellow arrowhead, strain: MG385). See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004248#pgen.1004248.s009" target="_blank">Movie 4 in supporting material</a>. (E) Kymographs showing only parallel traces of EB1 movement towards the cell tip when NIMA is not expressed compared to conditions when NIMA is expressed which causes EB1 comets to move both towards and away from the cell tip (strain: MG385). Bars, 5 μm.</p

<i>A. nidulans</i> orthologues of genes that are synthetically lethal with Δ<i>kin3</i> in <i>S. cerevisiae</i>.

a<p>Designation given by the <i>A. nidulans</i> Genome database.</p>b<p>Refers to the growth phenotype of the deletion strains on complete media (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004248#pgen-1004248-g001" target="_blank">Figure 1</a>, Supplementary <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004248#pgen.1004248.s001" target="_blank">Figure S1</a>, and data not shown).</p>c<p>Values obtained by BLASTp using the <i>S. cerevisiae</i> protein to query the <i>A. nidulans</i> Genome database at AspGD.</p>d<p>This study.</p

Multiple ESCRT pathway genes become essential when NIMA function is partially reduced.

<p>Colonies were grown from spores of the indicated genotypes for 96-permissive temperature for <i>nimA7</i> (35°C), the double mutants lacking either <i>An-vps28</i>, <i>An-vps36</i>, <i>An-vps24</i>, or An-<i>vps4</i> do not show any colony formation indicating a synthetic lethal interaction between <i>nimA7</i> and these deletion alleles. WT  =  R153, <i>nimA7</i>  =  MG71, <i>ΔAn-vps28</i>  =  MGH53, <i>nimA7</i> + <i>ΔAn-vps28</i>  =  MGH55, <i>ΔAn-vps24</i>  =  MGH49, <i>nimA7</i> + <i>ΔAn-vps24</i>  =  MGH51, <i>ΔAn-vps36</i>  =  MGH57, <i>nimA7</i> +<i>ΔAn-vps36</i>  =  MGH59, <i>ΔAn-vps4</i>  =  MGH45, <i>nimA7</i> + <i>ΔAn-vps4</i>  =  MGH47.</p

The NIMA kinase localizes to growing cell tips.

<p>(A) NIMA-GFP exhibits a dome-shaped localization at the tips of growing hyphal cells (strain: KF45). See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004248#pgen.1004248.s006" target="_blank">Movie 1 in supporting material</a>. (B) The average percentage signal intensity for NIMA-GFP (KF45) or GFP-S-Tag (CDS1068) along a ROI drawn across the cell tip. The background fluorescence signal from untagged cells was subtracted and the signal was normalized to the signal inside the cell (taking the first value on the X axis as 100%). (C) Increase in NIMA-GFP signal seen at 70% of the cell tips after depolymerization of actin by latrunculin B (strain: KF45). (D) Quantitation of the percentage of NIMA-GFP signal in cells that exhibited an increase in NIMA at the cell tips after treatment with latrunculin B (n = 19). Normalization of GFP signal was done as in (B). Bars, 5 μm.</p

Induction of ectopic NIMA results in defects in tip cell morphology.

<p>(A) Hyphae of wildtype cells or (B) cells carrying <i>alcA</i> driven expression of full length NIMA (<i>alcA</i>::NIMA-GFP, strain: CDS683) or (C) the C-terminal regulatory domain (<i>alcA</i>::NIMA-RegD-GFP, strain: CDS131) were grown under non-inducing conditions and expression of the respective NIMA constructs was induced by the addition of threonine. Representative images of cells after 6 hours of induction are shown. Breakdown of apical dominance and cell tip swelling in NIMA-GFP expressing cells are indicated by blue and green arrows respectively. (D) Quantitation of tip growth defects. PI  =  Pre-induction, I  =  <i>alcA</i> induced for 2, 4 or 6 hours as indicated. Bar, 5 μm.</p

Partial inhibition of NIMA results in alteration of microtubule dynamics and EB1 behavior.

<p>(A) Kymograph showing that in WT cells (strain: MG395), EB1-CR typically moves unidirectionally towards the cell tip. (B) When NIMA function is partially impaired (strain: MG397), the movement of EB1-CR is altered to give instances of EB1 comets moving away from the cell tip. (D) Microtubule tracks in cells with partial NIMA function bend and push against the cell tip wall, a phenotype not typical of WT cells (C). See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004248#pgen.1004248.s010" target="_blank">Movie 5</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004248#pgen.1004248.s011" target="_blank">6</a> in supporting material. (E) Model illustrating the effect of partial inhibition of NIMA on microtubule dynamics. Bars, 5 μm.</p

Ectopic NIMA-GFP displays a distinctive microtubule-dependent localization.

<p>(A) Ectopic NIMA-GFP (Strain: CDS683) forms dynamic cytoplasmic comets, some of which move to and around the cell tip (yellow arrowheads) when <i>alcA</i> driven NIMA expression is induced by germinating cells in glycerol. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004248#pgen.1004248.s007" target="_blank">Movie 2 in supporting material</a>. (B) Ectopic NIMA-GFP comets often exhibit bi-directional movement near the cell tip as revealed in the kymograph. (C) Upon addition of the microtubule poison benomyl, ectopic NIMA-GFP disperses from the comets throughout the cytoplasm. Bars, 5 μm.</p