CDC25B-induced centrin 2 foci have the capacity to function as microtubule organising centres.

<p>Microtubule re-growth assay on asynchronous U2OS-HA-CDC25B cells, which were incubated in the absence of tetracycline for 5 days, and then either fixed (Control), or incubated on ice for 30 minutes, and then fixed (Cold Shock) or incubated in warm media for 1 minute (Re-growth) to allow re-assembly of microtubules. Cells were co-stained for centrin 2 (red), and α-tubulin (green), and DNA co-stained with DAPI (blue). Magnifications of the centrosomes/ centrin foci (indicated by white boxes) are shown on the right hand side. Bar = 10 µm.</p

CDC25B protects centrosomal centrin from proteasome-mediated degradation.

<p>HeLa cells were either mock-transfected or treated with scrambled control or CDC25B siRNA duplexes for 44 hours followed by 4 hour treatment with 20µM MG132. (A) Examples of cells treated (+) or not (-) with MG132, and stained for CDC25B (CDC25B-S230P, red) and centrin 2 (green). Bar = 10 µm. (B) Percentage of cells treated with control or CDC25B siRNA duplexes in which centrin 2 and CDC25B-S230P (CDC25B siRNA only) were not detectable or substantially diminished at the centrosomes. Bars represent means of at least 200 cells counted from three independent experiments +/- SD. (C) Western blots of siRNA-depleted cells treated with (+) or without (-) MG132 and immunoblotted for total CDC25B, CDC25B-S230P, γ-tubulin and α-tubulin.</p

CDC25B-induced centrin 2 foci accumulate other PCM components.

<p>Asynchronous U2OS-HA-CDC25B cells were incubated in the absence of tetracycline for 48-72 hours and then fixed and co-stained for centrin 2 (red), DAPI (blue) and either Ninein (A), Pericentrin (B), γ-tubulin (C), Nedd1 (D), PCM-1 (E) or polyglutamylated (polyglut.) tubulin (F), as indicated. Images were taken using an Appotome microscope, except for C, lower panel, which is a maximum intensity projection of a z-stack taken using a laser scanning confocal microscope, to enhance the detection of γ-tubulin at distant foci. Bar = 10 µm.</p

CDK2 inhibition blocks CDC25B-induced centrin 2 foci formation.

<p>(A) Asynchronous U2OS-HA-CDC25B cells were incubated in the presence (tet +) or absence (tet -) of tetracycline with either 9µM CDK1 inhibitor RO-3306 [26] or 1µM CDK2 inhibitor PHA533533 [25] for 24 hours. (A) Graph represents the percentages of cells with excess centrin 2 foci. Data shown are the means of at least 200 cells counted from three independent experiments +/- SD. (B) Western blot analyses on soluble protein extracts from cells treated as in (A). Membranes were probed for HA-tagged CDC25B, centrin 2, inactive CDK (CDK-pY15P), total CDK2-cyclin E and its substrate nucleophosmin (NPM), which is phosphorylated by CDK2-cyclin E on T199. α-tubulin was used as loading control.</p

Image quality improvement in depth.

<p>(a) Single planes images of nuclei and mitotic figures at various depths within the MCTS resolved with AO or not (w/o AO). Images are acquired by using the same excitation intensity at 595 nm and same exposure time (300 ms). Scale Bar 5 µm. (b) The corresponding <i>IG_ROI</i> Ratio image. (c) The corresponding intensity plots along the line indicated.</p

HA-CDC25B overexpression induces the formation of extra-numerary centrin 2 “foci”.

<p>Asynchronous U2OS-HA-CDC25B cells were incubated in the continued presence (+ tet) or absence (- tet) of tetracycline as in Figure 1, fixed 48 hours later and co-stained for HA-CDC25B (red), centrin 2 (green) and DAPI (blue). (A) Examples of centrin 2 foci in cells expressing (- tet, arrows) HA-CDC25B in comparison to control cells (+ tet). Bar = 10 µm. (B) Histogram plot showing the percentage of cells harbouring excess centrin 2 “foci” following 48 hours HA-CDC25B expression. Bars represent means of at least 200 cells counted from three independent experiments +/- SD.</p

HA-CDC25B overexpression increases the level of centrin 2 at the centrosome.

<p>Asynchronous U2OS-HA-CDC25B cells were incubated in the continued presence (+tet) or absence (-tet) of tetracycline, fixed 24 hours later and co-stained for HA-CDC25B (red), centrin 2 (green) and DAPI (blue). (A) Examples of centrin fluorescence levels at the centrosome of cells expressing (-tet, arrow) or not (+tet) HA-CDC25B. Bar = 10 µm. (B, C) Analyses of centrin 2 fluorescence levels in cells expressing (-tet) or not (+tet) HA-CDC25B. (B) Cells were co-stained for centrin 2 (green) and Nek2 (red) as a marker for centrosomes. Insets are magnifications of centrosome regions (indicated by white boxes). Circles represent centrosome boundaries used to quantitate fluorescence intensities of centrin 2 and Nek2 at the centrosomes (C), using MetaMorph imaging software. (C) Box plots of the ratio of centrin 2 fluorescence intensity / Nek2 fluorescence intensity at the centrosomes of cells expressing or not HA-CDC25B. G1/S phase cells, identified as cells with a single centrosome (B, +tet) or duplicating centrosomes that were not yet separated (B, -tet), or G2 phase cells, identified as cells with 2 centrosomes that were separated, were all imaged on the same day using identical microscope and software settings. Box plots were prepared using the Prism software package and statistical analyses performed using unpaired student’s t-tests (**** P<0.0001; ns not significant, n=76-170 centrosomes). Data shown are from 1 experiment and are representative of those obtained from four independent experiments. (D) Western blot analyses of HA-CDC25B and centrin 2 levels in cells expressing or not HA-CDC25B. α-tubulin was used as a loading control.</p

CDC25B-induced centrin foci are dependent on the activity of Mps1 kinase.

<p>Asynchronous U2OS-HA-CDC25B cells were transfected with pEGFP or pEGFP-tagged wild-type (Mps1 WT), kinase-dead (Mps1 KD) or non-degradable (Mps1 Δ12/13) forms of Mps1 and incubated in the continued presence (+ tet) or absence (- tet) of tetracycline, fixed 48 hours later and co-stained for centrin 2 (red) and DAPI (blue). (A) Examples of transfected cells cultured in the absence of tetracycline. Insets are magnifications of centrosome regions (indicated by white boxes) of the transfected cell in each case. Bar = 10 µm. (B) Histogram plot showing the percentage of cells harbouring excess centrin 2 “foci” following 48 hours transfection and HA-CDC25B expression. Bars represent means of at least 200 cells counted from four independent experiments +/- SD. (C) Model for the role of CDC25B in regulating centrin 2 protein stability through its activity towards CDK2. </p

Scheme of the waoSPIM setup.

<p>In our experimental setup (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035795#pone.0035795.s005" target="_blank">Fig. S1</a>), a cylindrical lens focuses the light to a horizontal line (light sheet) that is imaged into the back focal plane of an illumination objective (10× NA 0.25) (yellow path). The sample is positioned in the light sheet inside a physiological chamber filled with aqueous medium. The emitted light is collected (red path) by an immersion objective (20× NA 0.5) fitted to the physiological chamber. AOTF, acousto-optic tunable filter; T1–T3, telescopes; M1–M5, mirrors; CL, cylindrical lens; DM, deformable mirror; D1 and D2, dichroic mirrors; HSWF, Hartmann–Shack wavefront sensor; B, Lens system for DM-HSWF pupil conjugation; TL, tube lens; CCD 1 and CCD2, coupled charged devices.</p

Signal and contrast improvement.

<p>Maximum projection of a 3D stack of 100 images (z spacing 1 µm) of a large MCTS expressing a fluorescent nuclear protein, H2B–HcRed, without (w/o AO) and with AO (AO). Scale bar, 50 µm. The asterisk marks the bead used as the point source emitter located at a depth of 150 µm. Both images were acquired by using the same excitation intensity at 595 nm and a 300 ms exposure time. (b) Magnified views of the bead. Scale bar, 5 µm. (c) Intensity profiles along the lines 1 and 2 indicated in (a). (d) FWHM (µm), RMS and Strelh ratio values for bead images in (b). (e) <i>IG_ROI</i> mapping images calculated from images in (a) and <i>IG_ROI</i> Ratio image calculated as</p><p></p>where is mapping images calculated from images obtained with AO and is mapping images calculated from images obtained without AO.<p></p