Roles of Pom2 in sporulation.

<p>(A) Localization of Pom2-3YFP during mating (the second column), cell fusion (third), and sporulation (fourth). The first column is wild-type control. Pom2-3YFP (JW1606×JW2166) and wild-type strains (JW81×JW729) with opposite mating types were grown in YE5S liquid medium for 12 h and then crossed on SPA5S plate at 25°C. Pairs of fluorescence and DIC images were taken 24 h after the crosses. (B and C) Defective sporulation in <i>pom2Δ</i> cells. Wild-type strains JW81 and JW729 (left) or <i>pom2Δ</i> strains JW210 and JW213 (right) were crossed on an SPA5S plate at 25°C and examined after 48 h. (B) Asci imaged using DIC. (C) Quantification of spore numbers in each ascus (n>500 asci for each cross). Bars, 5 µm.</p

The Pom2 kinase domain complements the Pom1 kinase domain.

<p>(A) Sequence alignment of the kinase domains of <i>S. pombe</i> DYRK family members Pom1 and Pom2. Protein sequences were aligned using the Vector NTI program. Identical and similar (D/E, I/L/V, K/R, N/Q, S/T) amino acids are shaded in yellow and green, respectively. (B) Differential-interference-contrast (DIC) images and (C) quantification of septum positioning defects in <i>pom1</i> mutants. Cells of wild type (strain JW81), <i>pom1Δ</i> (JB109), <i>pom1-pom2K</i> (JW1802), and <i>pom1-ΔK</i> (JW1745) were grown at 25°C. (C) >200 cells for each strain were quantified as described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028000#pone.0028000-Bhler1" target="_blank">[6]</a>. Septum position was scored by dividing cells into five equal sections (see examples in B). Septum angle was relative to the cell long axis. Septa with angles of 80° to 90° were counted as orthogonal, and septa with smaller angles as tilted. Septa with very aberrant appearance or placed longitudinally were counted as irregular. (D) Localization of Pom1-mECitrine (strain JW1836) and Pom1-pom2K-mECitrine (JW1837). Bars, 5 µm.</p

Pom2 localizes to mitochondria throughout the cell cycle and to the division site during cytokinesis.

<p>(A) Pom2 localizes to mitochondria. Cells expressing Pom2-tdTomato (strain JW1607) stained with mitochondrial dye DiOC₆(3) (top) or unstained (middle) and DiOC₆(3) stained wild-type cells (bottom, strain JW81) were imaged using exactly the same settings. (B) Pom2 co-localizes with Rlc1 in the contractile ring. Cells expressing Pom2-3GFP (bottom, strain JW1605), Rlc1-tdTomato (middle, JW1341), or both (top, JW1714) were imaged using exactly the same settings. Pom2 rings are marked by arrowheads. (C and D) Timing of Pom2 localization to the division site during cytokinesis. Temperature-sensitive <i>cdc25-22 pom2-3YFP sad1-mEGFP</i> cells (JW2646) were arrested at late G2 by growing at 36°C for 4 h before release to 25°C at time zero. Samples were taken every ten minutes, imaged with seven Z-sections spaced at 0.8 µm and quantified. (C) Maximum intensity projection of whole cells with Pom2-3YFP ring signals marked by arrowheads. (D) Graph of the time course of the fraction of cells with Pom2-3YFP ring signals (left Y-axis) and the mean spindle pole bodies (SPB) separation (right Y-axis). (E) Mildly overexpressed Pom2 localizes to the contractile ring. Wild type 972 and <i>41nmt1-GFP-pom2</i> (JW40) strains were grown in YE5S (repressing condition) at 25°C for 33 h before imaging. Images shown are maximum-intensity projections of 28 Z-sections with 0.2-µm spacing. Arrowheads indicate the contractile rings. Bars, 5 µm.</p

List of <i>S. pombe</i> strains used in this study.

<p>List of <i>S. pombe</i> strains used in this study.</p

Structure and Reactivity of Chromium(VI) Alkylidenes

Bis­(arylimido)­Cr­(VI) dialkyls lacking β-hydrogen decompose by α-hydrogen abstraction and, upon trapping with triphenylphosphine, yield isolable alkylidene complexes. Two such complexes, namely (ArN)₂CrCHR­(PPh₃) (R = ^tBu, SiMe₃), have been structurally characterized. The coordinatively unsaturated alkylidene intermediates are highly reactive; they effect CH activation of saturated hydrocarbons and they react with olefins to produce metallacyclobutanes

Meiosis and FSM formation defects in <i>pom2Δ</i> cells.

<p>(A) Representative DIC and fluorescence micrographs showing defects in meiosis and FSM formation in <i>pom2Δ</i> cells. <i>pom2⁺</i> (JW2619 and JW2620) or <i>pom2Δ</i> strains (JW2617 and JW2618) were crossed on SPA5S plates to induce meiosis and sporulation for 11 h before imaging. In merged images, nuclear membrane (Cut11-mRFP), FSM (GFP-Psy1), and nuclear DNA are in red, green, and blue, respectively. Representative images are shown. Images a-g are examples of asci with other than 4 nuclei, h-k are asci with 4 nuclei. (B) Quantification of meiosis and FSM formation defects in <i>pom2⁺</i> or <i>pom2Δ</i> cells as shown in (A). Asci with 4 well-separated DNA masses and a normal sphere-shaped FSM enclosing each DNA mass were classified as no defect; asci with other than 4 DNA masses but normal FSM morphology with each FSM enclosing one DNA mass, or 4 DNA masses with abnormal nuclei segregation pattern but normal FSM morphology were classified as meiosis defect only; spore membrane formation defect only includes asci with 4 DNA masses but FSM defects: either one or more DNA masses were not enclosed by FSM or two DNA masses were enclosed within one FSM; other asci were classified as defects in both meiosis and FSM formation. Bar, 3 µm.</p

Cytokinesis defects in <i>pom2</i> deletion and overexpression cells.

<p>(A and B) DIC and fluorescence images of wild type (strain JW81), <i>pom2Δ</i> (JW213), and <i>3nmt1-pom2</i> (JW216) cells. Cells were grown in EMM5S liquid medium for 30 h at 25°C. (B) Cells were stained with Hoechst to visualize nuclei and Calcofluor to visualize septa. (C) Quantification the septated cells (left) and the numbers of nuclei (right) in strains used in (B) (n>200 for each strain). (D and E) Effects of Pom2 overexpression on contractile-ring formation and constriction. Time course of contractile-ring assembly (D) and constriction (E) during cytokinesis in wild type (JW1568) and <i>41nmt1-pom2</i> (JW1626) cells. Cells were induced in EMM5S medium for 24 h. Seven Z-sections spaced at 0.8 µm were taken at each time point. The time point of SPB separation (D) or SPB reaching cell tips (E) is defined as time zero, respectively. Bars, 5 µm.</p

Abnormal mitochondrial morphology in <i>pom2</i> mutants and microtubule independence of Pom2 localization to mitochondria.

<p>Cells of <i>3nmt1-pom2</i> (JW216) were grown in repressing condition and then shifted to inducing condition of EMM5S for 24 h at 25°C. (A) Morphology of mitochondria in wild-type (JW81), <i>pom2Δ</i> (JW213), and <i>3nmt1-pom2</i> (JW216) cells stained with DiOC₆(3). (B) Quantification of mitochondrial morphology shown in (A) (n>300 cells for each strain). The morphologies were categorized into four classes: tubular (one to four branched tubules extended from one end of the cell to the other), moderately fused (net-like mitochondria), tightly fused (mitochondria accumulated at one side of the cell along the long axis), and aggregated (mitochondrion formed one or several big aggregates at or close to the cell middle). (C) Microtubule independence of Pom2 localization to mitochondria. DiOC₆(3) stained wild-type cells (middle, strain JW81), cells expressing tubulin GFP-Atb2 (left, JW1804), or Pom2-3YFP (right, JW1606) were treated with equal amount of microtubule depolymerizing drug TBZ (lower panel) or DMSO (upper panel). (D) Pom2 does not obviously affect microtubule cytoskeleton. Cells expressing GFP-Atb2 in wild type (left, strain JW1804) and in <i>pom2Δ</i> (right, strain JW1955) were imaged using the same settings. Bars, 5 µm.</p

Structure and Reactivity of Chromium(VI) Alkylidenes

Bis­(arylimido)­Cr­(VI) dialkyls lacking β-hydrogen decompose by α-hydrogen abstraction and, upon trapping with triphenylphosphine, yield isolable alkylidene complexes. Two such complexes, namely (ArN)₂CrCHR­(PPh₃) (R = ^tBu, SiMe₃), have been structurally characterized. The coordinatively unsaturated alkylidene intermediates are highly reactive; they effect CH activation of saturated hydrocarbons and they react with olefins to produce metallacyclobutanes