The above results show that Cam1 also localizes to the SPB during meiosis (Fig

The above results show that Cam1 also localizes to the SPB during meiosis (Fig. essential role in forespore membrane formation by stably maintaining Spo15, and thus Spo2 and Spo13, at the SPB in meiotic cells. Calmodulin is GSK690693 usually a calcium-binding protein that is ubiquitously distributed and highly conserved among eukaryotes. It contains four EF-hand Ca2+-binding sites, which are required for function. Calmodulin controls a variety of cellular processes mostly related to calcium signaling. When bound to calcium, calmodulin undergoes a characteristic conformational change to an active configuration. Activated calmodulin then binds effector proteins and transmits the signal to downstream regulators. Yeast is usually a genetically tractable model organism suitable for studying the biological function of calmodulin, using conditional-lethal calmodulin mutants (4). In the budding yeast gene (5). Cmd1p is usually implicated in a wide variety of cellular processes, including initiation of budding and mitotic spindle formation (24). The fission yeast has a common calmodulin encoded by the on SPB modification and FSM formation. MATERIALS AND METHODS Yeast strains, media, and culture conditions. The strains used in this study are listed in Table 1. The complete medium YEA (yeast extract agar) was used for growth. Malt extract agar (MEA) medium and GSK690693 synthetic sporulation media (MM-N and SSA) were used for mating and sporulation. These media have been described in Moreno et al. (17). cells were produced and sporulated at 28C. Table 1. strains used in this study [pREP1(ade6)]This studyAI3002 (FY19940)[pREP1(ade6)cam1+]This studyAI3003 (FY19941)[pREP1(ade6)cam1-22]This studyAI3004 (FY19942)[pREP1(ade6)]This studyAI3005 (FY19943)[pREP1(ade6)cam1+]This studyAI3006 (FY19944)[pREP1(ade6)cam1-22,117]This studyAI3007 (FY19945)[pREP1(ade6)]This studyAI3008 (FY19946)[pREP1(ade6)cam1+]This studyAI3009 (FY19947)[pREP1(ade6)cam1-EF2]This studyAI3010 (FY19948)[pREP1(ade6)]This studyAI3011 (FY19949)[pREP1(ade6)cam1+]This studyAI3012 (FY19950)[pREP1(ade6)cam1-EF3]This studyMFP7 (FY19951)strains constructed in this study have been deposited with the YGRC/NBRP under the accession numbers shown here. b? means that gene is usually integrated at gene promoter16pIL2in pREP1(ade6)This studypREP1(ade6)gfp-cam1-22,117in pREP1(ade6)This studypREP1(ade6)gfp-cam1-EF2in pREP1(ade6)This studypREP1(ade6)gfp-cam1-EF3in pREP1(ade6)This studypBR322(spo13-gfp)in pBR(leu1)This studypBR(cam1-22,117)in pBR(leu1)This studypBR(cam1-EF1)in pBR(leu1)This studypBR(cam1-EF2)in pBR(leu1)This studypBR(cam1-EF3)in pBR(leu1)This studypBR(cam1-EF4)in pBR(leu1)This study Open in a separate Icam2 windows The locus (19). The promoter and a coding region for GFP-Cam1 was linearized by restricting it with BamHI near the center of the strain (YN24 GSK690693 and YN104). The resulting integrant strains, AI248 and AI210, were cultured in MM-N sporulation medium. At intervals, portions of the culture were sampled, and crude cell extracts were prepared as described by Masai et al. (15). Polypeptides were resolved by SDS-polyacrylamide gel electrophoresis GSK690693 on 7.5% gels (for Spo15) or 10% gels (for GFP-Cam1) and then transferred onto polyvinylidene difluoride membranes (Millipore, Bedford, MA). Membranes were probed with anti-Spo15 antibody (23) at a 1:100 dilution or with mouse anti-GFP antibody (Roche, Basel, Switzerland) at a 1:200 dilution. Blots were also probed with anti–tubulin antibody, TAT1 (32), to normalize protein loading. Immunoreactive bands were visualized by chemiluminescence (NEN Life Sciences, Boston, MA) using horseradish peroxidase-conjugated goat anti-mouse IgG (GE Healthcare, Little Chalfont, Buckinghamshire, United Kingdom). Fluorescence microscopy. The SPB was visualized by immunofluorescence microscopy. Cells were fixed with glutaraldehyde and paraformaldehyde (8). The SPB was visualized by using rabbit anti-Sad1 antibody (a gift from O. Niwa, Kazusa DNA Research Institute) and Alexa Fluor 546- or 488-conjugated secondary antibody (Molecular Probes, Eugene, OR). Sad1 is usually a major SPB component required for bipolar spindle formation (7). The nuclear chromatin region was stained with 4,6-diamidino-2-phenylindole (DAPI) at 1 g/ml or Hoechst 33342 at 1 g/ml (Nakarai Tesque, Kyoto, Japan). Stained cells were observed under a fluorescence microscope (model BX50; Olympus, Tokyo, Japan). To assess SPB modification quantitatively, stained cells were observed under a fluorescence microscope (model IX-71; Olympus), and SPB width was measured by AQUACOSMOS software (Hamamatsu Photonics, Shizuoka, Japan). The FSM was observed by GFP-Psy1 fluorescence imaging (20). Psy1 is usually a plasma membrane-resident t-SNARE protein that is homologous to budding yeast Sso1 and Sso2 (1) and human syntaxin-1 (2). The fusion gene was integrated at the locus on chromosome III (14). Electron microscopy. Samples for electron microscopy were prepared as described previously (33), and sections were viewed on an electron microscope (H-7600; Hitachi, Tokyo, Japan) at 100 kV. Mutants of Cam1 Ca2+-binding sites. Cam1 contains four EF-hand Ca2+-binding sites (18). Each of the four EF-hand motifs was mutated by replacing a conserved glutamic acid residue with valine. The wild-type mutagenesis kit was used (QuikChange; Stratagene, La Jolla, CA). The.