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1 HEREDITAS (Beijing) , 35(8): ISSN 研究报告 DOI: /SP.J 裂殖酵母 Cnb1 参与胞质分裂过程范洁琼, 邓小龙, 冯碧薇, 王继峰, 余垚, 吕红,, 丝 / 苏氨酸特异性钙调磷酸酶 (Calcineurin, CN) 是一种在真核生物中广泛存在的蛋白, 是参与转录调控的重要分子裂殖酵母中的 CN 是由催化亚基 Ppb1 和调节亚基 Cnb1 组成的异源二聚体文章报道了裂殖酵母中 cnb1 + 的缺失引起细胞生长速度缓慢, 产生多隔膜现象, 胞质分裂受阻滞胞质分裂过程中, Cnb1 与 Ppb1 组成 CN 复合物, 与收缩环在分裂平面上共定位, 并与收缩环一起收缩 cnb1δ 菌株的隔膜成熟过程存在缺陷, 微管出现纵穿隔膜的现象上述结果说明 Cnb1 可能参与隔膜的成熟过程此外, 还检测了 cnb1δ 菌株中胞裂蛋白的信号胞裂蛋白包括 Spn1 Spn2 Spn3 和 Spn4, 它们是引导隔膜降解的重要分子结果显示, 在 cnb1δ 菌株中, 80% 左右的细胞在隔膜处缺失 Spn2 和 Spn3 的信号, 20% 左右的细胞缺失 Spn1 和 Spn4 的信号由于胞裂蛋白的蛋白表达量在 cnb1δ 中没有降低, 因此胞裂蛋白信号的消失不是转录缺陷引起的, 这暗示 Cnb1 可能采用了不依赖转录的方式来调控胞裂蛋白环的稳定性以上结果提示, Cnb1 可能通过影响隔膜的成熟及胞裂蛋白环的稳定性参与调节裂殖酵母的胞质分裂过程裂殖酵母 ; Cnb1; 胞质分裂 ; 胞裂蛋白 ; 收缩环 Cnb1 involved in cytokinesis in Schizosaccharomyces pombe FAN Jie-Qiong, DENG Xiao-Long, FENG Bi-Wei, WANG Ji-Feng, YU Yao, LV Hong State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai , China Abstract: Serine/Threonine-specific calcineurin (CN) is highly conserved in eukaryotes, which plays an important role in transcriptional regulation. In Schizosaccharomyces pombe, CN exists as a heterodimer composed by catalytic subunit Ppb1 and regulatory subunit Cnb1. Deletion of cnb1 + reduced the growth rate of cells, and caused a chained phenotype, and had delay in cytokinesis. In cytokinesis, Cnb1 could form CN complex with Ppb1 and could colocalize and constrict with the contractile ring at division plane. Tubulin could cross the septum in cnb1δ strain, suggesting that the septum is not fully matured. These results suggest Cnb1 might be involved in maturation of septum. The signals of septins in cnb1δ strain were also analyzed. Septins include Spn1, Spn2, Spn3, and Spn4. Septins help to guide hydrolytic enzymes for septum degradation. Eighty percent of cnb1δ cells lacked the signals of Spn2 or Spn3 at septum, and twenty percent of cnb1δ cells lacked the signals of Spn1 or Spn4 at septum. The reduction of the septin signals was not due to impaired transcription of septins, 收稿日期 : ; 修回日期 : 基金项目 : (973 ) ( 2009CB825601) ( ) 作者简介 :,, @fudan.edu.cn 通讯作者 :,,, honglv@fudan.edu.cn 网络出版时间 : :55:16 URL:

2 8 : Cnb since the protein levels of septins in the cnb1δ cells were not decreased. These results imply that Cnb1 might regulate the stability of septin ring in a transcription-independent manner. In general, our study showed that Cnb1 contributes to the maturation of septum and the stability of septin ring and is important in the cytokinesis. Keywords: Schizosaccharomyces pombe; Cnb1; cytokinesis; septin; contractile ring, (Cytokinesis), ;, [1] (Schizosaccharomyces pombe), Mid1, Mid1,,, [2,3], Mid1,, (Contractile ring) Rng2 Cdc15 Cdc12 Cdc3 [4~6], (Septin) Mid2 (Septin ring) [7, 8] Spn1 Spn2 Spn3 Spn4 [9] (Septation initiation network, SIN) [1],,, (Septum),, Eng1 Agn1,, [10] / (Calcineurin, CN) [11, 12], [13] CN, T NFAT CDRE Crz1p [14] Ppb1 Prz1 [15], [15~21] S. pombe, cnb1 + CN, ppb1 + CN Cnb1 CN, Ppb1 Ca 2+, (Calmodulin, CaM), CN, CN [22], Cnb1 CN, cnb1 +, CN [23], S. pombe, CN Ppb1 Ppb1 (Ppb1ΔC) SIN, CN SIN, [24] ppb1δ (PI(4)P5K), PI(4)P5K, Ppb1 PI(4)P5K [25] Cnb1 CN, Cnb1, Cnb1, Cnb1 [26], Cnb1 CN, FKBP12- FK506 [27] Cnb1, cnb1 +,,, Cnb1, cnb1δ,, Cnb1,, cnb1δ 80% Spn2 Spn3, 20% Spn1 Spn4 cnb1δ,, Cnb1,

3 1032 HEREDITAS (Beijing) Cnb1, E. coli DH5α CHGL FAML FAMU 3 pclonehyg1 [28], (Hyg R ) leu1 + ura4 + [30] 同源敲除质粒及基因敲除菌株构建 CHGL-Cnb1 Cnb1- KO-Up-F Cnb1-KO-Up-R cnb1 + (Up ), KpnⅠ/Bgl ; Cnb1-KO-Dw-F Cnb1-KO-Dw-R cnb1 + (Dw ), Sac /BamH Up Dw, CHGL, cnb1 + CHGL-Cnb1 1, 2 Cnb1-GFP cnb1 + S. pombe [28] Kpn /BamH CHGL-Cnb1, ( 1) S. pombe, [28] cnb1δ 稀释实验 YES S. pombe OD OD , 表 1 本研究所用裂殖酵母菌株 F274 wt h + ade6-m210 leu1-32 ura4-d18 JLP1590 wt h - ade6-m210 leu1-32 ura4-d18 FJQ01 cnb1δ h + cnb1::hyg R ade6-m210 leu1-32 ura4-d18 FJQ02 Cnb1-GFP h + cnb1::cnb1-gfp-hyg R ade6-m210 leu1-32 ura4-d18 FJQ03 Myo2-mCherry h + myo2::myo2-mcherry-ura4 + ade6-m210 leu1-32 ura4-d18 FJQ04 Cnb1-GFP h + cnb1::cnb1-gfp-hyg R cdc4::cdc4-mcherry-ura4 + ade6-m210 leu1-32 Cdc4-mCherry ura4-d18 FJQ05 cnb1δ Myo2-mCherry h + cnb1::hyg R myo2::myo2-mcherry-ura4 + ade6-m210 leu1-32 ura4-d18 FJQ07 Atb2-GFP Sad1-mCherry h - atb2::gfp-atb2-kan R sad1::sad1-mcherry-ura4 + leu1-32 ura4-d18 FJQ08 cnb1δ Atb2-GFP h - cnb1::hyg R atb2::gfp-atb2-kan R sad1::sad1-mcherry-ura4 + leu1-32 ura4- Sad1-mCherry D18 FJQ09 cnb1δ Spn1-GFP h - spn1-gfp::kan R cnb1::hyg R ura4-d18 FJQ10 cnb1δ Spn2-GFP h90 spn2::spn2-gfp-ha-kan R cnb1::hyg R ade6-216 leu1-32 lys1-131 ura4-d18 FJQ11 cnb1δ Spn3-GFP h90 spn3::spn3-gfp-ha-kan R cnb1::hyg R ade6-216 leu1-32 lys1-131 ura4-d18 FJQ12 cnb1δ Spn4-GFP h90 spn4::spn4-gfp-ha-kan R cnb1::hyg R ade6-216 leu1-32 lys1-131 ura4-d18 FJQ13 Spn1-GFP h - spn1-gfp::kan R ura4-d18 [7] FJQ14 Spn2-GFP h90 spn2::spn2-gfp-ha-kan R ade6-216 leu1-32 lys1-131 ura4-d18 [29] FJQ15 Spn3-GFP h90 spn3::spn3-gfp-ha-kan R ade6-216 leu1-32 lys1-131 ura4-d18 [29] FJQ16 Spn4-GFP h90 spn4::spn4-gfp-ha-kan R ade6-216 leu1-32 lys1-131 ura4-d18 [29] FJQ17 Mid2-GFP h - mid2::gfp-mid2-kan R ade6-m210 leu1-32 ura4-d18 FJQ18 cnb1δ Mid2-GFP h - cnb1::hyg R mid2::gfp-mid2-kan R ade6-m210 leu1-32 ura4-d18 FJQ19 Cnb1-GFP h + cnb1::cnb1-gfp-hyg R ppb1::ppb1-mcherry-leu1 + ade6-m210 leu1-32 Ppb1-mCherry ura4-d18 FJQ20 Ppb1-GFP h + ppb1::ppb1-gfp-hyg R ade6-m210 leu1-32 ura4-d18 FJQ21 ppb1δ Spn1-GFP h - spn1-gfp::kan R ppb1::hyg R ura4-d18 FJQ22 ppb1δ Spn2-GFP h90 spn2::spn2-gfp-ha-kan R ppb1::hyg R ade6-216 leu1-32 lys1-131 ura4-d18 FJQ23 ppb1δ Spn3-GFP h90 spn3::spn3-gfp-ha-kan R ppb1::hyg R ade6-216 leu1-32 lys1-131 ura4-d18 FJQ24 ppb1δ Spn4-GFP h90 spn4::spn4-gfp-ha-kan R ppb1::hyg R ade6-216 leu1-32 lys1-131 ura4-d18

4 8 : Cnb 表 2 构建 CHGL-Cnb1 质粒所用引物 Cnb1-KO-Up-F Cnb1-KO-Up-R Cnb1-KO-Dw-F Cnb1-KO-Dw-R (5 3 ) CCCGGTACCATTCTCAGATTTTGCTTCCTTCAT CCCAGATCTGAGAGGGAAACACAATAATATGAAAT CCCGAGCTCTGAGCATACTTAAAATTAAAACTG CCCTCTAGAGGATCCCAAATGAAAGTTCTTAATCTAATG, (DIC) YES, ml YES, OD DNA BLM, 0.5 mu/ml 32 4 h, Calcofluor, X51 ( ) YES, 32, r/min, 2 μl Calcofluor, X51 ( ) 蛋白质免疫印迹检测 (Western Blot) Western blot [31] Anti-GFP (ABMart ), Anti-Actin (ABMart ) 激光共聚焦延时拍摄图 1 利用同源重组的方法在裂殖酵母中敲除 cnb1 + 基因 5 5 μl YES 0.75 mu/ml BLM 10 μmol/l CPT 20 μg/ml TBZ 7.5 mmol/l HU 0.01% MMS 2.5 mmol/l H 2 O ~3 d, 37 5 μl YES 37 2~3 d, 生长曲线及菌体形态观察 F274(wt) FJQ01(cnb1Δ) YES, ml YES, OD , 4 h OD 600, X51 ( ) YES, 32, OD ~0.6, 32 3~4 h 30~50 μl YES,, 2~3 μl,, (, LSM700), cnb1 + cnb1δ, cnb1 + ( 2A), cnb1δ ( 2B) (0~8 h),

5 1034 HEREDITAS (Beijing) 图 2 cnb1 + 基因的缺失对裂殖酵母生长及细胞表型的影响 A OD 600 F274(wt) FJQ01(cnb1Δ) ( 3 ); B cnb1δ (DIC) ; C 4 h 8 h 12 h 16 h 20 h, F274(wt) FJQ01(cnb1Δ) >400 cnb1δ 40%, 18% (16~20 h), cnb1δ 20%, ( 2C),, cnb1δ cnb cnb1 + DNA cnb1 + DNA,, cnb1δ, DNA ( 3A), 2.5 mmol/l H 2 O 2 (37 ) Cnb1 cnb1δ 0.5 mu/ml BLM, ( 3B), BLM,, 10% cnb1δ ( 3B), Cnb1 2.3 Cnb1 Cnb1, 图 3 cnb1 + 的缺失对环境胁迫及 DNA 损伤条件下的裂殖酵母细胞生长和形态的影响 A 7.5 mmol/l HU 0.75 mu/ml BLM 20 μg/ml TBZ 0.01% MMS 10 μmol/l CPT 2.5 mmol/l H 2 O 2 YES, 32 2 d 37, 32 ; B 0.5 mu/ml BLM F274(wt) FJQ01(cnb1Δ) 4 h,, Calcofluor

6 8 : Cnb Cnb1 1, GFP cnb1 + C, Cnb1-GFP, FJQ02(Cnb1-GFP), Cnb1, Cnb1 ( 4A) Cnb1, 內 Cnb1-GFP Cdc4-mCherry (FJQ04), Cdc4, Cnb1 Cdc4 ( 4B), Cnb1, CN Ppb1, Ppb1 ( 4C), Ppb1 Cnb1 ( 4D), Cnb1 Ppb1 CN Cnb1, cnb1δ Myo2 cnb1 + Myo2 Myo2 ( 4E) Cnb1, Cnb1, Cnb1 2.4 Cnb1, SIN 图 4 Cnb1 及 Ppb1 与收缩环组分在分裂平面上共定位 A FJQ02(Cnb1-GFP), 0~50 min Cnb1 ; B FJQ04(Cnb1-GFP Cdc4-mCherry), Cnb1 Cdc4 ; C FJQ20(Ppb1-GFP), Ppb1 ; D FJQ19(Cnb1-GFP Ppb1-mCherry), Ppb1 Cnb1 ; E FJQ03(Myo2-mCherry) FJQ05(cnb1Δ Myo2-mCherry), cnb1 + Myo2

7 1036 HEREDITAS (Beijing) [32],, β-1,3-, α-1,3- β(1,3)- β(1,6)- [33],, [24], Atb2,, cnb1 +, Atb2 ( 5), ( 5) Cnb1,, Cnb1 2.5 Cnb1 [34],, cnb1 +,, Cnb1 Cnb1, cnb1δ 4,, ( 6A 6B) cnb1δ, 80% Spn2 Spn3, 20% Spn1 Spn4 ( 6A 6B) Cnb1, ppb1δ 4, ppb1 + cnb1 +, ( 6B), Western blot cnb1δ,, cnb1δ 4 ( 6C) cnb1δ,, cnb1 +, Mid2 Mid2, Mid2,, [34, 35], cnb1 +, Mid2 ( 6A) Mid2 cnb1 + 3 CN, Cnb1 Ppp3r1 75%,, Cnb1,, cnb1 +,, Cnb1 Ppb1,, Cnb1 Ppb1 CN, cnb1 +, 图 5 cnb1 + 的缺失影响隔膜的成熟 FJQ07(Atb-GFP Sad1-mCherry) FJQ08(cnb1Δ Atb-GFP Sad1-mCherry), Atb2 Sad1 Sad1

8 8 : Cnb 图 6 cnb1 + 的缺失对裂殖酵母胞裂蛋白的定位及其表达的影响 A FJQ09(Spn1-GFP) FJQ10(Spn2-GFP) FJQ11(Spn3-GFP) FJQ12(Spn4-GFP) FJQ17(Mid2-GFP) FJQ13(cnb1Δ Spn1-GFP) FJQ14(cnb1Δ Spn2-GFP) FJQ15(cnb1Δ Spn3-GFP) FJQ16(cnb1Δ Spn4-GFP) FJQ18(cnb1Δ Mid2-GFP) Spn1 Spn2 Spn3 Spn4 Mid2 ; B cnb1 + ppb1 +, Spn1 Spn2 Spn3 Spn4 ( >100 ); C Western blot FJQ09(Spn1-GFP) FJQ10(Spn2-GFP) FJQ11(Spn3-GFP) FJQ12(Spn4-GFP) FJQ13(cnb1Δ Spn1-GFP) FJQ14(cnb1Δ Spn2-GFP) FJQ15(cnb1Δ Spn3-GFP) FJQ16(cnb1Δ Spn4-GFP), GFP, (Actin) Cnb1 cnb1 +,,, ppb1 + [24] Cnb1, CN cnb1δ 4, 80% Spn2 Spn3 Mid2 cnb1δ, cnb1δ Cnb1, ppb1 +, ppb1 + cnb1 + ( 6B) Cnb1 Ppb1 CN, ppb1 + cnb1 +, Cnb1 Ppb1 CN,, Cnb1 CN,

9 1038 HEREDITAS (Beijing) CN,, PI3K, p85 p110, p85 [36] CN 参考文献 (References): [1] Goya A, Takaine M, Simanis V, Nakano K. Dividing the spoils of growth and the cell cycle: the fission yeast as a model for the study of cytokinesis. Cytoskeleton, 2011, 68(2): DOI [2] Almonacid M, Paoletti A. Mechanisms controlling division-plane positioning. Semin Cell Dev Biol, 2010, 21(9): DOI [3] Guertin DA, Trautmann S, Mccollum D. Cytokinesis in eukaryotes. Microbiol Mol Biol Rev, 2002, 66(2): DOI [4] Rajagopalan S, Wachtler V, Balasubramanian M. Cytokinesis in fission yeast: a story of rings, rafts and walls. Trends Genet, 2003, 19(7): DOI [5] Feierbach B, Chang F. Cytokinesis and the contractile ring in fission yeast. Curr Opin Microbiol, 2001, 4(6): DOI [6] Bathe M, Chang F. Cytokinesis and the contractile ring in fission yeast: towards a systems-level understanding. Trends Microbiol, 2010, 18(1): DOI [7] Wu JQ, Ye YF, Wang N, Pollard TD, Pringle JR. Cooperation between the septins and the actomyosin ring and role of a cell-integrity pathway during cell division in fission yeast. Genetics, 2010, 186(3): DOI [8] Kinoshita M. The septins. Genome Biol, 2003, 4(11): 236. DOI [9] An HM, Morrell JL, Jennings JL, Link AJ, Gould KL. Requirements of fission yeast septins for complex formation, localization, and function. Mol Biol Cell, 2004, 15(12): DOI [10] Martin-Cuadrado AB, Morrell JL, Konomi M, An HB, Petit C, Osumi M, Balasubramanian M, Gould KL, Del RF, de Aldana CR. Role of septins and the exocyst complex in the function of hydrolytic enzymes responsible for fission yeast cell separation. Mol Biol Cell, 2005, 16(10): DOI [11] Yoshida T, Toda T, Yanagida M. A calcineurin-like gene ppb1 + in fission yeast: mutant defects in cytokinesis, cell polarity, mating and spindle pole body positioning. J Cell Sci, 1994, 107(Pt7): DOI [12] Li HM, Rao A, Hogan PG. Interaction of calcineurin with substrates and targeting proteins. Trends Cell Biol, 2011, 21(2): DOI [13] Klee CB, Draetta GF, Hubbard MJ. Calcineurin. Adv Enzymol Relat Areas Mol Biol, 1988, 61: DOI [14] Stathopoulos AM, Cyert MS. Calcineurin acts through the CRZ1/TCN1-encoded transcription factor to regulate gene expression in yeast. Genes Dev, 1997, 11(24): DOI [15] Hirayama S, Sugiura R, Lu YB, Maeda T, Kawagishi K, Yokoyama M, Tohda H, Giga-Hama Y, Shuntoh H, Kuno T. Zinc finger protein Prz1 regulates Ca 2+ but not Cl - homeostasis in fission yeast. Identification of distinct branches of calcineurin signaling pathway in fission yeast. J Biol Chem, 2003, 278(20): DOI [16] Aramburu J, Heitman J, Crabtree GR. Calcineurin: a central controller of signalling in eukaryotes. EMBO Rep, 2004, 5(4): DOI [17] Hogan PG, Chen L, Nardone J, Rao A. Transcriptional regulation by calcium, calcineurin, and NFAT. Genes Dev, 2003, 17(18): DOI [18] Ruiz A, Yenush L, Arino J. Regulation of ENA1 Na + -ATPase gene expression by the Ppz1 protein phosphatase is mediated by the calcineurin pathway. Eukaryot Cell, 2003, 2(5): DOI [19] Stathopoulos-Gerontides A, Guo JJ, Cyert MS. Yeast calcineurin regulates nuclear localization of the Crz1p transcription factor through dephosphorylation. Genes Dev, 1999, 13(7): DOI [20] Viladevall L, Serrano R, Ruiz A, Domenech G, Giraldo J, Barceló A, Ariño J. Characterization of the calcium-mediated response to alkaline stress in Saccharomyces cerevisiae. J Biol Chem, 2004, 279(42): DOI [21] Yoshimoto H, Saltsman K, Gasch AP, Li HX, Ogawa N, Botstein D, Brown PO, Cyert MS. Genome-wide analysis of gene expression regulated by the calcineurin/crz1p signaling pathway in Saccharomyces cerevisiae. J Biol Chem, 2002, 277(34): DOI [22] Perrino BA, Ng LY, Soderling TR. Calcium regulation of calcineurin phosphatase activity by its B subunit and calmodulin. Role of the autoinhibitory domain. J Biol Chem, 1995, 270(1): DOI [23] Sio SO, Suehiro T, Sugiura R, Takeuchi M, Mukai H, Kuno T. The role of the regulatory subunit of fission yeast calcineurin for in vivo activity and its relevance to FK506 sensitivity. J Biol Chem, 2005, 280(13): DOI [24] Lu YB, Sugiura R, Yada T, Cheng H, Sio SO, Shuntoh H, Kuno T. Calcineurin is implicated in the regulation of the

10 8 : Cnb septation initiation network in fission yeast. Genes Cells, 2002, 7(10): DOI [25] Zhang YJ, Sugiura R, Lu YB, Asami M, Maeda T, Itoh T, Takenawa T, Shuntoh H, Kuno T. Phosphatidylinositol 4-phosphate 5-kinase Its3 and calcineurin Ppb1 coordinately regulate cytokinesis in fission yeast. J Biol Chem, 2000, 275(45): DOI [26] Matsuyama A, Arai R, Yashiroda Y, Shirai A, Kamata A, Sekido S, Kobayashi Y, Hashimoto A, Hamamoto M, Hiraoka Y, Horinouchi S, Yoshida M. ORFeome cloning and global analysis of protein localization in the fission yeast Schizosaccharomyces pombe. Nat Biotechnol, 2006, 24(7): DOI [27] Fox DS, Cruz MC, Sia RA, Ke HM, Cox GM, Cardenas ME, Heitman J. Calcineurin regulatory subunit is essential for virulence and mediates interactions with FKBP12- FK506 in Cryptococcus neoformans. Mol Microbiol, 2001, 39(4): DOI [28] Gregan J, Rabitsch PK, Rumpf C, Novatchkova M, Schleiffer A, Nasmyth K. High-throughput knockout screen in fission yeast. Nat Protoc, 2006, 1(5): DOI [29] Hayashi A, Ding DQ, Tsutsumi C, Chikashige Y, Masuda H, Haraguchi T, Hiraoka Y. Localization of gene products using a chromosomally tagged GFP-fusion library in the fission yeast Schizosaccharomyces pombe. Genes Cells, 2009, 14(2): DOI [30] Forsburg SL, Rhind N. Basic methods for fission yeast. Yeast, 2006, 23(3): DOI [31] Chen JQ, Li Y, Pan X, Lei BK, Chang C, Liu ZX, Lu H. The fission yeast inhibitor of growth (ING) protein Png1p functions in response to DNA damage. J Biol Chem, 2010, 285(21): DOI [32] Wolfe BA, Gould KL. Split decisions: coordinating cytokinesis in yeast. Trends Cell Biol, 2005, 15(1): DOI [33] Jin QW, Zhou MA, Bimbo A, Balasubramanian MK, Mccollum D. A role for the septation initiation network in septum assembly revealed by genetic analysis of sid2-250 suppressors. Genetics, 2006, 172(4): DOI [34] Berlin A, Paoletti A, Chang F. Mid2p stabilizes septin rings during cytokinesis in fission yeast. J Cell Biol, 2003, 160(7): DOI [35] Tasto JJ, Morrell JL, Gould KL. An anillin homologue, Mid2p, acts during fission yeast cytokinesis to organize the septin ring and promote cell separation. J Cell Biol, 2003, 160(7): DOI [36] García Z, Silio V, Marqués M, Cortés I, Kumar A, Hernandez C, Checa AI, Serrano A, Carrera AC. A PI3K activity-independent function of p85 regulatory subunit in control of mammalian cytokinesis. EMBO J, 2006, 25(20): DOI

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