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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 董桂書(Kuei-Shu Tung) | |
| dc.contributor.author | Yi-Hsin Chen | en |
| dc.contributor.author | 陳怡欣 | zh_TW |
| dc.date.accessioned | 2021-06-17T04:25:40Z | - |
| dc.date.available | 2023-08-19 | |
| dc.date.copyright | 2018-08-19 | |
| dc.date.issued | 2018 | |
| dc.date.submitted | 2018-08-15 | |
| dc.identifier.citation | Adames, N.R., Oberle, J.R., and Cooper, J.A. (2001). The surveillance mechanism of the spindle position checkpoint in yeast. J. Cell Biol. 153, 159-168.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/70311 | - |
| dc.description.abstract | 我們實驗室在研究Hsp26蛋白於出芽酵母菌孢子形成過程中所扮演的角色時，發現有一個和Hsp26有關的紡錘體檢控點 (Hsp26-dependent spindle checkpoint)，監控第二次減數分裂到孢子形成過程中紡錘體的形態是否適合形成孢子，藉此確保孢子的存活。先前發現Tem1和Ady3這兩個和Hsp26有交互作用的蛋白也參與此調控機制，Tem1蛋白是有絲分裂中mitotic exit network (MEN) pathway的重要成員，扮演類似感應器的角色，而Ady3蛋白則位於原生孢子膜 (prospore membrane) 前端，協助孢子形成。我們推測Tem1蛋白在Hsp26-dependent spindle checkpoint中可能也扮演著類似感應器的角色去偵測第二次減數分裂中紡錘體的形態是否適合形成孢子，並透過Hsp26蛋白來影響Ady3和Ssp1兩個前緣蛋白質 (leading edge protein) 的位置，以調控孢子形成。
為了瞭解其機制，本篇論文利用benomyl干擾紡錘絲的形成，發現不論在野生型酵母菌還是在hsp26突變細胞中，原生孢子膜的形成都不會受到影響，由此結果推論檢控點並不會調控原生孢子膜的生成，而是直接調控Ady3和Ssp1蛋白的定位以控制孢子可否形成。另外觀察到無論是Ady3、Ssp1或是Hsp26蛋白本身的表現並不會受到 benomyl處理影響，顯示其他蛋白質的共同參與此檢控點機制。 之前發現當細胞進行減數分裂時Tem1蛋白會被磷酸化，本實驗也發現此磷酸化並不受benomyl處理影響。綜合所有結果，本篇研究排除一些常見調控方式的可能性，幫助確定未來對此檢控點機制的研究方向。 | zh_TW |
| dc.description.abstract | Previous studies in our laboratory confirmed that there is a novel Hsp26-dependent spindle checkpoint during meiosis. The checkpoint monitors meiotic II spindle formation and regulates spore formation. In addition, two Hsp26-interacting proteins, Tem1 and Ady3, are involved in the checkpoint. Tem1 is an important component in the mitotic exit network (MEN) pathway, it plays a role of sensor for proper spindle positioning in a dividing cell. Ady3 is a component of the leading edge complex (LEP), associates with the growing prospore membrane (PSM) to assist proper spore formation. It was proposed that Tem1 might act as a sensor in the Hsp26-dependent spindle checkpoint to regulate the localization of Ady3 and Ssp1 into LEP.
In order to clarify the mechanism of the checkpoint, I used benomyl to interfere meiotic II spindle and found that PSM formation was not affected in the wild type, neither in the hsp26 mutant, indicating that PSM formation was not a target of the Hsp26-dependent spindle checkpoint. In addition, there was no detectable difference in the expression of Ady3, Ssp1 or Hsp26 after benomyl treatment, suggesting that there is other protein(s) involved in the checkpoint. It has been reported that Tem1 was phosphorylated during meiosis; however, phosphorylation of Tem1 was not affected in responding to spindle defects. In this study, I excluded some possibilities for certain regulatory mechanisms and provided useful clues for studying the mechanism of the Hsp26-dependent spindle checkpoint in the future. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-17T04:25:40Z (GMT). No. of bitstreams: 1 ntu-107-R04b43029-1.pdf: 1823345 bytes, checksum: e498c222da77db07578835acb0e97c4a (MD5) Previous issue date: 2018 | en |
| dc.description.tableofcontents | 中文摘要 ------------------------------- i
ABSTRACT ---------------------------- ii TABLE OF CONTENTS --------------------- iv LIST OF TABLES ---------------------------- vii LIST OF FIGURES --------------------------- ix CHAPTER 1. INTRODUCTION --------------------------- 1 I. Vegetative cell division --------------------- 1 A. The spindle position checkpoint --------------------------- 2 B. Mitosis exit network -------------------------------------------------------------- 2 II. Sporulation --------------------------------------------------------------------------- 4 A. Meiosis ----------------------------------------------------------------------------- 4 B. Spore formation ------------------------------------------------------------------- 5 1. Prospore formation ------------------------------------------------------------- 5 (1) Modification of the spindle pole body ---------------------------------- 6 (2) Prospore membrane formation ------------------------------------------- 7 (3) Leading edge complex ---------------------------------------------------- 8 2. Spore wall synthesis ---------------------------------------------------------- 10 III. Yeast small heat shock proteins ------------------------------------------------- 11 IV. Specific aims ---------------------------------------------------------------------- 14 CHAPTER 2. MATERIALS AND METHODS ----------------------------------- 15 I. Culture conditions ------------------------------------------------------------------- 15 II. DNA preparation and transformation -------------------------------------------- 16 III. Plasmids constructions -------------------------------------------------------------- 17 IV. Yeast strains constructions ------------------------------------------------------- 23 V. Benomyl treatment ----------------------------------------------------------------- 29 VI. Protein extraction and Western blot analysis ---------------------------------- 29 VII. Cytology analyses ---------------------------------------------------------------- 31 A. Immunofluorescence with yeast cells ---------------------------------------- 31 B. Image analysis ------------------------------------------------------------------- 32 VIII. Yeast two-hybrid assay --------------------------------------------------------- 33 CHAPTER 3. RESULTS -------------------------------------------------------------- 34 I. Hsp26-interacting proteins -------------------------------------------------------- 34 A. The interaction of Hsp26 with Ady3 and Tem1 ----------------------------- 34 B. Tem1 had no interaction with Ady3 or Ssp1 --------------------------------- 35 II. Analysis of Hsp26 expression ---------------------------------------------------- 35 A. Epitope tagging of Hsp26 ------------------------------------------------------ 35 B. Complementation test of HSP26-3xmyc ------------------------------------- 36 C. Expression of Hsp26 during sporulation ------------------------------------- 37 D. There was no detectable modification of Hsp26 after benomyl treatment --- 38 III. Regulation of Ady3 and Ssp1 by the Hsp26-dependent spindle checkpoint -- 39 A. The expression of Ady3 and Ssp1 after benomyl treatment -------------------- 39 B. Constructions of HSP26 ADY3 and HSP26 SSP1 double tagged strains ---- 40 C. Benomyl trial of HSP26-3xmyc 3xHA-ADY3 and HSP26-3xmyc 3xHA-SSP1------------------------------- 41 IV. Analyses of the prospore membrane protein Spo20 ------------------------- 42 A. Epitope tagging of Spo20 ------------------------------------------------------ 42 B. 3xmyc-Spo20 and 6xmyc-Spo20 were detectable through Western blot analysis ------------------------------------------------------------------------------------------- 43 C. Benomyl trial of the SPO20 tagged strains ---------------------------------- 44 D. The Hsp26-dependent spindle checkpoint did not regulate PSM formation------------------------------------------------------------------------------------------- 45 V. Tem1 expression during sporulation ------------------------------------------- 46 A. pCLB2-TEM1-3xHA was shut down in meiosis ---------------------------- 46 B. TEM1-3xHA from its own promoter ------------------------------------------ 47 C. Phosphorylation of Tem1 did not respond to the benomyl treatment ---- 48 D. Stability of Tem1 is not dependent on Hsp26 ------------------------------- 49 CHAPTER 4. DISCUSSIONS ------------------------------------------------------------- 50 I. The Hsp26-dependent spindle checkpoint does not regulate the expression of Hsp26, Ady3 and Ssp1 ------------------------------------------------------------------ 50 II. The Hsp26-dependent spindle checkpoint might respond to spindle defects during PSM growth ---------------------------------------------------------------------- 51 III. The function of Tem1 in meiosis is subtle ------------------------------------------ 51 REFERENCES ------------------------------------------------------------------------------- 53 | |
| dc.language.iso | en | |
| dc.subject | 原生孢子膜 | zh_TW |
| dc.subject | 減數分裂 | zh_TW |
| dc.subject | 紡錘絲檢控點 | zh_TW |
| dc.subject | Hsp26蛋白 | zh_TW |
| dc.subject | Hsp26 | en |
| dc.subject | spindle checkpoint | en |
| dc.subject | prospore membrane | en |
| dc.subject | meiosis | en |
| dc.title | 酵母菌產生孢子過程中Hsp26相關紡錘體檢控點的調控機制 | zh_TW |
| dc.title | The regulatory mechanism of the Hsp26-dependent spindle checkpoint in yeast sporulation | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 106-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 王廷方,蔡皇龍 | |
| dc.subject.keyword | Hsp26蛋白,紡錘絲檢控點,原生孢子膜,減數分裂, | zh_TW |
| dc.subject.keyword | Hsp26,spindle checkpoint,prospore membrane,meiosis, | en |
| dc.relation.page | 98 | |
| dc.identifier.doi | 10.6342/NTU201803216 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2018-08-15 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 分子與細胞生物學研究所 | zh_TW |
| 顯示於系所單位： | 分子與細胞生物學研究所 | |
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