指導教授：吳益群臺灣大學：分子與細胞生物學研究所陳羿吟Chen, Yi-YinYi-YinChen2014-11-252018-07-062014-11-252018-07-062014http://ntur.lib.ntu.edu.tw//handle/246246/261089型態發生(morphogenesis)是多細胞生物個體發育出完整外觀樣貌的重要步驟。在線蟲(Caenorhabditis elegans)中，表皮組織跟肌肉組織的發育為調控形態發生的主要環節。在先前的遺傳實驗中，我們找到一個klp-11(tp8)的突變株，會在身體表面產生圓弧狀突起的異常身體型態，在此我們將之稱為bulge。klp-11屬於第二類kinesin驅動蛋白，tp8會導致其高度保守性驅動區域的第兩百五十一個甘胺酸突變成天門冬胺酸。利用RNAi的方式將klp-11抑制，會導致klp-11(tp8)這個突變株的腫脹凸起的比例下降，但不會造成野生型的線蟲產生腫脹凸起的缺陷，據此推測tp8可能是一個具有新功能的等位基因。因為腫脹突起的位置是發生於線蟲身體表面，而位於線蟲身體外層的細胞類型是表皮細胞與肌肉細胞，因此我們利用會表現在特定組織的螢光蛋白觀察，發現在腫脹突起區域確實存在表皮與肌肉細胞的表現，表示tp8可能是影響到這些細胞而導致腫脹凸起的現象產生。接下來我們更進一步的利用穿透式電子顯微鏡了解klp-11(tp8)突變株腫脹凸起的地方或是其他區域是否有異常。與先前螢光蛋白觀察的結果相符的是在腫脹凸起的地方會有表皮與肌肉細胞的存在。除此之外，我們還觀察到表皮細胞與神經細胞會累積在腫脹區域的底部，某些表皮細胞甚而往下生長至肌肉細胞的下方，造成彼此鑲嵌的現象。這些現象顯示klp-11(tp8)是一個在許多細胞類型中具有多重缺陷的突變。我們觀察到在腫脹凸起的地方會有表皮、肌肉、神經細胞與流體囊泡的存在，所以我們推測造成腫脹的原因可能是因為囊泡的運送發生問題或是這些細胞產生不正常的增生。我們表現正常的klp-11基因接上會專一表現在肌肉細胞的啟動子可以部份挽救腫脹凸起的產生，顯示tp8可以藉由影響肌肉細胞而導致腫脹，但除此之外，亦有其他細胞種類參與其中。此外，因為KLP-11會與KLP-20及KAP-1兩個蛋白質形成一個異源三聚體並在感覺神經元的鞭毛中的纖毛上進行物質的運送，因此我們推測klp-11(tp8)可能影響到進行運送物質的功能，使得運送物質異常大量累積於負向端進而使得線蟲身體的型態產生向外腫脹凸起的異常外觀。Morphogenesis is an essential process for a multi-cellular organism to develop into its characteristic body shape. In C. elegans, this process is largely controlled by the development of hypodermis and muscle tissues. In a genetic screen, we isolated the klp-11(tp8) mutant with an abnormal body shape, which has an arcuate swell in the body surface, referred as “bulge” here. klp-11 encodes a type II kinesin motor protein subunit. tp8 caused an amino acid substitution from glycine to aspartate in a highly conserved residue in the motor domain. Deletion of klp-11 by RNA interference caused no effect in the wild-type but suppressed the bulge phenotype in the klp-11(tp8) mutant, suggesting that tp8 may be a neomorphic allele of klp-11. We found that cells in the bulge consists of hypodermal, muscle cells or neurons using cell-specific fluorescence reporters. Consistently, klp-11 was found to be expressed in these cells. It is possible that tp8 may elicit an abnormal function of a kinesin complex to cause bulge formation from these cells. We next examined the ultrastructural defects in the bulge and other part(s) of the klp-11(tp8) mutant, using the transmission electron microscopy (TEM), and find muscle cells and neurons here within the bulge region, consistent with the fluorescent microscopy result. We also found the hypodermal cells grow under the muscle cells in the klp-11(tp8) mutant. These data show that tp8 causes multiple defects in various cell types. We observed many vesicles, neurons, muscle and hypodermal cells accumulated in the bulge region, so we hypothesized that the bulge may be caused by mis-regulation of vesicle transport and/or cell proliferation. We expressed wild-type klp-11 under the control of muscle-specific promoter could partially rescue the bulge phenotype indicated that klp-11 may function at least in muscle cells to prevent the bulge formation. In addition, because KLP-11 forms a heterotrimer with KLP-20 and KAP-1 and function as intraflagellar transport (IFT) in ciliated sensory neurons, we speculate that klp-11(tp8) may influence cargo transport by abnormal cargo accumulation in the minus end or absence of cargo in the plus end to cause bulge phenotype.致謝 i 中文摘要 ii Abstract iii Tables of content 1 Introduction 4 Materials and methods 8 C. elegans strains 8 RNA interference (RNAi) 8 Transgenic worms 9 Microscopy 9 Transmission electron microscopy (TEM) 9 Results 11 The klp-11(tp8) single mutant has bulged phenotype 11 Bulge is an allele-specific phenotype in klp-11(tp8) mutant 12 klp-11(tp8) is likely a neomorphic allele to cause bulge formation 12 klp-11 may cause the bulge formation by abnormal cargo accumulation 13 klp-11 was expressed in neurons、hypodermal and muscle cells 14 Bulged region contains hypodermal and muscle cells, sometimes a neuron 15 Muscle-expressed klp-11 can partially rescue the bulge phenotype 15 Transmission electron microscopy (TEM) datas indicate bulged region contains some cell types 16 Discussion 17 klp-11(tp8) has a new function to produce the abnormal cargos accumulation 17 The klp-11(tp8) mutant may affects hypodermal and muscle cells to cause bulge 18 The abnormal existence of multivesicular body may lead to bulged phenotype 19 References 22 Figures 26 Fig-1 KLP11 and KLP20 form a heterodimer that C-terminally associates with the cargo binding subunit, KAP-1 to transport cargo with OSM-3 26 Fig-2 The grp-1(gm350); klp-11(tp8) mutant shows abnormal body morphology 28 Fig-3 The klp-11(tp8) single mutant has body bulged phenotype 29 Fig-4 The expression of klp-11 in the hypodermal cells 30 Fig-5 The expression of klp-11 in the muscle cells 32 Fig-6 The expression of klp-11 in the neurons 33 Fig-7 Bulged region contains both hypodermal and muscle cells 34 Fig-8 Some klp-11(tp8) mutants show abnormal expression pattern 35 Fig-9 The bulged region contains muscle nucleus 36 Fig-10 The bulged region sometimes contains a neuron 37 Fig-11 The cross section of klp-11(tp8) shows some defects 39 Fig-12 The bulged region contains neurons and muscle cells 40 Fig-12 There are many multivesicular bodies(MVB) in the bulged region 42 Fig-14 The model of bulge formation 43 Tables 44 Table 1 Bulge is an allele-specific phenotype in klp-11(tp8) mutant 44 Table 2 Bulges are often observed between the mid body to anus at the dorsal side of the klp-11(tp8) mutant 45 Table 3 Both klp-20 and kap-1 mutants show no bulged phenotype 46 Table 4 klp-11(tp8) is a recessive mutant allele 47 Table 5 Overexpression of klp-11 promoter-driven klp-11 cDNA can rescue the bulged phenotype of klp-11(tp8) mutant 48 Table 6 klp-11(tp8) is likely a neomorphic allele to cause bulge formation 49 Table 7 The bulge formation in klp-11(tp8) mutant may be the outcome of cargo accumulation 50 Table 8 Bulged phenotype in klp-11(tp8) mutant may be the consequence of dynein cargo accumulation in the minus end of microtubule 51 Table 9 Muscle-expression klp-11 can partially rescue the bulged phenotype 52 Supplemental figures 53 Fig-S1 Screen for mutant with abnormal tail morphology in the grp-1 mutant 53 Supplemental tables 54 Table S1 klp-11(tp8) is likely a neomorphic allele to cause bulge formation 547591109 bytesapplication/pdf論文公開時間：2024/12/31論文使用權限：同意有償授權(權利金給回饋學校)型態異常驅動蛋白thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/261089/1/ntu-103-R01b43009-1.pdf