請用此 Handle URI 來引用此文件:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/7784
標題: | NRIP藉由調控F-actin參與肌細胞融合 NRIP regulates F-actin for myoblast fusion |
作者: | Ya-Ju Han 韓雅如 |
指導教授: | 陳小梨 |
關鍵字: | 核受體結合蛋白,肌動蛋白,肌細胞融合,肌細胞生成素,WD40 repeats, NRIP,actin,myoblast fusion,myogenin,WD40 repeats, |
出版年 : | 2019 |
學位: | 碩士 |
摘要: | 核受體結合蛋白(Nuclear receptor interaction protein, NRIP)又名IQWD1或DCAF6,是一個鈣離子依賴性的攜鈣素(calmodulin)結合蛋白,NRIP由860個胺基酸所組成,其中包含七個WD 40 repeats及一個IQ motif。在本實驗室先前的研究中,我們發現在注射心臟毒素(cardiotoxin)造成肌肉受損所產生的肌肉再生中,全身性NRIP基因剔除鼠與正常小鼠相比,會產生較高比例的小尺寸的肌纖維(myofiber),顯示NRIP可能會調控肌細胞融合(myoblast fusion)。先前已有許多研究指出肌細胞的融合與肌動蛋白結合蛋白(actin-binding protein)所調控之肌動蛋白細胞骨架重塑有密切關係。此外,我們實驗室先前的研究發現在in vitro的情況下,NRIP和肌動蛋白有交互作用,因此在本篇實驗中我們進一步探討NRIP是否會藉由調控肌動蛋白參與在肌細胞融合中。
首先,利用細胞的免疫沉澱法(immunoprecipitation)我們確認在in vivo的情況下,NRIP和肌動蛋白彼此間也會有交互作用;除了肌動蛋白結合蛋白以外,許多膜蛋白也被報導會促進肌細胞的融合,因此我們做了細胞質、膜蛋白分離的實驗,並發現NRIP分布在細胞核及質中的比例是差不多的。 接著,我們進一步探討NRIP是否會調控肌小管形成(myotube formation),利用NRIP基因剔除肌細胞KO19,我們發現與正常肌細胞C2C12比起來,KO19形成肌小管的能力嚴重受損,並伴隨著肌細胞分化及融合能力的下降,顯示NRIP確實會調控肌小管形成,接著我們進一步在C2C12及KO19中大量送入NRIP,發現不管在C2C12或KO19中,外送NRIP皆能促進肌小管的形成。 在肌細胞融合過程中,由肌動蛋白所構成類似足體的構造(podosome-like structure)對於融合孔的產生是重要的,首先我們觀察到NRIP會聚集在肌細胞接合處,顯示NRIP確實可能會調控肌細胞融合,接著我們利用顯微影像曠時攝影技術(time-lapse microscopy)觀察到NRIP與肌動蛋白共定位(colocalize)且聚集在細胞質中形成焦點並往細胞膜外突出形成類似足體的構造,顯示其可能參與在肌細胞融合侵入的過程中。 此外我們利用不同NRIP的片段探討NRIP的哪一段會和肌動蛋白結合,結果發現NRIP可能會透過WD67直接與肌動蛋白連結,或是利用IQ motif間接經由輔肌動蛋白異構體(α-actinin2, ACTN2)與肌動蛋白連結,我們並進一步探討NRIP與肌動蛋白連結是否與肌細胞融合有關,結果顯示在KO19中大量送入WD67可以促進肌細胞融合,代表NRIP與肌動蛋白連結與肌細胞融合是有關連的。 最後,我們探討了由NRIP所調控之肌細胞生成素(myogenin)主導的肌小管形成,我們先前的研究結果顯示NRIP藉由與調鈣素結合,活化下游鈣調磷酸酶(calcineurin)及鈣調蛋白肌酶II (calmodulin kinase II)進而促進肌細胞生成素的表現,我們進一步將C2C12中NRIP基因減弱(gene knockdown),發現肌細胞生成素及肌球蛋白重鏈(myosin heavy chain)的RNA及蛋白質表現皆較控制組下降,表示NRIP會經由調控肌細胞生成素參與在肌小管形成中。總體來說,我們發現了NRIP可以透過和肌動蛋白結合進而去調控肌細胞融合外,也可以透過調控肌細胞生成素參與在肌小管形成的過程中。 Nuclear receptor interaction protein (NRIP) also named as IQWD1 and DCAF6 is a Ca2+-dependent calmodulin-binding protein. NRIP consists of 860 amino acids, with seven WD40 repeats and one IQ motif. In previous study, we observe that NRIP global knockout (gKO) mice show higher frequency of small myofiber size distribution than wild-type mice at day 21 post cardiotoxin-induced injury during muscle regeneration, indicating that NRIP may regulate myoblast fusion to form myofiber. Myoblast fusion has been reported to be strongly associated with actin cytoskeleton remodeled by actin-binding proteins. Moreover, our unpublished results reveal that NRIP can interact with F-actin in vitro. Therefore, in this study, we investigated whether NRIP through interacting with actin to regulate myoblast fusion during myogenesis. First, we investigated whether NRIP interacts with actin in vivo. By performing immunoprecipitation assay, the results revealed that NRIP and actin interacted reciprocally in vivo. Not only actin-binding proteins but also membrane-bound proteins have been reported to promote myoblast fusion. Therefore, we next performed cytosolic and membrane protein fractionation and found that NRIP expressed in both cytoplasm and membrane with nearly equal amount. Next, we examined whether NRIP is responsible for myotube formation. We generated NRIP-null C2C12 myoblast—KO19. By conducted immunofluorescence assay, we observed the severely impaired the ability of myotube formation in KO19 with both reduced differentiation and fusion index compared to C2C12, indicating that NRIP is responsible for myoblast differentiation and fusion. We further overexpressed NRIP in C2C12, and the results showed that NRIP can enhance the ability of myotube formation in C2C12. Moreover, overexpression of NRIP in KO19 could rescue myotube formation. These results indicate that NRIP is responsible for myotube formation. During myoblast fusion, the F-actin-enriched podosome-like structure is essential for fusion pore formation. We first observed that NRIP concentrated at the site of cell-cell contact, implying that NRIP may play a role in fusion. Next, we performed time-lapse microscopy and discovered that NRIP and actin colocalized and concentrated to form NRIP- and actin-enriched foci in cytoplasm and then protruded toward cell membrane to form podosome-like structure which might promote invasion during myoblast fusion. We also performed cell fusion assay and verified that NRIP could enhance myoblast fusion. Realizing NRIP is an actin-binding protein, we further investigate which domain of NRIP is responsible for binding with actin. We performed immunoprecipitation assay and the results suggested that NRIP might directly interact with actin through WD67 domain and indirectly bind with actin through IQ motif in an ACTN2-dependent manner. To examine whether NRIP binding with actin is correlated with myoblast fusion, we overexpressed WD67 domains which can bind with actin in KO19 and examined myotube formation. The fusion index was significantly higher in KO19/WD67 than KO19/vector, indicating that the WD67 domain of NRIP binding with actin is correlated to myoblast fusion. Apart from NRIP-regulated actin for myotube formation, we also investigated NRIP-regulated myogenin for myotube formation. Our unpublished results reveal that NRIP regulates myogenin through binding with Calmodulin (CaM) to activate calcineurin (CaN) and calmodulin kinase II (CaMKII) signaling pathway. We further performed Ad-shNRIP to knockdown C2C12 during myogenesis, and found that the protein and RNA expression levels of myogenin and MyHC decreased, suggesting that NRIP also through myogenin to regulate myotube formation. Collectively, NRIP not only acts through interacting with actin to regulate myoblast fusion but also through regulating myogenin to take part in myotube formation. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/7784 |
DOI: | 10.6342/NTU201902210 |
全文授權: | 同意授權(全球公開) |
顯示於系所單位: | 微生物學科所 |
文件中的檔案:
檔案 | 大小 | 格式 | |
---|---|---|---|
ntu-108-1.pdf | 2.99 MB | Adobe PDF | 檢視/開啟 |
系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。