摘要或動機

Adducin蛋白在細胞骨架的調節上扮演著重要的角色。然而，近來有許多研究指出，骨架蛋白也會出現在細胞核並參與轉錄調控，因此本研究的目的即在探討adducin蛋白是否會進入細胞核中，並參與轉錄調控或具有其他功能。在本研究中，我們將綠色螢光蛋白(GFP)標示的adducin質體DNA，利用轉染技術送入老鼠纖維母細胞株NIH3T3中表現。NIH3T3細胞原本並無adducin蛋白的表現，在共軛焦顯微鏡下觀察，野生型的GFP-adducin蛋白會表現於細胞核與細胞質中。由於adducin蛋白尾端序列攜有可能往核內運輸的訊號，於是將位在此一訊號中的離胺酸718及離胺酸719進行突變，結果發現此一突變株只能在細胞質中表現。此外，蛋白磷酸脢C(protein kinase C)已知能磷酸化adducin蛋白在絲胺酸716及絲胺酸726的位置，於是假設其磷酸化是否與其在細胞內的分布有關。將adducin的絲胺酸726置換成丙胺酸，並不影響其在細胞內的分布。然而將絲胺酸716置換成丙胺酸後，則完全只在細胞核中表現。由於adducin可分布於細胞核，因此我們懷疑adducin蛋白可能與細胞分裂有關，於是本研究利用流式細胞儀分析adducin轉染後NIH3T3細胞的細胞週期。流式細胞儀的分析結果顯示，攜有GFP-adducin或其突變株的細胞與未經轉染的NIH3T3細胞的細胞週期並沒有顯著差異。其次，為了避免因轉染的效率不高而造成統計上的誤差，我們利用顯微鏡追蹤技術觀察攜有GFP-adducin的細胞株，結果顯示攜有adducin突變株的NIH3T3細胞株仍能正常分裂。再者，因為adducin能與細胞骨架中的肌動蛋白結合，所以adducin不同的分布位置可能影響細胞附著與細胞展延的效率。細胞展延試驗的結果顯示，adducin及其突變株對細胞附著與細胞展延的效率並無明顯的影響。本研究的結果證明，adducin的確帶有往核內運輸的訊號，其在細胞質中的分布可能也同時受到絲胺酸716磷酸化的影響。然而adducin的功用似乎與纖維母細胞的分裂與展延無明顯的關聯性。Adducin, an actin binding protein, is known to play an important role in the regulation of the membrane cortical cytoskeleton. More and more evidence indicates that proteins involved in the cytoskeletal regulation could also reside in the nucleus and participate in gene regulation. Thus, the goal of this study is to examine whether
adducin is expressed in the nucleus and involved in certain nuclear events. In this study, adducin and its various mutants were fused with green fluorescent protein (GFP) and transfected into mouse NIH3T3 fibroblasts which do not have endogenous adducin for monitoring their subcellular distribution under a laser scanning confocal microscope. The wild-type GFP-adducin was found to be present both in the nucleus and in the cytoplasm. The COOH-tail of adducin contains a motif analogous to the nuclear localization signal (NLS). Mutation of two lysine residues (lysine 718 and lysine 719) located within this motif abolished the nuclear localization of adducin. Moreover, adducin is known to be phosphorylated by protein kinase C at serine 716 and 726. Substitution of adducin serine 726 with alanine had no effect on its subcellular localization. In contrast, substitution of adducin serine 716 with alanine led to only nuclear expression. Nuclear localization of adducin renders it possible that adducin may be involved in the regulation of cell division cycle. For cell cycle analysis, flow cytometry was applied. The results of flow cytometry indicated that expression of adducin and its mutants in NIH3T3 fibroblasts did not affect their cell cycle progression. To further examine the effect of adducin on cell division, NIH3T3 cells transiently transfected by adducin were monitored by time lapse video microscopy. The video clearly showed that the cells with GFP-adducin underwent cell division to generate two daughter cells. Since adducin is well known to bind to actin and thereby regulate microfilaments, we wondered that expression of adducin in NIH3T3 cells might affect their adhesion and spreading onto extracellular matrix proteins. The results of cell spreading assays showed that adducin appeared not to affect cell spreading. In conclusion, our results demonstrate that the subcellular distribution of adducin is likely regulated by two signals, one is the nuclear localization signal and the other is the phosphorylation status of the serine 716. However, enforced expression of exogenous adducin in fibroblasts such as NIH3T3 cells does not alter their cell cycle or cell spreading on fibronectin.