建立抑制dynein和dynactin在胞內交互作用之光活化平台

Abstract

Dynein和dynactin會形成複合蛋白，在微管細胞骨架中扮演蛋白質及胞器運送的角色。Dynactin由許多單體所組成，其中一個單體為p150glued，在上面有一個α螺旋片段簡稱CC1，已知會和dynein中的單體dynein intermediate chain (DIC)連接形成複合蛋白。從我們實驗驗證當大量表現CC1會與內生型的dynactin去競爭dynein，破壞兩運送蛋白間的交互作用，進而影響細胞內胞器的運送，例如高基氏體的型態和微管束的運送。為了想要瞭解在特定位置破壞兩運送蛋白交互作用後對細胞產生的影響，我們將CC1與一個可以被光所調控的蛋白LOV連接，希望在黑暗的環境中能阻擋CC1與DIC之連接。而在受藍光激發後，透過瓦解LOV的結構，讓CC1能夠與DIC連接，並進而破壞dynein-dynactin的交互作用。然而，從目前實驗中我們發現在黑暗的環境下，PA-CC1就會影響部分的高基氏體型態或微管束的運送。而受藍光激發後，PA-CC1也沒有更進一步的影響高基氏體型態或微管束的運送。這些結果顯示現階段PA-CC1無法按照我們的設計影響dynein-dynactin的相互作用。為了更進一步探討其中的問題，我們用免疫共沉澱的方法，直接量化PA-CC1與DIC的作用，我們在CC1及PA-CC1的N端接上EGFP，並用GFP的抗體進行共沉澱。從實驗結果中，發現EGFP-CC1與EGFP-PA-CC1皆無法偵測到DIC的訊號。我們猜測可能原因為CC1與內生型DIC的鍵結能力太弱，導致無法在現階段的條件看到DIC的訊號。

Cytoplasmic dynein and dynactin form a large protein complex that functions as a MTs-based motor. Dynactin is a multisubunit complex that is required for most types of cytoplasmic dynein activity in the eukaryotic cells. p150glued is the subunit of the dynactin complex which binds to the dynein complex. This interaction is mediated by the coil-coiled 1 region (CC1) of the p150glued subunit, which binds to the dynein intermediate chain (DIC) in the dynein complex. We showed that overexpressing CC1 in cells can disrupt dynein-driven cellular organization (such as Golgi apparatus organization and microtubule bundle transportation). To observe the cell morphology by local disrupting dynein-dynactin interaction without altering overall dynein activity, we fused CC1 to the photoactive LOV domain from phototropin in hope to create a photoactivatable CC1 (PA-CC1) that would block CC1-DIC interactions in the dark. Upon blue light irradiation, the α-helix linking LOV to CC1 unwinds and this should allow PA-CC1 to interact with the DIC. Our preliminary result showed that overexpressing PA-CC1 in the dark already disrupted Golgi apparatus organization and microtubule bundle transportation. Light irradiation did further disrupt Golgi apparatus organization or microtubule bundle transportation. These data suggested that our PA-CC1 construct did not regulate dynein-dynactin interaction according to our design at the current stage. To gain insight into the problem, we quantified the interaction between DIC and our construct directly using co-immunoprecipitation. We fused EGFP to the N-terminus of CC1 (EGFP-CC1) and PA-CC1 (EGFP-PA-CC1), then used anti-GFP antibody to perform co-immunoprecipitation assay. However, neither EGFP-CC1 nor EGFP-PA-CC1 interacted with the endogenous DIC in our co-immunoprecipitation assay. We hypothesized that this is due to the low binding affinity between CC1 and the endogenous DIC.