鈣粘蛋白為位於細胞膜的跨膜蛋白，為連結細胞的重要分子。早期研究中已知癌細胞要開始轉移時，鈣粘蛋白在膜上的表現會消失，失去細胞間的連結助於細胞移動。但近期發現，當癌細胞為群體移動時，鈣粘蛋白並不會完全的消失，而是有重新排列現象發生，其中相關機制尚有許多未了解之處。原被認為是細胞間連結分子的鈣粘蛋白，為什麼在群體細胞移動過程中不會完全消失?在此功能是什麼?我們提出鈣粘蛋白在細胞群體移動的過程中可能是一個助力的假設。在我的研究中，會利用黑腹果蠅當作模式生物。而其卵形成的過程中有6-8顆邊界會從卵的前端穿越養護細胞移動到卵細胞的邊界，鈣粘蛋白一樣會在細胞移動中有重新排列的現象，因此我們可以藉此來觀察鈣粘蛋白在細胞移動時是怎麼重新排列的。我們在此用兩個不同的方法來進行實驗：FRAP以及split GFP。透過FRAP可以知道鈣粘蛋白在膜上的流動狀況及囊泡運送的狀況。透過split GFP可以知道邊界細胞跟養護細胞之間的鈣粘半白是否真的有連結。過去我們已知蜕皮激素會調控鈣粘蛋白的分佈，實驗中發現蜕皮激素似乎是透過調控囊泡運送改變鈣粘蛋白的分佈。我們已經建立好實驗的條件，在未來能夠更精準的對鈣粘蛋白在膜上的狀況進行分析。

SUMMARY

E-cadherin (Epithelial cadherin, E-cad) is a main important adhesion junction (AJ) molecule. AJ maintain cell conserved junction, establish cell polarity and involve tissue development. Before cell initial to move, cell must lose E-cad to remove the junction. Cell migration occur in development, wound healing and cancer cell metastasis. In the past, lot scientists consider that during cancer cell invasion cell almost loss E-cad. However, in cluster cancer cells migration has been found that E-cad still expression and reorganized the distribution pattern. How the E-cad role in invasion cell invasion in is still unclear. In order to, study the reason of E-cad change the pattern during a group cell moving? Therefore, we use the Drosophila border cells migration to be a collective cells movement model. There is a group border cell (epithelial cell) form a cluster on anterior and start migration at 9 stage. The border cells cluster pass through nurse cells (germline) until to reach oocyte border. This movement process is like cancer cells cluster migration and reorganize the pattern of E-cad. We can analysis How are different expression patterns of E-cadherin achieved during migration. Here, we use two approach to study different way of E-cad. One is FRAP (fluorescence recovery after photobleaching) to analysis the membrane dynamic of E-cad. The other way is split GFP to analysis the E-cad homophilic interaction between cell-cell junction during cell migration. In the result, we observe the recovery rate of E-cad on the membrane. And expected the split GFP can work during E-cad homophilic interaction (Split GFP was work in the S2 cells test).
Key words: E-cadherin, FRAP and split GFP

INTRODUCTION

E-cadherin (E-cad) is a transmembrane protein and an important molecule in adhesion junctions. In the past, many scholars discovered cell loss E-cad in cancer metastasis. Loss of cell-cell junction may be beneficial for cell migration. However, in recent studies, E-cad was not lost in cluster tumor migration, but recombination was observed in the expression pattern of E-cad. The role of E-cad in cells migration is unclear. Here, we want to know how different expression patterns of E-cadherin are implemented? We use two technology to analyze this question. One is Fluorescence recovery after photobleaching (FRAP), we want to know the transport of E-cad or E-cad membrane fluidity during migration. In order to conducive to the progress of the experiment, we chose the border cell collective cell migration as a model system. we will study the E-cadherin dynamic in Drosophila border cell migration. On the other hand, we will also use the split GFP method to explore the E-cad interaction between the cells. Another important question is whether the pattern change of E-cad is regulated by other factors? We will also explore ecdysone, the only steroid hormone in insects. In the past, we knew that ecdysone is the temporal control of border cell migration. Finally, we would like to know more about how ecdysone regulates E-cad.

MATERIALS AND METHODS

In the first experiment, we utilized Fluorescence recovery after photobleaching (FRAP) to bleach E-cad::GFP between different cell junction during border cell migration. The cell junctions we bleached include border cells and border cells, border cells and nurse cells and follicle cells (the cell did not move). Then we calculated E-cad recovery and half-life values to analyze E-cad transport in the membrane. In the second experiment, to analyze the connection of E-cadherin, more precisely between border cells and nurse cells, we used a split GFP technique. We fused the GFP1-10 and GFP11 to the E-cad N’ terminal, respectively. We expressed GFP1-10-E-cad in the border cells through slbo promoter and expressed GFP11-E-cad in the nurse cells through UAS-GAL4 system. During border cells migration, we can detect GFP signals between border cell and nurse cell if E-cad is homophilic interaction. We tested split GFP in Drosophila S2 cells prior to the Drosophila in vivo experiment. In the cells test we generated two construction, one was pAc5.1-gfp1-10-E-cad-V5 and the other was pAc5.1-Flag-gfp11-E-cad. Here we used the pAc5.1 vector because it contains the actin 5c promoter that can turn on gene in S2 cells. We transfected these two structures into S2 cells, respectively. S2 cells were then co-cultured for two days to observe whether split GFP was reconstituted when fuse with E-cad.

RESULTS AND DISCUSSION

First, in the FRAP experiment, we bleached three junctions and observe the recovery rate in WT, separately. One is the between follicle cell without the ability to move. Second is between border cells, in the moving cluster but forming a conserve junction. The third is the between border cell and nurse cell, the junction between the moving cluster and the non-moving cell. We found recovery half-time about eight seconds in border cells and follicle cells. And about 40 seconds between border cells and nurse cells. The average of all three recovers is 40%. In the group of Ecdysone hyperactive form, we bleached in border cells and between border cells and nurse cells, the recovery of half-life was 70 seconds. Significantly longer than WT. Second, in the split GFP experiment, we pertest in S2 cell, Drosophila cell to make sure the experimental concept is work. After we complete two constructions, pAc5.1-GFP1-10-E-cad-V5 and pAc5.1-Flag-GFP11-E-cad-V5. We perform transfection the two construction, separately. Then co-culture one day. We can see from the fix sample that split GFP will reconstituted when E-cad homophilic interaction. Next, we also complete the remaining two constructs, pslbo-GFP1-10-E-cad and pUASp-GFP11-E-cad. After microinjection, we have got GFP11-E-cad Fly, and GFP1-10 is almost done. After completion, the split GFP observation of Drosophila oogenesis can be performed.

CONCLUSION

Through FRAP observation, the speed of E-cadherin recovery on the membrane is slow under the condition of ecdysone hyperactive form. Ecdysone may cause E-cad expression different on the membrane to change pattern by regulate E-cad transport. On the other hand, split GFP fusion with E-cad is also demonstrated, GFP reconstitution is not affected by E-cad interaction. Then we can apply the experiment in Drosophila in vivo.

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