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Title: | 微小管和動肌蛋白交聯因子(MACF1)在細胞爬行中與細胞動態連結的交互作用 Interactions between microtubule-actin crosslinking factor 1 (MACF1) and cell junction during cancer cell migration |
Authors: | TIEN-TSAI LIN 林天財 |
Advisor: | 蔡丰喬(Feng-Chiao Tsai) |
Keyword: | 微小管和動肌蛋白交聯因子(MACF1), microtubule-actin crosslinking factor 1 (MACF1), |
Publication Year : | 2020 |
Degree: | 碩士 |
Abstract: | 微管-肌動蛋白交聯因子1(MACF1)是與F-肌動蛋白和微管相互作用的細胞骨架交聯蛋白。在其N 端有兩個與Ca2+結合的EF hand,是否Ca2+會調控MACF1 與Microtubule 結合的相互作用仍然是未解的疑問。因此,我們探討細胞內和細胞外Ca2+如何分別調節細胞骨架重塑和細胞粘附。在細胞內,我們使用GFP 標籤和N 末端截短MACF1 蛋白MACF1 Tail(其EF hand 有各種突變或缺失)的螢光活細胞成像瞭解Ca2+如何調節MACF1 及微管間相互作用。EFhand 缺失的蛋白顯示MACF1 對微管尖端EB1 結合不須鈣離子調控。排除內生性MACF1 影響後,我們發現相較於微管lattice,MACF1 對微管尖端結構EB1具有較高親和力。在不同的Ca2+濃度下,MACF1 Tail 顯現微管結構間的結合轉移。在細胞外部分,隨機爬行試驗表明敲除癌細胞中的MACF1 除了能降低速度外,還可以增強細胞間的協調性。我們發現MACF1 藉由αE-catenin 影響細胞間的協調性。而粘附連接adherens junctions 在MACF1 調節細胞間的協調性卻並非主要的角色。連同文獻報導,抑制MACF1 能藉由微管調控影響緊密連接蛋白Tight Junction 的動態變化,我們假設MACF1 直接透過細胞骨架影響Tight Junction 的動態變化達到調節細胞間的協調性。通過這些方法,我們將闡明MACF1 如何調節細胞骨架和細胞黏附,希望在不久的將來開發出針對EMT和/或黏附相關疾病的新穎治療策略。 Interactions between microtubule-actin crosslinking factor 1 (MACF1) and cell junction during cancer cell migration Microtubule-actin crosslinking factor 1 (MACF1) is a cytoskeletal crosslinking protein that interacts with F-actin and microtubules. Although there are two putative Ca2+-binding EF hands at its C-terminus, whether and how Ca2+ interacts with MACF1 remains elusive. We thus explored how intracellular and extracellular Ca2+ regulated cytoskeletal remodeling and cell adhesion respectively. In the intracellular aspect, we studied how Ca2+ regulated MACF1-microtubule interaction using GFPtagged N terminus truncated MACF1 proteins with various mutations or deletions on their EF hands. SAS cells expressing MACF1 tail with or without EF-truncation showed similar distribution patterns in microtubule lattice and microtubule tips, indicating that EF-hand might not be mandatory for the binding of MACF1 to microtubule tip. However, affinities of MACF1 to microtubule tips might be higher than that to microtubule lattices. Moreover, the transfer of MACF1 binding from microtubule tips to microtubule lattices might depend on Ca2+. In the extracellular part, our random migration assays demonstrated that MACF1 knockdown in cancer cells increased cell-cell coordination in addition to their speed reduction. Interestingly, α-Ecatenin knockdown abolished above effect of MACF1 on cell-cell coordination. Our MACF1-CDH1 double knockdown experiment revealed that adherens junctions might not contribute to the effect of MACF1 on cell-cell coordination. Together with literature reports that MACF1 knockdown reduced tight junction dynamics through microtubule, we hypothesized that MACF1 induced tight junction internalization via directly switching the balance from cell-cell adhesion to cell-matrix adhesion. To verify this hypothesis, we are currently using immunofluorescence to examine how MACF1 alters the dynamics of cell-cell adhesion. With these approaches we will elucidate how MACF1 regulate cytoskeletons and cell adhesion, with the hope to develop novel therapeutic strategies against EMT and/ or adhesion-related diseases in the near future. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/69594 |
DOI: | 10.6342/NTU202003935 |
Fulltext Rights: | 有償授權 |
Appears in Collections: | 藥理學科所 |
Files in This Item:
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U0001-1808202011164300.pdf Restricted Access | 5.23 MB | Adobe PDF |
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