Golgi-derived microtubule (MT) arrays are essential to directionally persistent cell migration and vesicle transport. In this study, we have examined MT nucleation sites in two breast cancer cell lines, MDA-MB-231 and MCF-7, with the hypothesis that only the migratory invasive MDA-MB-231 cells exhibit MTs originating from the Golgi. MTs were disassembled and allowed to slightly regrow so individual nucleation sites could then be observed via fluorescently tagged antibodies (α-tubulin, cis-Golgi marker GM130, and EB1—a MT plus-end binding protein) and confocal microscopy. To determine if MT nucleation at the Golgi is more apparent during active migration compared to when cells are stationary, cells were treated with the chemoattractant epidermal growth factor (EGF) and examined for colocalizations between the Golgi, α-tubulin, and γ-tubulin. Images were analyzed qualitatively for color overlap, and quantitatively using Manders Colocalization Coefficients. Differences between groups were tested for significance using one-way analysis of variances and Tukey's post hoc test. Significantly higher colocalization values (coloc) in the highly invasive MDA-MB-231 cells (α-tubulin coloc GM130 = 0.39, GM130 coloc α-tubulin = 0.82, GM130 coloc EB1 = 0.24, and EB1 coloc GM130 = 0.38) compared to the weakly invasive MCF-7 cells (0.15, 0.08, 0.02, and 0.16, respectively) were observed. EGF-treated cells exhibited higher colocalization values than control cells for three of the four protein combinations tested, but EGF-treated MDA-MB-231 cells exhibited significantly higher values (α-tubulin coloc GM130 = 0.20, GM130 coloc α-tubulin = 0.89, and γ-tubulin coloc GM130 = 0.47) than both control groups as well as the EGF-treated MCF-7 cells. Results support the hypothesis that MT nucleation at the Golgi occurs more frequently in the invasive MDA-MB-231 cell line compared to the weakly invasive MCF-7 cells. The presence or absence of Golgi-derived MTs may help to explain the difference in migratory potential commonly exhibited by these two cell lines.

中文翻译：

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乳腺癌细胞高尔基体微管成核的证据

高尔基体衍生的微管 (MT) 阵列对于定向持续细胞迁移和囊泡运输至关重要。在这项研究中，我们检查了两种乳腺癌细胞系 MDA-MB-231 和 MCF-7 中的 MT 成核位点，并假设只有迁移侵袭性 MDA-MB-231 细胞才表现出源自高尔基体的 MT。MT 被拆卸并允许稍微重新生长，因此可以通过荧光标记抗体（α-微管蛋白、顺式高尔基体标记 GM130 和 EB1（一种 MT 加端结合蛋白）和共聚焦显微镜观察各个成核位点。为了确定与细胞静止时相比，主动迁移期间高尔基体的 MT 成核是否更明显，用趋化剂表皮生长因子 (EGF) 处理细胞，并检查高尔基体、α-微管蛋白和 γ-微管蛋白之间的共定位。对图像的颜色重叠进行定性分析，并使用曼德斯共定位系数进行定量分析。使用单向方差分析和 Tukey 事后检验检验组间差异的显着性。与高侵袭性 MDA-MB-231 细胞相比，共定位值 (coloc) 显着更高（α-微管蛋白 coloc GM130 = 0.39、GM130 coloc α-微管蛋白 = 0.82、GM130 coloc EB1 = 0.24 和 EB1 coloc GM130 = 0.38）观察到弱侵袭性 MCF-7 细胞（分别为 0.15、0.08、0.02 和 0.16）。对于测试的四种蛋白质组合中的三种，EGF 处理的细胞表现出比对照细胞更高的共定位值，但 EGF 处理的 MDA-MB-231 细胞表现出显着更高的值（α-微管蛋白 coloc GM130 = 0.20，GM130 coloc α-微管蛋白 = 0.89 ，和 γ-微管蛋白 coloc GM130 = 0.47）比两个对照组以及 EGF 处理的 MCF-7 细胞。结果支持这样的假设：与弱侵袭性 MCF-7 细胞相比，侵袭性 MDA-MB-231 细胞系中高尔基体 MT 成核更频繁地发生。高尔基体衍生的 MT 的存在或不存在可能有助于解释这两种细胞系通常表现出的迁移潜力的差异。