组成图示
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传感器类型
综述或非传感器论文
检测对象
活性 RhoC（GTP-RhoC）；样品基质：活细胞（MTLn3 大鼠乳腺腺癌细胞）内 invadopodia 周围胞质/膜区
检测原理
该研究采用 FRET 型 RhoC 荧光生物传感器检测活细胞内 RhoC 活性。传感器由 Cerulean 供体、RhoC 活性感应/结合模块（PBD）和 Venus 受体串联组成。当 GTP 结合的活性 RhoC 与 PBD 模块结合时，供体与受体间距离或取向改变，Venus/Cerulean FRET 比值升高，从而反映局部 RhoC 活性。在 invadopodia 处，p190RhoGEF 位于突起外周并激活 RhoC，p190RhoGAP 位于核心内并水解 GTP 使 RhoC 失活，形成外高内低的活性环。RhoC 激活 ROCK-LIMK 通路，磷酸化 cofilin S3，抑制其切割 F-actin，使 cofilin 活性集中于核心，促进 barbed ends 生成和聚焦突起。
检测灵敏度
原文未报告 LOD、线性范围、灵敏度斜率、相关系数。
效应效果
传感器特异性由 RhoC-PBD、RhoCF39A、RhoCQ63L、RhoCG14V 突变体验证，突变体在 invadopodia 周围无可测活性升高；RhoA 传感器无类似模式。表达传感器不影响 invadopodia 降解或数量。RhoC siRNA 降低 Transwell 侵袭和突起深度，但增加每个 invadopodium 降解面积并形成分支突起。EGF 使 pCofilinS3 约升 2 倍，RhoC 敲低后无显著增加；RhoC-Q63L 使 pCofilinS3 升 >2 倍，可被 H-1152（5 μM）或 Y-27632（10 μM）阻断。作者认为该机制聚焦 cofilin 活性，增强肿瘤侵袭，提示 RhoC/ROCK/LIMK 为抗转移靶点。
传感器的构成
- 表达基底：MTLn3 大鼠乳腺腺癌细胞，Tet-off 诱导系统稳定表达 RhoC 生物传感器。
- 荧光供体：Cerulean，作为 FRET 供体，提供激发与供体荧光。
- 识别元件：RhoC-PBD 活性感应/结合模块，结合 GTP-RhoC 并引起构象变化。
- 荧光受体：Venus，作为 FRET 受体，活性 RhoC 结合后改变 FRET 效率。
- 对照突变体：RhoCF39A、RhoCQ63L、RhoCG14V，用于验证传感器特异性。
- 读出系统：共聚焦/荧光显微镜，测量 Venus/Cerulean FRET 比值。
中文摘要
背景：Rho GTPases 参与癌症转移调控。侵袭性癌细胞形成 invadopodia，即富含 F-actin 的基质降解突起，对肿瘤侵袭和血管内侵入重要。invadopodia 处 actin 动态调控关键，需要 cofilin 切割活性产生无 actin 的 barbed ends。cofilin 切割活性受多种机制严格调控，包括 Rho GTPases 调控 cofilin 丝氨酸磷酸化，但 invadopodia 处哪种 Rho GTPase 调控 cofilin 磷酸化状态未知。结果：本文首次展示 RhoC 激活如何在 invadopodia 受控，并调控 cofilin 磷酸化以控制 actin-free barbed ends 生成。荧光 RhoC 生物传感器活细胞成像显示 RhoC 活性空间局限于 invadopodia 周围区域。这种时空限制由空间上不同的调控元件控制，将 RhoC 激活限制在该区室。p190RhoGEF 定位于 invadopodia 周围激活 RhoC，而 p190RhoGAP 定位于 invadopodia 内部使 GTPase 失活。RhoC 激活增强 invadopodia 外 cofilin 磷酸化。结论：结果显示 RhoC 活性在 invadopodia 处由 p190RhoGEF 和 p190RhoGAP 空间调控。invadopodia 周围 RhoC 激活将 cofilin 活性限制在 invadopodium 核心内，形成聚焦的 invadopodial 突起。该机制可能增强转移过程中肿瘤细胞侵袭。
英文摘要
BACKGROUND: RhoGTPases have been implicated in the regulation of cancer metastasis. Invasive carcinoma cells form invadopodia, F-actin-rich matrix-degrading protrusions that are thought to be important for tumor cell invasion and intravasation. Regulation of actin dynamics at invadopodial protrusions is crucial to drive invasion. This process requires the severing activity of cofilin to generate actin-free barbed ends. Previous work demonstrates that cofilin's severing activity is tightly regulated through multiple mechanisms, including regulation of cofilin serine phosphorylation by Rho GTPases. However, it is not known which Rho GTPase is involved in regulating cofilin's phosphorylation status at invadopodia.
RESULTS: We show here, for the first time, how RhoC activation is controlled at invadopodia and how this activation regulates cofilin phosphorylation to control cofilin's generation of actin-free barbed ends. Live-cell imaging of fluorescent RhoC biosensor reveals that RhoC activity is spatially confined to areas surrounding invadopodia. This spatiotemporal restriction of RhoC activity is controlled by "spatially distinct regulatory elements" that confine RhoC activation within this compartment. p190RhoGEF localizes around invadopodia to activate RhoC, whereas p190RhoGAP localizes inside invadopodia to deactivate the GTPase within the structure. RhoC activation enhances cofilin phosphorylation outside invadopodia.
CONCLUSION: These results show how RhoC activity is spatially regulated at invadopodia by p190RhoGEF and p190RhoGAP. RhoC activation in areas surrounding invadopodia restricts cofilin activity to within the invadopodium core, resulting in a focused invadopodial protrusion. This mechanism likely enhances tumor cell invasion during metastasis.