Quantitative investigation of negative membrane curvature sensing and generation by I-BARs in filopodia of living cells
Quantitative investigation of negative membrane curvature sensing and generation by I-BARs in filopodia of living cells
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DOI：
10.1039/c9sm01185d
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发表时间：
2019-12-28
期刊：
影响因子：
3.4
通讯作者：
Stamou, Dimitrios
中科院分区：
文献类型：
--
作者：
Breuer, Artu;Lauritsen, Line;Stamou, Dimitrios
膜曲率最近被认为是细胞功能的主动调节剂，几个蛋白质家族被鉴定为膜曲率的传感器和发生器。其中，反向Bin/Amphiphysin/Rvs（I-BAR）结构域家族涉及具有负膜曲率的膜结构（例如丝状伪足或树突棘）的感测和产生。然而，迄今为止，I-BAR结构域的定量生物物理研究主要发生在重建中。在这里，我们使用荧光显微镜定量研究膜曲率传感和生成I-BAR在丝状伪足的活细胞。作为模型系统，我们选择了I-BAR家族的两个原型成员，胰岛素受体底物p53和转移中的缺失。我们的数据表明I-BAR如何在活细胞的复杂环境中感测负膜曲率，揭示了它们对膜的结合亲和力和饱和密度对膜曲率的依赖性。负膜曲率的蛋白质分选的非单调依赖性使我们能够应用先前开发的热力学模型来提供在质膜上结合的两个I-BAR的有效固有曲率和弯曲刚度的估计。我们的研究结果与胰岛素受体底物p53在重建进行的研究。为了定量I-BAR产生的膜曲率，我们测量了它们的过表达如何降低丝状伪足的大小分布的峰值和宽度，从而导致丝状伪足群体具有更小和更均匀的直径。我们的研究结果提供了一个定量的生物物理洞察能力的I-BAR的感觉和产生负膜曲率在拥挤的环境中的活细胞。
Membrane curvature has recently been recognized as an active regulator of cellular function, with several protein families identified as sensors and generators of membrane curvature. Amongst them, the inverse Bin/Amphiphysin/Rvs (I-BAR) domain family has been implicated in the sensing and generation of membrane structures with negative membrane curvature e.g. filopodia or dendritic spines. However, to date, quantitative biophysical investigations of I-BAR domains have mostly taken place in reconstitution. Here, we use fluorescence microscopy to quantitatively investigate membrane curvature sensing and generation by I-BARs in filopodia of living cells. As a model system, we selected two prototypic members of the I-BAR family, the insulin receptor substrate p53 and missing-in-metastasis. Our data demonstrated how I-BARs sense negative membrane curvature in the complex environment of live cells by revealing a dependence on membrane curvature for both their binding affinity to membranes and their saturation density. The non-monotonic dependence of protein sorting with negative membrane curvature allowed us to apply previously developed thermodynamic models to provide estimates of the effective intrinsic curvature and bending rigidity of the two I-BARs bound at the plasma membrane. Our results agree with studies performed on the insulin receptor substrate p53 in reconstitution. To quantitate membrane curvature generation by I-BARs we measured how their overexpression reduces the peak and the width of the size distribution of filopodia, resulting in filopodia populations with smaller and more uniform diameters. Our findings provide a quantitative biophysical insight in the ability of I-BARs to sense and generate negative membrane curvature in the crowded environment of living cells.