Ioannou Ion A, Brooks Nickolas J, Kuimova Marina K, Elani Yuval
Department of Chemistry, Imperial College London, Molecular Sciences Research Hub, London W12 0BZ, U.K.
Department of Chemical Engineering, Imperial College London, South Kensington, London SW7 2AZ, U.K.
JACS Au. 2024 Apr 30;4(5):2041-2049. doi: 10.1021/jacsau.4c00237. eCollection 2024 May 27.
The actin cytoskeleton and its elaborate interplay with the plasma membrane participate in and control numerous biological processes in eukaryotic cells. Malfunction of actin networks and changes in their dynamics are related to various diseases, from actin myopathies to uncontrolled cell growth and tumorigenesis. Importantly, the biophysical and mechanical properties of actin and its assemblies are deeply intertwined with the biological functions of the cytoskeleton. Novel tools to study actin and its associated biophysical features are, therefore, of prime importance. Here we develop a new approach which exploits fluorescence lifetime imaging microscopy (FLIM) and environmentally sensitive fluorophores termed molecular rotors, acting as quantitative microviscosity sensors, to monitor dynamic viscoelastic properties of both actin structures and lipid membranes. In order to reproduce a minimal actin cortex in synthetic cell models, we encapsulated and attached actin networks to the lipid bilayer of giant unilamellar vesicles (GUVs). Using a cyanine-based molecular rotor, DiSC(3), we show that different types of actin bundles are characterized by distinct packing, which can be spatially resolved using FLIM. Similarly, we show that a lipid bilayer-localized molecular rotor can monitor the effects of attaching cross-linked actin networks to the lipid membrane, revealing an increase in membrane viscosity upon actin attachment. Our approach bypasses constraints associated with existing methods for actin imaging, many of which lack the capability for direct visualization of biophysical properties. It can therefore contribute to a deeper understanding of the role that actin plays in fundamental biological processes and help elucidate the underlying biophysics of actin-related diseases.
肌动蛋白细胞骨架及其与质膜的复杂相互作用参与并控制真核细胞中的众多生物学过程。肌动蛋白网络的功能异常及其动力学变化与多种疾病相关，从肌动蛋白肌病到细胞生长失控和肿瘤发生。重要的是，肌动蛋白及其组装体的生物物理和机械特性与细胞骨架的生物学功能紧密相连。因此，研究肌动蛋白及其相关生物物理特征的新型工具至关重要。在此，我们开发了一种新方法，该方法利用荧光寿命成像显微镜（FLIM）和称为分子转子的环境敏感荧光团（作为定量微粘度传感器）来监测肌动蛋白结构和脂质膜的动态粘弹性特性。为了在合成细胞模型中重现最小的肌动蛋白皮层，我们将肌动蛋白网络包裹并附着到巨型单层囊泡（GUV）的脂质双层上。使用基于花青的分子转子DiSC(3)，我们表明不同类型的肌动蛋白束具有不同的堆积特征，可通过FLIM在空间上进行分辨。同样，我们表明脂质双层定位的分子转子可以监测将交联的肌动蛋白网络附着到脂质膜上的效果，揭示肌动蛋白附着后膜粘度增加。我们的方法绕过了与现有肌动蛋白成像方法相关的限制，其中许多方法缺乏直接可视化生物物理特性的能力。因此，它有助于更深入地理解肌动蛋白在基本生物学过程中所起的作用，并有助于阐明肌动蛋白相关疾病的潜在生物物理学。
