通过相关光谱法在黏附组装和解聚过程中测量桩蛋白动力学。
Paxillin dynamics measured during adhesion assembly and disassembly by correlation spectroscopy.
作者信息
Digman Michelle A, Brown Claire M, Horwitz Alan R, Mantulin William W, Gratton Enrico
机构信息
Laboratory for Fluorescence Dynamics, Department of Biomedical Engineering, University of California, Irvine, California, USA.
出版信息
Biophys J. 2008 Apr 1;94(7):2819-31. doi: 10.1529/biophysj.107.104984. Epub 2007 Nov 9.
Paxillin is an adaptor molecule involved in the assembly of focal adhesions. Using different fluorescence fluctuation approaches, we established that paxillin-EGFP is dynamic on many timescales within the cell, ranging from milliseconds to seconds. In the cytoplasmic regions, far from adhesions, paxillin is uniformly distributed and freely diffusing as a monomer, as determined by single-point fluctuation correlation spectroscopy and photon-counting histogram analysis. Near adhesions, paxillin dynamics are reduced drastically, presumably due to binding to protein partners within the adhesions. The photon-counting histogram analysis of the fluctuation amplitudes reveals that this binding equilibrium in new or assembling adhesions is due to paxillin monomers binding to quasi-immobile structures, whereas in disassembling adhesions or regions of adhesions, the equilibrium is due to exchange of large aggregates. Scanning fluctuation correlation spectroscopy and raster-scan image correlation spectroscopy analysis of laser confocal images show that the environments within adhesions are heterogeneous. Relatively large adhesions appear to slide transversally due to a treadmilling mechanism through the addition of monomeric paxillin at one side and removal of relatively large aggregates of proteins from the retracting edge. Total internal reflection microscopy performed with a fast acquisition EM-CCD camera completes the overall dynamic picture and adds details of the heterogeneous dynamics across single adhesions and simultaneous bursts of activity at many adhesions across the cell.
桩蛋白是一种参与粘着斑组装的衔接分子。我们使用不同的荧光涨落方法确定,桩蛋白 - 增强绿色荧光蛋白(paxillin - EGFP)在细胞内从毫秒到秒的多个时间尺度上都是动态的。通过单点涨落相关光谱和光子计数直方图分析确定,在远离粘着斑的细胞质区域,桩蛋白以单体形式均匀分布并自由扩散。在粘着斑附近,桩蛋白的动力学显著降低,推测是由于与粘着斑内的蛋白质伴侣结合。对涨落幅度的光子计数直方图分析表明,新形成或正在组装的粘着斑中的这种结合平衡是由于桩蛋白单体与准固定结构结合,而在正在解体的粘着斑或粘着斑区域,平衡是由于大聚集体的交换。激光共聚焦图像的扫描涨落相关光谱和光栅扫描图像相关光谱分析表明,粘着斑内的环境是异质的。相对较大的粘着斑似乎通过一种踏车机制横向滑动,即在一侧添加单体桩蛋白,同时从回缩边缘去除相对较大的蛋白质聚集体。使用快速采集的电子倍增电荷耦合器件(EM - CCD)相机进行的全内反射显微镜观察完善了整体动态图像,并增加了单个粘着斑异质动力学以及细胞内多个粘着斑同时爆发活动的细节。