Paxillin dynamics measured during adhesion assembly and disassembly by correlation spectroscopy
Paxillin dynamics measured during adhesion assembly and disassembly by correlation spectroscopy
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DOI：
10.1529/biophysj.107.104984
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发表时间：
2008-04-01
影响因子：
3.4
通讯作者：
Gratton, Enrico
中科院分区：
文献类型：
--
作者：
Digman, Michelle A.;Brown, Claire M.;Gratton, Enrico
桩蛋白是参与粘着斑组装的衔接分子。使用不同的荧光波动方法，我们确定paxillin-EGFP在细胞内的许多时间尺度上是动态的，从毫秒到秒。在细胞质区域，远离粘连，桩蛋白是均匀分布和自由扩散的单体，确定由单点波动相关光谱和光子计数直方图分析。在粘连附近，桩蛋白动力学急剧降低，可能是由于与粘连内的蛋白质伴侣结合。的波动幅度的光子计数直方图分析表明，这种结合平衡在新的或组装的粘连是由于桩蛋白单体结合到准固定结构，而在拆卸粘连或粘连区域，平衡是由于交换大的聚集体。激光共聚焦图像的扫描起伏相关光谱和光栅扫描图像相关光谱分析表明，粘连内的环境是异质性的。相对较大的粘连似乎横向滑动，这是由于通过在一侧添加单体桩蛋白并从回缩边缘去除相对较大的蛋白质聚集体而形成的双研磨机制。全内反射显微镜与快速采集EM-CCD相机完成整体动态图片，并增加了跨单个粘连和同时爆发的活动在许多粘连在整个细胞的异质动态的细节。
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.