Jaqaman Khuloud, Galbraith James A, Davidson Michael W, Galbraith Catherine G
Department of Biophysics, UT Southwestern Medical Center, Dallas, TX 75390-8816
OHSU Center for Spatial Systems Biomedicine and Department of Biomedical Engineering, Portland, OR 97201-5042
Mol Biol Cell. 2016 May 15;27(10):1561-9. doi: 10.1091/mbc.E16-01-0018. Epub 2016 Mar 23.
Recent advances in light microscopy permit visualization of the behavior of individual molecules within dense macromolecular ensembles in live cells. It is now conceptually possible to relate the dynamic organization of molecular machinery to cellular function. However, inherent heterogeneities, as well as disparities between spatial and temporal scales, pose substantial challenges in deriving such a relationship. New approaches are required to link discrete single-molecule behavior with continuous cellular-level processes. Here we combined intercalated molecular and cellular imaging with a computational framework to detect reproducible transient changes in the behavior of individual molecules that are linked to cellular behaviors. Applying our approach to integrin transmembrane receptors revealed a spatial density gradient underlying characteristic molecular density increases and mobility decreases, indicating the subsequent onset of local protrusive activity. Integrin mutants further revealed that these density and mobility transients are separable and depend on different binding domains within the integrin cytoplasmic tail. Our approach provides a generalizable paradigm for dissecting dynamic spatiotemporal molecular behaviors linked to local cellular events.
光学显微镜技术的最新进展使得在活细胞中密集的大分子聚集体内可视化单个分子的行为成为可能。从概念上讲，现在可以将分子机器的动态组织与细胞功能联系起来。然而，固有的异质性以及空间和时间尺度之间的差异，在推导这种关系时带来了重大挑战。需要新的方法来将离散的单分子行为与连续的细胞水平过程联系起来。在这里，我们将插入式分子和细胞成像与一个计算框架相结合，以检测与细胞行为相关的单个分子行为中可重复的瞬态变化。将我们的方法应用于整合素跨膜受体，揭示了特征性分子密度增加和流动性降低背后的空间密度梯度，表明随后局部突出活动的开始。整合素突变体进一步揭示，这些密度和流动性瞬变是可分离的，并且取决于整合素细胞质尾部内的不同结合域。我们的方法为剖析与局部细胞事件相关的动态时空分子行为提供了一个可推广的范例。
