School of Biomedical Engineering, McMaster University, Hamilton, ON, Canada.
Department of Physics and Astronomy, McMaster University, Hamilton, ON, Canada.
Sci Rep. 2020 Nov 9;10(1):19375. doi: 10.1038/s41598-020-76272-2.
The diffusion of membrane receptors is central to many biological processes, such as signal transduction, molecule translocation, and ion transport, among others; consequently, several advanced fluorescence microscopy techniques have been developed to measure membrane receptor mobility within live cells. The membrane-anchored receptor cluster of differentiation 14 (CD14) and the transmembrane toll-like receptor 2 (TLR2) are important receptors in the plasma membrane of macrophages that activate the intracellular signaling cascade in response to pathogenic stimuli. The aim of the present work was to compare the diffusion coefficients of CD14 and TLR2 on the apical and basal membranes of macrophages using two fluorescence-based methods: raster image correlation spectroscopy (RICS) and single particle tracking (SPT). In the basal membrane, the diffusion coefficients obtained from SPT and RICS were found to be comparable and revealed significantly faster diffusion of CD14 compared with TLR2. In addition, RICS showed that the diffusion of both receptors was significantly faster in the apical membrane than in the basal membrane, suggesting diffusion hindrance by the adhesion of the cells to the substrate. This finding highlights the importance of selecting the appropriate membrane (i.e., basal or apical) and corresponding method when measuring receptor diffusion in live cells. Accurately knowing the diffusion coefficient of two macrophage receptors involved in the response to pathogen insults will facilitate the study of changes that occur in signaling in these cells as a result of aging and disease.
膜受体的扩散对于许多生物过程至关重要，例如信号转导、分子易位和离子转运等；因此，已经开发了几种先进的荧光显微镜技术来测量活细胞内膜受体的流动性。膜锚定的分化抗原 14（CD14）和跨膜 toll 样受体 2（TLR2）是巨噬细胞质膜中重要的受体，它们可以在受到病原体刺激时激活细胞内信号级联反应。本研究的目的是使用两种基于荧光的方法：光栅图像相关光谱（RICS）和单颗粒跟踪（SPT）来比较巨噬细胞顶膜和底膜上 CD14 和 TLR2 的扩散系数。在底膜中，SPT 和 RICS 获得的扩散系数相当，并且发现 CD14 的扩散速度明显快于 TLR2。此外，RICS 表明，两种受体在顶膜中的扩散速度明显快于底膜，这表明细胞与底物的黏附阻碍了扩散。这一发现强调了在活细胞中测量受体扩散时选择适当膜（即顶膜或底膜）和相应方法的重要性。准确了解参与对病原体侵袭反应的两种巨噬细胞受体的扩散系数将有助于研究由于衰老和疾病导致这些细胞中信号转导发生的变化。
