High-Speed Single-Molecule Tracking of CXCL13 in the B-Follicle.
High-Speed Single-Molecule Tracking of CXCL13 in the B-Follicle.
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DOI：
10.3389/fimmu.2018.01073
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发表时间：
2018
影响因子：
7.3
通讯作者：
Leake MC
中科院分区：
文献类型：
--
作者：
Miller H;Cosgrove J;Wollman AJM;Taylor E;Zhou Z;O'Toole PJ;Coles MC;Leake MC
可溶性因子是细胞与环境沟通的重要手段。然而，许多分子很容易与细胞外基质成分相互作用，导致多种扩散模式。因此，原位扩散的分子量化是一个具有挑战性的成像前沿，需要非常高的空间和时间分辨率。克服这一方法障碍是了解调节免疫功能的细胞外因子的精确空间模式的关键。为了解决这一问题，我们开发了一种高速光学显微镜系统，能够在体外组织样本中进行毫秒取样，并在受控体外样本中进行亚毫秒取样，以表征一系列复杂微环境中的分子扩散。我们证明，在确定分子迁移率的荧光相关光谱(FCS)和光漂白后荧光恢复(FRAP)用于评估扩散的竞争工具中，该方法优于竞争工具。然后，我们应用这种方法来研究趋化因子CXCL13，它是淋巴组织结构的关键决定因素，以及B细胞介导的免疫。利用荧光标记的CCL19和CXCL13在胶原基质中的超分辨单分子跟踪研究了趋化因子迁移行为的异质性，结果表明这两个分子都有一个固定部分和一个可移动部分，其独特的扩散速率分别为8.4 ± 0.2和6.2 ± 0.3 µM_2s−1。为了更好地了解原位迁移行为，我们分析了CXCL13-AF647在小鼠淋巴结组织切片中的扩散，观察到固定组分和可移动组分，扩散系数为6.6 ± 0.4 µM2−1，这表明卵泡内的迁移也是多峰的。在分子水平的运动行为的定量研究中，我们对CXCL13在卵泡内的生物利用度有了更深入的了解。我们的高速单分子跟踪方法提供了一个新的视角，从这个角度来理解与免疫系统相关的可溶性因子的流动性。
Soluble factors are an essential means of communication between cells and their environment. However, many molecules readily interact with extracellular matrix components, giving rise to multiple modes of diffusion. The molecular quantification of diffusion in situ is thus a challenging imaging frontier, requiring very high spatial and temporal resolution. Overcoming this methodological barrier is key to understanding the precise spatial patterning of the extracellular factors that regulate immune function. To address this, we have developed a high-speed light microscopy system capable of millisecond sampling in ex vivo tissue samples and submillisecond sampling in controlled in vitro samples to characterize molecular diffusion in a range of complex microenvironments. We demonstrate that this method outperforms competing tools for determining molecular mobility of fluorescence correlation spectroscopy (FCS) and fluorescence recovery after photobleaching (FRAP) for evaluation of diffusion. We then apply this approach to study the chemokine CXCL13, a key determinant of lymphoid tissue architecture, and B-cell-mediated immunity. Super-resolution single-molecule tracking of fluorescently labeled CCL19 and CXCL13 in collagen matrix was used to assess the heterogeneity of chemokine mobility behaviors, with results indicating an immobile fraction and a mobile fraction for both molecules, with distinct diffusion rates of 8.4 ± 0.2 and 6.2 ± 0.3 µm2s−1, respectively. To better understand mobility behaviors in situ, we analyzed CXCL13-AF647 diffusion in murine lymph node tissue sections and observed both an immobile fraction and a mobile fraction with an example diffusion coefficient of 6.6 ± 0.4 µm2s−1, suggesting that mobility within the follicle is also multimodal. In quantitatively studying mobility behaviors at the molecular level, we have obtained an increased understanding of CXCL13 bioavailability within the follicle. Our high-speed single-molecule tracking approach affords a novel perspective from which to understand the mobility of soluble factors relevant to the immune system.
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DOI：
10.1073/pnas.73.12.4594
发表时间：
1976-01-01
影响因子：
11.1
作者：
AXELROD, D;RAVDIN, P;PODLESKI, TR
通讯作者：
PODLESKI, TR
影响因子：
8
作者：
Fischer, H;Polikarpov, I;Craievich, AF
通讯作者：
Craievich, AF
影响因子：
56.9
作者：
Balzarotti, Francisco;Eilers, Yvan;Hell, Stefan W.
通讯作者：
Hell, Stefan W.
影响因子：
56.9
作者：
Asbury, CL;Fehr, AN;Block, SM
通讯作者：
Block, SM
影响因子：
2.3
作者：
EHRENBER.M;RIGLER, R
通讯作者：
RIGLER, R