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High-speed single-molecule tracking of CXCL13 in the B-Follicle

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Publication details

JournalFrontiers in immunology
DateAccepted/In press - 30 Apr 2018
DateE-pub ahead of print - 22 May 2018
DatePublished (current) - 22 May 2018
Number of pages15
Early online date22/05/18
Original languageEnglish


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
sub-millisecond 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 µm2s-1 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 a 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.

Bibliographical note

© 2018 Miller, Cosgrove, Wollman, Taylor, Zhou, O’Toole, Coles and Leake.

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