Miller, Helen Louise, Cosgrove, Jason, Wollman, Adam orcid.org/0000-0002-5501-8131 et al. (5 more authors) (2018) High-speed single-molecule tracking of CXCL13 in the B-Follicle. Frontiers in immunology. 1073. p. 1073. ISSN 1664-3224
Abstract
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.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | Funding Information: This work was supported by the Biological Physical Sciences Institute (BPSI), MRC grants MR/K01580X/1 (to PT and ML), MC_PC_15073 (MC and ML), and BBSRC grant BB/N006453/1 (AW and ML). JC is supported by a studentship from the Wellcome Trust 4-year PhD programme (WT095024MA): Combating Infectious Disease: Computational Approaches in Translation Science. AW was supported by the Wellcome Trust [ref: 204829] through the Centre for Future Health (CFH) at the University of York, UK. The authors thank Jo Marrison and Andrew Leech (Bioscience Technology Facility, University of York) for technical assistance with FCS and FRAP microscopy, and for SEC-MALLs, respectively, Chris Power (Carl Zeiss Microscopy) for help with FCS, and Anne Theury for providing lymph node tissue sections. Publisher Copyright: © 2018 Miller, Cosgrove, Wollman, Taylor, Zhou, O'Toole, Coles and Leake. |
Keywords: | Algorithms,B-Lymphocytes/immunology,Biomarkers,Cell Tracking/methods,Chemokine CCL19/genetics,Chemokine CXCL13/genetics,Collagen/metabolism,Humans,Image Processing, Computer-Assisted,Lymph Nodes/metabolism,Single Molecule Imaging/methods,Spectrometry, Fluorescence/methods |
Dates: |
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Institution: | The University of York |
Academic Units: | The University of York > Faculty of Sciences (York) > Health Sciences (York) The University of York > Faculty of Sciences (York) > Biology (York) The University of York > Faculty of Sciences (York) > Physics (York) The University of York > Faculty of Sciences (York) > Centre for Immunology and Infection (CII) (York) |
Depositing User: | Pure (York) |
Date Deposited: | 02 May 2018 08:50 |
Last Modified: | 04 Dec 2024 00:14 |
Published Version: | https://doi.org/10.3389/fimmu.2018.01073 |
Status: | Published |
Refereed: | Yes |
Identification Number: | 10.3389/fimmu.2018.01073 |
Related URLs: | |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:130321 |
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