High-Speed Single-Molecule Tracking of CXCL13 in the B-Follicle

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 is thus a challenging imaging frontier, re...

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Vydáno v:Frontiers in immunology Ročník 9; s. 1073
Hlavní autoři: Miller, Helen, Cosgrove, Jason, Wollman, Adam J. M., Taylor, Emily, Zhou, Zhaokun, O’Toole, Peter J., Coles, Mark C., Leake, Mark C.
Médium: Journal Article
Jazyk:angličtina
Vydáno: Switzerland Frontiers Media S.A 22.05.2018
Témata:
Algorithms
B-Lymphocytes - immunology
B-Lymphocytes - metabolism
Biomarkers
biophysics
Cell Tracking - methods
Chemokine CCL19 - genetics
Chemokine CCL19 - metabolism
Chemokine CXCL13 - genetics
Chemokine CXCL13 - metabolism
chemokines
Collagen - metabolism
Humans
Image Processing, Computer-Assisted
Immunology
Lymph Nodes - metabolism
lymphoid tissues
Single Molecule Imaging - methods
single-molecule imaging
single-molecule tracking
Spectrometry, Fluorescence - methods
single-molecule tracking
chemokines
biophysics
single-molecule imaging
lymphoid tissues
ISSN:1664-3224, 1664-3224
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Shrnutí: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 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 tissue samples and submillisecond sampling in controlled 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 µm s , respectively. To better understand mobility behaviors , 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 µm s , 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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Specialty section: This article was submitted to Cytokines and Soluble Mediators in Immunity, a section of the journal Frontiers in Immunology
Reviewed by: Hideki Nakano, National Institute of Environmental Health Sciences (NIEHS), United States; Gerhard J. Schütz, Technische Universität Wien, Austria
Edited by: Jorge Bernardino De La Serna, Science and Technology Facilities Council, United Kingdom
Co-first authors
ISSN:1664-3224
1664-3224
DOI:10.3389/fimmu.2018.01073