Abstract
Fibronectin fibrils within the extracellular matrix play central roles in physiological and pathological processes, yet many structural details about their hierarchical and molecular assembly remain unknown. Here we combine site-specific protein labelling with single-molecule localization by stepwise photobleaching or direct stochastic optical reconstruction microscopy (dSTORM), and determine the relative positions of various labelled sites within native matrix fibrils. Single end-labelled fibronectin molecules in fibrils display an average end-to-end distance of ∼133 nm. Sampling of site-specific antibody epitopes along the thinnest fibrils (protofibrils) shows periodic punctate label patterns with ∼95 nm repeats and alternating N- and C-terminal regions. These measurements suggest an antiparallel 30–40 nm overlap between N-termini, suggesting that the first five type I modules bind type III modules of the adjacent molecule. Thicker fibres show random bundling of protofibrils without a well-defined line-up. This super-resolution microscopy approach can be applied to other fibrillar protein assemblies of unknown structure.
Highlights
Fibronectin fibrils within the extracellular matrix play central roles in physiological and pathological processes, yet many structural details about their hierarchical and molecular assembly remain unknown
To investigate the hierarchical structure of Fn fibrils within native extracellular matrix (ECM) by Single-molecule localization microscopy (SMLM), normal human dermal fibroblasts (NHDFs) were cultured on Fn-coated glass coverslips for 17–20 h in medium supplemented with 50 mg ml À 1 plasma Fn (Fig. 1a)
The cell-assembled Fn matrix was mainly composed of plasma Fn (pFn) due to the short culture duration and the excess of pFn in the medium (Supplementary Fig. 1)
Summary
Fibronectin fibrils within the extracellular matrix play central roles in physiological and pathological processes, yet many structural details about their hierarchical and molecular assembly remain unknown. Sampling of site-specific antibody epitopes along the thinnest fibrils (protofibrils) shows periodic punctate label patterns with B95 nm repeats and alternating N- and C-terminal regions. These measurements suggest an antiparallel 30–40 nm overlap between N-termini, suggesting that the first five type I modules bind type III modules of the adjacent molecule. SMLM comprises a number of related ‘super-resolution’ techniques like photoactivated localization microscopy (PALM)[35], stochastic optical reconstruction microscopy (STORM)[36], direct stochastic optical reconstruction microscopy (dSTORM)[37] or stepwise photobleaching[38,39] These utilize different mechanisms to switch the fluorescence of labels on and off over time and sequentially measure the position of single fluorophores. Incomplete and inaccurate labelling, as well as the stochastic nature of most
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