A new mechanism of fibronectin fibril assembly revealed by live imaging and super-resolution microscopy

Abstract

Fn1 fibrils have long been viewed as continuous fibers composed of extended, periodically aligned Fn1 molecules. However, our live imaging and single-molecule localization microscopy (SMLM) are inconsistent with this traditional view and show Fn1 fibrils composed of roughly spherical nanodomains containing 6-11 Fn1 dimers. As they move toward the cell center, Fn1 nanodomains become organized into linear arrays, wherein nanodomains are spaced at the average periodicity of 105±17 nm. Periodical Fn1 nanodomain arrays can be visualized between cells in culture and within tissues; they are resistant to deoxycholate treatment and retain nanodomain periodicity in the absence of cells. The nanodomain periodicity in fibrils remained constant when probed with antibodies recognizing distinct Fn1 epitopes or combinations of antibodies recognizing epitopes spanning the length of Fn1. FUD, a peptide that binds Fn1 N-terminus and disrupts Fn1 fibrillogenesis, blocks the organization of Fn1 nanodomains into periodical arrays. These studies establish a new paradigm of Fn1 fibrillogenesis.

Initiation of fibronectin fibrillogenesis is an enzyme-dependent process

“…In line with this notion, we found that in cultured cells expressing GFP-tagged FN 24 , FN fibrils are observed under β1-integrin adhesions and LoxL3 is distributed at nascent adhesion sites (Figure 1A). Notably, LoxL3 is expressed in neural crest cells (Figure S1A) and following its deletion (LoxL3 Δ/Δ ) the mutant embryos display a cleft palate phenotype and reduced neural crest cell numbers at branchial arches (Figure S1B-H), both of which are FN-associated phenotypes 25,26 .…”

“…Cell culture: MEF FN-GFP were described previously 24 Adhesion, cell area and fibronectin analysis: All of the adhesion and fibronectin fibrils analyses shown in this paper were performed using the trainable Weka segmentation plugin 45 of ImageJ (NIH) software.…”

“…To explore whether these mutations affect also FN fibrillogenesis, we first tested whether cells fibrilize their own secreted FN. To that end, we coated coverslips with Alexa 647labelled FN and cultured MEF FN-GFP (in which GFP is knocked-in to both alleles of the FN gene 24 ) for 48 hours, followed by imaging of the FN fibrils. All FN fibrils we observed were GFP labelled, demonstrating that cells use their own secreted FN in forming the initial fibrils (Figure S3B-C).…”

Robust and fast multicolor Single Molecule Localization Microscopy using spectral separation and demixing

“…Yet for the two optimized strategies we tested here, 2-color SD-STORM is not slower than S2C-PAINT, because the loss in maximum density is more than compensated by the faster blinking and lower exposure times possible with STORM compared to PAINT. For 2-color acquisitions, we thus would favor SD-STORM over S2C-PAINT as it is compatible with initial observation before super-resolved acquisition, faster, straightforward to implement with a commercially available setup (Jackson et al, 2022; Tomer et al, 2022; Gazzola et al, 2023) and readily extensible to 3-color imaging. A key advantage of S2C-PAINT in the future is that implementing successive rounds of PAINT imaging would provide a straightforward way to extend it to multiplexing with 4 or more channels, with a significant gain in speed compared to single-color Exchange PAINT.…”

Assessing crosstalk in simultaneous multicolor single-molecule localization microscopy

“…Yet for the two optimized strategies we tested here, two-color SD-dSTORM is not slower than S2C-DNA-PAINT, because the loss in maximum density is more than compensated by the faster blinking and lower exposure times possible with dSTORM compared with DNA-PAINT ( Table S1 ). For two-color acquisitions, we thus would favor SD-dSTORM over S2C-DNA-PAINT as it is compatible with initial observation before super-resolved acquisition, faster, straightforward to implement with a commercially available setup, 56 , 57 , 58 and readily extensible to three-color imaging. A key advantage of S2C-DNA-PAINT in the future is that implementing successive rounds of PAINT imaging would provide a straightforward way to extend it to multiplexing with four or more channels, with the possibility of combining it with recent faster DNA-PAINT approaches 16 , 59 for a significant gain in speed compared with single-color exchange-PAINT.…”