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

Abstract

Fibronectin (Fn1) is an essential ECM glycoprotein important for embryonic development and homeostasis. The functions of Fn1 in regulating cell fate decisions, morphogenesis and cellular responses to injury are intimately linked to the process of Fn1 fibrillogenesis. Therefore, understanding the mechanisms by which Fn1 proteins assemble into fibrils is necessary to gain insights into diverse functions of Fn1. Using CRISPR/Cas9 mutagenesis, we generated mice and cell lines wherein a sequence encoding a fluorescent protein (FP) was knocked into the Fn1 locus replacing the termination codon, resulting in the expression of Fn1-FP proteins subject to endogenous regulation. Live imaging and super-resolution microscopy revealed that Fn1 fibrils are not continuous fibers as was thought before, instead, they are comprised of a discontinuous array of small nanodomains. Live imaging showed that Fn1 nanodomains are mobile and that they become arranged into progressively longer linear arrays as they move toward the nucleus in parallel with the rearward actin flow. The organization of Fn1 nanodomains into linear fibrillar arrays but not the formation of Fn1 nanodomains is regulated by the interactions mediated by the Fn1 N-terminal assembly domain. The nanodomain architecture of Fn1 fibrils is observed in multiple contexts: in three-dimensional ECM in vivo, on substrata of different composition and stiffness, and is retained when the linkage of Fn1 fibrils to cells is disrupted. The modular assembly and structure of Fn1 fibrils bears important implications for mechanisms of ECM remodeling and signal transduction.

Initiation of fibronectin fibrillogenesis is an enzyme-dependent process

“…Our previous finding that members of the Lysyl oxidase enzyme family bind FN, that LOX-Like 3 (LOXL3) oxidizes it, and that its enzymatic activity induces FN-dependent integrin activation 23 , raised the possibility of an enzyme-dependent initiation of fibrillogenesis , a force-independent mechanism which relies on enzymes and their substrates’ availability. 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 .…”

“…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 647-labelled 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).…”

“…MEF FN-GFP were described previously 24 . MEF Paxillin-GFP cells were kindly provided by B. Geiger (Weizmann institute of Science, Israel).…”

Collagen Assembly at the Cell Surface: Dogmas Revisited

“…With improved resolution of microscopic techniques, we will also be able to obtain more detailed information. Use of super-resolution microscopy has recently shown that the FN fibrils are not continuous but consist of nanodomains [ 156 ]. As we learn more, it is likely that the unexpected will continue to surprise us.…”