Number and brightness analysis in live cells reveals that NCAM and FGF2 elicit different assembly and dynamics of FGFR1

Abstract

Both fibroblast growth factor-2 (FGF2) and neural cell adhesion molecule (NCAM) trigger FGF receptor 1 (FGFR1) signaling; however, they induce remarkably distinct receptor trafficking and cellular responses. The molecular basis of such a dichotomy and the role of distinct types of ligand-receptor interaction remain elusive. Number of molecules and brightness (N&B) analysis revealed that FGF2 and NCAM promote different FGFR1 assembly and dynamics at the plasma membrane. NCAM stimulation elicits long-lasting cycles of short-lived FGFR1 monomers and multimers, a behavior that might reflect a rapid FGFR1 internalization and recycling. FGF2, instead, induces stable dimerization at the dose that stimulates cell proliferation. Reducing the occupancy of FGFR1 in response to low FGF2 doses causes a switch towards cyclically exposed and unstable receptor dimers, consistently with previously reported biphasic response to FGF2 and with the divergent signaling elicited by different ligand concentrations. Similar instability was observed upon altering the endocytic pathway. Thus, FGF2 and NCAM induce differential FGFR1 clustering at the cell surface, which might account for the distinct intracellular fate of the receptor and, hence, for the different signaling cascades and cellular responses.

DDR1 autophosphorylation is a result of aggregation into dense clusters

“…Various collagenous ligands are known to induce different degrees of receptor clustering for the platelet collagen receptor GPVI 21 . For other RTKs, ligands that lead to different cellular responses were recently shown to affect the strength of dimers (EGFR) or differential clustering (FGFR1) 22,23 .…”

NCAM1/FGF module serves as a putative pleuropulmonary blastoma therapeutic target

“…Since the antitumorigenic effect of huN901-DM1 has been attributed to its cytotoxic conugate DM1, a member of the maytansinoids mitotic inhibitors 23 , our results probably reflect the killing of NCAM1-high cells by the cytotoxic component rather than direct effect of anti CD56 on downregulation of self-renewal genes including FGF pathway. Nevertheless, due to the known role of NCAM in regulating the FGF pathway through its association with FGFR that results in FGFR recycling at the cell surface 45,46 , sustained signaling 36 , and cancer progression 47 , we believe that downregulation of NCAM1 by the antibody interferes with the NCAM/FGFR interplay which may result in reduction of tumor aggressiveness and metastatic properties as was shown in other cancers 47 . Finally, our work highlights the translational relevance of this model in identifying targetable genes in general and of the anti-NCAM1 strategy in particular, implicating NCAM1 as a therapeutic target.…”

The timing of Start is determined primarily by increased synthesis of the Cln3 activator rather than dilution of the Whi5 inhibitor

“…In their Letter, Schmoller et al (2022) stated that they are not familiar with 2psN&B and thus cannot judge the approach. 2psN&B is a well-established and widely used fluorescence fluctuation-based method for quantitative single-cell imaging (e.g., Nagy et al, 2010;Hellriegel et al, 2011;Moutin et al, 2014;Bourges et al, 2017;Cutrale et al, 2019; Volume 33 May 1, 2022 Cln3 synthesis activates Start | 5 Zamai et al, 2019). 2psN&B can provide absolute protein concentrations, protein complex stoichiometry, and/or dynamic information on diffusion rates and has several advantages over wide-field fluorescence imaging.…”