Ghelfi, Elisa

Summary Physical forces shape cell behavior, yet how they integrate with signaling to control fate and disease remains unclear. The alveolar epithelium is patterned by FGF signaling and mechanical stretch, but how these cues specify AT1 and AT2 cells is poorly understood. Here we show that cell membrane tension (CMT) is a conserved regulator of epithelial fate in mouse and human lungs. CMT drops before differentiation and is spatially patterned, defining where bipotent progenitors acquire AT1 or AT2 identity. Lower CMT enhances FGFR2 endocytosis and ERK signaling to drive AT2 differentiation and permits architectural remodeling that enables stretch-mediated YAP/TAZ nuclear entry for AT1 maturation. β-catenin elevates CMT cell-intrinsically independent of its role in canonical WNT transcription, while embedding, osmotic compression, or fibroblast wrapping elevate CMT extrinsically. Combined intrinsic and extrinsic tension traps alveolar epithelial cells in a KRT8⁺ transitional state seen in fibrotic lungs. Membrane tension thus integrates physical and molecular cues linking morphogenesis to fibrosis. Graphical Abstract In Brief We identify a conserved drop in cell membrane tension (CMT) that gates epithelial fate transitions across mouse and human lungs. In bipotent progenitors, reduced CMT promotes Alveolar Type 2 (AT2) fate via FGFR2 endocytosis and ERK signaling, as well as Alveolar Type 1 (AT1) fate by enabling architectural remodeling required for YAP/TAZ nuclear entry. We further show that intrinsic β-catenin and extrinsic confinement cues, including mesenchymal contact, converge to elevate CMT, restricting differentiation and—in adult AT2s—inducing a KRT8⁺ transitional state associated with fibrosis. Highlights Cell membrane tension drops before differentiation and is required for AT1 and AT2 fate acquisition. Reduced tension enhances FGFR2 endocytosis and ERK signaling to drive AT2 specification. Reduced tension permits architectural remodeling and YAP/TAZ nuclear entry required for AT1 maturation. Intrinsic β-catenin and extrinsic confinement elevate CMT to restrict differentiation and together strongly induce a KRT8⁺ transitional state associated with fibrosis.

Abstract RATIONALE: The alveolar epithelium is primarily comprised of two cell types – the flat alveolar type I (AT1) cell responsible for gas exchange, and the cuboidal AT2 cell responsible for surfactant secretion, alveolar defense and acts as facultative stem cells in injury repair. Both cell types emerge from a population of bipotent distal progenitors (DPs). While the FGF signaling involved in directing AT2 fate has been identified, the mechanism by which AT2 differentiation is precisely timed is not well understood. In embryonic stem cells, it has been demonstrated that differentiation is regulated by a biophysical parameter, cell membrane tension (CMT), that acts upstream of FGF signaling by suppressing receptor endocytosis. Given this, we sought to investigate whether and how CMT plays a role in timing the alveolar epithelial differentiation, particularly focusing on how CMT might regulate FGF signaling. METHODS: DPs were isolated from E15.5 mice and plated on top of or embedded in Matrigel. DPs were treated with small molecules, to rise CMT (CHIR22901 and Methyl Beta cyclodextrin; MBCD), inhibiting either endocytosis (Dynasore) or pathways downstream of FGF signaling (FR180204, Akt VIII, KRpep-2d, NSC 74859). CMT is measured both with optical trap technology and FlipTR dye, which uses fluorescence lifetime imaging microscopy. Tissues and cells were immunostained and imaged using markers for AT1 and AT2 cells, Fgfr2, and the endocytosis marker EEA1. The transgenic mice for this project are in a C57B6 strain background and include a pan-lung epithelial Cre (TgNkx2.1-Cre) bred to Cre-dependent ERK reporter (Rosa26SAE). RESULTS: We found that CMT reduces immediately prior to alveolar differentiation, and that increasing CMT with CHIR and MBCD blocks differentiation of DPs in culture. As CMT reduces, we observe an increase in endocytosis and internalization of Fgfr2 in both cultured cells and in vivo at E17.5. Further, scRNAseq analysis showed an upregulation of ERK target genes and we have validated higher ERK activity in nAT2s in the reporter line. Further, we validated that CMT regulates FGF signaling, as decrease in CMT results in more Fgfr2 endocytosis that likely activates ERK and Akt signaling. Our small molecule inhibition data suggests that ERK and Akt signaling are required for AT2 fate selection and we propose that they are the downstream targets of FGF signaling that are activated following lowered CMT and increased Fgfr2 endocytosis. Our findings are of fundamental importance for understanding how cell mechanics regulates biochemical signaling and fate selection during lung development.

Abstract RATIONALE: Alveoli form late in development and, for first breath, alveolar type 1 (AT1) as well as AT2 epithelial cell types must be functional. While prior studies have mapped the cellular and transcriptional programs underlying this process and identified AT fate inductive cues, questions remain as to the timing and regulation of their emergence. Thus, we sought to identify when, where, and how AT2 cells first arise and motivated by recent work in embryonic stem cells, we investigated whether the biophysical parameter cell membrane tension (CMT) plays a role. METHODS: Single-cell RNA-sequencing (scRNAseq) datasets sampling mouse lung development were processed using cluster correlation, velocity, and gene ontology analysis. Marker expression and AT2 morphology was assessed by immunostaining, and their migration confirmed by time-lapse confocal microscopy on embryonic precision-cut lung slices (PCLS). CMT was measured via optical tweezers (OT) on isolated cells as well as fluorescence lifetime (FLIM) on organoids and PCLS. Progenitors (E15.5) and AT2s (E16.5- >PN50) were isolated from distal region of the mouse lung, as well as human AT2s recently isolated, expanded, and banked in Mayo Clinic's recently established patient lung cell biorepository. CMT was raised using methyl-β-cyclodextran (MBCD) or polyethylene glycol (PEG). Small molecules CHIR and ICG-001 were used in culture to raise and lower β-catenin levels, respectively. Fluorescently labeled mesenchymal cells were isolated, expanded, and added to organoids in suspension for co-association experiments. RESULTS: No evidence of distal progenitors selecting either AT fate was found until E16.5, at which time we identified a distinct nascent AT2 (nAT2) state that ontology analysis detects upregulation of single-cell migration genes. Resistin-like molecule alpha (RELM-α), a type 2 inflammation regulator, labels nAT2s – which first individually emerged around E16.5 (although instances exist at E15.5) at intermediate zones between the branch tips and stalks of the distal epithelium. Following molecular emergence, we observed nAT2s extruded basally and can subsequently interact with nearby but anatomically distinct lumens, a behavior we provisionally name interlumenal junctioning and whose dynamics we validated by timelapse microscopy. Regarding CMT, we found its reduction in distal progenitors occurs prior to, and is required for, differentiation. We observed CMT reduction is sensitive to the manner of organoid culture, with on top culture resulting in more reduction and differentiation. Finally, we observed CMT is regulated cell-intrinsically via the adaptor protein β-catenin, as well as -extrinsically through osmotic (via PEG) or mesenchymal cell interactions, both of which reduce membrane fluidity.

Abstract RATIONALE: The alveolar epithelium consists of two cell types (AT2 and AT1) derived from a common Distal Progenitor (DP). Understanding how the epithelium of alveoli functions and becomes differentiated is essential for treatment of lung diseases. One possible regulator of cell fate is cell membrane tension (CMT), a biophysical property measured by the energy required to deform the membrane and known to be an important regulator of cell movement and endocytic processes. AT1 fate selection is largely driven and reinforced by YAP (Yes-associated protein), a mechano-sensitive transcription factor gated from entering the nucleus by the size of nuclear pores, which increases upon stretch. Here, we investigated whether and how CMT regulates the transduction of stretch-dependent YAP signaling and the remodeling into AT1 cells. METHODS: DPs were isolated from E15.5 mice and cultured on Matrigel for use in differentiation experiments. Immunostaining for AT1 and AT2 markers, Phalloidin, DAPI and YAP were used to assess YAP localization and architectural changes. We generated transgenic mice containing a pan-epithelial Cre (TgNkx2.1-Cre), a Cre-dependent driver of ltta (TigreAi140), and a TetOff regulated construct expressing YAP1 (Rosa26teto-Yap1), resulting in constitutive Yap1 expression in Cre-expressing DPs. CMT is measured both with optical trap technology and with the use of FlipTR dye, which uses Fluorescence Lifetime Imaging Microscopy (FLIM) to determine CMT. RESULTS: To demonstrate the role of CMT in timing AT1 fate, we found that CMT lowers in DPs concomitantly with differentiation, and that flattening cells (likely AT1s) have even lower CMT than round cells (likely AT2s). Using organotypic culture, we found that raising CMT with MBCD, PEG, or Matrigel embedding largely stops differentiation of DPs. Surprisingly, even when YAP is overdriven in mice in E15.5 distal progenitors, early precocious AT1 differentiation is not observed, suggesting YAP signaling is not alone sufficient to drive AT1 fate selection in DPs with higher CMT. We reproduced this finding when culturing DPs under high CMT conditions-promoting high YAP/TAZ levels via LATS inhibition was also not sufficient to select for AT1 fate. Finally, we found that high CMT disrupts nuclear YAP localization, nuclear-actin attachment, and cytoskeletal remodeling that is typically when DPs remodel towards AT1 fate. We propose that high baseline CMT in DPs prevents cellular remodeling, specifically that it suppresses the formation of actin-nuclear associations that imbue the nucleus with stretch sensitivity, thereby preventing an increased sensitivity to stretch and thus higher YAP signaling.