Han, Y.

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.