Arp2/3 complex activity is necessary for mouse ESC differentiation, times formative pluripotency, and enables lineage specification.

Mouse embryonic stem cells (mESCs), a model for differentiation into primed epiblast-like cells (EpiLCs), have revealed transcriptional and epigenetic control of early embryonic development. The control and significance of morphological changes, however, remain less defined. We show marked changes in morphology and actin architectures during differentiation that depend on Arp2/3 complex but not formin activity. Inhibiting Arp2/3 complex activity pharmacologically or genetically does not block exit from naive pluripotency, but attenuates increases in EpiLC markers. We find that inhibiting Arp2/3 complex activity delays formative pluripotency and causes globally defective lineage specification as indicated by RNA sequencing, with significant effects on TBX3-depedendent transcriptional programs. We also identify two previously unreported indicators of mESC differentiation, namely, MRTF and FHL2, which have inverse Arp2/3 complex-dependent nuclear translocation. Our findings on Arp2/3 complex activity in differentiation and the established role of formins in EMT indicate that these two actin nucleators regulate distinct modes of epithelial plasticity.

pHLARE: a new biosensor reveals decreased lysosome pH in cancer cells.

Many lysosome functions are determined by a lumenal pH of ∼5.0, including the activity of resident acid-activated hydrolases. Lysosome pH (pHlys) is often increased in neurodegenerative disorders and predicted to be decreased in cancers, making it a potential target for therapeutics to limit the progression of these diseases. Accurately measuring pHlys, however, is limited by currently used dyes that accumulate in multiple intracellular compartments and cannot be propagated in clonal cells for longitudinal studies or used for in vivo determinations. To resolve this limitation, we developed a genetically encoded ratiometric pHlys biosensor, pHLARE (pH Lysosomal Activity REporter), which localizes predominantly in lysosomes, has a dynamic range of pH 4.0 to 6.5, and can be stably expressed in cells. Using pHLARE we show decreased pHlys with inhibiting activity of the mammalian target of rapamycin complex 1 (mTORC1). Also, cancer cells from different tissue origins have a lower pHlys than untransformed cells, and stably expressing oncogenic RasV12 in untransformed cells is sufficient to decrease pHlys. pHLARE is a new tool to accurately measure pHlys for improved understanding of lysosome dynamics, which is increasingly considered a therapeutic target.

Formin-dependent TGF-ß signaling for epithelial to mesenchymal transition.

The role of distinct actin filament architectures in epithelial plasticity remains incompletely understood. We therefore determined roles for formins and the Arp2/3 complex, which are actin nucleators generating unbranched and branched actin filaments, respectively, in the process of epithelial to mesenchymal transition (EMT). In clonal lung, mammary, and renal epithelial cells, the formin activity inhibitor SMIFH2 but not the Arp2/3 complex activity inhibitor CK666 blocked EMT induced by TGF-ß. SMIFH2 prevented the proximal signal of increased Smad2 phosphorylation and hence also blocked downstream EMT markers, including actin filament remodeling, decreased expression of the adherens junction protein E-cadherin, and increased expression of the matrix protein fibronectin and the transcription factor Snail. The short hairpin RNA silencing of formins DIAPH1 and DIAPH3 but not other formins phenocopied SMIFH2 effects and inhibited Smad2 phosphorylation and changes in Snail and cadherin expression. Formin activity was not necessary for the cell surface expression or dimerization of TGF-ß receptors, or for nuclear translocation of TAZ, a transcription cofactor in Hippo signaling also regulated by TGF-ß. Our findings reveal a previously unrecognized role for formin-dependent actin architectures in proximal TGF-ß signaling that is necessary for Smad2 phosphorylation but not for cross-talk to TAZ.