Phospholipids are major components of biological membranes. They play an essential role in intracellular signaling and organelle dynamics; however, the availability of suitable lipid-specific probes is limited, which has hindered studies on their spatial distribution and functional dynamics in living cells. Previously, we demonstrated that octadecyl rhodamine B chloride (R18) is transported to the endoplasmic reticulum via nonvesicular membrane transport. In this study, we showed that R18 is internalized in a phosphatidylethanolamine (PE)-dependent manner in vivo. The internalization of R18 in Saccharomyces cerevisiae is blocked in PE-deficient mutants, but restored by ethanolamine supplementation, which suggests strict PE dependence. Moreover, R18 delivered to vacuoles through autophagy was not terminally retained, but underwent Pep4- and Atg15-dependent export from the vacuoles. The exported R18 was then redirected to endosomes following prolonged autophagy. These results suggest that R18 may serve as an indicator of PE dynamics and vacuole–endosome lipid transport, which contributes to lipid homeostasis inside vacuoles.

Key words: autophagy, in vivo lipid dynamics, octadecyl rhodamine B (R18), phospholipase, phospholipid, vacuole, yeast

During early embryogenesis, gastrulation occurs within a specific region of the pluripotent epiblast, where cells undergo significant changes in their context. The induction of these cellular transformations in particular cell populations suggests the involvement of non-diffusible factors that activate signaling pathways in a spatiotemporal manner. Syntaxin4 (Stx4), a type IV membrane protein that functions as an intravesicular fusion mediator, often translocates across membranes to perform a latent extracellular role in locally regulating cellular behaviors. Through the culture of mouse embryonic egg cylinders isolated from E6.0 embryos and embryonic stem cells (ESCs), we demonstrate that the membrane translocation of Stx4 may play a crucial role in this early stage of development. Using membrane-impermeable antagonistic peptides against extracellular Stx4, along with several small-molecule inhibitors and activators, we found that cells with extracellular Stx4 deactivate focal adhesion kinase (FAK), which then impacts AKT/PI3K signaling and results in increased expression of P-cadherin, ultimately inducing the expression of the gastrulation marker brachyury. Activation of this signaling pathway also triggers Rho/ROCK signaling in ESCs, leading to morphological changes. These findings offer important insights into gastrulation by shedding light on the molecular mechanisms that initiate the spatiotemporal changes in the uniform pluripotent cell sheet.

Key words: gastrulation, FAK, P-cadherin, Rho/ROCK, membrane flip