Significance Ras small GTPases including ARFs act as molecular switches to modulate signaling pathways involved in hematopoiesis. Decreased guanine nucleotide exchange factor activity or increased GTPase-activating protein activation are associated with many leukemias, myelodysplastic syndromes, and myeloproliferative disorders. Drosophila is a good model to address questions related to aberrant hematopoiesis. Using Drosophila genetics and gene expression analysis we show tissue-specific function of the ubiquitously expressed endocytic protein, Drosophila ARF1 by interaction of ARF1–GTP with a blood-cell–expressed endocytic protein Asrij. The ARF1–Asrij axis brings about endosomal regulation of multiple signaling pathways in hematopoiesis.

SummaryEndocytosis is implicated in the maintenance of embryonic stem cell (ESC) pluripotency, although its exact role and the identity of molecular players remain poorly understood. Here, we show that the clathrin heavy chain (CLTC), involved in clathrin-mediated endocytosis (CME), is vital for maintaining mouse ESC (mESC) pluripotency. Knockdown of Cltc resulted in a loss of pluripotency accompanied by reduced E-cadherin (E-CAD) levels and increased levels of transforming growth factor β (TGF-β) and extracellular signal-regulated kinase (ERK) signaling. We demonstrate that both E-CAD and TGF-β receptor type 1 (TGF-βR1) are internalized through CME in mESCs. While E-CAD is recycled, TGF-βR1 is targeted for lysosomal degradation thus maintaining inverse levels of these molecules. Finally, we show that E-CAD interacts with ERK, and that the decreased pluripotency upon CME loss can be rescued by inhibiting TGF-βR, MEK, and GSK3β, or overexpressing E-CAD. Our results demonstrate that CME is critical for balancing signaling outputs to regulate ESC pluripotency, and possibly cell fate choices in early development.

Cell fate determination in the early mammalian embryo is regulated by multiple mechanisms. Recently, genes involved in vesicular trafficking have been shown to play an important role in cell fate choice, although the regulation of their expression remains poorly understood. Here we demonstrate for the first time that multiple endocytosis associated genes (EAGs) are repressed through a novel, dual mechanism in mouse embryonic stem cells (mESCs). This involves the action of the Polycomb Repressive Complex, PRC2, as well as post-transcriptional regulation by the ESC-specific cell cycle-regulating (ESCC) family of microRNAs. This repression is relieved upon differentiation. Forced expression of EAGs in mESCs results in a decrease in pluripotency, highlighting the importance of dual repression in cell fate regulation. We propose that endocytosis is critical for cell fate choice, and dual repression may function to tightly regulate levels of endocytic genes.

Mouse embryonic stem cells (mESCs) display unique mechanical properties, including low cellular stiffness in contrast to differentiated cells which are stiffer. We have previously shown that mESCs lacking the clathrin heavy chain (Cltc), an essential component for clathrin-mediated endocytosis (CME), display a loss of pluripotency and an enhanced expression of differentiation markers. However, it is not known whether physical properties such as cellular stiffness also change upon loss of Cltc, similar to what is seen in differentiated cells, and if so, how these altered properties specifically impact pluripotency. Using atomic force microscopy (AFM), we demonstrate that mESCs lacking Cltc display higher Young’s modulus, indicative of greater cellular stiffness, in comparison to wild-type mESCs. The increase in stiffness was accompanied by the presence of actin stress fibres and accumulation of the inactive, phosphorylated, actin binding protein COFILIN. Treatment of Cltc knockdown mESCs with actin polymerization inhibitors resulted in a decrease in the Young’s modulus to values similar to those obtained with WT mESCs. However, a rescue in the expression profile of pluripotency factors was not obtained. Additionally, while WT mouse embryonic fibroblasts could be reprogrammed to a state of pluripotency, this was inhibited in the absence of Cltc. This indicates that the presence of active CME is essential for the pluripotency of embryonic stem cells. Additionally, while physical properties may serve as a simple readout of the cellular state, they may not always faithfully recapitulate the underlying molecular fate.