A device that traps cells in micrometer-sized capture cups efficiently achieves cell pairing and subsequent chemically or electrically induced fusion. The authors show that fused NIH 3T3 cells continue to be viable and morphologically normal off the chip, and they also show reprogramming of mouse embryonic fibroblasts after fusion with embryonic stem cells. Cell fusion has been used for many different purposes, including generation of hybridomas and reprogramming of somatic cells. The fusion step is the key event in initiation of these procedures. Standard fusion techniques, however, provide poor and random cell contact, leading to low yields. We present here a microfluidic device to trap and properly pair thousands of cells. Using this device, we paired different cell types, including fibroblasts, mouse embryonic stem cells and myeloma cells, achieving pairing efficiencies up to 70%. The device is compatible with both chemical and electrical fusion protocols. We observed that electrical fusion was more efficient than chemical fusion, with membrane reorganization efficiencies of up to 89%. We achieved greater than 50% properly paired and fused cells over the entire device, fivefold greater than with a commercial electrofusion chamber and observed reprogramming in hybrids between mouse embryonic stem cells and mouse embryonic fibroblasts.

Mice expressing a constitutively active form of RagA are unable to inhibit mTORC1 after birth and to trigger autophagy, and succumb perinatally. The mTOR complex 1 (mTORC1) pathway is a major regulator of growth in eukaryotes and a drug target for common diseases including cancer and neurodegeneration. It is known that mTORC1 senses amino acids through the Rag family of GTPases, but their physiological importance is unknown. David M. Sabatini and colleagues show that following birth, which stops the maternal nutrient supply, mTORC1 is inhibited in mice in a Rag-dependent fashion. This inhibition triggers autophagy, which promotes the release of amino acids needed to sustain plasma glucose levels via gluconeogenesis between birth and suckling. Thus the Rag pathway acts as a general nutrient sensor, and through its regulation of mTORC1, helps maintain nutrient homeostasis and survival in neonates. The mechanistic target of rapamycin complex 1 (mTORC1) pathway regulates organismal growth in response to many environmental cues, including nutrients and growth factors1. Cell-based studies showed that mTORC1 senses amino acids through the RagA–D family of GTPases2,3 (also known as RRAGA, B, C and D), but their importance in mammalian physiology is unknown. Here we generate knock-in mice that express a constitutively active form of RagA (RagAGTP) from its endogenous promoter. RagAGTP/GTP mice develop normally, but fail to survive postnatal day 1. When delivered by Caesarean section, fasted RagAGTP/GTP neonates die almost twice as rapidly as wild-type littermates. Within an hour of birth, wild-type neonates strongly inhibit mTORC1, which coincides with profound hypoglycaemia and a decrease in plasma amino-acid concentrations. In contrast, mTORC1 inhibition does not occur in RagAGTP/GTP neonates, despite identical reductions in blood nutrient amounts. With prolonged fasting, wild-type neonates recover their plasma glucose concentrations, but RagAGTP/GTP mice remain hypoglycaemic until death, despite using glycogen at a faster rate. The glucose homeostasis defect correlates with the inability of fasted RagAGTP/GTP neonates to trigger autophagy and produce amino acids for de novo glucose production. Because profound hypoglycaemia does not inhibit mTORC1 in RagAGTP/GTP neonates, we considered the possibility that the Rag pathway signals glucose as well as amino-acid sufficiency to mTORC1. Indeed, mTORC1 is resistant to glucose deprivation in RagAGTP/GTP fibroblasts, and glucose, like amino acids, controls its recruitment to the lysosomal surface, the site of mTORC1 activation. Thus, the Rag GTPases signal glucose and amino-acid concentrations to mTORC1, and have an unexpectedly key role in neonates in autophagy induction and thus nutrient homeostasis and viability.

Mir-290 through mir-295 ( mir-290–295 ) is a mammalian-specific microRNA (miRNA) cluster that, in mice, is expressed specifically in early embryos and embryonic germ cells. Here, we show that mir-290–295 plays important roles in embryonic development as indicated by the partially penetrant lethality of mutant embryos. In addition, we show that in surviving mir-290–295 -deficient embryos, female but not male fertility is compromised. This impairment in fertility arises from a defect in migrating primordial germ cells and occurs equally in male and female mutant animals. Male mir-290–295 ^−/− mice, due to the extended proliferative lifespan of their germ cells, are able to recover from this initial germ cell loss and are fertile. Female mir-290–295 ^−/− mice are unable to recover and are sterile, due to premature ovarian failure.