TOR is a serine-threonine kinase that was originally identified as a target of rapamycin in Saccharomyces cerevisiae and then found to be highly conserved among eukaryotes. In Drosophila melanogaster, inactivation of TOR or its substrate, S6 kinase, results in reduced cell size and embryonic lethality, indicating a critical role for the TOR pathway in cell growth control. However, the in vivo functions of mammalian TOR (mTOR) remain unclear. In this study, we disrupted the kinase domain of mouse mTOR by homologous recombination. While heterozygous mutant mice were normal and fertile, homozygous mutant embryos died shortly after implantation due to impaired cell proliferation in both embryonic and extraembryonic compartments. Homozygous blastocysts looked normal, but their inner cell mass and trophoblast failed to proliferate in vitro. Deletion of the C-terminal six amino acids of mTOR, which are essential for kinase activity, resulted in reduced cell size and proliferation arrest in embryonic stem cells. These data show that mTOR controls both cell size and proliferation in early mouse embryos and embryonic stem cells. TOR (target of rapamycin) was originally identified in two mutantSaccharomyces cerevisiae strains, TOR1-1 and TOR2-1, that are resistant to the growth-inhibiting effect of the immunophilin-immunosuppressant complex FKBP (FK506 binding protein) and rapamycin (17). TOR1 and TOR2 are large proteins (Ϸ280 kDa) and are Ϸ70% identical (26,28 (21, 48). mTOR and other members of this family, including ATM, ATR/FPR, and DNA-PKcs, contain C-terminal regions with high similarity to the catalytic domains of phosphoinositide (PI)-3 kinase and PI-4 kinase (26, 28). However, PIKK members are not lipid kinases but rather function as serine-threonine kinases (4, 20). The PIKK proteins contain a short segment at the extreme C terminus that is essential for protein kinase activity and is not present in PI-3 and PI-4 kinases (51).Cell culture studies have demonstrated that mTOR controls protein synthesis, in part by phosphorylating downstream substrates, including p70 s6 kinase (p70 S6K1 ) (3, 5, 20) and eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E-BP1) (4, 5, 13, 15). p70 S6K phosphorylates the 40S ribosomal protein S6 and is proposed to play a crucial role in the translation of 5Ј-terminal oligopyrimidine tract mRNAs, which primarily encode ribosomal proteins and components of the translation apparatus (22, 23). Phosphorylation of 4E-BP1 disrupts its binding to eIF4E, a protein that binds the 5Ј cap structure of mRNA. Released eIF4E then forms a functional translation initiation complex with eIF4G, eIF4A, and eIF3 ribosomes, enhancing translation (29, 45). Inactivation of 4E-BP1 and family proteins has a profound effect on translation of mRNAs with complex 5Ј untranslated regions, which often encode regulatory proteins such as protooncogenes (45). The recent discoveries of a 150-kDa binding partner of mTOR, named raptor (regulatory-associated protein of mTOR) (14,27), and its Saccharomyces cerevisi...

Recent advances have suggested that direct induction of neural stem cells (NSCs) could provide an alternative to derivation from somatic tissues or pluripotent cells. Here we show direct derivation of stably expandable NSCs from mouse fibroblasts through a curtailed version of reprogramming to pluripotency. By constitutively inducing Sox2, Klf4, and c-Myc while strictly limiting Oct4 activity to the initial phase of reprogramming, we generated neurosphere-like colonies that could be expanded for more than 50 passages and do not depend on sustained expression of the reprogramming factors. These induced neural stem cells (iNSCs) uniformly display morphological and molecular features of NSCs, such as the expression of Nestin, Pax6, and Olig2, and have a genome-wide transcriptional profile similar to that of brain-derived NSCs. Moreover, iNSCs can differentiate into neurons, astrocytes, and oligodendrocytes. Our results demonstrate that functional NSCs can be generated from somatic cells by factor-driven induction.

Cell-penetrating peptides (CPPs) are capable of introducing a wide range of cargoes into living cells. Descriptions of the internalization process vary from energy-independent cell penetration of membranes to endocytic uptake. To elucidate whether the mechanism of entry of CPP constructs might be influenced by the properties of the cargo, we used time lapse confocal microscopy analysis of living mammalian cells to directly compare the uptake of the well-studied CPP TAT fused to a protein (>50 amino acids) or peptide (<50 amino acids) cargo. We also analyzed various constructs for their subcellular distribution and mobility after the internalization event. TAT fusion proteins were taken up largely into cytoplasmic vesicles whereas peptides fused to TAT entered the cell in a rapid manner that was dependent on membrane potential. Despite their accumulation in the nucleolus, photobleaching of TAT fusion peptides revealed their mobility. The bioavailability of internalized TAT peptides was tested and confirmed by the strong inhibitory effect on cell cycle progression of two TAT fusion peptides derived from the tumor suppressor p21(WAF/Cip) and DNA Ligase I measured in living cells.