E mbryonic stem (ES) cells are derived from early mammalian embryos and display characteristics of totipotency, i.e., after transfer to a suitable in vivo environment they contribute to the primary germ layers (ectoderm, endoderm, and mesoderm) and populate the germline of mice (1, 2). ES cells can be propagated in an undifferentiated state and genetically manipulated in vitro. Thus, transgenic animals can be generated by introducing foreign genes into ES cells, followed by transplantation of the ES cells into embryos and germ-line transmission.The first reports of genetic manipulation of ES cells demonstrated that vectors derived from retroviruses can infect ES cells and that the integrated virus (provirus) is transmitted through the germline (3, 4). Furthermore, it was shown that retroviral vectors are able to infect preimplantation embryos, giving rise to transgenic animals that transmit the proviral DNA to offspring (5-8). However, further analysis revealed that both infected ES cells and preimplantation embryos lack significant provirus transcription. Two major mechanisms have been identified for retrovirus silencing (see references in ref. 9): trans-acting factors that bind to the viral promoters in the long terminal repeats (LTRs) and methylation of the integrated retroviral genome and flanking host DNA sequences. According to the organization of their genome (for review see ref. 10), one can distinguish simple retroviruses, such as the prototypic murine leukemia virus, from complex retroviruses like the lentiviruses. HIV type 1 (HIV-1) is one of the best-studied complex retroviruses and has the ability to infect nondividing cells presumably by import of the viral DNA through the nuclear pore and subsequent integration into the host genome (references in ref. 11). Vectors derived from lentiviruses can transduce a broad spectrum of terminally differentiated, nondividing cells, as well as, hematopoietic stem cells of multiple mammalian species (references in ref. 11).In this communication, we show that (i) unlike traditional oncoretroviral vectors, expression of transgenes introduced by lentiviral vectors into murine or human ES cells is not silenced.(ii) Transgene expression is not ''shut off'' during differentiation, and the transgene is expressed in multiple tissues of chimeric animals generated by transfer of lentivector-transduced ES cells in blastocysts. (iii) Germ-line transmission of transgenes introduced into ES cells by lentiviral vectors and (iv) preimplantation embryos at morula stage can be successfully transduced with lentiviral vectors, and the resulting progeny express the transgene. We therefore conclude that lentiviral vectors will be excellent tools for generating transgenic animals. Materials and MethodsVirus Production. LV-green fluorescent protein (GFP) was constructed by cloning the CAG promoter into the ClaI and BamHI sites of the vector LV-pGFP (12), thereby replacing the phosphoglycerate kinase (PGK) promoter. LV-Lac was cloned by introducing the LacZ-woodchuck hepatitis virus frag...

Faithful execution of developmental programs relies on the acquisition of unique cell identities from pluripotent progenitors, a process governed by combinatorial inputs from numerous signaling cascades that ultimately dictate lineage-specific transcriptional outputs. Despite growing evidence that metabolism is integrated with many molecular networks, how pathways that control energy homeostasis may affect cell fate decisions is largely unknown. Here, we show that AMP-activated protein kinase (AMPK), a central metabolic regulator, plays critical roles in lineage specification. Although AMPK-deficient embryonic stem cells (ESCs) were normal in the pluripotent state, these cells displayed profound defects upon differentiation, failing to generate chimeric embryos and preferentially adopting an ectodermal fate at the expense of the endoderm during embryoid body (EB) formation. AMPK −/− EBs exhibited reduced levels of Tfeb, a master transcriptional regulator of lysosomes, leading to diminished endolysosomal function. Remarkably, genetic loss of Tfeb also yielded endodermal defects, while AMPK-null ESCs overexpressing this transcription factor normalized their differential potential, revealing an intimate connection between Tfeb/lysosomes and germ layer specification. The compromised endolysosomal system resulting from AMPK or Tfeb inactivation blunted Wnt signaling, while up-regulating this pathway restored expression of endodermal markers. Collectively, these results uncover the AMPK pathway as a novel regulator of cell fate determination during differentiation.

The acute cellular response to stress generates a subpopulation of reversibly stress-tolerant cells under conditions that are lethal to the majority of the population. Stress tolerance is attributed to heterogeneity of gene expression within the population to ensure survival of a minority. We performed whole transcriptome sequencing analyses of metastatic human breast cancer cells subjected to the chemotherapeutic agent paclitaxel at the single-cell and population levels. Here we show that specific transcriptional programs are enacted within untreated, stressed, and drugtolerant cell groups while generating high heterogeneity between single cells within and between groups. We further demonstrate that drug-tolerant cells contain specific RNA variants residing in genes involved in microtubule organization and stabilization, as well as cell adhesion and cell surface signaling. In addition, the gene expression profile of drug-tolerant cells is similar to that of untreated cells within a few doublings. Thus, single-cell analyses reveal the dynamics of the stress response in terms of cell-specific RNA variants driving heterogeneity, the survival of a minority population through generation of specific RNA variants, and the efficient reconversion of stress-tolerant cells back to normalcy.

The actin cytoskeleton plays multiple critical roles in cells, from cell migration to organelle dynamics. The small and transient actin structures regulating organelle dynamics are difficult to detect with fluorescence microscopy. We developed an approach using fluorescent protein-tagged actin nanobodies targeted to organelle membranes to enable live cell imaging of previously undetected sub-organellar actin dynamics with high spatiotemporal resolution. These probes reveal that ER-associated actin drives fission of multiple organelles including mitochondria, endosomes, lysosomes, peroxisomes, and the Golgi.. CC-BY-NC-ND 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under

Despite being the frontline therapy for type 2 diabetes, the mechanisms of action of the biguanide drug metformin are still being discovered. In particular, the detailed molecular interplays between the AMPK and the mTORC1 pathway in the hepatic benefits of metformin are still ill defined. Metformin-dependent activation of AMPK classically inhibits mTORC1 via TSC/RHEB, but several lines of evidence suggest additional mechanisms at play in metformin inhibition of mTORC1. Here we investigated the role of direct AMPK-mediated serine phosphorylation of RAPTOR in a new Raptor AA mouse model, in which AMPK phospho-serine sites Ser722 and Ser792 of RAPTOR were mutated to alanine. Metformin treatment of primary hepatocytes and intact murine liver requires AMPK regulation of both RAPTOR and TSC2 to fully inhibit mTORC1, and this regulation is critical for both the translational and transcriptional response to metformin. Transcriptionally, AMPK and mTORC1 were both important for regulation of anabolic metabolism and inflammatory programs triggered by metformin treatment. The hepatic transcriptional response in mice on high-fat diet treated with metformin was largely ablated by AMPK deficiency under the conditions examined, indicating the essential role of this kinase and its targets in metformin action in vivo.