AMPK regulation of Raptor and TSC2 mediate metformin effects on transcriptional control of anabolism and inflammation.

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 RaptorAA 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.

AMPK governs lineage specification through Tfeb-dependent regulation of lysosomes.

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.

Actin chromobody imaging reveals sub-organellar actin dynamics.

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 challenging to detect with fluorescence microscopy, making it difficult to determine whether actin filaments are directly associated with specific membranes. To address these limitations, we developed fluorescent-protein-tagged actin nanobodies, termed 'actin chromobodies' (ACs), targeted to organelle membranes to enable high-resolution imaging of sub-organellar actin dynamics.

Single-cell analyses of transcriptional heterogeneity during drug tolerance transition in cancer cells by RNA sequencing.

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 drug-tolerant 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.

INF2-mediated actin polymerization at ER-organelle contacts regulates organelle size and movement.

Proper regulation of organelle dynamics and inter-organelle contacts is critical for cellular health and function. Both the endoplasmic reticulum (ER) and actin cytoskeleton are known to regulate organelle dynamics, but how, when, and where these two subcellular components are coordinated to control organelle dynamics remains unclear. Here, we show that ER-associated actin consistently marks mitochondrial, endosomal, and lysosomal fission sites. We also show that actin polymerization by the ER-anchored isoform of the formin protein INF2 is a key regulator of the morphology and mobility of these organelles. Together, our findings establish a mechanism by which INF2-mediated polymerization of ER-associated actin at ER-organelle contacts regulates organelle dynamics.

INF2-mediated actin polymerization at ER-organelle contacts regulates organelle size and movement.

Proper regulation of organelle dynamics is critical for cellular function, but the mechanisms coordinating multiple organelles remain poorly understood. Here we show that actin polymerization mediated by the endoplasmic reticulum (ER)-anchored formin INF2 acts as a master regulator of organelle morphology and movement. Using high-resolution imaging, we demonstrate that INF2-polymerized actin filaments assemble at ER contact sites on mitochondria, endosomes, and lysosomes just prior to their fission. Genetic manipulation of INF2 activity alters the size, shape and motility of all three organelles. Our findings reveal a conserved mechanism by which the ER uses actin polymerization to control diverse organelles, with implications for understanding organelle dysfunction in neurodegenerative and other diseases. This work establishes INF2-mediated actin assembly as a central coordinator of organelle dynamics and inter-organelle communication.

AAV-mediated rescue of Eps8 expression in vivo restores hair-cell function in a mouse model of recessive deafness.

The transduction of acoustic information by hair cells depends upon mechanosensitive stereociliary bundles that project from their apical surface. Mutations or absence of the stereociliary protein EPS8 cause deafness in humans and mice, respectively. Eps8 knockout mice (Eps8 -/- ) have hair cells with immature stereocilia and fail to become sensory receptors. Here, we show that exogenous delivery of Eps8 using Anc80L65 in P1-P2 Eps8 -/- mice in vivo rescued the hair bundle structure of apical-coil hair cells. Rescued hair bundles correctly localize EPS8, WHIRLIN, MYO15, and BAIAP2L2, and generate normal mechanoelectrical transducer currents. Inner hair cells with normal-looking stereocilia re-expressed adult-like basolateral ion channels (BK and KCNQ4) and have normal exocytosis. The number of hair cells undergoing full recovery was not sufficient to rescue hearing in Eps8 -/- mice. Adeno-associated virus (AAV)-transduction of P3 apical-coil and P1-P2 basal-coil hair cells does not rescue hair cells, nor does Anc80L65-Eps8 delivery in adult Eps8 -/- mice. We propose that AAV-induced gene-base therapy is an efficient strategy to recover the complex hair-cell defects in Eps8 -/- mice. However, this therapeutic approach may need to be performed in utero since, at postnatal ages, Eps8 -/- hair cells appear to have matured or accumulated damage beyond the point of repair.

Transgenesis by lentiviral vectors: lack of gene silencing in mammalian embryonic stem cells and preimplantation embryos.

The introduction of foreign genes into early mouse embryos and embryonic stem (ES) cells is invaluable for the analysis of gene function and regulation in the living animal. The use of vectors derived from retroviruses as gene transfer vehicles in this setting has had limited success because of silencing of transgene expression. Here, we show that vectors derived from lentiviruses, which are complex retroviruses, can efficiently deliver genes to murine ES cells and that transgene expression is stable during proliferation of undifferentiated ES cells. The transgene is expressed during differentiation of ES cells in vitro (embryoid bodies) and in vivo (teratomas). Transfer of lentivector-transduced ES cells into blastocysts resulted in chimeric animals that expressed the transgene in multiple tissues. Embryos derived from crossings of chimeric mice expressed the transgene, indicating successful germ-line transmission. Infection of murine preimplantation embryos at morula stage with lentiviral vectors resulted in stable transduction and expression of the transgene in mouse embryos and in newborn mice. Finally, human ES cells were transduced by lentiviral vectors and expressed the transgene over several passages. Thus, lentiviral vectors represent a significant improvement over oncoretroviral vectors used previously for gene transfer into murine ES cells and preimplantation embryos. Ability to transfer foreign genes into human ES cells has potential relevance for the development of gene and cell-based therapies.