RagA, but Not RagB, Is Essential for Embryonic Development and Adult Mice

The mechanistic target of rapamycin complex 1 (mTORC1) integrates cues from growth factors and nutrients to control metabolism. In contrast to the growth factor input, genetic disruption of nutrient-dependent activation of mTORC1 in mammals remains unexplored. We engineered mice lacking RagA and RagB genes, which encode the GTPases responsible for mTORC1 activation by nutrients. RagB has limited expression, and its loss shows no effects on mammalian physiology. RagA deficiency leads to E10.5 embryonic death, loss of mTORC1 activity, and severe growth defects. Primary cells derived from these mice exhibit no regulation of mTORC1 by nutrients and maintain high sensitivity to growth factors. Deletion of RagA in adult mice is lethal. Upon RagA loss, a myeloid population expands in peripheral tissues. RagA-specific deletion in liver increases cellular responses to growth factors. These results show the essentiality of nutrient sensing for mTORC1 activity in mice and its suppression of PI3K/Akt signaling.United States. National Institutes of Health (R01 CA129105)United States. National Institutes of Health (R01 CA103866)United States. National Institutes of Health (R01 AI047389)United States. National Institutes of Health (R21 AG042876)American Federation for Aging ResearchStarr FoundationDavid H. Koch Institute for Integrative Cancer Research at MIT. Frontier Research ProgramEllison Medical FoundationUnited States. National Institutes of Health (AG041765)National Cancer Institute (U.S.) (F31CA167872

Identification of novel proteins that regulate the amino acid-sensitive mTORC1 pathway

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2016.Cataloged from PDF version of thesis. "February 2016."Includes bibliographical references.mTOR is a serine-threonine kinase that, as the catalytic subunit of mTORC1, promotes growth and anabolism. Due to its central role in metabolism, the local and systemic environment surrounding the cell tightly regulate mTORC1. Growth factors and nutrients are each required to activate mTORC1 and promote growth. Activation of mTORC1 by growth factors has been well-elucidated, but it is only recently becoming clear how nutrients, specifically amino acids, activate mTORC1. The presence of amino acids leads to the recruitment of mTORC1 from the cytosol to the surface of the lysosomal membrane, allowing it to be activated downstream of growth factors. This amino acid-induced translocation is mediated by the Rag GTPases and Ragulator (the scaffold for the Rag GTPases and mTORC1 on the lysosomal membrane). Here we describe the identification of two new components of Ragulator, HBXIP and c7orf59, that are required for the lysosomal localization of both the Rag GTPases and mTORC1 and that allowed us to identify new functions that Ragulator fulfills. We also characterized RagA-null mice and RagA-null mouse embryonic fibroblasts (MEFs). RagA is required for embryonic development, and, surprisingly, its deletion in adult mice leads to an expansion of monocytes. MEFs derived from RagA-null embryos display atypical, nutrient-insensitive mTORC1 activation. Finally, we identified c17orf59, a new Ragulator-interacting protein that inhibits the interaction between the Rag GTPases and Ragulator, inhibiting mTORC1 activation by amino acids. We report here our progress in characterizing the components of the amino acid-sensitive mTORC1 pathway and their physiological roles and we discuss the many open questions that remain to be studied regarding how amino acid sufficiency promotes the lysosomal localization of mTORC1.by Lawrence D. Schweitzer.Ph. D

Disruption of the Rag-Ragulator Complex by c17orf59 Inhibits mTORC1

mTORC1 controls key processes that regulate cell growth, including mRNA translation, ribosome biogenesis, and autophagy. Environmental amino acids activate mTORC1 by promoting its recruitment to the cytosolic surface of the lysosome, where its kinase is activated downstream of growth factor signaling. mTORC1 is brought to the lysosome by the Rag GTPases, which are tethered to the lysosomal membrane by Ragulator, a lysosome-bound scaffold. Here, we identify c17orf59 as a Ragulator-interacting protein that regulates mTORC1 activity through its interaction with Ragulator at the lysosome. The binding of c17orf59 to Ragulator prevents Ragulator interaction with the Rag GTPases, both in cells and in vitro, and decreases Rag GTPase lysosomal localization. Disruption of the Rag-Ragulator interaction by c17orf59 impairs mTORC1 activation by amino acids by preventing mTOR from reaching the lysosome. By disrupting the Rag-Ragulator interaction to inhibit mTORC1, c17orf59 expression may represent another mechanism to modulate nutrient sensing by mTORC1

Measuring Surface Bulk Elemental Composition on Venus

The extreme surface environment (462 C, 93 bars pressure) of Venus makes subsurface measurements of its bulk elemental composition extremely challenging. Instruments landed on the surface of Venus must be enclosed in a pressure vessel. The high surface temperatures also require a thermal control system to keep the instrumentation temperatures within their operational range for as long as possible. Since Venus surface probes can currently operate for only a few hours, it is crucial that the lander instrumentation be able to make statistically significant measurements in a short time. An instrument is described that can achieve such a measurement over a volume of thousands of cubic centimeters of material by using high energy penetrating neutron and gamma radiation. The instrument consists of a Pulsed Neutron Generator (PNG) and a Gamma-Ray Spectrometer (GRS). The PNG emits isotropic pulses of 14.1 MeV neutrons that penetrate the pressure vessel walls, the dense atmosphere and the surface rock. The neutrons induce nuclear reactions in the rock to produce gamma rays with energies specific to the element and nuclear process involved. Thus the energies of the detected gamma rays identify the elements present and their intensities provide the abundance of each element. The GRS spectra are analyzed to determine the Venus elemental composition from the spectral signature of individual major, minor, and trace radioactive elements. As a test of such an instrument, a Schlumberger Litho Scanner oil well logging tool was used in a series of experiments at NASA's Goddard Space Flight Center. The Litho Scanner tool was mounted above large (1.8 m x 1.8 m x.9 m) granite and basalt monuments and made a series of one-hour elemental composition measurements in a planar geometry more similar to a planetary lander measurement. Initial analysis of the results shows good agreement with target elemental assay

Performance of a RT-PCR Assay in Comparison to FISH and Immunohistochemistry for the Detection of ALK in Non-Small Cell Lung Cancer

Patients with lung cancers harboring an activating anaplastic lymphoma kinase (ALK) rearrangement respond favorably to ALK inhibitor therapy. Fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) are validated and widely used screening tests for ALK rearrangements but both methods have limitations. The ALK RGQ RT-PCR Kit (RT-PCR) is a single tube quantitative real-time PCR assay for high throughput and automated interpretation of ALK expression. In this study, we performed a direct comparison of formalin-fixed paraffin-embedded (FFPE) lung cancer specimens using all three ALK detection methods. The RT-PCR test (diagnostic cut-off ΔCt of ≤8) was shown to be highly sensitive (100%) when compared to FISH and IHC. Sequencing of RNA detected full-length ALK transcripts or EML4-ALK and KIF5B-ALK fusion variants in discordant cases in which ALK expression was detected by the ALK RT-PCR test but negative by FISH and IHC. The overall specificity of the RT-PCR test for the detection of ALK in cases without full-length ALK expression was 94% in comparison to FISH and sequencing. These data support the ALK RT-PCR test as a highly efficient and reliable diagnostic screening approach to identify patients with non-small cell lung cancer whose tumors are driven by oncogenic ALK.Medicine, Faculty ofMedical Oncology, Division ofMedicine, Department ofPathology and Laboratory Medicine, Department ofReviewedFacult

Cooperative nutrient accumulation sustains growth of mammalian cells

The coordination of metabolic processes to allow increased nutrient uptake and utilization for macromolecular synthesis is central for cell growth. Although studies of bulk cell populations have revealed important metabolic and signaling requirements that impact cell growth on long time scales, whether the same regulation influences short-term cell growth remains an open question. Here we investigate cell growth by monitoring mass accumulation of mammalian cells while rapidly depleting particular nutrients. Within minutes following the depletion of glucose or glutamine, we observe a growth reduction that is larger than the mass accumulation rate of the nutrient. This indicates that if one particular nutrient is depleted, the cell rapidly adjusts the amount that other nutrients are accumulated, which is consistent with cooperative nutrient accumulation. Population measurements of nutrient sensing pathways involving mTOR, AKT, ERK, PKA, MST1, or AMPK, or pro-survival pathways involving autophagy suggest that they do not mediate this growth reduction. Furthermore, the protein synthesis rate does not change proportionally to the mass accumulation rate over these time scales, suggesting that intracellular metabolic pools buffer the growth response. Our findings demonstrate that cell growth can be regulated over much shorter time scales than previously appreciated.National Cancer Institute (U.S.) (Koch Institute Support (Core) Grant P30-CA14051)National Cancer Institute (U.S.). Physical Sciences Oncology Center (U54CA143874)National Institutes of Health (U.S.) (Contract R01GM085457)National Cancer Institute (U.S.) (Fellowship F31CA167872)National Institutes of Health (U.S.) (Interdepartmental Biotechnology Training Program 5T32GM008334