Continuous sensing of nutrients and growth factors by the mTORC1-TFEB axis

mTORC1 senses nutrient and growth factor status and phosphorylates downstream targets, including the transcription factor TFEB, to coordinate metabolic supply and demand. The molecular mechanisms of mTORC1 activation are thought to enforce a strict requirement for simultaneous amino acid and growth factor stimuli, but this model has not been evaluated with quantitative or single-cell methods. Here, we develop a series of fluorescent protein-TFEB fusions and investigate how combinations of stimuli jointly regulate signaling from mTORC1 to TFEB at the single-cell level. Live-cell imaging of individual cells revealed that mTORC1-TFEB signaling responds with graded changes to individual amino acid and growth factor inputs, rather than behaving as a logical AND gate. We find that mTORC1 inputs can be sequentially sensed, with responses that vary between mTORC1 substrates and are amplified by input from other kinases, including GSK3β. In physiologically relevant concentrations of amino acids, we observe fluctuations in mTORC1-TFEB signaling that indicate continuous responsiveness to nutrient availability. Our results clarify how the molecular regulation of mTORC1 enables homeostatic processes at the cellular level and provide a more precise understanding of its behavior as an integrator of multiple inputs.

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Analysis at single-cell resolution identifies a stable mammalian tRNA-mRNA interface and increased translation efficiency in neurons

10.1101/2021.06.28.450167 ◽

2021 ◽

Author(s):

William Gao ◽

Carlos J Gallardo-Dodd ◽

Claudia Kutter

Keyword(s):

Amino Acid ◽

Codon Usage ◽

Single Cell ◽

Adult Mouse ◽

Trna Gene ◽

Supply And Demand ◽

Cell Types ◽

Ribosome Profiling ◽

Translation Efficiency ◽

Gene Usage

The correlation between codon and anticodon pools influences the efficiency of translation, but whether differences exist in these pools across individual cells is unknown. We determined that codon usage and amino acid demand are highly stable across different cell types using single-cell RNA-sequencing atlases of adult mouse and fetal human. After demonstrating the robustness of ATAC-sequencing for analysis of tRNA gene usage, we quantified anticodon usage and amino acid supply in adult mouse and fetal human single-cell ATAC-seq atlases. We found that tRNA gene usage is overall coordinated across cell types, except in neurons which clustered separately from other cell types. Integration of these datasets revealed a strong and statistically significant correlation between amino acid supply and demand across almost all cell types. Neurons have an enhanced translation efficiency over other cell types, driven by an increased supply of Ala-AGC anticodons. This results in faster decoding of the Ala-GCC codon, as determined by cell-type specific ribosome profiling, and a reduction of Ala-AGC anticodon pools may be implicated in neurological pathologies. This study, the first such examination of codon usage, anticodon usage, and translation efficiency at single-cell resolution, identifies conserved features of translation elongation across mammalian cellular diversity and evolution.

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Review of Single-Cell RNA Sequencing in the Heart

International Journal of Molecular Sciences ◽

10.3390/ijms21218345 ◽

2020 ◽

Vol 21 (21) ◽

pp. 8345

Author(s):

Shintaro Yamada ◽

Seitaro Nomura

Keyword(s):

Single Cell ◽

Rna Sequencing ◽

Molecular Mechanisms ◽

Clinical Medicine ◽

Basic Research ◽

Cellular Level ◽

Cardiovascular Development ◽

Biological Mechanisms ◽

Technological Advances ◽

Single Cell Rna Sequencing

Single-cell RNA sequencing (scRNA-seq) technology is a powerful, rapidly developing tool for characterizing individual cells and elucidating biological mechanisms at the cellular level. Cardiovascular disease is one of the major causes of death worldwide and its precise pathology remains unclear. scRNA-seq has provided many novel insights into both healthy and pathological hearts. In this review, we summarize the various scRNA-seq platforms and describe the molecular mechanisms of cardiovascular development and disease revealed by scRNA-seq analysis. We then describe the latest technological advances in scRNA-seq. Finally, we discuss how to translate basic research into clinical medicine using scRNA-seq technology.

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Rheb and Rags come together at the lysosome to activate mTORC1

Biochemical Society Transactions ◽

10.1042/bst20130037 ◽

2013 ◽

Vol 41 (4) ◽

pp. 951-955 ◽

Cited By ~ 48

Author(s):

Marlous J. Groenewoud ◽

Fried J.T. Zwartkruis

Keyword(s):

Amino Acids ◽

Growth Factors ◽

Amino Acid ◽

Cell Growth ◽

Mammalian Target Of Rapamycin ◽

Maturation Process ◽

Phospholipase D1 ◽

Strict Requirement ◽

Mtorc1 Activation ◽

Dependent Interaction

mTORC1 (mammalian target of rampamycin complex 1) is a highly conserved protein complex regulating cell growth and metabolism via its kinase mTOR (mammalian target of rapamycin). The activity of mTOR is under the control of various GTPases, of which Rheb and the Rags play a central role. The presence of amino acids is a strict requirement for mTORC1 activity. The heterodimeric Rag GTPases localize mTORC1 to lysosomes by their amino-acid-dependent interaction with the lysosomal Ragulator complex. Rheb is also thought to reside on lysosomes to activate mTORC1. Rheb is responsive to growth factors, but, in conjunction with PLD1 (phospholipase D1), is also an integral part of the machinery that stimulates mTORC1 in response to amino acids. In the present article, we provide a brief overview of novel mechanisms by which amino acids affect the function of Rags. On the basis of existing literature, we postulate that Rheb is activated at the Golgi from where it will travel to lysosomes. Maturation of endosomes into lysosomes may be required to assure a continuous supply of GTP-bound Rheb for mTORC1 activation, which may help to drive the maturation process.

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Grb2 Regulates Internalization of EGF Receptors through Clathrin-coated Pits

Molecular Biology of the Cell ◽

10.1091/mbc.e02-08-0532 ◽

2003 ◽

Vol 14 (3) ◽

pp. 858-870 ◽

Cited By ~ 209

Author(s):

Xuejun Jiang ◽

Fangtian Huang ◽

Andriy Marusyk ◽

Alexander Sorkin

Keyword(s):

Growth Factor ◽

Hela Cells ◽

Binding Sites ◽

Molecular Mechanisms ◽

Fluorescent Protein ◽

Yellow Fluorescent Protein ◽

Growth Factor Receptor ◽

Receptor Internalization ◽

Sequence Motifs ◽

Coated Pits

The molecular mechanisms of clathrin-dependent internalization of epidermal growth factor receptor (EGFR) are not well understood and, in particular, the sequence motifs that mediate EGFR interactions with coated pits have not been mapped. We generated a panel of EGFR mutants and stably expressed these mutants in porcine aortic endothelial (PAE) cells. Interestingly, mutations of tyrosine phosphorylation sites 1068 and 1086 that interact with growth-factor-receptor-binding protein Grb2 completely abolished receptor internalization in PAE cells. Quantitative analysis of colocalization of EGF-rhodamine conjugate and coated pits labeled with yellow-fluorescent-protein–tagged β2 subunit of clathrin adaptor complex AP-2 revealed that EGFR mutants lacking Grb2 binding sites do not efficiently enter coated pits. The depletion of Grb2 from PAE as well as HeLa cells expressing endogenous EGFRs by RNA interference substantially reduced the rate of EGFR internalization through clathrin-dependent pathway, thus providing the direct evidence for the important role of Grb2 in this process. Overexpression of Grb2 mutants, in which the SH3 domains were either deleted or inactivated by point mutations, significantly inhibited EGFR internalization in both PAE and HeLa cells. These findings indicate that Grb2, in addition to its key function in signaling through Ras, has a major regulatory role at the initial steps of EGFR internalization through clathrin-coated pits. Furthermore, the EGFR mutant lacking Grb2 binding sites did not efficiently recruit c-Cbl and was not polyubiquitinated. The data are consistent with the model whereby Grb2 participates in EGFR internalization through the recruitment of Cbl to the receptor, thus allowing proper ubiquitylation of EGFR and/or associated proteins at the plasma membrane.

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Secretion of hepatoma-derived growth factor is regulated by N-terminal processing

Biological Chemistry ◽

10.1515/bc.2010.147 ◽

2010 ◽

Vol 391 (12) ◽

Cited By ~ 11

Author(s):

Ketan Thakar ◽

Tim Kröcher ◽

Soniya Savant ◽

Doron Gollnast ◽

Sørge Kelm ◽

...

Keyword(s):

Growth Factor ◽

Molecular Mechanisms ◽

Enhanced Green Fluorescent Protein ◽

Fluorescent Protein ◽

Critical Role ◽

Disulfide Bridge ◽

Terminal Part ◽

N Terminus ◽

Regulation Of Secretion ◽

Green Fluorescent

Abstract Hepatoma-derived growth factor (HDGF) was first purified as a growth factor secreted by hepatoma cells. It promotes angiogenesis and has been related to tumorigenesis. To date, little is known about the molecular mechanisms of HDGF functions and especially its routes or regulation of secretion. Here we show that secretion of HDGF requires the N-terminal 10 amino acids and that this peptide can mediate secretion of other proteins, such as enhanced green fluorescent protein, if fused to their N-terminus. Our results further demonstrate that cysteine residues at positions 12 and 108 are linked via an intramolecular disulfide bridge. Surprisingly, phosphorylation of serine 165 in the C-terminal part of HDGF plays a critical role in the secretion process. If this serine is replaced by alanine, the N-terminus is truncated, the intramolecular disulfide bridge is not formed and the protein is not secreted. In summary, these observations provide a model of how phosphorylation, a disulfide bridge and proteolytic cleavage are involved in HDGF secretion.

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Single cell detection of latent cytomegalovirus reactivation in host tissue

Journal of General Virology ◽

10.1099/vir.0.029827-0 ◽

2011 ◽

Vol 92 (6) ◽

pp. 1279-1291 ◽

Cited By ~ 36

Author(s):

Anja Marquardt ◽

Stephan Halle ◽

Christof K. Seckert ◽

Niels A. W. Lemmermann ◽

Tibor Z. Veres ◽

...

Keyword(s):

Single Cell ◽

Molecular Mechanisms ◽

Fluorescent Protein ◽

Single Cells ◽

Cell Detection ◽

Cytomegalovirus Reactivation ◽

Single Cell Detection ◽

Latently Infected ◽

Organ Tissue

The molecular mechanisms leading to reactivation of latent cytomegalovirus are not well understood. To study reactivation, the few cells in an organ tissue that give rise to reactivated virus need to be identified, ideally at the earliest possible time point in the process. To this end, mouse cytomegalovirus (MCMV) reporter mutants were designed to simultaneously express the red fluorescent protein mCherry and the secreted Gaussia luciferase (Gluc). Whereas Gluc can serve to assess infection at the level of individual mice by measuring luminescence in blood samples or by in vivo imaging, mCherry fluorescence offers the advatage of detection of infection at the single cell level. To visualize cells in which MCMV was being reactivated, precision-cut lung slices (PCLS) that preserve tissue microanatomy were prepared from the lungs of latently infected mice. By day 3 of cultivation of the PCLS, reactivation was revealed by Gluc expression, preceding the detection of infectious virus by approximately 4 days. Reactivation events in PCLS could be identified when they were still confined to single cells. Notably, using fractalkine receptor–GFP reporter mice, we never observed reactivation originating from CX3CR1+ monocytes or pulmonary dendritic cells derived therefrom. Furthermore, latent viral genome in the lungs was not enriched in sorted bone-marrow-derived cells expressing CD11b. Taken together, these complementary approaches suggest that CD11b+ and CX3CR1+ subsets of the myeloid differentiation lineage are not the main reservoirs and cellular sites of MCMV latency and reactivation in the lungs.

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