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1.  Dynamics of mTORC1 activation in response to amino acids 
eLife  null;5:e19960.
Amino acids are essential activators of mTORC1 via a complex containing RAG GTPases, RAGULATOR and the vacuolar ATPase. Sensing of amino acids causes translocation of mTORC1 to lysosomes, an obligate step for activation. To examine the spatial and temporal dynamics of this translocation, we used live imaging of the mTORC1 component RAPTOR and a cell permeant fluorescent analogue of di-leucine methyl ester. Translocation to lysosomes is a transient event, occurring within 2 min of aa addition and peaking within 5 min. It is temporally coupled with fluorescent leucine appearance in lysosomes and is sustained in comparison to aa stimulation. Sestrin2 and the vacuolar ATPase are negative and positive regulators of mTORC1 activity in our experimental system. Of note, phosphorylation of canonical mTORC1 targets is delayed compared to lysosomal translocation suggesting a dynamic and transient passage of mTORC1 from the lysosomal surface before targetting its substrates elsewhere.
eLife digest
Cells in all organisms must constantly measure the amount of nutrients available to them in order to be healthy and grow properly. For example, cells use a complex sensing system to measure how many amino acids – the building blocks of proteins – are available to them. One enzyme called mTOR alerts the cell to amino acid levels. When amino acids are available, mTOR springs into action and turns on the production of proteins in the cell. However, when amino acids are scarce, mTOR turns off, which slows down protein production and causes the cell to begin scavenging amino acids by digesting parts of itself.
Studies of mTOR have shown that the protein cannot turn on until it visits the surface of small sacks in the cell called lysosomes. These are the major sites within cell where proteins and other molecules are broken down. Scientists know how mTOR gets to the lysosomes, but not how quickly the process occurs.
Now, Manifava, Smith et al. have used microscopes to record live video of the mTOR enzyme as it interacts with amino acids revealing the whole process takes place in just a few minutes. In the experiments, a fluorescent tag was added to part of mTOR to make the protein visible under a microscope. The video showed that, in human cells supplied with amino acids, mTOR reaches the lysosomes within 2 minutes of the amino acids becoming available. Then, within 3-4 minutes the mTOR turns on and leaves the lysosome. Even though the mTOR has left the lysosome, it somehow remembers that amino acids are available and stays active.
The experiments show that mTOR’s brief interaction with the lysosome switches it on and keeps it on even after mTOR leaves. Future studies will be needed to determine exactly how mTOR remembers its interaction with the lysosome and stays active afterwards.
PMCID: PMC5059141  PMID: 27725083
amino acids; mtor; signaling; Human
2.  Autophagy initiation by ULK complex assembly on ER tubulovesicular regions marked by ATG9 vesicles 
Nature Communications  2016;7:12420.
Autophagosome formation requires sequential translocation of autophagy-specific proteins to membranes enriched in PI3P and connected to the ER. Preceding this, the earliest autophagy-specific structure forming de novo is a small punctum of the ULK1 complex. The provenance of this structure and its mode of formation are unknown. We show that the ULK1 structure emerges from regions, where ATG9 vesicles align with the ER and its formation requires ER exit and coatomer function. Super-resolution microscopy reveals that the ULK1 compartment consists of regularly assembled punctate elements that cluster in progressively larger spherical structures and associates uniquely with the early autophagy machinery. Correlative electron microscopy after live imaging shows tubulovesicular membranes present at the locus of this structure. We propose that the nucleation of autophagosomes occurs in regions, where the ULK1 complex coalesces with ER and the ATG9 compartment.
The ULK1 complex is required during autophagosome nucleation, but where autophagic membranes initiate is unknown. Here the authors use super-resolution microscopy to propose that autophagosomes originate from tubulovesicular structures in the ER that align with ATG9 vesicles and recruit ULK1.
PMCID: PMC4987534  PMID: 27510922
3.  Coronavirus nsp6 proteins generate autophagosomes from the endoplasmic reticulum via an omegasome intermediate 
Autophagy  2011;7(11):1335-1347.
Autophagy is a cellular response to starvation which generates autophagosomes to carry cellular organelles and long-lived proteins to lysosomes for degradation. Degradation through autophagy can provide an innate defense against virus infection, or conversely autophagosomes can promote infection by facilitating assembly of replicase proteins. We demonstrate that the avian coronavirus, infectious bronchitis virus (IBV), activates autophagy. A screen of individual IBV nonstructural proteins (nsps) showed that autophagy was activated by IBV nsp6. This property was shared with nsp6 of mammalian coronaviruses mouse hepatitis virus, and severe acute respiratory syndrome virus, and the equivalent nsp5–7 of the arterivirus porcine reproductive and respiratory syndrome virus. These multiple-spanning transmembrane proteins located to the endoplasmic reticulum (ER) where they generated Atg5 and LC3II -positive vesicles, and vesicle formation was dependent on Atg5 and class III PI3 kinase. The vesicles recruited double-FYVE-domain containing protein (DFCP) indicating localized concentration of phosphatidylinositol 3 phosphate, and therefore shared many features with omegasomes formed from the ER in response to starvation. Omegasomes induced by viral nsp6 matured into autophagosomes that delivered LC3 to lysosomes and therefore recruited and recycled the proteins needed for autophagosome nucleation, expansion, cellular trafficking and delivery of cargo to lysosomes. The coronavirus nsp6 proteins activated omegasome and autophagosome formation independently of starvation, but activation did not involve direct inhibition of mTOR signaling, activation of sirtuin 1 or induction of ER stress.
PMCID: PMC3242798  PMID: 21799305
autophagy; omegasome; endoplasmic reticulum; coronavirus; nonstructural proteins
4.  Rhabdomere biogenesis in Drosophila photoreceptors is acutely sensitive to phosphatidic acid levels 
The Journal of Cell Biology  2009;185(1):129-145.
Phosphatidic acid (PA) is postulated to have both structural and signaling functions during membrane dynamics in animal cells. In this study, we show that before a critical time period during rhabdomere biogenesis in Drosophila melanogaster photoreceptors, elevated levels of PA disrupt membrane transport to the apical domain. Lipidomic analysis shows that this effect is associated with an increase in the abundance of a single, relatively minor molecular species of PA. These transport defects are dependent on the activation state of Arf1. Transport defects via PA generated by phospholipase D require the activity of type I phosphatidylinositol (PI) 4 phosphate 5 kinase, are phenocopied by knockdown of PI 4 kinase, and are associated with normal endoplasmic reticulum to Golgi transport. We propose that PA levels are critical for apical membrane transport events required for rhabdomere biogenesis.
PMCID: PMC2700502  PMID: 19349583
5.  Autophagosome formation from membrane compartments enriched in phosphatidylinositol 3-phosphate and dynamically connected to the endoplasmic reticulum 
The Journal of Cell Biology  2008;182(4):685-701.
Autophagy is the engulfment of cytosol and organelles by double-membrane vesicles termed autophagosomes. Autophagosome formation is known to require phosphatidylinositol 3-phosphate (PI(3)P) and occurs near the endoplasmic reticulum (ER), but the exact mechanisms are unknown. We show that double FYVE domain–containing protein 1, a PI(3)P-binding protein with unusual localization on ER and Golgi membranes, translocates in response to amino acid starvation to a punctate compartment partially colocalized with autophagosomal proteins. Translocation is dependent on Vps34 and beclin function. Other PI(3)P-binding probes targeted to the ER show the same starvation-induced translocation that is dependent on PI(3)P formation and recognition. Live imaging experiments show that this punctate compartment forms near Vps34-containing vesicles, is in dynamic equilibrium with the ER, and provides a membrane platform for accumulation of autophagosomal proteins, expansion of autophagosomal membranes, and emergence of fully formed autophagosomes. This PI(3)P-enriched compartment may be involved in autophagosome biogenesis. Its dynamic relationship with the ER is consistent with the idea that the ER may provide important components for autophagosome formation.
PMCID: PMC2518708  PMID: 18725538

Results 1-5 (5)