mTORC1 plays a crucial role in protein synthesis and cell proliferation and growth. It is activated by growth factors and amino acids, including essential amino acids (EAAs), such as leucine; Leu enters cells via the Leu transporter LAT1–4F2hc (also known as SLC7A5–SLC3A2) and potentially via endocytosis. Here, we investigated the contribution of the different routes of Leu entry into cells to mTORC1 activation using pharmacological inhibitors and cells that lack LAT1 or dynamin-1, -2 and -3. Our results show that LAT1 is the major route of Leu entry into cells and mTORC1 activation (∼70%), whereas dynamin-dependent endocytosis and macropinocytosis contribute minimally to both (5–15%). However, macropinocytosis contributes significantly (∼40%) to activation of mTORC1 by other EAAs. Surprisingly, the dynamin inhibitors dynasore and Dyngo 4A, which minimally inhibited Leu uptake, abolished mTORC1 activation independently of dynamin. Instead, dynasore inhibited RagA binding to Raptor, reduced mTORC1 recruitment to the lysosome, and inhibited Akt activation and TSC2-S939 phosphorylation; this resulted in inhibition of Rheb and mTORC1 activity. Our results suggest that these commonly used inhibitors of dynamin and endocytosis are potent suppressors of mTORC1 activation via off-target effects and not via dynamin inhibition.
Mechanistic (mammalian) target of rapamycin complex 1 (mTORC1) is a protein complex implicated in the promotion of protein, lipid and nucleic acid synthesis, and inhibition of autophagy, and it is inhibited by rapamycin (Bar-Peled and Sabatini, 2012; Bar-Peled and Sabatini, 2014). mTORC1 comprises the Ser/Thr kinase mTOR, its regulatory components Raptor and mLST8, and also includes two inhibitory proteins, Deptor and PRAS40 (Saxton et al., 2016). Several upstream signaling pathways activate the mTORC1 pathway, including growth factors and nutrients (amino acids), especially essential amino acids (EAAs) such as leucine (Jewell et al., 2013; Nicklin et al., 2009). In starved cells, mTORC1 resides in the cytoplasm, but upon EAA stimulation it is recruited to the lysosomal membrane where it is activated by the GTPase Rheb (Sancak et al., 2010). This recruitment is facilitated by intralysosomal amino acid sensing by the Ragulator, which activates RagA/C at the lysosomal membrane to recruit mTORC1 to that membrane (Sancak et al., 2010). Leucine that enters the cell has been reported to activate mTORC1 by either directly binding the cytosolic sensor sestrin-2, a negative regulator of GATOR2, the suppressor of RagA/C (Wolfson et al., 2016), and/or by entering the lysosome and activating mTORC1 via the Ragulator and the V-ATPase, by an inside-out mechanism (Zoncu et al., 2011); this last step also involves the Arg transporter SLC38A9 (Rebsamen et al., 2015; Wang et al., 2015).
Leu entry into cells could be mediated by different routes: (1) Leu entry via the Leu transporter, LAT1–4F2hc (also known as SLC7A5–SLC3A2), which imports Leu in exchange for Gln (Mastroberardino et al., 1998; Verrey, 2003). The intracellular Gln used to propel Leu entry by LAT1–4F2hc itself enters cells through the Gln transporter ASCT2 (SLC1A5) (Nicklin et al., 2009) and SNAT family of transporters (Bröer et al., 2016). LAT1–4F2hc activity is reversible, depending on Gln and Leu gradients (Verrey, 2003); (2) Leu entry via endocytosis, either by fluid-phase endocytosis (macropinocytosis) that results in the accumulation of amino acids in lysosomes (Yoshida et al., 2015); or by (3) clathrin- or caveolin-mediated endocytosis, which are dependent on dynamin. The relative contribution of the different routes of Leu entry into cells to the activation of mTORC1 is not clear, and thus investigated here.
In the current study, we inhibited LAT1–4F2hc with BCH (2-amino-2-norbornanecarboxylic acid) or by using LAT1 knockout (KO) cells. We inhibited clathrin- and caveolin-dependent endocytosis by blocking dynamin with its inhibitors dynasore (Macia et al., 2006) or Dyngo 4a (McCluskey et al., 2013), or by using dynamin-1, -2, -3 triple knockout (TKO) cells and inhibited fluid-phase endocytosis with latrunculin A (Park et al., 2013). We then examined Leu-mediated mTORC1 activation using these techniques. Our results show that Leu-dependent activation of mTORC1 is primarily mediated by influx of Leu into cells by LAT1–4F2hc, with a smaller contribution of endocytosis. Moreover, we found that while depletion (KO) of dynamin from cells did not reduce mTORC1 activation by Leu or essential amino acids, dynasore and Dyngo 4a abolished it by interfering with several upstream effectors of mTORC1, and by partially attenuating fluid-phase endocytosis.
Routes of Leu entry into cells
EAAs, primarily Leu, are known to activate mTORC1 (Bar-Peled and Sabatini, 2012). It was previously shown that Leu enters the cell via the Leu transporter (LAT1–4F2hc) to facilitate mTORC1 activation (Nicklin et al., 2009). To evaluate the possible contribution of endocytosis of Leu (dynamin-mediated or via fluid-phase macropinocytosis) towards the activation of mTORC1, we first tested the effect of inhibiting clathrin and caveolin-mediated endocytosis (and some forms of fluid-phase endocytosis; Cao et al., 2007) with dynamin inhibitors on [3H]Leu entry into LS174T (colon adenocarcinoma) cells. Our results show that [3H]Leu uptake (at 5 min) into cells was only minimally (∼15%) reduced when treated with dynasore, a well-known dynamin inhibitor (Macia et al., 2006) that blocks clathrin- or caveolin-dependent endocytosis (Fig. 1A); in contrast, it was dramatically reduced by the LAT1 inhibitor BCH, or in LAT1-knockout LS174T cells (Fig. 1) generated using zinc finger nucleases (Fig. 1A), in agreement with previous studies (Cormerais et al., 2016; Nicklin et al., 2009). Likewise, Leu entry into dynamin TKO mouse embryonic fibroblasts (MEFs; Park et al., 2013), revealed ∼15% reduction in Leu uptake relative to that in wild-type (WT, uninduced TKO) MEFs, but ∼70% reduction after BCH treatment (Fig. 1B). Similarly, adding latrunculin A (LTR, an actin polymerization inhibitor that inhibits micropinocytosis; Park et al., 2013) had only a small effect on Leu uptake (reduction of ∼10%) (Fig. 1C). Collectively, these data suggest that Leu uptake into cells is primarily mediated via the Leu transporter (Fig. S1A).
Contribution of different routes of Leu uptake to mTORC1 activation
To analyze the contribution of the different routes of Leu uptake to mTORC1 activation, we used both the LS174T-LAT1 KO cells (Fig. 2) and dynamin TKO MEFs (Fig. 3) described above, as well as pharmacological inhibitors, to analyze pT389-S6K1 (pp70) phosphorylation (pp70/p70 ratio). Fig. 2A–D shows a strong reduction (but not ablation) of mTORC1 activation by EAA in LS174T–LAT1 KO cells, or in LS174T WT cells treated with BCH. mTORC1 activation by Leu was almost completely eliminated when Leu was used to activate mTORC1 (Fig. 2E,F), suggesting that other amino acids in EAA contribute to mTORC1 activation. Interestingly, LTR further reduced the remaining EAA-mediated activation of mTORC1 in the LS174T–LAT1 KO cells (Fig. 2C,D), suggesting a contribution of micropinocytosis (Fig. S1B). Notably, dynasore completely abolished mTORC1 activation by either EAA or Leu, in LS174T WT or LAT1 KO cells (Fig. 2A,B) (see below).
To analyze the contribution of dynamin-mediated endocytosis to mTORC1 activation we tested EAA- or Leu-induced activation of mTORC1 in WT and dynamin TKO MEFs (Fig. 3). Surprisingly, loss of dynamin-1,-2 and -3 led to enhanced mTORC1 activation by both EAA and Leu (Fig. 3), suggesting that dynamin normally suppresses mTORC1. LTR partially reduced mTORC1 activation (Fig. 3C–E) and dynasore ablated mTORC1 activation by both EAA and Leu (Fig. 3). In cells stimulated with Leu, mTORC1 activity was markedly reduced by BCH, suggesting that in both WT and dynamin TKO MEFs Leu uptake via the Leu transporter plays a major role in activating mTORC1 (Fig. S1B).
Dynasore minimally inhibits Leu uptake, but strongly reduces mTORC1 activation by Leu or EAA
Given the modest reduction in Leu uptake following dynasore treatment (Fig. 1), we were surprised to find a complete ablation of mTORC1 activation in cells stimulated with EAA or Leu (Figs 2 and 3). Dynasore and one of its close structural analogs, Dyngo4A (McCluskey et al., 2013), also abolished mTORC1 activation in HeLa, HEK293T and NALM-6 cell lines (Fig. S2A–C), suggesting that the results are not unique to LS174T (WT or LAT1 KO) or MEFs [WT (un-induced) or dynamin TKO] cells. Dynasore inhibits mTORC1 activation within 15 min in a dose-dependent manner at concentrations that also inhibit transferrin receptor endocytosis (Fig. S2D–G) and are commonly used to inhibit endocytosis. Since dynasore abolished mTORC1 activation in cells that either have or lack dynamin-1, -2 and -3 (Fig. 3), it is likely that its inhibition of mTORC1 is not dependent on its ability to inhibit dynamin. Together, these results demonstrate that dynamin-1, -2 and -3 do not promote activation of mTORC1 by amino acids, and that the abolishment of mTORC1 activation upon dynasore or Dyngo 4a treatment arises from the off-target effect of these inhibitors.
Effect of dynasore on macropinocytosis
It was previously shown that dynasore inhibits macropinocytosis (Park et al., 2013). Our results confirm these observations, by showing that uptake of Alexa-Fluor-488 dextran (10 kDa) into lysosomes was impaired by dynasore in cells lacking dynamin (dynamin TKO MEFs). This impairment was the same as that achieved by using LTR, an actin polymerization inhibitor commonly used to inhibit macropinocytosis (Park et al., 2013) (Fig. 4A,B). Whereas both dynasore (Fig. 1A) and LTR (at either 2 or 10 µM; Fig. 1C) only marginally reduced Leu uptake, LTR inhibited ∼5% and ∼40% of Leu and EAA-induced mTORC1 activation, respectively (Figs 2 and 3, and Fig. S1B), and dynasore abolished it completely (Fig. 3). These data suggest that LTR, possibly via inhibiting macropinocytosis (although we cannot preclude the possibility it has other deleterious cellular effects), partially inhibits Leu- or EAA-dependent mTORC1 activation, but does not abolish it completely as seen with dynasore. Therefore, in addition to partially inhibiting macropinocytosis, the ablation of mTORC1 by dynasore may arise from its additional off-target effects. Our subsequent experiments below were aimed at identifying at least some of these effects.
Decreased binding of RagA to Raptor mediated by dynasore leads to impaired recruitment of mTORC1 to lysosomes and impaired mTORC1 activation
Dynasore functions as a non-competitive inhibitor of the GTPase activity of dynamin (Macia et al., 2006). Since dynasore and its close structural analog Dyngo 4A inhibit mTORC1 activation in cells independently of inhibition of dynamin, we searched for effectors within the mTORC1 pathway that might be alternative targets of dynasore. It is well established that upon amino acid stimulation, mTORC1 re-localizes from the cytosol to the lysosomal membrane, the site of mTORC1 activation (Sancak et al., 2010). To determine if dynasore affects mTORC1 sub-cellular localization, HeLa cells were serum starved and stimulated with EAA in the presence or absence of dynasore and were co-stained with antibodies against endogenous mTOR and the lysosomal marker LAMP1. This revealed that dynasore drastically reduced the amino-acid-dependent localization of mTOR to the lysosome (Fig. 5A). Quantification of these results revealed decreased co-localization of mTOR- and LAMP1-positive puncta per cell in cells treated with dynasore relative to control cells stimulated with amino acids (Fig. 5B).
The amino-acid-stimulated translocation of mTORC1 to the lysosomal surface is dependent upon binding of the Rag subfamily of Ras small GTP-binding proteins to the Raptor component of the mTORC1 complex (Sancak et al., 2008, 2010; Sekiguchi et al., 2001). Moreover, Rag proteins function as obligate heterodimers, which consist of either RagA or RagB bound to RagC or RagD, and the simultaneous GTP loading of RagA/B and GDP loading of RagC/D is crucial to promote the interaction with Raptor and mTORC1 recruitment to the lysosomal surface (Sancak et al., 2010; Tsun et al., 2013). Given that we observed a defect in the recruitment of mTORC1 to the lysosome (in the presence of amino acids) upon dynasore treatment, we hypothesized that dynasore may reduce the ability of the Rag proteins to associate with Raptor. To test this, we co-expressed Raptor with mutant forms of RagA (Q66L) and RagC (S75L), which maintain them in the constitutively GTP- and GDP-bound states, respectively (Bar-Peled et al., 2012; Tsun et al., 2013), in HeLa cells under starved conditions. These Rag mutants represent a functional hetero-dimer that maximally binds to Raptor (Tsun et al., 2013). Our results show that dynasore attenuated the binding of raptor to RagAQ66L by 70%, while its binding to RagCS75L was unaffected (Fig 5C,D). This suggests that dynasore acts by preventing the binding of RagA to Raptor, and thus the translocation of mTORC1 to the lysosomal membrane.
We also tested whether dynasore can inhibit the GTPase activity of RagA in an in vitro GTPase assay. We investigated this by immunoprecipitation (IP) of epitope-tagged dynamin-1, RagA (WT or Q66L) or Rheb transfected into mammalian (HeLa) cells, and incubating them in the GTPase assay in the presence of dynasore. Although dynasore did not inhibit the GTPase activity of Rheb, it inhibited the GTPase activity of the both WT and mutant RagA proteins (Fig 5E; Fig. S3), suggesting that dynasore may also target the GTPase activity of RagA as an additional off-target effect.
RagAQ66L and RagCS75L also render mTORC1 signaling insensitive to amino acid starvation (Sancak et al., 2008; Tsun et al., 2013). To investigate whether we could rescue the effect of dynasore on mTORC1 activation using these mutants, HeLa cells were transfected with RagAQ66L and RagCS75L, and serum starved in the presence of dynasore. Our results show that both RagAQ66L and RagAQ66L/Rag CS75L could not rescue mTORC1 activation under these conditions in the presence of dynasore (Fig. 5F,G), supporting our above results that demonstrate dynasore-mediated inhibition of the RagA–Raptor association.
Dynasore inhibits Rheb by suppressing the Akt pathway and activating TSC2
Our results show that although dynasore reduces the binding of RagA to Raptor by 70%, it completely abolishes amino-acid-dependent mTORC1 activation. We thus considered that dynasore may be playing additional role(s) in inhibiting mTORC1. Diverse cellular signals upstream of mTORC1 activation converge on the TSC1/TSC2 (TSC1/2) tumor suppressor complex. This complex negatively regulates the Rheb GTPase, which potently activates the protein kinase activity of mTORC1 (Brugarolas et al., 2004; Castro et al., 2003; Zhang et al., 2003). To investigate whether dynasore affects mTORC1 activation along this signaling axis, we performed rescue experiments in HeLa cells co-transfected with RagAQ66L, RagCS75L and a constitutively active Rheb (RhebN153T), and treated them with dynasore in the absence of amino acids and serum. Fig. 6A,B shows that although co-expression of RagAQ66L, RagCS75L and RhebN153T in HeLa cells resulted in a strong increase in S6K1 phosphorylation, treatment of these cells with dynasore attenuated (∼70%) mTORC1 activation, but did not abolish it completely compared with dynasore treated cells co-expressing only RagAQ66L and RagCS75L. This suggests that dynasore may also be acting along the TSC1/2–Rheb axis to inhibit mTORC1 activation; an effect partially rescued by a dominant active Rheb. Our results in Fig. 5E show that dynasore does not directly inhibit the GTPase activity of Rheb. Instead, immunoblotting with antibodies against p-AKT (S473) and the Akt phosphorylation site on TSC2 (S939) revealed that cells treated with dynasore exhibit attenuated Akt activation (pAkt) and drastically reduced Akt-dependent TSC2 phosphorylation (Fig. 6A). Since dynasore also blocked Akt phosphorylation and mTORC1-mediated activation by myrisoylated, constitutively active Akt [Akt(myr)] or DA-Akt, (Fig. S4), it is likely that dynasore blocks an upstream component of the Akt pathway.
To determine whether the loss of p-Akt in dynasore-treated cells was a consequence of dynamin inhibition, we used the dynamin TKO MEFs. Our results show that triple knockout of dynamin in these MEFs led to elevated pAkt (S473) levels relative to the WT (un-induced) control, and treatment with dynasore abolished this activation (Fig. 6C,D). Taken together, these data show that dynasore also inhibits Akt activation (probably indirectly), thus inhibiting Rheb function via TSC2 activation, which can be partially rescued with a constitutively active Rheb.
Dynasore and dynamin inhibit autophagy
It is well established that inhibition of mTORC1 strongly induces autophagy (Bar-Peled and Sabatini, 2012). To investigate whether dynasore-mediated mTORC1 inhibition also promotes autophagy, we serum starved HeLa cells in the presence of dynasore and analyzed the stabilization of LC3-II protein levels in the presence of bafilomycin A to inhibit the lysosome. Immunoblotting using antibodies against LC3 shows that although serum-starved cells had increased LC3-II proteins levels in the presence of bafilomycin A (relative to cells grown in serum), cells treated with dynasore had no increase in LC3-II levels regardless of their growth medium (Fig. 7A). Quantification of the LC3-II/LC3-I ratio, a measure of autophagic flux, also showed a decrease in autophagy in cells treated with dynasore (Fig. 7B). This effect was also seen using HEK293T cells, suggesting that the effect of dynasore on autophagy is conserved in other cell lines (Fig. S5A,B).
To investigate whether the dynasore-mediated inhibition of autophagy is due to its effect on dynamin, we performed similar autophagy experiments to those described above using the dynamin TKO MEF cells. As seen in Fig. 7C,D knockout of dynamin reduced autophagy under starved conditions, leading to the accumulation of LC3-II independent of bafilomycin A. This suggests that dynamin is normally required for autophagy. Altogether, our data show that mTORC1 inhibition by dynasore does not promote autophagy as expected, but rather inhibits it, probably at least partly because of its inhibitory effect on dynamin. These data also support a role for dynamin in promoting autophagy.
Our work here provides two important and interrelated findings: (1) Leu entry into cells to activate mTORC1 is primarily mediated by the Leu transporter LAT1–4F2hc, with a smaller contribution from fluid-phase endocytosis (macropinocytosis) and no contribution from dynamin-dependent endocytosis (see below); (2) dynasore strongly inhibits mTORC1 activation by Leu or EAA independently of its inhibitory effect on dynamin.
It was previously reported that treatment of cells with D-Phe (a Leu transporter inhibitor) or depletion of LAT1 strongly reduces mTORC1 activation (Cormerais et al., 2016; Nicklin et al., 2009), in agreement with our results here, which quantified the contribution of the Leu transporter and the major endocytic routes to mTORC1 activation using pharmacological and genetic approaches. Our data show that LAT1–4F2hc contributed ∼70% of Leu entry into cells and mTORC1 activation, clathrin- and caveolin-mediated (dynamin-dependent) endocytosis contributes only ∼10–15% to Leu uptake but did not enhance mTORC1 activation, while fluid-phase endocytosis (LTR sensitive) had a very small effect on Leu uptake and mTORC1 activation (∼5%). However, LTR has an intermediate effect (40%) on mTORC1 activation by EAA, suggesting that other essential amino acids contribute to mTORC1 activation in addition to Leu (Fig. S1). We currently do not know, however, if the effect of LTR is solely caused by inhibition of macropinocytosis, as it inhibits actin polymerization, which could have many deleterious effects on the cell (unfortunately, currently there are no perfect ways to specifically or exclusively inhibit macropinocytosis). It is worth noting that a recent paper by the Swanson lab (Yoshida et al., 2015) showed that amino acid entry into cells via macropinocytosis is required for growth-factor-stimulated activation of mTORC1 by amino acids. In contrast, Palm et al. (2015) reported that when cells depleted of amino acids utilize extracellular proteins as a source of amino acids, mTORC1 activation results in inhibition of cell proliferation, which is opposite to its stimulatory effect under replete amino acid conditions. Thus, it is important to quantify the contribution of intracellular versus lysosomal amino acids to mTORC1 activation under various conditions, a task that is currently challenging given our inability to measure concentrations of individual amino acids in lysosomes and their fluxes in and out of this organelle. Moreover, while cytosolic Leu can activate mTORC1 by inhibiting sestrin-2 in the cytoplasm (Saxton et al., 2016), and intra-lysosomal Leu/EAA can activate mTORC1 via an inside-out mechanism (Zoncu et al., 2011), the relationship between these two events and their possible coordination are unknown. Our recent identification of sorting of LAT1–4F2hc to the lysosomal membrane by LAPTM4b (Milkereit et al., 2015) and the fact that the activity of LAT1–4F2hc is reversible depending on the Leu/Gln gradient (Verrey, 2003), might suggest a mechanism to allow exchange of cytoplasmic and intralysosomal Leu to enhance communication between these cellular compartments, and hence coordinate mTORC1 activation at the lysosomal membrane.
A surprising observation in our current study was that knockout of dynamin-1, -2 and -3 led to a marked enhancement of mTORC1 activation, suggesting that dynamin normally suppresses this complex. While we do not know the reason for this, one possibility is that it is mediated by the elevated Akt activation (pAkt) that we observed in the dynamin TKO cells (Fig. 5). The reason for the elevated pAkt levels in these cells is also unknown, although one possibility is that inhibition of clathrin-mediated endocytosis increases cell surface stability of activated growth factor receptors, which continue to signal to activate their downstream targets (Sousa et al., 2012), including PI3K–Akt pathway components. A cross-talk between Akt and dynamin-1 was proposed recently (Liberali et al., 2014; Reis et al., 2015).
Our findings here demonstrate a dramatic inhibition of mTORC1 activation by the commonly used endocytosis inhibitors, dynasore and dyngo 4a, independently of dynamin. These observations point to off-target effects of these related compounds. These off-target effects include at least some proteins or pathways known to regulate mTORC1, thus causing: (1) interference with the ability of RagA to bind Raptor, leading to attenuated recruitment of mTORC1 to the lysosomal membrane; (2) inhibition of Akt phosphorylation/activation (probably indirectly) and phosphorylation of its downstream effector TSC2, thus reducing mTORC1 activation downstream of growth factor (and other) signaling; and (3) a possible reduction in macropinocytosis. It is likely that there are other off-target effects of these compounds as well, and some have been reported already, such as a reduction in labile cholesterol in membranes (Preta et al., 2015), or disruption of membrane ruffling and fluid-phase endocytosis (Park et al., 2013). Although the identification of a new class of compounds or drugs that inhibit mTORC1 may be attractive for the development of new therapies for cancer or metabolic diseases, a more comprehensive understanding of their global cellular targets is first needed to fully understand the possible side-effects of these (and other) compounds.
Another unexpected observation in our studies was that dynasore inhibited autophagy, despite its strong inhibition of mTORC1 activation, which should have led to enhanced autophagy. We suspect that, in this case, inhibition of dynamin itself by dynasore might have contributed to the blockade of autophagy. Indeed, a role for dynamin in autophagic maturation and function was proposed recently (Fang et al., 2016; Schulze et al., 2013). In summary, our work here quantified the contribution of Leu entry into cells, via different uptake routes, to mTORC1 activation by Leu and essential amino acids, and discovered dynasore as a strong inhibitor of mTORC1 activation independently of its inhibition of dynamin.
MATERIALS AND METHODS
Cell lines, reagents and antibodies
Antibodies used in the experiments are: anti-LAMP1 (Abcam, H4A3; 1:1000); anti-β-actin (Sigma, A2228; 1:10,000); anti-FLAG (Sigma, F1804; 1:10,000); anti-hemagglutinin (HA) (Covance, MMS-101R; 1:1000); anti-pS6K (pp70) (Thr389) (Cell Signaling, 9234S; 1:1000); anti-4E-BP1 (Cell Signaling, 9452; 1:1000); anti-p4E-BP1 (T37/46) (Cell Signaling, 9459S, 1:1000); anti-pAkt (T473) (Cell Signaling, 9271S, 1:1000); anti-pTSC2 (T939) Cell Signaling, 3615S, 1:1000); anti-LAT1 (Cell Signaling, #5347S, 1:1000); anti-S6K (p70) (Santa Cruz, SC-8418, 1:1000); anti-GFP (Neomarkers, MS-1288-P, 1:2000); anti-Akt (BD Transduction Lab, 610860, 1:2000); anti-dynamin (BD Biosciences, 610245, 1:10,000) and anti-LC3 (Novus Biologicals, NB600-1384, 1:2000).
Anti-FLAG M2 affinity agarose (A2220) was purchased from Sigma. Secondary antibodies conjugated to horseradish peroxidase (1:10,000) were purchased from Molecular Probes. Reagents used for immunofluorescence imaging were: normal goat serum (Jackson ImmunoResearch Laboratories), Alexa-Fluor-488/647 goat anti-mouse or anti-rabbit secondary antibody (Invitrogen) all used at 1:1000, Alexa-Fluor-488-conjugated dextran (Molecular Probes, D22910) and 4,6-diamidino-2-phenylindole (DAPI, Molecular Probes, 1:5000). Radioactive leucine (Leu) uptake was monitored with [3H]-Leu (PerkinElmer). Dynasore (sc-202592) and Dyngo 4A (ab120689) were obtained from Chem Cruz and Abcam, respectively; 2-amino-2-norbornanecarboxylic acid (BCH) (A7902) and 4-hydroxytamoxifen (H7904) were purchased from Sigma; Bafilomacin A1 (1334) was purchased from Tocris. Latrunculin A (10010630) was purchased from Cayman Chemical. The in vitro ATPase/GTPase activity assay kit (MAK113) was purchased from Sigma. HeLa and HEK293T cells were maintained in full medium [DMEM supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin]; LS174T WT and LAT1 KO cells were generated by Dr Jacques Pouyssegur, as described (Cormerais et al., 2016) and were maintained in EMEM (Eagle's minimum essential medium) supplemented as for the other cells. The Dynamin TKO MEFs were a generous gift from Dr Pietro De Camilli, Yale University (Park et al., 2013), and were maintained in full medium (DMEM) as above, and supplemented with 2 mM L-glutathione. The NALM6 cells were grown in RPMI 1640 medium supplemented with 10% FBS. Cells were transfected using Polyjet reagent (Signagen). For stimulation experiments, cells were serum and nutrient starved overnight in RPMI 1640 medium without amino acids, sodium phosphate powder (#R8999-04A, US Biological) and stimulated with RPMI supplemented with 1× MEM or essential amino acids (EAAs), Life Technologies, which includes L-Arg, L-Cys, L-His, L-Ile, L-Leu L-Lys, L-Met, L-Phe, L-Thr, L-Trp, L-Tyr and L-Val (www.lifetechnologies.com/ca/en/home/technical-resources/media-formulation.164.html).
LS174T cells or dynamin TKO MEFs were grown in six-well plates and serum starved for 2 h in RPMI. They were then stimulated with RPMI supplemented with EAA and 4 μCi [3H]Leu per 5 ml per well for 5 min in the presence of 80 μM dynasore (1 h) or 10 mM 2-amino-2-norbornanecarboxylic acid (BCH) (2 h) or 2 μM Latrunculin A (30 min), as indicated. The wells were washed three times with cold PBS and lysed in lysis buffer [50 mM HEPES, pH 7.5, 150 mN NaCl, 1% Triton X-100, 10% glycerol, 1.5 mM MgCl2, 1.0 mM EGTA, supplemented with 10 μg/ml of each leupeptin, aprotinin and pepstatin, and 1 mM phenylmethanesulfonylfluoride (PMSF)]. The cell lysates were transferred to scintillation vials and [3H]Leu radioactivity was measured with a scintillation counter (Hidex 300 SL, Southern Scientific) using MiKrowin 2000 software (Mikrotek Laborsystem GmbH). The radioactive counts were normalized to the respective lysate concentrations.
Amino acid stimulation and mTORC1 activation
Cells were serum starved for 2 h in RPMI 1640 medium without amino acids and were stimulated for 15 min with RPMI supplemented with EAA; for activation by L-Leu, cells were pre-treated for 1 h with 2 mM glutamine before stimulation for 15 min with 0.4 mM L-Leu. For drug inhibition experiments, cells were treated with either dynasore (80 μM, unless otherwise indicated) or Dyngo 4A (30 μM) for 1 h, BCH (10 mM) for 2 h or Latrunculin A (2 μM) for 30 min, prior to amino acid stimulation. For RagAQ66L, RagCS75L, RhebN153T or Akt (mys-Akt) rescue experiments, HeLa cells were transfected with the indicated plasmids for 48 h, starved for 2 h and stimulated with EAAs (15 min) with or without dynasore treatment (1 h). For mTORC1 activation experiments in dynamin TKO MEFs, cells were treated with 4-hydroxytamoxifen (4-OHT) (as described in Park et al., 2013) to induce knockout before starving the cells and treatment with EAAs or dynasore, as described above. Control WT MEFs were un-induced TKO MEFs. Cells were lysed in lysis buffer and mTORC1 activation was monitored using anti-pT389-S6K1 and anti-p4E-BP1 antibodies. All blots were imaged using the Odyssey Imaging system and quantified using Image Studio version 3.1.4 (LI-COR). Each experiment was performed at least three times.
Immunofluorescence confocal microscopy
HeLa cells were cultured on poly-D-lysine-coated coverslips in six-well plates, serum and nutrient starved for 2 h and then stimulated with EAAs for 15 min. For dynasore treatment, cells were treated with 80 μM dynasore (or DMSO control) for 1 h before stimulating with amino acid. At 24 h post transfection, wells were washed three times with cold 1 ml PBS and fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 and incubated with 1:100 normal goat serum in 3% skimmed milk (30 min). Slides were stained for 1 h with rabbit anti-mTORC (1:1000) and mouse anti-human LAMP1 (1:1000) in 3% skimmed milk. After three PBS washes, cells were incubated with goat anti-mouse or anti-rabbit Alexa-Fluor-488/647-conjugated secondary antibody, and briefly stained with 4,6-diamidino-2-phenylindole (DAPI). Coverslips were mounted with Dako Cytomation. Images were acquired using a Quorum WAveFX-X1 spinning-disk confocal microscope at ×60 magnification with an Olympus S-Apo ×60/1.35 oil objective (Quorum Technologies, Guelph, Canada). Quantification of mTOR-positive puncta that co-localized with LAMP1 was assessed by Volocity 6.0.1 (PerkinElmer).
For dextran uptake, WT or dynamin TKO MEFs were cultured on poly-D-lysine-coated coverslips in six-well plates and incubated for 30 min at 37°C in the presence of 0.4 mg/ml Alexa-Fluor-488-conjugated dextran (10 kDa). The wells were washed three times with cold 1 ml PBS and fixed with 4% paraformaldehyde and imaged as described above. Dextran uptake in cells (N=60) was quantified using the Volocity 6.0.1 software and normalized as a measure of fluorescent dextran intensity per cell area.
HeLa cells were co-transfected with the specified cDNA constructs, serum starved for 2 h in the presence or absence of 80 μM dynasore or 30 μM Dyngo4A for 1 h and lysed in lysis buffer. Co-IP of FLAG–Raptor, GFP–RagA, GST-HA–RagC was determined by IP of FLAG–Raptor from 1 mg of cleared cell lysate with anti-FLAG M2 affinity beads and immunoblotting with either anti-HA or anti-GFP antibodies. All blots were imaged using the Odyssey Imaging system and co-IP of FLAG–Raptor with either GFP–RagA or GST-HA–RagC was quantified using Image Studio.
Dynamin TKO MEFs were treated with 4-OHT (as described in Park et al., 2013) to induce knock out of dynamin-1, -2 and -3 before starving the cells for 2 h in the presence of 80 μM dynasore (1 h) and 0.1 μM bafilomycin A (2 h), as indicated. The cells were lysed in lysis buffer as described above and autophagic rate (flux) was monitored using anti-LC3 antibodies. All blots were imaged using the Odyssey Imaging system and quantified using Image Studio.
In vitro GTPase assay
HeLa cells were transfected with the either GFP–RagA (WT or Q66L), GFP–dynamin-1 or FLAG–Rheb, as indicated, for 48 h and immunoprecipitated. The immunoprecipitates were washed three times with IP wash buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, 10% glycerol and 0.1% Triton X-100) and incubated in GTPase assay in the presence or absence of dynasore following the protocol outlined in the kit. The absorbance at 600 nm was read with a visible light plate reader (Molecular Devices) using the SoftMax Pro software, and normalized to the amount of protein immunoprecipitated as analyzed by western blotting and quantified as described above.
We thank Dr Pietro De Camilli for the dynamin triple knockout MEFs and Sergio Grinstein for helpful advice.
Conceptualization: D.R., A.P.; Methodology: A.P.; Validation: A.P.; Formal analysis: A.P.; Investigation: A.P.; Resources: Y.C., J.P.; Data curation: A.P.; Writing - original draft: D.R., A.P.; Writing - review & editing: D.R., Y.C., J.P.; Supervision: D.R.; Funding acquisition: D.R.
This work was supported by the Canadian Institute of Health Research (CIHR grant MOP-142711) to D.R. D.R. is holds a Canada Research Chair (CRC, Tier I) from the Canadian Foundation for Innovation. J.P. and Y.C. were supported by the Centre Scientifique de Monaco (CSM), a grant from Gemluc and by Université Côte d'Azur (UCA), Institute for Research on Cancer and Aging, Nice (IRCAN), Centre National de la Recherche Scientifique (CNRS) and Centre A. Lacassagne.
The authors declare no competing or financial interests.