The mechanical activation of mTOR signaling: an emerging role for late endosome/lysosomal targeting

Abstract

It is well recognized that mechanical signals play a critical role in the regulation of skeletal muscle mass, and the maintenance of muscle mass is essential for mobility, disease prevention and quality of life. Furthermore, over the last 15 years it has become established that signaling through a protein kinase called the mammalian (or mechanistic) target of rapamycin (mTOR) is essential for mechanically-induced changes in protein synthesis and muscle mass, however, the mechanism(s) via which mechanical stimuli regulate mTOR signaling have not been defined. Nonetheless, advancements are being made, and an emerging body of evidence suggests that the late endosome/lysosomal (LEL) system might play a key role in this process. Therefore, the purpose of this review is to summarize this body of evidence. Specifically, we will first explain why the Ras homologue enriched in brain (Rheb) and phosphatidic acid (PA) are considered to be direct activators of mTOR signaling. We will then describe the process of endocytosis and its involvement in the formation of LEL structures, as well as the evidence which indicates that mTOR and its direct activators (Rheb and PA) are all enriched at the LEL. Finally, we will summarize the evidence that has implicated the LEL in the regulation of mTOR by various growth regulatory inputs such as amino acids, growth factors and mechanical stimuli.

Figure 1. Endocytosis and the Late Endosome/Lysosomal System

Endocytosis begins with the uptake of plasma membrane into primary endocytic vesicles. These vesicles are then delivered to the early endosome which is characterized by the presence of Rab5 on its cytosolic membrane. The early endosome sorts the endocytosed material and returns the majority of endocytosed material to the plasma membrane either directly, or with the assistance of the recycling endosome which is characterized by the presence of Rab11(van Ijzendoorn 2006). The remaining material that has been targeted for degradation is delivered by the early endosome to the late endosome. The late endosome is characterized by the presence of Rab7 and multivesicular bodies. It also contains the lysosomal membrane protein LAMP2 which is delivered, along with endocytosed material, to the lysosome through the formation of a hybrid organelle. The hybrid organelle degrades the endocytosed material and the lysosome then reforms (Huotari and Helenius 2011).

Figure 2. Forced Targeting of Raptor to the LEL is Sufficient to Activate mTOR Signaling

Electroporation was used to co-transfect mouse Tibialis Anterior muscles with 30 μg of plasmid DNA encoding various forms of HA-tagged Raptor and 2 μg of Myc-tagged p70^S6k (Goodman et al. 2013; Sancak et al. 2010). At 7 days post transfection, the muscles were collected, homogenized, and then the Myc-tagged p70^S6k was immunopurified and subjected to western blot analysis for p70^S6k phosphorylated on the Threonine 389 residue [P-p70(T389)] and total p70^S6k as previously described (Goodman et al. 2010). The values at the top of the blot represent the ratio of P-p70(T389) to total p70^S6k (a marker of mTOR signaling) and were expressed relative to the values obtained in the WT-Raptor group. * Significantly different from WT-Raptor, P ≤ 0.05.

Figure 3. Insulin Abolishes the Association of TSC2 with the LEL

Mice were given an intraperitoneal injection of 20 U/kg insulin or the vehicle as a control condition and then collected 30 min later. (A–F) Cross-sections of the Tibialis Anterior muscles from control (A–C), or insulin (D–F), stimulated mice were then subjected to immunohistochemistry for LAMP2 and TSC2 as previously described (Jacobs et al. 2013). (B, E) Grayscale images of the signals for LAMP2. (C, F) Grayscale images of the signals for TSC2. Scale bars in B and E represent 10 μm. (G) Colocalization of the large and intense TSC2 positive puncta with LAMP2 was quantified as detailed previously (Jacobs et al. 2013). Values in the graph represent the means + SEM and were obtained from n = 14–46 randomly selected images per group. * Significantly different from control, P ≤ 0.05.

Figure 4. Mechanical Stimulation Induces an Increase in the Number of LAMP2 and mTOR Positive Puncta

The Tibialis Anterior (TA) muscles from mice were subjected to mechanical stimulation (MCH) via a bout of eccentric contractions as previously described (Jacobs et al. 2013). (A–F) Cross-sections from control (A–C), or MCH (D–F) muscles were then subjected to immunohistochemistry for LAMP2, mTOR, and type 2b myosin heavy chain (MHC2b), as previously described (Jacobs et al. 2013; Goodman et al. 2012). (B, E) Grayscale images of the signals for LAMP2. (C, F) Grayscale images of the signals for mTOR. Scale bars in C and F represent 20 μm. (G) The number of LAMP2 and mTOR puncta/μm² in randomly selected MHC2b positive fibers where manually counted by an investigator that was blind of the sample identifications. Values in the graph represents the mean + SEM and were obtained from a total of n = 126–133 fibers per group. * Significantly different from control, P ≤ 0.05.

Figure 5. Conceptual Model of How Mechanical Stimuli Could Regulate mTOR Signaling at the LEL

In this model, the LEL in skeletal muscles serve as a major regulatory center for controlling mTOR signaling. In response to the mechanically-induced signaling events (shown with arrows), mTOR signaling transitions to its active state. See the summary section for details. Adapted and reprinted from (Jacobs et al. 2013) with permission from the publisher (John Wiley and Sons).