mTOR controls endoplasmic reticulum-Golgi apparatus trafficking of VSVg in specific cell types.

BACKGROUND: Mammalian/mechanistic target of rapamycin (mTOR) complexes are essential for cell proliferation, growth, differentiation, and survival. mTORC1 hyperactivation occurs in the tuberous sclerosis complex (TSC). mTORC1 localizes to the surface of lysosomes, where Rheb activates it. However, mTOR was also found on the endoplasmic reticulum (ER) and Golgi apparatus (GA). Recent studies showed that the same inputs regulate ER-to-GA cargo transport and mTORC1 (e.g., the level of amino acids or energy status of the cell). Nonetheless, it remains unknown whether mTOR contributes to the regulation of cargo passage through the secretory pathway. METHODS: The retention using selective hooks (RUSH) approach was used to image movement of model cargo (VSVg) between the ER and GA in various cell lines in which mTOR complexes were inhibited. We also investigated VSVg trafficking in TSC patient fibroblasts. RESULTS: We found that mTOR inhibition led to the overall enhancement of VSVg transport through the secretory pathway in PC12 cells and primary human fibroblasts. Also, in TSC1-deficient cells, VSVg transport was enhanced. CONCLUSIONS: Altogether, these data indicate the involvement of mTOR in the regulation of ER-to-GA cargo transport and suggest that impairments in exocytosis may be an additional cellular process that is disturbed in TSC.

An Insight of Scientific Developments in TSC for Better Therapeutic Strategy.

Tuberous sclerosis complex (TSC) is a rare genetic disease, which is characterized by noncancerous tumors in multi-organ systems in the body. Mutations in the TSC1 or TSC2 genes are known to cause the disease. The resultant mutant proteins TSC1 (hamartin) and TSC2 (tuberin) complex evade its normal tumor suppressor function, which leads to abnormal cell growth and proliferation. Both TSC1 and TSC2 are involved in several protein-protein interactions, which play a significant role in maintaining cellular homeostasis. The recent biochemical, genetic, structural biology, clinical and drug discovery advancements on TSC give a useful insight into the disease as well as the molecular aspects of TSC1 and TSC2. The complex nature of TSC disease, a wide range of manifestations, mosaicism and several other factors limits the treatment choices. This review is a compilation of the course of TSC, starting from its discovery to the current findings that would take us a step ahead in finding a cure for TSC.

HDAC6 differentially regulates autophagy in stem-like versus differentiated cancer cells.

Cancer stem-like cells (CSCs), a small population of pluripotent cells residing within heterogeneous tumor mass, remain highly resistant to various chemotherapies as compared to the differentiated cancer cells. It is being postulated that CSCs possess unique molecular mechanisms, such as autophagic homeostasis, that allow CSCs to withstand the therapeutic assaults. Here we demonstrate that HDAC6 inhibition differentially modulates macroautophagy/autophagy in CSCs as compared to that of differentiated cancer cells. Using human and murine CSC models and differentiated cells, we show that the inhibition or knockdown (KD) of HDAC6 decreases CSC pluripotency by downregulating major pluripotency factors POU5F1, NANOG and SOX2. This decreased HDAC6 expression increases ACTB, TUBB3 and CSN2 expression and promotes differentiation in CSCs in an apoptosis-independent manner. Mechanistically, HDAC6 KD in CSCs decreases pluripotency by promoting autophagy, whereas the inhibition of pluripotency via retinoic acid treatment, POU5F1 or autophagy-related gene (ATG7 and ATG12) KD in CSCs decreases HDAC6 expression and promotes differentiation. Interestingly, HDAC6 KD-mediated CSC growth inhibition is further enhanced in the presence of autophagy inducers Tat-Beclin 1 peptide and rapamycin. In contrast to the results observed in CSCs, HDAC6 KD in differentiated breast cancer cells downregulates autophagy and increases apoptosis. Furthermore, the autophagy regulator p-MTOR, upstream negative regulators of p-MTOR (TSC1 and TSC2) and downstream effectors of p-MTOR (p-RPS6KB and p-EIF4EBP1) are differentially regulated in CSCs versus differentiated cancer cells following HDAC6 KD. Overall these data identify the differential regulation of autophagy as a molecular link behind the differing chemo-susceptibility of CSCs and differentiated cancer cells.

mTORC1 Controls Phase Separation and the Biophysical Properties of the Cytoplasm by Tuning Crowding.

Macromolecular crowding has a profound impact on reaction rates and the physical properties of the cell interior, but the mechanisms that regulate crowding are poorly understood. We developed genetically encoded multimeric nanoparticles (GEMs) to dissect these mechanisms. GEMs are homomultimeric scaffolds fused to a fluorescent protein that self-assemble into bright, stable particles of defined size and shape. By combining tracking of GEMs with genetic and pharmacological approaches, we discovered that the mTORC1 pathway can modulate the effective diffusion coefficient of particles ≥20 nm in diameter more than 2-fold by tuning ribosome concentration, without any discernable effect on the motion of molecules ≤5 nm. This change in ribosome concentration affected phase separation both in vitro and in vivo. Together, these results establish a role for mTORC1 in controlling both the mesoscale biophysical properties of the cytoplasm and biomolecular condensation.