Diabetic nephropathy (DN) is among the most lethal complications that occur in type 1 and type 2 diabetics.Podocyte dysfunction is postulated to be a critical event associated with proteinuria and glomerulosclerosis in glomerular diseases including DN. However, molecular mechanisms of podocyte dysfunction in the development of DN are not well understood. Here we have shown that activity of mTOR complex 1 (mTORC1), a kinase that senses nutrient availability, was enhanced in the podocytes of diabetic animals. Further, podocytespecific mTORC1 activation induced by ablation of an upstream negative regulator (PcKOTsc1) recapitulated many DN features, including podocyte loss, glomerular basement membrane thickening, mesangial expansion, and proteinuria in nondiabetic young and adult mice. Abnormal mTORC1 activation caused mislocalization of slit diaphragm proteins and induced an epithelial-mesenchymal transition-like phenotypic switch with enhanced ER stress in podocytes. Conversely, reduction of ER stress with a chemical chaperone significantly protected against both the podocyte phenotypic switch and podocyte loss in PcKOTsc1 mice. Finally, genetic reduction of podocyte-specific mTORC1 in diabetic animals suppressed the development of DN. These results indicate that mTORC1 activation in podocytes is a critical event in inducing DN and suggest that reduction of podocyte mTORC1 activity is a potential therapeutic strategy to prevent DN.

Protein synthesis and autophagic degradation are regulated in an opposite manner by mammalian target of rapamycin (mTOR), whereas under certain conditions it would be beneficial if they occured in unison to handle rapid protein turnover. We observed a distinct cellular compartment at the trans-side of the Golgi apparatus, the ‘TOR-autophagy spatial coupling compartment’ (TASCC), where (auto)lysosomes and mTOR accumulated during Ras-induced senescence. mTOR recruitment to the TASCC was amino acid- and Rag guanosine triphosphatase (GTPase)-dependent, and disruption of mTOR localization to the TASCC suppressed interleukin-6/8 synthesis. TASCC-formation was observed during macrophage differentiation and in glomerular podocytes; both displayed increased protein secretion. The spatial coupling of cells’ catabolic and anabolic machinery could augment their respective functions and facilitate the mass synthesis of secretory proteins.

Autophagy is a highly conserved process that degrades cellular long-lived proteins and organelles. Accumulating evidence indicates that autophagy plays a critical role in kidney maintenance, diseases and aging. Ischemic, toxic, immunological, and oxidative insults can cause an induction of autophagy in renal epithelial cells modifying the course of various kidney diseases. This review summarizes recent insights on the role of autophagy in kidney physiology and diseases alluding to possible novel intervention strategies for treating specific kidney disorders by modifying autophagy.

Macropinosomes are large endocytic vesicles that form in ruffling regions of plasma membrane. To analyze signal organization relative to ruffle closure into circular ruffles and cup closure into macropinosomes, this study used quantitative microscopy to measure 3′ phosphoinositides and small-GTPase activities in a representative subset of forming macropinosomes. Macropinocytosis was stimulated by the addition of macrophage colony-stimulating factor (M-CSF) to macrophages expressing fluorescent reporter proteins. Ratiometric and fluorescence resonance energy transfer (FRET) microscopy determined that Rac1 activity and phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P3] levels increased transiently, peaking 26-30 seconds after ruffle closure. Three-dimensional reconstruction of cells labeled with the fluorescent dye FM4-64 showed that PtdIns(3,4,5)P3 was restricted to open, circular cups in the plasma membrane. Quantitative fluorescence microscopic methods determined the timing of cup closure, which followed 40-100 seconds after Rac1 and PtdIns(3,4,5)P3 deactivation and coincided with accumulation of phosphatidylinositol 3-phosphate and Rab5a. Thus, ruffle closure creates a circular domain of plasma membrane that localizes the activation and deactivation of Rac1 and phosphoinositide 3-kinase (PI3K), followed by recruitment of Rab5a and the contractile activities of cup closure.

Enteropathogenic Escherichia coli delivers a subset of effectors into host cells via a type III secretion system, and this step is required for the progression of disease. Here, we show that the type III effectors, EspG and its homolog Orf3, trigger actin stress fiber formation and the destruction of the microtubule networks beneath adherent bacteria. Both effectors were shown to possess the ability to interact with tubulins, and to stimulate microtubule destabilization in vitro. A recent study showed that microtubule-bound GEF-H1, a RhoA-specific guanine nucleotide exchange factor, was converted to its active form by microtubule destabilization, and this sequence of events resulted in RhoA stimulation. Indeed, EspG- and Orf3-induced stress fiber formation was inhibited by the expression of dominant-negative forms of GEF-H1 and RhoA, but not of Rac1 and Cdc42, and by treatment with a ROCK inhibitor. These results indicate that the impact of EspG/Orf3 on microtubule networks triggers the activation of the RhoA-ROCK signaling pathway via GEF-H1 activity. This report reveals for the first time that a pathogen can exploit the host factor GEF-H1.

The rapid activation of the mechanistic target of rapamycin complex-1 (mTORC1) by growth factors is increased by extracellular amino acids through yet-undefined mechanisms of amino acid transfer into endolysosomes. Because the endocytic process of macropinocytosis concentrates extracellular solutes into endolysosomes and is increased in cells stimulated by growth factors or tumor-promoting phorbol esters, we analyzed its role in amino acid–dependent activation of mTORC1. Here, we show that growth factor-dependent activation of mTORC1 by amino acids, but not glucose, requires macropinocytosis. In murine bone marrow–derived macrophages and murine embryonic fibroblasts stimulated with their cognate growth factors or with phorbol myristate acetate, activation of mTORC1 required an Akt-independent vesicular pathway of amino acid delivery into endolysosomes, mediated by the actin cytoskeleton. Macropinocytosis delivered small, fluorescent fluid-phase solutes into endolysosomes sufficiently fast to explain growth factor–mediated signaling by amino acids. Therefore, the amino acid–laden macropinosome is an essential and discrete unit of growth factor receptor signaling to mTORC1.

Mammalian target of rapamycin (mTOR) is a kinase that plays a key role in a wide array of cellular processes and exists in two distinct functional complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). Although mTORC2 is primarily activated by growth factors, mTORC1 is regulated by numerous extracellular and intracellular signals such as nutrients, growth factors, and cellular redox. Previous study has shown that cysteine oxidants sufficiently activate mTORC1 activity under amino acid-depleted conditions and that a reducing agent effectively suppresses amino acid-induced mTORC1 activity, thereby raising the possibility that redox-sensitive mechanisms underlie amino acid-dependent mTORC1 regulation. However, the molecular mechanism by which redox regulates mTORC1 activity is not well understood. In this study, we show that the redox-sensitive regulation of mTORC1 occurs via Rheb but not the Rag small GTPase. Enhancing cellular redox potential with cysteine oxidants significantly increases Rheb GTP levels. Importantly, modulation of the cellular redox potential with a cysteine oxidant or reducing agent failed to alter mTORC1 activity in TSC1 ؊/؊ or TSC2 ؊/؊ mouse embryonic fibroblast cells. Furthermore, a cysteine oxidant has little effect on mTOR localization but sufficiently activates mTORC1 activity in both p18 ؊/؊ and control mouse embryonic fibroblast cells, suggesting that the redox-sensitive regulation of mTORC1 occurs independent of the Ragulator⅐Rag complex. Taken together, our results suggest that the TSC complex plays an important role in redox-sensitive mTORC1 regulation and argues for the activation of mTORC1 in places other than the lysosome upon inhibition of the TSC complex.The mammalian target of rapamycin (mTOR) 2 belongs to the family of phosphatidylinositol 3-kinase (PI3K)-related kinases and shares high sequence similarity to PI3K despite possessing protein kinase activity (1). mTOR is an evolutionarily conserved protein kinase that forms two distinct functional complexes termed mTOR complex 1 and mTOR complex 2 (mTORC1 and mTORC2, respectively) (2-4). mTORC1 is known to be a rapamycin-sensitive complex that regulates a wide array of cellular processes such as cell growth and autophagy, whereas mTORC2 is known to be rapamycin-resistant and involved in the regulation of cell survival as well as cytoskeletal reorganization. Although mTORC2 activation is primarily mediated by growth factors, mTORC1 activation can be achieved by multiple inputs such as amino acids, growth factors, glucose, and oxidative stress (5, 6).Recent studies have shown that two small GTPases, Rheb and Rag, play an essential role in the regulation of mTORC1 activation (7-10). Rheb interacts with and activates mTORC1, whereas Rag small GTPases function as essential spatial regulators of mTORC1 localization (8, 11-13). The Rag small GTPases function as heterodimers formed between RagA/B and RagC/D and bind to the MP1⅐p14⅐p18 complex (Ragulator), which is predominantly expressed on the lysosomal membrane (12,14). Upon ...

Shigella deliver a subset of effectors into the host cell via the type III secretion system, that stimulate host cell signal pathways to modulate the actin dynamics required for invasion of epithelial cells. Here we show that one of the Shigella effectors, called VirA, can interact with tubulin to promote microtubule (MT) destabilization, and elicit protrusions of membrane ruf¯ing. Under in vitro conditions, VirA inhibited polymerization of tubulin and stimulated MT destabilization. Upon microinjection of VirA into HeLa cells, a localized membrane ruf¯ing was induced rapidly. Overexpression of VirA in host cells caused MT destruction and protruding membrane ruf¯es which were absent when VirA was co-expressed with a dominant-negative Rac1 mutant. Indeed, Shigella but not the virA mutant stimulated Rac1, including the formation of membrane ruf¯es in infected cells. Importantly, the MT structure beneath the protruding ruf¯ing was destroyed. Furthermore, drug-induced MT growth in HeLa cells greatly enhanced the Shigella entry. These results indicate that VirA is a novel type of bacterial effector capable of inducing membrane ruf¯ing through the stimulation of MT destabilization.