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In tuberous sclerosis complex (TSC), Tsc2 mutations are associated with more severe disease manifestations than Tsc1 mutations and the role of extracellular vesicles (EVs) in this context is not yet studied. We report a comparative analysis of EVs derived from isogenic renal cells except for Tsc1 or Tsc2 gene status and hypothesized that in spite of having similar physical characteristics, EVs modulate signaling pathways differently, thus leading to TSC heterogenicity. We used mouse inner medullary collecting duct (mIMCD3) cells with the Tsc1 (T1G cells) or Tsc2 (T2J cells) gene disrupted by CRISPR/CAS9. EVs were isolated from the cell culture media by size-exclusion column chromatography followed by detailed physical and chemical characterization. Physical characterization of EVs was accessed by tunable resistive pulse sensing and dynamic light scattering, revealing similar average sizes and zeta potentials (at pH 7.4) for EVs from mIMCD3 (123.5 ± 5.7 nm and −16.3 ± 2.1 mV), T1G cells (131.5 ± 8.3 nm and −19.8 ± 2.7 mV), and T2J cells (127.3 ± 4.9 nm and −20.2 ± 2.1 mV). EVs derived from parental mIMCD3 cells and both mutated cell lines were heterogeneous (>90% of EVs < 150 nm) in nature. Immunoblotting detected cilial Hedgehog signaling protein Arl13b; intercellular proteins TSG101 and Alix; and transmembrane proteins CD63, CD9, and CD81. Compared to Tsc2 deletion, Tsc1 deletion cells had reduced EV production and release rates. EVs from Tsc1 mutant cells altered mTORC1, autophagy, and β-catenin pathways differently than EVs from Tsc2 -mutated cells. Quantitative PCR analysis revealed the down regulation of miR-212a-3p and miR-99a-5p in EVs from Tsc2 -mutated cells compared to EVs from Tsc1 -mutant cells. Thus, EV-derived miR-212-3p and mIR-99a-5p axes may represent therapeutic targets or biomarkers for TSC disease. [ABSTRACT FROM AUTHOR]

The successful production of recombinant protein for biochemical, biophysical, and structural biological studies critically depends on the correct expression organism. Currently, the most commonly used expression organisms for structural studies are Escherichia coli (~70% of all PDB structures) and the baculovirus/ insect cell expression system (~5% of all PDB structures). While insect cell expression is frequently successful for large eukaryotic proteins, it is relatively expensive and time‐consuming compared to E. coli expression. Frequently the decision to carry out a baculovirus project means restarting cloning from scratch. Here we describe an integrated system that allows simultaneous cloning into E. coli and baculovirus expression vectors using the same PCR products. The system offers a flexible array of N‐ and C‐terminal affinity, solubilization and utility tags, and the speed allows expression screening to be completed in E. coli, before carrying out time and cost‐intensive experiments in baculovirus. Importantly, we describe a means of rapidly generating polycistronic bacterial constructs based on the hugely successful biGBac system, making InteBac of particular interest for researchers working on recombinant protein complexes. [ABSTRACT FROM AUTHOR]

Autophagy dysregulation has been implicated in numerous diseases and many therapeutic agents are known to modulate this pathway. Therefore, the ability to accurately monitor autophagy is critical to understanding its role in the pathogenesis and treatment of many diseases. Recently an imaging flow cytometry method measuring colocalization of microtubule associated protein 1B light chain 3 (LC3) and lysosomal signals via Bright Detail Similarity (BDS) was proposed which enabled evaluation of autophagic processing. However, since BDS only evaluates colocalization of LC3 and lysosomal signals, the number of autophagy organelles was not taken into account. We found that in cells classified as having Low BDS, there was a large degree of variability in accumulation of autophagosomes. Therefore, we developed a new approach wherein BDS was combined with number of LC3+ puncta, which enabled us to distinguish between cells having very few autophagy organelles versus cells with accumulation of autophagosomes or autolysosomes. Using this method, we were able to distinguish and quantify autophagosomes and autolysosomes in breast cancer cells cultured under basal conditions, with inhibition of autophagy using chloroquine, and with induction of autophagy using amino acid starvation. This technique yields additional insight into autophagy processing making it a useful supplement to current techniques. © 2015 International Society for Advancement of Cytometry [ABSTRACT FROM AUTHOR]

Lymphangioleiomyomatosis (LAM) is an often fatal disease primarily affecting young women in which tuberin (TSC2)-null cells metastasize to the lungs. The mechanisms underlying the striking female predominance of LAM are unknown. We report here that 17-β-estradiol (E2) causes a 3- to 5-fold increase in pulmonary metastases in male and female mice, respectively, and a striking increase in circulating tumor cells in mice bearing tuberin-null xenograft tumors. E2-induced metastasis is associated with activation of p42/44 MAPK and is completely inhibited by treatment with the MEK1/2 inhibitor, Cl-1040. In vitro, E2 inhibits anoikis of tuberin-null cells. Finally, using a bioluminescence approach, we found that E2 enhances the survival and lung colonization of intravenously injected tuberin-null cells by 3-fold, which is blocked by treatment with Cl-1040. Taken together these results reveal a new model for LAM pathogenesis in which activation of MEK-dependent pathways by E2 leads to pulmonary metastasis via enhanced survival of detached tuberin-null cells. [ABSTRACT FROM AUTHOR]

The most exciting advances in the tuberous sclerosis complex (TSC) field occurred in 1993 and 1997 with the cloning of the TSC2 and TSC1 genes, respectively, and in 2003 with the identification of Rheb as the target of tuberin's (TSC2) GTPase activating protein (GAP) domain. Rheb has a dual role: it activates mTOR and inactivates B-Raf. Activation of mTOR leads to increased protein synthesis through phosphorylation of p70S6K and 4E-BP1. Upon insulin or growth factor stimulation, tuberin is phosphorylated by several kinases, including AKT/PKB, thereby suppressing its GAP activity and activating mTOR. Phosphorylation of hamartin (TSC1) by CDK1 also negatively regulates the activity of the hamartin/tuberin complex. Despite these biochemical advances, exactly how mutations in TSC1 or TSC2 lead to the clinical manifestations of TSC is far from being understood. Two of the most unusual phenotypes in TSC are the apparent metastasis of benign cells carrying TSC1 and TSC2 mutations, resulting in pulmonary lymphangiomyomatosis, and the ability of cells with TSC1 or TSC2 mutations to differentiate into the separate components of renal angiomyolipomas (vessels, smooth muscle and fat). We will discuss how the TSC signaling pathways are affected by mutations in TSC1 or TSC2, focusing on how these mutations may lead to the renal and pulmonary manifestations of TSC.Oncogene (2005) 24, 7475–7481. doi:10.1038/sj.onc.1209090 [ABSTRACT FROM AUTHOR]

This review is focused on pathways and mechanisms that might provide molecular links between the pathogenesis of renal and pulmonary disease in tuberous sclerosis complex and the pathogenesis of the neurologic manifestations of tuberous sclerosis complex. Tuberous sclerosis complex is an autosomal dominant disorder in which the manifestations can include seizures; mental retardation; autism; benign tumors of the brain, retina, skin, and kidneys; and pulmonary lymphangiomyomatosis. Lymphangiomyomatosis is a life-threatening lung disease affecting almost exclusively young women. Genetic data have demonstrated that the cells giving rise to renal angiomyolipomas, the most frequent tumor type in patients with tuberous sclerosis complex, exhibit differentiation plasticity. Genetic studies have also shown that the benign smooth muscle cells of angiomyolipomas and pulmonary lymphangiomyomatosis have the ability to migrate or metastasize to other organs. These findings indicate that hamartin and tuberin play functional roles in the regulation of cell migration and differentiation. The biochemical pathways responsible for these effects are not yet fully understood but might involve dysregulation of the small guanosine triphosphatase Rho. Similar pathways might contribute to aberrant neuronal differentiation and migration in tuberous sclerosis complex. (J Child Neurol 2004;19:710-715). [ABSTRACT FROM AUTHOR]

Lymphangioleiomyomatosis (LAM) is a progressive lung disease affecting almost exclusively women. The reasons for this strong gender predisposition are poorly understood. Renal angiomyolipomas occur in 50–50% of sporadic LAM patients. The smooth muscle cells of pulmonary LAM and renal angiomyolipomas are nearly indistinguishable morphologically. Here, we report the first successful cell culture of a LAM-associated renal angiomyolipoma. The cells carded inactivating mutations in both alleles of the TSC2 gene and expressed estrogen receptor α, estrogen receptor β, and androgen receptor. To elucidate the cellular pathways through which steroid hormones influence LAM pathogenesis, we treated the cells with both estradiol and tamoxifen. Cell growth was stimulated by estradiol, associated with phosphorylation of p44/42 MAPK at 5 min and an increase in c-myc expression at 4 h. Tamoxifen citrate also stimulated cell growth, associated with increased phosphorylation of p44/42 MAPK and expression of c-myc, indicating that tamoxifen has agonist effects on angiomyolipoma cells. This response to tamoxifen in human angiomyolipoma cells differs from prior studies of Eker rat leiomyoma cells, possibly reflecting cell type or species differences in cells lacking tuberin. Our data provide the first evidence that estradiol stimulates the growth of angiomyolipoma cells, that tamoxifen has agonist effects in angiomyolipoma cells, and that estradiol and tamoxifen impact both genomic and nongenomic signaling pathways in angiomyolipoma cells. The responsiveness of angiomyolipoma cells to estradiol may be related to the underlying reasons that LAM affects primarily women. [ABSTRACT FROM AUTHOR]

The purpose of this paper is to examine the capabilities provided by an iterative – adaptive mesh redesign method developed for the Boundary Integral Equation (BIE) method for elastoplastic analysis. In previous papers [1, 2] the fundamentals of the method where presented in elasticity and elastoplasticity. It was shown there that the adaptive procedure converges fast and is specially developed to reveal the ability of the BIE to provide high accuracy with very economic deployment of quadratic boundary and interior elements. In this paper the results of method applied on particular problems in elastoplasticity are compared against well known experimental solutions. The ability of the adaptive scheme to converge fast is demonstrated, but more important finding is the fact that the progressive refinement of the discretisation gives more insight into the physical problem. [ABSTRACT FROM AUTHOR]

Tuberous sclerosis complex (TSC) is a tumor suppressor gene syndrome characterized by seizures, mental retardation, autism, and tumors of the brain, kidney, heart, retina, and skin. TSC is caused by mutations in either TSC1 or TSC2, both of which are tumor suppressor genes. Hamartin, the protein product of TSC1, was found to interact with the ezrin-radixin-moesin family of cytoskeletal proteins and to activate the small GTPase Rho. To determine whether tuberin, the TSC2 product, can also activate Rho, we stably expressed full-length human tuberin in two cell types: MDCK cells and ELT3 cells. ELT3 cells lack endogenous tuberin expression. We found that expression of human tuberin in both MDCK and ELT3 cells was associated with an increase in the amount of Rho-GTP, but not in Rac1-GTP or cdc42-GTP. Tuberin expression increased cell adhesion in both cell types, and decreased chemotactic cell migration in ELT3 cells. In MDCK cells, there was a decrease in the amount of total Focal Adhesion Kinase (FAK) and an increase in the fraction of phosphorylated FAK. These findings demonstrate for the first time that tuberin activates Rho and regulates cell adhesion and migration. Pathways involving Rho activation may have relevance to the clinical manifestations of TSC, including pulmonary lymphangioleiomyomatosis.Oncogene (2002) 21, 8470–8476. doi:10.1038/sj.onc.1205962 [ABSTRACT FROM AUTHOR]

Examines the relevance of antibody p27 and cell cycle regulation to the clinical manifestations of tuberous sclerosis complex (TSC). HEK293 cells overexpressing wild type tumor suppressor gene TSC1, wild type TSC2 or disease causing mutant forms of TSC2; Cell cycle effects of hamartin.

Presents information on a study which identified the somatic TSC2 mutations in five of seven angiomyolipomas from sporadic lymphangioleiomyomatosis (LAM) patients. Molecular mechanism of pulmonary smooth muscle proliferation in LAM; Materials and methods of the study; Results and discussion.

The tuberous sclerosis complex (Tsc) proteins regulate the conserved mTORC1 growth regulation pathway. We identified that loss of the Tsc2 gene in mouse inner medullary collecting duct (mIMCD) cells induced a greater than two-fold increase in extracellular vesicle (EV) production compared to the same cells having an intact Tsc axis. We optimized EV isolation using a well-established size exclusion chromatography method to produce high purity EVs. Electron microscopy confirmed the purity and spherical shape of EVs. Both tunable resistive pulse sensing (TRPS) and dynamic light scattering (DLS) demonstrated that the isolated EVs possessed a heterogenous size distribution. Approximately 90% of the EVs were in the 100–250 nm size range, while approximately 10% had a size greater than 250 nm. Western blot analysis using proteins isolated from the EVs revealed the cellular proteins Alix and TSG101, the transmembrane proteins CD63, CD81, and CD9, and the primary cilia Hedgehog signaling-related protein Arl13b. Proteomic analysis of EVs identified a significant difference between the Tsc2-intact and Tsc2-deleted cell that correlated well with the increased production. The EVs may be involved in tissue homeostasis and cause disease by overproduction and altered protein content. The EVs released by renal cyst epithelia in TSC complex may serve as a tool to discover the mechanism of TSC cystogenesis and in developing potential therapeutic strategies. [ABSTRACT FROM AUTHOR]

Tuberous sclerosis complex (TSC) is a tumor predisposition syndrome with significant renal cystic and solid tumor disease. While the most common renal tumor in TSC, the angiomyolipoma, exhibits a loss of heterozygosity associated with disease, we have discovered that the renal cystic epithelium is composed of type A intercalated cells that have an intact Tsc gene that have been induced to exhibit Tsc‐mutant disease phenotype. This mechanism appears to be different than that for ADPKD. The murine models described here closely resemble the human disease and both appear to be mTORC1 inhibitor responsive. The induction signaling driving cystogenesis may be mediated by extracellular vesicle trafficking. TSC renal cystic disease develops in about half of the patients. The disease appears to caused by an induction mechanism such that a small population of mutant cells can cause significant renal cystic disease comprised of mostly genetically normal cells. [ABSTRACT FROM AUTHOR]

Lack of high-quality antibodies against transmembrane proteins is a widely recognized hindrance in biomedical and cell biological research. Here we present a robust pipeline for the generation of polyclonal antibodies employing full-length membrane proteins as immunogens to overcome this 'antibody bottleneck'. We express transmembrane proteins fused to a MISTIC fragment that enhances expression of eukaryotic membrane proteins in E. coli. Purified membrane proteins are used as immunogen for rabbit injection employing standard immunizing protocols. The raised antibodies against membrane proteins of the endoplasmic reticulum and the nuclear envelope, which we use as test cases, function in a wide range of applications and are superior to ones produced against soluble domains as immunogens. [ABSTRACT FROM AUTHOR]