Severe progressive neurological paediatric disease mucopolysaccharidosis III type C is caused by mutations in the HGSNAT gene leading to deficiency of acetyl-CoA: α-glucosaminide N-acetyltransferase involved in the lysosomal catabolism of heparan sulphate. To understand the pathophysiology of the disease we generated a mouse model of mucopolysaccharidosis III type C by germline inactivation of the Hgsnat gene. At 6-8 months mice showed hyperactivity, and reduced anxiety. Cognitive memory decline was detected at 10 months and at 12-13 months mice showed signs of unbalanced hesitant walk and urinary retention. Lysosomal accumulation of heparan sulphate was observed in hepatocytes, splenic sinus endothelium, cerebral microglia, liver Kupffer cells, fibroblasts and pericytes. Starting from 5 months, brain neurons showed enlarged, structurally abnormal mitochondria, impaired mitochondrial energy metabolism, and storage of densely packed autofluorescent material, gangliosides, lysozyme, phosphorylated tau, and amyloid-β. Taken together, our data demonstrate for the first time that deficiency of acetyl-CoA: α-glucosaminide N-acetyltransferase causes lysosomal accumulation of heparan sulphate in microglial cells followed by their activation and cytokine release. They also show mitochondrial dysfunction in the neurons and neuronal loss explaining why mucopolysaccharidosis III type C manifests primarily as a neurodegenerative disease.

Activation of mast cells by aggregation of the high-affinity IgE receptors (FcεRI) initiates signaling events leading to the release of inflammatory and allergic mediators stored in cytoplasmic granules. A key role in this process play changes in concentrations of intracellular Ca2+ controlled by store-operated Ca2+ entry (SOCE). Although microtubules are also involved in the process leading to degranulation, the molecular mechanisms that control microtubule rearrangement during activation are largely unknown. In this study, we report that activation of bone marrow-derived mast cells (BMMCs) induced by FcεRI aggregation or treatment with pervanadate or thapsigargin results in generation of protrusions containing microtubules (microtubule protrusions). Formation of these protrusions depended on the influx of extracellular Ca2+. Changes in cytosolic Ca2+concentration also affected microtubule plus-end dynamics detected by microtubule plus-end tracking protein EB1. Experiments with knockdown or reexpression of STIM1, the key regulator of SOCE, confirmed the important role of STIM1 in the formation of microtubule protrusions. Although STIM1 in activated cells formed puncta associated with microtubules in protrusions, relocation of STIM1 to a close proximity of cell membrane was independent of growing microtubules. In accordance with the inhibition of Ag-induced Ca2+ response and decreased formation of microtubule protrusions in BMMCs with reduced STIM1, the cells also exhibited impaired chemotactic response to Ag. We propose that rearrangement of microtubules in activated mast cells depends on STIM1-induced SOCE, and that Ca2+ plays an important role in the formation of microtubule protrusions in BMMCs.

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The microtubule cytoskeleton is critically important for spatio-temporal organization of eukaryotic cells. The nucleation of new microtubules is typically restricted to microtubule organizing centers (MTOCs) and requires γ-tubulin that assembles into multisubunit complexes of various sizes. γ-Tubulin ring complexes (TuRCs) are efficient microtubule nucleators and are associated with large number of targeting, activating and modulating proteins. γ-Tubulin-dependent nucleation of microtubules occurs both from canonical MTOCs, such as spindle pole bodies and centrosomes, and additional sites such as Golgi apparatus, nuclear envelope, plasma membrane-associated sites, chromatin and surface of pre-existing microtubules. Despite many advances in structure of γ-tubulin complexes and characterization of γTuRC interacting factors, regulatory mechanisms of microtubule nucleation are not fully understood. Here, we review recent work on the factors and regulatory mechanisms that are involved in centrosomal and non-centrosomal microtubule nucleation.

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This paper reports on the results of chemical synthesis and biological assays performed on several new analogues of noscapine. We have successfully synthesized four noscapine analogues called 1a–4a, as well as their four corresponding enantiomers called 1b–4b. The chemical pathway consisted of three steps with yields in excess of 60% in each step. Subsequently, we have performed biological activity assays intended to reveal the mode of action of these compounds on microtubules in buffer and in cancer cell lines. We have assayed fluorescence quenching effects in microtubule polymerization experiments, cytotoxicity evaluation in breast cancer cell lines, as well as microtubule dynamicity assessments, for each of the synthesized compounds. Finally, we performed computational docking simulations to two binding sites on β-tubulin: (a) the colchicine binding site and (b) the noscapine binding site. Our results indicate that these compounds have relatively low cytotoxicity profile and less pronounced effects on microtubule dynamics compared with noscapine. Our computational results indicate that these compounds bind to both putative binding sites but have higher affinity for the colchicine site.

A glycolytic profile unifies a group of pheochromocytomas and paragangliomas (PHEOs/PGLs) with distinct underlying gene defects, including von Hippel-Lindau (VHL) and succinate dehydrogenase B (SDHB) mutations. Nevertheless, their tumor aggressiveness is distinct: PHEOs/PGLs metastasize rarely in VHL-, but frequently in SDHB-patients. To date, the molecular mechanisms causing the more aggressive phenotype in SDHB-PHEOs/PGLs remain largely unknown. Recently, however, an excellent model to study aggressive PHEOs (mouse tumor tissue (MTT) cells) has been developed from mouse PHEO cells (MPC). We employed this model for a proteomics based approach to identify changes characteristic for tumor aggressiveness, which we then explored in a homogeneous set of human SDHB- and VHL-PHEOs/PGLs. The increase of glucose transporter 1 in VHL, and of hexokinase 2 in VHL and SDHB, confirmed their glycolytic profile. In agreement with the cell model and in support of decoupling of glycolysis, the Krebs cycle and oxidative phosphorylation (OXPHOS), SDHB tumors showed increased lactate dehydrogenase levels. In SDHB-PGLs OXPHOS complex activity was increased at complex III and, as expected, decreased at complex II. Moreover, protein and mRNA expression of all tested OXPHOS-related genes were higher in SDHB- than in VHL-derived tumors. Although there was no direct evidence for increased reactive oxygen species production, elevated superoxide dismutase 2 expression may reflect elevated oxidative stress in SDHB-derived PHEOs/PGLs. For the first time, we show that despite dysfunction in complex II and evidence for a glycolytic phenotype, the Warburg effect does not seem to fully apply to SDHB-PHEOs/PGLs with respect to decreased OXPHOS. In addition, we present evidence for increased LDHA and SOD2 expression in SDHB-PHEOs/PGLs, proteins that have been proposed as promising therapeutic targets in other cancers. This study provides new insight into pathogenic mechanisms in aggressive human PHEOs/PGLs, which may lead to identifying new diagnostic and prognostic markers in the near future.

Ag-mediated activation of mast cells initiates signaling events leading to Ca2+ response, release of allergic mediators from cytoplasmic granules, and synthesis of cytokines and chemokines. Although microtubule rearrangement during activation has been described, the molecular mechanisms that control their remodeling are largely unknown. Microtubule nucleation is mediated by complexes that are formed by γ-tubulin and γ-tubulin complex proteins. In this study, we report that, in bone marrow–derived mast cells (BMMCs), γ-tubulin interacts with p21-activated kinase interacting exchange factor β (βPIX) and G protein–coupled receptor kinase-interacting protein (GIT)1. Microtubule regrowth experiments showed that the depletion of βPIX in BMMCs stimulated microtubule nucleation, whereas depletion of GIT1 led to the inhibition of nucleation compared with control cells. Phenotypic rescue experiments confirmed that βPIX and GIT1 represent negative and positive regulators of microtubule nucleation in BMMCs, respectively. Live-cell imaging disclosed that both proteins are associated with centrosomes. Immunoprecipitation and pull-down experiments revealed that an enhanced level of free cytosolic Ca2+ affects γ-tubulin properties and stimulates the association of GIT1 and γ-tubulin complex proteins with γ-tubulin. Microtubule nucleation also was affected by Ca2+ level. Moreover, in activated BMMCs, γ-tubulin formed complexes with tyrosine-phosphorylated GIT1. Further experiments showed that GIT1 and βPIX are involved in the regulation of such important physiological processes as Ag-induced chemotaxis and degranulation. Our study provides for the first time, to our knowledge, a possible mechanism for the concerted action of tyrosine kinases, GIT1/βPIX proteins, and Ca2+ in the propagation of signals leading to the regulation of microtubule nucleation in activated mast cells.