Aggregation of high-affinity IgE receptors (FcεRIs) on granulated mast cells triggers signaling pathways leading to a calcium response and release of inflammatory mediators from secretory granules. While microtubules play a role in the degranulation process, the complex molecular mechanisms regulating microtubule remodeling in activated mast cells are only partially understood. Here, we demonstrate that the activation of bone marrow mast cells induced by FcεRI aggregation increases centrosomal microtubule nucleation, with G protein-coupled receptor kinase-interacting protein 2 (GIT2) playing a vital role in this process. Both endogenous and exogenous GIT2 were associated with centrosomes and γ-tubulin complex proteins. Depletion of GIT2 enhanced centrosomal microtubule nucleation, and phenotypic rescue experiments revealed that GIT2, unlike GIT1, acts as a negative regulator of microtubule nucleation in mast cells. GIT2 also participated in the regulation of antigen-induced degranulation and chemotaxis. Further experiments showed that phosphorylation affected the centrosomal localization of GIT2 and that during antigen-induced activation, GIT2 was phosphorylated by conventional protein kinase C, which promoted microtubule nucleation. We propose that GIT2 is a novel regulator of microtubule organization in activated mast cells by modulating centrosomal microtubule nucleation.
Microtubules composed of αβ-tubulin dimers are dynamic cytoskeletal polymers that play key roles in essential cellular processes such as cell division, organelle positioning, intracellular transport, and cell migration. γ-Tubulin is a highly conserved member of the tubulin family that is required for microtubule nucleation. γ-Tubulin, together with its associated proteins, forms the γ-tubulin ring complex (γ-TuRC), that templates microtubules. Here we review recent advances in the structure of γ-TuRC, its activation, and centrosomal recruitment. This provides new mechanistic insights into the molecular mechanism of microtubule nucleation. Accumulating data suggest that γ-tubulin also has other, less well understood functions. We discuss emerging evidence that γ-tubulin can form oligomers and filaments, has specific nuclear functions, and might be involved in centrosomal cross-talk between microtubules and microfilaments.
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
ER distribution depends on microtubules, and ER homeostasis disturbance activates the unfolded protein response resulting in ER remodeling. CDK5RAP3 (C53) implicated in various signaling pathways interacts with UFM1-protein ligase 1 (UFL1), which mediates the ufmylation of proteins in response to ER stress. Here we find that UFL1 and C53 associate with γ-tubulin ring complex proteins. Knockout of UFL1 or C53 in human osteosarcoma cells induces ER stress and boosts centrosomal microtubule nucleation accompanied by γ-tubulin accumulation, microtubule formation, and ER expansion. C53, which is stabilized by UFL1, associates with the centrosome and rescues microtubule nucleation in cells lacking UFL1. Pharmacological induction of ER stress by tunicamycin also leads to increased microtubule nucleation and ER expansion. Furthermore, tunicamycin suppresses the association of C53 with the centrosome. These findings point to a novel mechanism for the relief of ER stress by stimulation of centrosomal microtubule nucleation.
Multimodal gadolinium fluoride nanoparticles belong to potential contrast agents useful for bimodal optical fluorescence and magnetic resonance imaging. However, the metallic nature of the nanoparticles, similarly to some paramagnetic iron oxides, might induce allergic and anaphylactic reactions in patients after administration. A reduction of these adverse side effects is a priority for the safe application of the nanoparticles. Herein, we prepared paramagnetic poly(4-styrenesulfonic acid-co-maleic acid) (PSSMA)-stabilized GdF3 nanoparticles with surface modified by Atto 488-labeled poly(styrene-grad-2-dimethylaminoethyl acrylate)-block-poly(2-dimethylaminoethyl acrylate) (PSDA-A488) with reactive amino groups for introduction of an additional imaging (luminescence) modality and possible targeting of anticancer drugs. The saturation magnetization of GdF3@PSSMA particles according to SQUID magnetometry reached 157 Am2 kg-1 at 2 K and magnetic field of 7 T. GdF3@PSSMA-PSDA-A488 nanoparticles were well tolerated by human cervical adenocarcinoma (HeLa), mouse bone marrow-derived mast cells (BMMC), and rat basophilic mast cells (RBL-2H3); the particles also affected cell morphology and protein tyrosine phosphorylation in mast cells. Moreover, the nanoparticles interfered with the activation of mast cells by multivalent antigens and inhibited calcium mobilization and cell degranulation. These findings show that the new multimodal GdF3-based nanoparticles possess properties useful for various imaging methods and might minimize mast cell degranulation incurred after future nanoparticle diagnostic administration.
- MeSH
- degranulace buněk MeSH
- krysa rodu rattus MeSH
- lidé MeSH
- mastocyty * MeSH
- myši MeSH
- nanočástice * MeSH
- polymery MeSH
- růstový diferenciační faktor 3 MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Profilin 1 is a crucial actin regulator, interacting with monomeric actin and several actin-binding proteins controlling actin polymerization. Recently, it has become evident that this profilin isoform associates with microtubules via formins and interferes with microtubule elongation at the cell periphery. Recruitment of microtubule-associated profilin upon extensive actin polymerizations, for example, at the cell edge, enhances microtubule growth, indicating that profilin contributes to the coordination of actin and microtubule organization. Here, we provide further evidence for the profilin-microtubule connection by demonstrating that it also functions in centrosomes where it impacts on microtubule nucleation.
- MeSH
- aktiny metabolismus MeSH
- Caco-2 buňky MeSH
- centrozom metabolismus MeSH
- forminy metabolismus MeSH
- genový knockout MeSH
- lidé MeSH
- melanom experimentální metabolismus patologie MeSH
- mikrofilamentové proteiny metabolismus MeSH
- mikrotubuly metabolismus MeSH
- myši MeSH
- nádory kůže metabolismus patologie MeSH
- polymerizace MeSH
- profiliny genetika metabolismus MeSH
- signální transdukce genetika MeSH
- transfekce MeSH
- tubulin metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Microtubules, polymers of the heterodimeric protein αβ-tubulin, are indispensable for many cellular activities such as maintenance of cell shape, division, migration, and ordered vesicle transport. In vitro assays to study microtubule functions and their regulation by associated proteins require the availability of assembly-competent purified tubulin. However, tubulin is a thermolabile protein that rapidly converts into a nonpolymerizing state. For this reason, it is usually stored at -80 °C or liquid nitrogen to preserve its conformation and polymerization properties. In this chapter, we describe a method for freeze-drying of assembly-competent tubulin in the presence of nonreducing sugar trehalose, and methods enabling the evaluation of tubulin functions in rehydrated samples.
- MeSH
- lidé MeSH
- lyofilizace MeSH
- stabilita proteinů MeSH
- trehalosa chemie MeSH
- tubulin chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
De novo heterozygous missense variants in the γ-tubulin gene TUBG1 have been linked to human malformations of cortical development associated with intellectual disability and epilepsy. Here, we investigated through in-utero electroporation and in-vivo studies, how four of these variants affect cortical development. We show that TUBG1 mutants affect neuronal positioning, disrupting the locomotion of new-born neurons but without affecting progenitors' proliferation. We further demonstrate that pathogenic TUBG1 variants are linked to reduced microtubule dynamics but without major structural nor functional centrosome defects in subject-derived fibroblasts. Additionally, we developed a knock-in Tubg1Y92C/+ mouse model and assessed consequences of the mutation. Although centrosomal positioning in bipolar neurons is correct, they fail to initiate locomotion. Furthermore, Tubg1Y92C/+ animals show neuroanatomical and behavioral defects and increased epileptic cortical activity. We show that Tubg1Y92C/+ mice partially mimic the human phenotype and therefore represent a relevant model for further investigations of the physiopathology of cortical malformations.
- MeSH
- centrozom metabolismus MeSH
- chování zvířat MeSH
- elektronová mikroskopie MeSH
- embryo savčí MeSH
- epilepsie genetika MeSH
- fibroblasty cytologie metabolismus ultrastruktura MeSH
- genetická predispozice k nemoci MeSH
- genový knockin MeSH
- HeLa buňky MeSH
- intravitální mikroskopie MeSH
- konfokální mikroskopie MeSH
- lidé MeSH
- malformace mozkové kůry genetika MeSH
- mikrotubuly genetika metabolismus MeSH
- missense mutace MeSH
- modely nemocí na zvířatech MeSH
- mozková kůra abnormality cytologie diagnostické zobrazování MeSH
- myši transgenní MeSH
- myši MeSH
- neurogeneze genetika MeSH
- neurony fyziologie MeSH
- pohyb buněk genetika MeSH
- tubulin genetika MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- audiovizuální média MeSH
- časopisecké články MeSH
- práce podpořená grantem MeSH
Mast cells play an effector role in innate immunity, allergy, and inflammation. Antigen-mediated activation of mast cells initiates signaling events leading to Ca2+ response and the release of inflammatory and allergic mediators from granules. Diseases associated with deregulated mast cell functions are hard to treat and there is an increasing demand for new therapeutic strategies. Miltefosine (hexadecylphosphocholine) is a new candidate for treatment of mast cell-driven diseases as it inhibits activation of mast cells. It has been proposed that miltefosine acts as a lipid raft modulator through its interference with the structural organization of surface receptors in the cell membrane. However, molecular mechanisms of its action are not fully understood. Here, we report that in antigen-activated bone marrow-derived mast cells (BMMCs), miltefosine inhibits degranulation, reorganization of microtubules, as well as antigen-induced chemotaxis. While aggregation and tyrosine phosphorylation of IgE receptors were suppressed in activated cells pre-treated with miltefosine, overall tyrosine phosphorylation levels of Lyn and Syk kinases, and Ca2+ influx were not inhibited. In contrast, lipid raft disruptor methyl-β-cyclodextrin attenuated the Ca2+ influx. Tagged-miltefosine rapidly localized into the cell interior, and live-cell imaging of BMMCs with labeled intracellular granules disclosed that miltefosine inhibited movement of some granules. Immunoprecipitation and in vitro kinase assays revealed that miltefosine inhibited Ca2+- and diacylglycerol-regulated conventional protein kinase C (cPKC) isoforms that are important for mast cell degranulation. Inhibition of cPKCs by specific inhibitor Ly333531 affected activation of BMMCs in the same way as miltefosine. Collectively, our data suggest that miltefosine modulates mast cells both at the plasma membrane and in the cytosol by inhibition of cPKCs. This alters intracellular signaling pathway(s) directed to microtubules, degranulation, and migration.
- Publikační typ
- časopisecké články MeSH
γ-Tubulin is essential for microtubule nucleation and also plays less understood roles in nuclear and cell-cycle-related functions. High abundancy of γ-tubulin in acentrosomal Arabidopsis cells facilitated purification and biochemical characterization of large molecular species of γ-tubulin. TEM, fluorescence, and atomic force microscopy of purified high molecular γ-tubulin forms revealed the presence of linear filaments with a double protofilament substructure, filament bundles and aggregates. Filament formation from highly purified γ-tubulin free of γ-tubulin complex proteins (GCPs) was demonstrated for both plant and human γ-tubulin. Moreover, γ-tubulin associated with porcine brain microtubules formed oligomers. Experimental evidence on the intrinsic ability of γ-tubulin to oligomerize/polymerize was supported by conservation of α- and β-tubulin interfaces for longitudinal and lateral interactions for γ-tubulins. STED (stimulated emission depletion) microscopy of Arabidopsis cells revealed fine, short γ-tubulin fibrillar structures enriched on mitotic microtubular arrays that accumulated at polar regions of acentrosomal spindles and the outer nuclear envelope before mitosis, and were also present in nuclei. Fine fibrillar structures of γ-tubulin representing assemblies of higher order were localized in cell-cycle-dependent manner at sites of dispersed γ-tubulin location in acentrosomal plant cells as well as at sites of local γ-tubulin enrichment after drug treatment. Our findings that γ-tubulin preserves the capability of prokaryotic tubulins to self-organize into filaments assembling by lateral interaction into bundles/clusters help understanding of the relationship between structure and multiple cellular functions of this protein species and suggest that besides microtubule nucleation and organization, γ-tubulin may also have scaffolding or sequestration functions.
- MeSH
- Arabidopsis chemie genetika MeSH
- cytoskelet chemie genetika MeSH
- mikrofilamenta chemie genetika ultrastruktura MeSH
- mikrotubuly chemie genetika MeSH
- mitóza genetika MeSH
- polymerizace MeSH
- proteinové agregáty genetika MeSH
- proteiny asociované s mikrotubuly chemie genetika MeSH
- tubulin chemie genetika ultrastruktura MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
