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.
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.
γ-Tubulin is assumed to be a typical cytosolic protein necessary for nucleation of microtubules from microtubule organizing centers. Using immunolocalization and cell fractionation techniques in combination with siRNAi and expression of FLAG-tagged constructs, we have obtained evidence that γ-tubulin is also present in nucleoli of mammalian interphase cells of diverse cellular origins. Immunoelectron microscopy has revealed γ-tubulin localization outside fibrillar centers where transcription of ribosomal DNA takes place. γ-Tubulin was associated with nucleolar remnants after nuclear envelope breakdown and could be translocated to nucleoli during mitosis. Pretreatment of cells with leptomycin B did not affect the distribution of nuclear γ-tubulin, making it unlikely that rapid active transport via nuclear pores participates in the transport of γ-tubulin into the nucleus. This finding was confirmed by heterokaryon assay and time-lapse imaging of photoconvertible protein Dendra2 tagged to γ-tubulin. Immunoprecipitation from nuclear extracts combined with mass spectrometry revealed an association of γ-tubulin with tumor suppressor protein C53 located at multiple subcellular compartments including nucleoli. The notion of an interaction between γ-tubulin and C53 was corroborated by pull-down and co-immunoprecipitation experiments. Overexpression of γ-tubulin antagonized the inhibitory effect of C53 on DNA damage G(2) /M checkpoint activation. The combined results indicate that aside from its known role in microtubule nucleation, γ-tubulin may also have nuclear-specific function(s).
- MeSH
- astrocyty metabolismus MeSH
- buněčné jadérko metabolismus MeSH
- buněčné jádro metabolismus MeSH
- časosběrné zobrazování MeSH
- fluorescenční protilátková technika MeSH
- glioblastom metabolismus MeSH
- hmotnostní spektrometrie MeSH
- imunoelektronová mikroskopie MeSH
- imunoprecipitace MeSH
- intracelulární signální peptidy a proteiny metabolismus MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- lidé MeSH
- mikrotubuly metabolismus MeSH
- mitóza fyziologie MeSH
- nádorové buněčné linie MeSH
- nádory mozku metabolismus MeSH
- proteiny nervové tkáně metabolismus MeSH
- transport proteinů fyziologie MeSH
- tubulin metabolismus MeSH
- tumor supresorové geny MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
γ-Tubulin is the key protein for microtubule nucleation. Duplication of the γ-tubulin gene occurred several times during evolution, and in mammals γ-tubulin genes encode proteins which share ∼97% sequence identity. Previous analysis of Tubg1 and Tubg2 knock-out mice has suggested that γ-tubulins are not functionally equivalent. Tubg1 knock-out mice died at the blastocyst stage, whereas Tubg2 knock-out mice developed normally and were fertile. It was proposed that γ-tubulin 1 represents ubiquitous γ-tubulin, while γ-tubulin 2 may have some specific functions and cannot substitute for γ-tubulin 1 deficiency in blastocysts. The molecular basis of the suggested functional difference between γ-tubulins remains unknown. Here we show that exogenous γ-tubulin 2 is targeted to centrosomes and interacts with γ-tubulin complex proteins 2 and 4. Depletion of γ-tubulin 1 by RNAi in U2OS cells causes impaired microtubule nucleation and metaphase arrest. Wild-type phenotype in γ-tubulin 1-depleted cells is restored by expression of exogenous mouse or human γ-tubulin 2. Further, we show at both mRNA and protein levels using RT-qPCR and 2D-PAGE, respectively, that in contrast to Tubg1, the Tubg2 expression is dramatically reduced in mouse blastocysts. This indicates that γ-tubulin 2 cannot rescue γ-tubulin 1 deficiency in knock-out blastocysts, owing to its very low amount. The combined data suggest that γ-tubulin 2 is able to nucleate microtubules and substitute for γ-tubulin 1. We propose that mammalian γ-tubulins are functionally redundant with respect to the nucleation activity.
- MeSH
- časové faktory MeSH
- down regulace MeSH
- embryonální vývoj genetika MeSH
- implantace embrya MeSH
- intracelulární prostor metabolismus MeSH
- lidé MeSH
- mikrotubuly metabolismus MeSH
- mitóza genetika MeSH
- myši inbrední C57BL MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- protein - isoformy nedostatek genetika metabolismus MeSH
- transport proteinů MeSH
- tubulin nedostatek genetika metabolismus MeSH
- vývojová regulace genové exprese MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
