The expansion of brain size in species with a large and gyrified cerebral cortex is triggered by a relative enlargement of the subventricular zone (SVZ) during development. Here, we uncover the key role of the novel interphase centrosome protein Akna in this process and show that it localizes mainly at subdistal appendages of the mother centriole in subtypes of neural stem and progenitor cells. Akna is necessary and sufficient to organize microtubules (MT) at the centrosome and regulate their polymerization. These processes show an unprecedented role of MT dynamics controlled by Akna in regulating entry to, and exit from, the SVZ by controlling delamination from the neuroepithelial ventricular zone and retention of cells in the SVZ. Importantly, Akna plays a similar role in mammary epithelial cells undergoing epithelial-to-mesenchymal transition (EMT), generalizing the importance of this new centrosomal protein in orchestrating MT polymerization to control cell delamination. Main Text Expansion of the SVZ is the developmental hallmark of enlarged and folded cerebral cortices, underpinning the importance of understanding the mechanisms that govern its formation. Epithelial-like neural stem cells (NSCs) divide in the ventricular zone (VZ), and mostly generate a new NSC and a committed progenitor cell at midneurogenesis. The latter delaminates and transforms into a basal progenitor (BP) which constitute the SVZ 1,2. Keeping cells for a defined temporal window in the SVZ is essential to control further amplification and fate determination 3,4. To identify novel regulators of these processes we compared the transcriptome of murine NSC sub-types that generate BPs from those that do not 5-7. We report here a novel and unexpected regulator of BP generation and SVZ formation, called Akna. Our work uncovers the function of this mis-annotated protein at the centrosome and reveals interphase centrosomal microtubule organizing center (MTOC) activity as a novel mechanism regulating EMT-like delamination of cells from the VZ to enter the SVZ and their retention therein. Akna is an integral component of the interphase centrosome In murine cerebral cortex, Akna mRNA levels correlate with the time of SVZ generation (low at embryonic day 11 (E11), high at E14, low at E18) and NSCs isolated at the peak of SVZ generation have higher Akna levels when transitioning to BPs 6 (Extended Data Fig. 1a,b). We therefore chose Akna as a candidate regulator of SVZ and BP generation and generated several rat and mouse monoclonal antibodies against Akna, validated by means of RNA interference, to test this hypothesis further (Extended Data Fig. 1c-f, information about clones Konno, D. et al. Neuroepithelial progenitors undergo LGN-dependent planar divisions to maintain self-renewability during mammalian neurogenesis. Nat Cell Biol 10, 93-101 (2008). Taverna, E., Gotz, M. & Huttner, W. B. The cell biology of neurogenesis: toward an understanding of the development and evolution of the neocortex. Annu Rev Cell Dev Biol 30, 465-502 (2014). Martinez-...
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.
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).
γ-Tubulins are highly conserved members of the tubulin superfamily essential for microtubule nucleation. Humans possess 2 γ-tubulin genes. It is thought that γ-tubulin-1 represents a ubiquitous isotype, whereas γ-tubulin-2 is found predominantly in the brain, where it may be endowed with divergent functions beyond microtubule nucleation. The molecular basis of the purported functional differences between γ-tubulins is unknown. We report discrimination of human γ-tubulins according to their electrophoretic and immunochemical properties. mutagenesis revealed that the differences in electrophoretic mobility originate in the C-terminal regions of the γ-tubulins. Using epitope mapping, we discovered mouse monoclonal antibodies that can discriminate between human γ-tubulin isotypes. Real time quantitative RT-PCR and 2-dimensional-PAGE showed that γ-tubulin-1 is the dominant isotype in fetal neurons. Although γ-tubulin-2 accumulates in the adult brain, γ-tubulin-1 remains the major isotype in various brain regions. Localization of γ-tubulin-1 in mature neurons was confirmed by immunohistochemistry and immunofluorescence microscopy on clinical samples and tissue microarrays. Differentiation of SH-SY5Y human neuroblastoma cells by all- retinoic acid, or oxidative stress induced by mitochondrial inhibitors, resulted in upregulation of γ-tubulin-2, whereas the expression of γ-tubulin-1 was unchanged. Fractionation experiments and immunoelectron microscopy revealed an association of γ-tubulins with mitochondrial membranes. These data indicate that in the face of predominant γ-tubulin-1 expression, the accumulation of γ-tubulin-2 in mature neurons and neuroblastoma cells during oxidative stress may denote a prosurvival role of γ-tubulin-2 in neurons.-Dráberová, E., Sulimenko, V., Vinopal, S., Sulimenko, T., Sládková, V., D'Agostino, L., Sobol, M., Hozák, P., Křen, L., Katsetos, C. D., Dráber, P. Differential expression of human γ-tubulin isotypes during neuronal development and oxidative stress points to γ-tubulin-2 prosurvival function.
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.
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