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Abstract Background Primary cilia on the surface of eukaryotic cells serve as sensory antennas for the reception and transmission in various cell signaling pathways. They are dynamic organelles that rapidly form during differentiation and cell cycle exit. Defects in these organelles cause a group of wide-ranging disorders called ciliopathies. Tonicity-responsive enhancer-binding protein (TonEBP) is a pleiotropic stress protein that mediates various physiological and pathological cellular responses. TonEBP is well-known for its role in adaptation to a hypertonic environment, to which primary cilia have been reported to contribute. Furthermore, TonEBP is involved in a wide variety of other signaling pathways, such as Sonic Hedgehog and WNT signaling, that promote primary ciliogenesis, suggesting a possible regulatory role. However, the functional relationship between TonEBP and primary ciliary formation remains unclear. Methods TonEBP siRNAs and TonEBP-mCherry plasmids were used to examine their effects on cell ciliation rates, assembly and disassembly processes, and regulators. Serum starvation was used as a condition to induce ciliogenesis. Results We identified a novel pericentriolar localization for TonEBP. The results showed that TonEBP depletion facilitates the formation of primary cilia, whereas its overexpression results in fewer ciliated cells. Moreover, TonEBP controlled the expression and activity of aurora kinase A, a major negative regulator of ciliogenesis. Additionally, TonEBP overexpression inhibited the loss of CP110 from the mother centrioles during the early stages of primary cilia assembly. Finally, TonEBP regulated the localization of PCM1 and AZI1, which are necessary for primary cilia formation. Conclusions This study proposes a novel role for TonEBP as a pericentriolar protein that regulates the integrity of centriolar satellite components. This regulation has shown to have a negative effect on ciliogenesis. Investigations into cilium assembly and disassembly processes suggest that TonEBP acts upstream of the aurora kinase A - histone deacetylase 6 signaling pathway and affects basal body formation to control ciliogenesis. Taken together, our data proposes previously uncharacterized regulation of primary cilia assembly by TonEBP.

Background: Primary cilia on the surface of eukaryotic cells serve as sensory antennas for the reception and transmission in various cell signaling pathways. They are dynamic organelles that rapidly form during differentiation and cell cycle exit. Defects in these organelles cause a group of wide-ranging disorders called ciliopathies. Tonicity-responsive enhancer-binding protein (TonEBP) is a pleiotropic stress protein that mediates various physiological and pathological cellular responses. TonEBP is well-known for its role in adaptation to a hypertonic environment, to which primary cilia have been reported to contribute. Furthermore, TonEBP is involved in a wide variety of other signaling pathways, such as Sonic Hedgehog and WNT signaling, that promote primary ciliogenesis, suggesting a possible regulatory role. However, the functional relationship between TonEBP and primary ciliary formation remains unclear. Methods: TonEBP siRNAs and TonEBP-mCherry plasmids were used to examine their effects on cell ciliation rates, assembly and disassembly processes, and regulators. Serum starvation was used as a condition to induce ciliogenesis. Results: We identified a novel pericentriolar localization for TonEBP. The results showed that TonEBP depletion facilitates the formation of primary cilia, whereas its overexpression results in fewer ciliated cells. Moreover, TonEBP controlled the expression and activity of aurora kinase A, a major negative regulator of ciliogenesis. Additionally, TonEBP overexpression inhibited the loss of CP110 from the mother centrioles during the early stages of primary cilia assembly. Finally, TonEBP regulated the localization of PCM1 and AZI1, which are necessary for primary cilia formation. Conclusions: This study proposes a novel role for TonEBP as a pericentriolar protein that regulates the integrity of centriolar satellite components. This regulation has shown to have a negative effect on ciliogenesis. Investigations into cilium assembly and disassembly processes suggest that TonEBP acts upstream of the aurora kinase A - histone deacetylase 6 signaling pathway and affects basal body formation to control ciliogenesis. Taken together, our data proposes previously uncharacterized regulation of primary cilia assembly by TonEBP. [ABSTRACT FROM AUTHOR]

During development, neurons achieve a stereotyped neuron typespecific morphology, which relies on dynamic support by microtubules (MTs). An important player is the augmin complex (hereafter augmin), which binds to existing MT filaments and recruits the γ-tubulin ring complex (γ-TuRC), to form branched MTs. In cultured neurons, augmin is important for neurite formation. However, little is known about the role of augmin during neurite formation in vivo. Here, we have revisited the role of mammalian augmin in culture and then turned towards the class four Drosophila dendritic arborization (c4da) neurons. We show that MT density is maintained through augmin in cooperation with the γ-TuRC in vivo. Mutant c4da neurons show a reduction of newly emerging higher-order dendritic branches and in turn also a reduced number of their characteristic space-filling higher-order branchlets. Taken together, our data reveal a cooperative function for augmin with the γ-TuRC in forming enough MTs needed for the appropriate differentiation of morphologically complex dendrites in vivo. [ABSTRACT FROM AUTHOR]

Microtubule Organizing Centers (MTOC) are subcellular structures in eukaryotic cells where nucleation of microtubules (MTs) takes place and represents the filament's minus end. Their localization depends on the species, cell type, and cell cycle stage. Along the fungal kingdom, the Spindle Pole Body (SPB) in the nucleus (an equivalent to Centrosomes in animal cells) is the principal MTOC. Other MTOCs have been identified in filamentous fungi, such as the Spitzenkörper in the hyphal tips of Schizosaccharomyces pombe or the septal pore of Aspergillus nidulans. However, in the fungal-model organism Neurospora crassa, these alternative MTOCs have not been recognized. Here, we present a Mass spectrometry-based dataset of proteins interacting with four MTOC components of N. crassa tagged with fluorescent proteins: γ-Tubulin-sGFP (main nucleator at the SPB), MZT-1-sGFP (structural SPB microprotein), APS-2-dRFP (septal protein and recognized SPB component), and SPA-10-sGFP (septal MTOC protein). A WT and a cytosolic GFP expressing strain were included as controls. The protein interactors were pulled down by Co-IP1, using GFP-Magnetic agarose that captures recombinant GFP proteins (including GFP-derivatives) in their native state. Bounded proteins were separated by SDS-PAGE and identified by nano LC-MS/MS2. The protein annotation was done using the N. crassa protein database.

γ-Tubulin typically forms a ring-shaped complex with 5 related γ-tubulin complex proteins (GCP2 to GCP6), and this γ-tubulin ring complex (γTuRC) serves as a template for microtubule (MT) nucleation in plants and animals. While the γTuRC takes part in MT nucleation in most eukaryotes, in fungi such events take place robustly with just the γ-tubulin small complex (γTuSC) assembled by γ-tubulin plus GCP2 and GCP3. To explore whether the γTuRC is the sole functional γ-tubulin complex in plants, we generated 2 mutants of the GCP6 gene encoding the largest subunit of the γTuRC in Arabidopsis thaliana. Both mutants showed similar phenotypes of dwarfed vegetative growth and reduced fertility. The gcp6 mutant assembled the γTuSC, while the wild-type cells had GCP6 join other GCPs to produce the γTuRC. Although the gcp6 cells had greatly diminished γ-tubulin localization on spindle MTs, the protein was still detected there. The gcp6 cells formed spindles that lacked MT convergence and discernable poles; however, they managed to cope with the challenge of MT disorganization and were able to complete mitosis and cytokinesis. Our results reveal that the γTuRC is not the only functional form of the γ-tubulin complex for MT nucleation in plant cells, and that γ-tubulin-dependent, but γTuRC-independent, mechanisms meet the basal need of MT nucleation. Moreover, we show that the γTuRC function is more critical for the assembly of spindle MT array than for the phragmoplast. Thus, our findings provide insight into acentrosomal MT nucleation and organization.

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The regulation and recruitment of γ-TuRCs, the prime nucleator of microtubules, to the centrosome are still thrust areas of research. The interaction of fodrin, a sub-plasmalemmal cytoskeletal protein, with γ-tubulin is a new area of interest. To understand the cellular significance of this interaction, we show that depletion of α-fodrin brings in a significant reduction of γ-tubulin in neural cell centrosomes making it functionally under-efficient. This causes a loss of nucleation ability that cannot efficiently form microtubules in interphase cells and astral microtubules in mitosis. Fluorescence Recovery after Photobleaching (FRAP) experiment implies that α-fodrin is important in the recruitment of γ-tubulin to the centrosome resulting in the aforementioned effects. Further, our experiments indicate that the interaction of α-fodrin with certain pericentriolar matrix proteins such as Pericentrin and CDK5RAP2 are crucial for the recruitment of γ-tubulin to the centrosome. Earlier we reported that α-fodrin limits the nucleation potential of γ-TuRC. In that context, this study suggests that α-fodrin is a γ-tubulin recruiting protein to the centrosome thus preventing cytoplasmic microtubule nucleation and thereby compartmentalizing the process to the centrosome for maximum efficiency. Summary statementα-fodrin is a γ-tubulin interacting protein that controls the process of γ-tubulin recruitment to the centrosome and thereby regulates the microtubule nucleation capacity spatially and temporally. [ABSTRACT FROM AUTHOR]

Oocyte meiotic errors can cause infertility, miscarriage, and birth defects. Here the role and the underlying mechanism of p21 activated kinase 4 (PAK4) in mouse oocyte meiosis is evaluated. It is found that PAK4 expression and its phosphorylation are detected in high level at germinal vesicle (GV) stage, and gradually decreased after meiotic resumption in oocytes. PAK4 has direct physical interaction with both mitogen-activated protein kinases 1/2 (MEK1/2) and Paxillin, they are colocalized on the spindle structure during metaphases I and II. Phospho-PAK4 is distributed beneath the cytoplasmic membrane and on the chromosomes, and colocalized with the microtubule organizing center (MTOC) proteins, Pericentrin and γ-tubulin, as well as phosphor-MEK1/2 and phosphor-Paxillin on spindle poles. PAK4 inhibition by chemical inhibitor LCH-7749944, specific Pak4 morpholino oligo or the dominant negative mutant Pak4 K350, 351 M influence the meiotic resumption, spindle assembly and its cortical migration, and associated with the downregulation in the dephosphorylation of cyclin dependent kinase 1 (CDK1) and the levels of Cyclin B1, MEK1/2, Paxillin, g-tubulin, acetylated a-tubulin, Arp3, and Cofilin phosphorylation in oocytes. In sum, PAK4 functions to sustain the rational levels of Cyclin B1, MEK1/2, Paxillin, y-tubulin, acetylated a-tubulin, Arp3, and phosphor-Cofilin in mouse oocytes, thereby promotes the meiotic resumption, spindle assembly, and migration during meiotic maturation., (© 2024 Wiley‐VCH GmbH.)

In contrast to well-studied fungal and animal cells, plant cells assemble bipolar spindles that exhibit a great deal of plasticity in the absence of structurally defined microtubule-organizing centers like the centrosome. While plants employ some evolutionarily conserved proteins to regulate spindle morphogenesis and remodeling, many essential spindle assembly factors found in vertebrates are either missing or not required for producing the plant bipolar microtubule array. Plants also produce proteins distantly related to their fungal and animal counterparts to regulate critical events such as the spindle assembly checkpoint. Plant spindle assembly initiates with microtubule nucleation on the nuclear envelope followed by bipolarization into the prophase spindle. After nuclear envelope breakdown, kinetochore fibers are assembled and unified into the spindle apparatus with convergent poles. Of note, compared to fungal and animal systems, relatively little is known about how plant cells remodel the spindle microtubule array during anaphase. Uncovering mitotic functions of novel proteins for spindle assembly in plants will illuminate both common and divergent mechanisms employed by different eukaryotic organisms to segregate genetic materials. [ABSTRACT FROM AUTHOR]

Colobanthus quitensis is one of the two angiosperm plant species commonly spread in the Antarctic. The species has been extensively analyzed at morphological, anatomical and physiological levels, but information regarding its genetic vari-ability remains limited. The aim of the study was to identify molecular genetic differences between C. quitensis populations in one of the Antarctic localities, the Argentine Islands region by estimating the intron length polymorphism of actin, α- and γ-tubulin genes. Samples of C. quitensis from different Antarctic natural populations were collected during the season of the 24th and previous Ukrainian Antarctic expeditions. Total DNA was isolated using the QIAGEN DNeasy Plant Mini Kit. The polymerase chain reaction was carried out with our own degenerate primers. The resulting amplicons were separated and visualized using polyacrylamide gel electrophoresis followed by silver nitrate staining. Molecular genetic analysis of natural populations of C. quitensis was performed using three DNA-marker systems based on the detection of intron length polymor-phism of actin, α- and γ-tubulin genes. A low level of genetic polymorphism of C. quitensis in the studied region by these types of markers was established. By assessing the intron length polymorphism of actin genes of the studied C. quitensis populations it was possible to establish that the populations of Skua Island had unique amplicons characteristic only for this location. This indicates the possibility of further use of the analysis of intron length polymorphism of actin genes for the study of the molecu-lar genetic diversity of the Antarctic pearlwort. At the same time, the results of analysis of the intron length polymorphism of α- and γ-tubulin genes induce selection of more specific primers, taking into account the structure of the C. quitensis genome. C. quitensis in the study region has a low level of genetic variability in intron length polymorphism of actin, α- and γ-tubulin genes. Overall, the results indicate that DNA markers based on gene structure analysis of highly conserved cytoskeletal pro-teins, namely, actin, α- and γ-tubulin, as additional sources of information, can be used for molecular genetic analysis of C. quitensis populations in the Antarctic.

Objectives: Histone deacetylase 8 (HDAC8) is one of the class I HDAC family proteins, which participates in the neuronal disorders, parasitic/viral infections, tumorigenesis and many other biological processes. However, its potential function during female germ cell development has not yet been fully understood. Materials and methods: HDAC8‐targeting siRNA was microinjected into GV oocytes to deplete HDAC8. PCI‐34051 was used to inhibit the enzyme activity of HDAC8. Immunostaining, immunoblotting and fluorescence intensity quantification were applied to assess the effects of HDAC8 depletion or inhibition on the oocyte meiotic maturation, spindle/chromosome structure, γ‐tubulin dynamics and acetylation level of α‐tubulin. Results: We observed that HDAC8 was localized in the nucleus at GV stage and then translocated to the spindle apparatus from GVBD to M II stages in porcine oocytes. Depletion of HDAC8 led to the oocyte meiotic failure by showing the reduced polar body extrusion rate. In addition, depletion of HDAC8 resulted in aberrant spindle morphologies and misaligned chromosomes due to the defective recruitment of γ‐tubulin to the spindle poles. Notably, these meiotic defects were photocopied by inhibition of HDAC8 activity using its specific inhibitor PCI‐34051. However, inhibition of HDAC8 did not affect microtubule stability as assessed by the acetylation level of α‐tubulin. Conclusions: Collectively, our findings demonstrate that HDAC8 acts as a regulator of spindle assembly during porcine oocyte meiotic maturation. [ABSTRACT FROM AUTHOR]

Microtubule nucleation is mainly mediated by the γ‐tubulin ring complex (γTuRC), whose core components are γ‐tubulin and γ‐tubulin complex proteins GCP2–6. A substantial fraction of γ‐tubulin also exists with GCP2 and GCP3 in a tetramer called the γ‐tubulin small complex (γTuSC). To date, the mechanisms underlying the turnover of γ‐tubulin and GCPs have remained unclear. Here, we show that γ‐tubulin, GCP2, and GCP3 are proteolyzed by the ubiquitin‐proteasome system, and we identify cullin 1, cullin 4A, and cullin 4B as the E3 ligases that mediate the ubiquitination and, consequently, the degradation of γ‐tubulin. Notably, we found that γTuSC disassembly promotes the degradation of γ‐tubulin, GCP2, and GCP3, which indicates a role for γTuSCs in the stabilization of its components. [ABSTRACT FROM AUTHOR]

Abstract Background Resveratrol and its natural stilbene-containing derivatives have been extensively investigated as potential chemotherapeutic agents. The synthetic manipulation of the stilbene scaffold has led to the generation of new analogues with improved anticancer activity and better bioavailability. In the present study we investigated the anticancer activity of a novel trimethoxystilbene derivative (3,4,4′-trimethoxylstilbene), where two methoxyl groups are adjacent on the benzene ring (ortho configuration), and compared its activity to 3,5,4′-trimethoxylstilbene, whose methoxyl groups are in meta configuration. Results We provide evidence that the presence of the two methoxyl groups in ortho configuration renders 3,4,4′-trimethoxystilbene more efficient than the meta isomer in inhibiting cell proliferation and producing apoptotic death in colorectal cancer cells. Confocal microscopy of α- and γ-tubulin staining shows that the novel compound strongly depolymerizes the mitotic spindle and produces fragmentation of the pericentrosomal material. Computer assisted docking studies indicate that both molecules potentially interact with γ-tubulin, and that 3,4,4′-trimethoxystilbene is likely to establish stronger interactions with the protein. Conclusions These findings demonstrate the ortho configuration confers higher specificity for γ-tubulin with respect to α-tubulin on 3,4,4′ trimethoxystilbene, allowing it to be defined as a new γ-tubulin inhibitor. A strong interaction with γ-tubulin might be a defining feature of molecules with high anticancer activity, as shown for the 3,4,4′ isomer.

Cryo-electron microscopy recently resolved the structure of the vertebrate γ-tubulin ring complex (γ-TuRC) purified from Xenopus laevis egg extract and human cells to near-atomic resolution. These studies clarified the arrangement and stoichiometry of γ-TuRC components and revealed that one molecule of actin and the small protein MZT1 are embedded into the complex. Based on this structural census of γ-TuRC core components, we developed a recombinant expression system for the reconstitution and purification of human γ-TuRC from insect cells. The recombinant γ-TuRC recapitulates the structure of purified native γ-TuRC and has similar functional properties in terms of microtubule nucleation and minus end capping. This recombinant system is a central step towards deciphering the activation mechanisms of the γ-TuRC and the function of individual γ-TuRC core components.

α/β-Tubulin inhibitors that alter microtubule (MT) dynamics are commonly used in cancer therapy, however, these inhibitors also cause severe side effects such as peripheral neuropathy. γ-Tubulin is a possible target as antitumor drugs with low side effects, but the antitumor effect of γ-tubulin inhibitors has not been reported yet. In this study, we verified the antitumor activity of gatastatin, a γ-tubulin specific inhibitor. The cytotoxicity of gatastatin was relatively weak compared with that of the conventional MT inhibitors, paclitaxel and vinblastine. To improve the cytotoxicity, we screened the chemicals that improve the effects of gatastatin and found that BI 2536, a Plk1 inhibitor, greatly increases the cytotoxicity of gatastatin. Co-treatment with gatastatin and BI 2536 arrested cell cycle progression at mitosis with abnormal spindles. Moreover, mitotic cell death induced by the combined treatment was suppressed by the Mps1 inhibitor, reversine. These findings suggest that co-treatment with Plk1 and γ-tubulin inhibitors causes spindle assembly checkpoint-dependent mitotic cell death by impairing centrosome functions. These results raise the possibility of Plk1 and γ-tubulin inhibitor co-treatment as a novel cancer chemotherapy.

Urothelial carcinoma (UC) comprises two subtypes, low grade (LG-UC) and high grade (HG-UC), with different pathological and clinical behavior. LG-UC and HG-UC are classified based on cellular and structural atypia of pathological findings. The mechanisms responsible for maintaining structural atypia, such as the disturbance of nuclear polarity, remain unclear. In this study, we studied microtubule-organizing center (MTOC)-mediated nuclear polarity in UC subtypes. We evaluated six cases with normal urothelium (NU), 10 LG-UC cases, and 10 HG-UC cases by double immunofluorescence staining of γ-tubulin as a marker of MTOC and E-cadherin as a marker of each cell border. The number and position of γ-tubulin dots of expression in more than 100 cells per case were assessed using the spatial relationship with the nucleus and surface-basal axis. We found one γ-tubulin dot in most normal and tumor cells, and more than two γ-tubulin dots in 4.6% of NU cells, 6.1% of LG-UC cells, and 9.8% of HG-UC cells. More than three γ-tubulin dots were found only in 1.2% of HG-UC cells. Surface side positioning of γ-tubulin was found in 77.4% of normal urothelial cells, 63.8% of LG-UC cells, and 39.2% of HG-UC cells, whereas aberrant lateral and basal side positioning of γ-tubulin was found in 22.6% of normal urothelial cells, 36.1% of LG-UC cells, and 60.8% of HG-UC cells. We concluded that numerical and positional aberrations of MTOC in UC cases were strongly correlated with both cellular and structural atypia as well as abnormal cell proliferation. [ABSTRACT FROM AUTHOR]

One of the defining features of apicomplexan parasites is their cytoskeleton composed of alveolar vesicles, known as the inner membrane complex (IMC) undergirded by intermediate-like filament network and an array of subpellicular microtubules (SPMTs). In Toxoplasma gondii , this specialized cytoskeleton is involved in all aspects of the disease-causing lytic cycle, and notably acting as a scaffold for parasite offspring in the internal budding process. Despite advances in our understanding of the architecture and molecular composition, insights pertaining to the coordinated assembly of the scaffold are still largely elusive. Here, T. gondii tachyzoites were dissected by advanced, iterative expansion microscopy (pan-ExM) revealing new insights into the very early sequential formation steps of the tubulin scaffold. A comparative study of the related parasite Sarcocystis neurona revealed that different MT bundling organizations of the nascent SPMTs correlate with the number of central and basal alveolar vesicles. In absence of a so far identified MT nucleation mechanism, we genetically dissected T. gondii γ-tubulin and γ-tubulin complex protein 4 (GCP4). While γ-tubulin depletion abolished the formation of the tubulin scaffold, a set of MTs still formed that suggests SPMTs are nucleated at the outer core of the centrosome. Depletion of GCP4 interfered with the correct assembly of SPMTs into the forming daughter buds, further indicating that the parasite utilizes the γ-tubulin complex in tubulin scaffold formation .

During development, neurons achieve a stereotyped neuron type-specific morphology, which relies on dynamic support by microtubules (MTs). An important player is the augmin complex (hereafter augmin), which binds to existing MT filaments and recruits the γ-tubulin ring complex (γ-TuRC), to form branched MTs. In cultured neurons, augmin is important for neurite formation. However, little is known about the role of augmin during neurite formation in vivo. Here, we have revisited the role of mammalian augmin in culture and then turned towards the class four Drosophila dendritic arborization (c4da) neurons. We show that MT density is maintained through augmin in cooperation with the γ-TuRC in vivo. Mutant c4da neurons show a reduction of newly emerging higher-order dendritic branches and in turn also a reduced number of their characteristic space-filling higher-order branchlets. Taken together, our data reveal a cooperative function for augmin with the γ-TuRC in forming enough MTs needed for the appropriate differentiation of morphologically complex dendrites in vivo., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Understanding the molecular mechanism of drug resistance helps to identify an effective target for breast cancer therapy. In this study we investigated the regulatory role of Obg-like ATPase 1 which is involved in multiple uses of drug resistance against breast cancer. Paclitaxel resistant cell line (MCF-7-PTR) was developed by a continuous increasing paclitaxel concentration. MTT assay was used to validate either acquired resistant or OLA1 modified cell lines. qRT-PCR, western blotting, apoptosis, and cell cycle assays were executed to evaluate gene and protein expression in cell lines. A series of in vitro assays was performed in the cells with RNAi-mediated knockdown to expound the regulatory function of OLA1 in breast cancer. We demonstrated that OLA1 was highly correlated with either acquired or intrinsic resistance of breast cancer. Further study showed that escalated expression of OLA1 promoted the EMT process in tumor cells through TGF-β/Smad signaling cascades, resulting in the enhanced expression of anti-apoptosis-related proteins (cleaved caspase3, Bax, Bcl-2) and the strengthening depolymerization of microtubules in tumor cells. Our findings revealed that OLA1 enhanced the anti-apoptotic ability and elucidated a regulatory role of OLA1 in promoting chemotherapy resistance of breast cancer. Chemo-sensitivity of the disease can be thus enhanced significantly by knocked down OLA1, which led to the inactivation of the TGF-β/Smad signaling cascades, polymerized microtubules, and promoted cell apoptosis. Our data suggest that OLA1 may be developed as a potential target to improve chemotherapy of patients with breast cancer.

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.

Filamentous mammalian orthoreovirus (MRV) viral factories (VFs) are membrane-less cytosolic inclusions in which virus transcription, replication of dsRNA genome segments, and packaging of virus progeny into newly synthesized virus cores take place. In infected cells, the MRV μ2 protein forms punctae in the enlarged region of the filamentous VFs that are co-localized with γ-tubulin and resistant to nocodazole treatment, and permitted microtubule (MT)-extension, features common to MT-organizing centers (MTOCs). Using a previously established reconstituted VF model, we addressed the functions of MT-components and MTOCs concerning their roles in the formation of filamentous VFs. Indeed, the MTOC markers γ-tubulin and centrin were redistributed within the VF-like structures (VFLS) in a μ2-dependent manner. Moreover, the MT-nucleation centers significantly increased in numbers, and γ-tubulin was pulled-down in a binding assay when co-expressed with histidine-tagged-μ2 and μNS. Thus, μ2, by interaction with γ-tubulin, can modulate MTOCs localization and function according to viral needs. • μ2 punctae in filamentous reovirus viral factories (VF) behave as MTOCs. • MT-nucleating centers increase in number and localize within μ2/μNS VF-like structures. • γ-tubulin was pulled-down in co-expression with His 6X -μ2 and μNS. • MT-overexpression impairs MT-nucleating center localization in VF-like structures. • μ2 by interaction with γ-tubulin, can modulate MTOCs localization and function. [ABSTRACT FROM AUTHOR]

Understanding the molecular mechanism of drug resistance helps to identify an effective target for breast cancer therapy. In this study we investigated the regulatory role of Obg-like ATPase 1 which is involved in multiple uses of drug resistance against breast cancer. Paclitaxel resistant cell line (MCF-7-PTR) was developed by a continuous increasing paclitaxel concentration. MTT assay was used to validate either acquired resistant or OLA1 modified cell lines. qRT-PCR, western blotting, apoptosis, and cell cycle assays were executed to evaluate gene and protein expression in cell lines. A series of in vitro assays was performed in the cells with RNAi-mediated knockdown to expound the regulatory function of OLA1 in breast cancer. We demonstrated that OLA1 was highly correlated with either acquired or intrinsic resistance of breast cancer. Further study showed that escalated expression of OLA1 promoted the EMT process in tumor cells through TGF-β/Smad signaling cascades, resulting in the enhanced expression of anti-apoptosis-related proteins (cleaved caspase3, Bax, Bcl-2) and the strengthening depolymerization of microtubules in tumor cells. Our findings revealed that OLA1 enhanced the anti-apoptotic ability and elucidated a regulatory role of OLA1 in promoting chemotherapy resistance of breast cancer. Chemo-sensitivity of the disease can be thus enhanced significantly by knocked down OLA1, which led to the inactivation of the TGF-β/Smad signaling cascades, polymerized microtubules, and promoted cell apoptosis. Our data suggest that OLA1 may be developed as a potential target to improve chemotherapy of patients with breast cancer. [ABSTRACT FROM AUTHOR]

Centrosome amplification leads to aberrant mitosis, giving rise to aneuploidy and it has been associated with poor prognosis in human cancers. This study aimed to evaluate the relationship between polyploidy, centrosome abnormalities, and response to endocrine treatment in progestin-induced mouse mammary carcinomas. We found cells with three or more centrosomes in the polyploid tumors. The endocrine unresponsive tumors showed a higher average number of centrosomes per cell than the responsive tumors. The results suggest an association between polyploidy and centrosome amplification with the resistance to endocrine therapy in this luminal breast cancer model. [ABSTRACT FROM AUTHOR]

γ-Tubulin is associated with microtubule nucleation, but evidence is accumulating in eukaryotes that it also functions in nuclear processes and in cell division control independently of its canonical role. We found that in Arabidopsis thaliana , γ-tubulin interacts specifically with E2FA, E2FB, and E2FC transcription factors both in vitro and in vivo. The interaction of γ-tubulin with the E2Fs is not reduced in the presence of their dimerization partners (DPs) and, in agreement, we found that γ-tubulin interaction with E2Fs does not require the dimerization domain. γ-Tubulin associates with the promoters of E2F-regulated cell cycle genes in an E2F-dependent manner, probably in complex with the E2F–DP heterodimer. The up-regulation of E2F target genes PCNA , ORC2 , CDKB1;1 , and CCS52A under γ-tubulin silencing suggests a repressive function for γ-tubulin at G1/S and G2/M transitions, and the endocycle, which is consistent with an excess of cell division in some cells and enhanced endoreduplication in others in the shoot and young leaves of γ-tubulin RNAi plants. Altogether, our data show ternary interaction of γ-tubulin with the E2F–DP heterodimer and suggest a repressive role for γ-tubulin with E2Fs in controlling mitotic activity and endoreduplication during plant development. [ABSTRACT FROM AUTHOR]

Centrosomes are important microtubule-organizing centers (MTOCs) in animal cells. In addition, non-centrosomal MTOCs (ncMTOCs) are found in many cell types. Their composition and structure are only poorly understood. Here, we analyzed nuclear MTOCs (spindle-pole bodies, SPBs) and septal MTOCs in Aspergillus nidulans. They both contain γ-tubulin along withmembers of the family of γ-tubulin complex proteins (GCPs). Our data suggest that SPBs consist of γ-tubulin small complexes (γ-TuSCs) at the outer plaque, and larger γ-tubulin ring complexes (γ-TuRC) at the inner plaque. We show that the MztA protein, an ortholog of the human MOZART protein (also known as MZT1), interacted with the inner plaque receptor PcpA (the homolog of fission yeast Pcp1) at SPBs, while no interaction nor colocalizationwas detected between MztA and the outer plaque receptor ApsB (fission yeastMto1). SeptalMTOCs consist of γ-TuRCs including MztA but are anchored through AspB and Spa18 (fission yeast Mto2). MztA is not essential for viability, although abnormal spindles were observed frequently in cells lacking MztA.Quantitative PALM imaging revealed unexpected dynamics of the protein composition of SPBs, with changing numbers of γ-tubulin complexes over time during interphase and constant numbers during mitosis. [ABSTRACT FROM AUTHOR]

Microtubule Organizing Centers (MTOC) are subcellular structures in eukaryotic cells where nucleation of microtubules (MTs) takes place and represents the filament's minus end. Their localization depends on the species, cell type, and cell cycle stage. Along the fungal kingdom, the Spindle Pole Body (SPB) in the nucleus (an equivalent to Centrosomes in animal cells) is the principal MTOC. Other MTOCs have been identified in filamentous fungi, such as the Spitzenkörper in the hyphal tips of Schizosaccharomyces pombe or the septal pore of Aspergillus nidulans . However, in the fungal-model organism Neurospora crassa , these alternative MTOCs have not been recognized. Here, we present a Mass spectrometry-based dataset of proteins interacting with four MTOC components of  N. crassa tagged with fluorescent proteins: γ-Tubulin-sGFP (main nucleator at the SPB), MZT-1-sGFP (structural SPB microprotein), APS-2-dRFP (septal protein and recognized SPB component), and SPA-10-sGFP (septal MTOC protein). A WT and a cytosolic GFP expressing strain were included as controls. The protein interactors were pulled down by Co-IP 1 , using GFP-Magnetic agarose that captures recombinant GFP proteins (including GFP-derivatives) in their native state. Bounded proteins were separated by SDS-PAGE and identified by nano LC-MS/MS 2 . The protein annotation was done using the N. crassa protein database., (© 2024 The Authors. Published by Elsevier Inc.)

Members of the tubulin superfamily are GTPases; the activities of GTPases are necessary for life. The members of the tubulin superfamily are the constituents of the microtubules and the γ-tubulin meshwork. Mutations in members of the tubulin superfamily are involved in developmental brain disorders, and tubulin activities are the target for various chemotherapies. The intricate functions (game) of tubulins depend on the activities of the GTP-binding domain of α-, β-, and γ-tubulin. This review compares the GTP-binding domains of γ-tubulin, α-tubulin, and β-tubulin and, based on their similarities, recapitulates the known functions and the impact of the γ-tubulin GTP-binding domain in the regulation of the γ-tubulin meshwork and cellular homeostasis.

SnRK1 protein kinases are integral components of cell signaling involved in stress response, regulation of energy metabolism, seed germination and maturation, autophagy, and other processes. Nevertheless, many functions of these protein kinases remain unknown. In order to study the intracellular localization of KIN10 (catalytic subunit of the SnRK1 protein kinase complexes), protoplasts of the wild A. thaliana ecotype (Col-0) were transformed with a construct that included the KIN10-RFP fusion gene. It was established that the chimeric KIN10-RFP protein was diffusely distributed in the cytoplasm; however, it was mainly localized on the cell periphery, in the region adjacent to the plasma membrane. The diffuse distribution of KIN10 and γ-tubulin in the cytoplasm of A. thaliana root cells was demonstrated by immunofluorescence microscopy. The patterns of γ-tubulin intracellular localization in the root cells of the kin10 and kin11 knockout mutants and wild type A. thaliana were shown to be different. The intensity of γ-tubulin fluorescence in both mutants was lower than in the wild type-plants: this may be indicative of certain impairments in the formation of γ-tubulin complexes in the mutants. A lower intensity of γ-tubulin fluorescence was also recorded in cells of all lines cultivated under the conditions of energy shortage. In particular, the lowest level of fluorescence was recorded in the kin10 and kin11 mutants exposed to this type of stress, and this may be indicative of the synergistic influence of simultaneous dysfunction of one of these genes and energy deficiency on the formation of γ-tubulin complexes (γTuSC and γTuRC). The results obtained demonstrate the possible involvement of SnRK1 (KIN10 and KIN11) in the physiological response of plants to energy stress. With the data on the possible phosphorylation of the Ser131 residue in plant γ-tubulin by KIN10 taken into account, it can be assumed that SnRK1 protein kinases are involved in the regulation of microtubule polymerization in plants. [ABSTRACT FROM AUTHOR]

ContentsSummaryI.IntroductionII.MT arrays in plant cellsIII.γ‐Tubulin and MT nucleationIV.MT nucleation sites or flexible MTOCs in plant cellsV.MT‐dependent MT nucleationVI.Generating new MTs for spindle assemblyVII.Generation of MTs for phragmoplast expansion during cytokinesisVIII.MT generation for the cortical MT arrayIX.MT nucleation: looking forward AcknowledgementsReferences Summary: Cytoskeletal microtubules (MTs) have a multitude of functions including intracellular distribution of molecules and organelles, cell morphogenesis, as well as segregation of the genetic material and separation of the cytoplasm during cell division among eukaryotic organisms. In response to internal and external cues, eukaryotic cells remodel their MT network in a regulated manner in order to assemble physiologically important arrays for cell growth, cell proliferation, or for cells to cope with biotic or abiotic stresses. Nucleation of new MTs is a critical step for MT remodeling. Although many key factors contributing to MT nucleation and organization are well conserved in different kingdoms, the centrosome, representing the most prominent microtubule organizing centers (MTOCs), disappeared during plant evolution as angiosperms lack the structure. Instead, flexible MTOCs may emerge on the plasma membrane, the nuclear envelope, and even organelles depending on types of cells and organisms and/or physiological conditions. MT‐dependent MT nucleation is particularly noticeable in plant cells because it accounts for the primary source of MT generation for assembling spindle, phragmoplast, and cortical arrays when the γ‐tubulin ring complex is anchored and activated by the augmin complex. It is intriguing what proteins are associated with plant‐specific MTOCs and how plant cells activate or inactivate MT nucleation activities in spatiotemporally regulated manners. [ABSTRACT FROM AUTHOR]

Intramolecular electrostatic attraction and repulsion strongly influence the conformational sampling of intrinsically disordered proteins and domains (IDPs). In order to better understand this complex relationship, we have used nuclear magnetic resonance to measure side chain pKa values and pH‐dependent translational diffusion coefficients for the unstructured and highly acidic carboxyl‐terminus of γ‐tubulin (γ‐CT), providing insight into how the net charge of an IDP relates to overall expansion or collapse of the conformational ensemble. Many of the pKa values in the γ‐CT are shifted upward by 0.3–0.4 units and exhibit negatively cooperative ionization pH profiles, likely due to the large net negative charge that accumulates on the molecule as the pH is raised. pKa shifts of this magnitude correspond to electrostatic interaction energies between the affected residues and the rest of the charged molecule that are each on the order of 1 kcal mol−1. Diffusion of the γ‐CT slowed with increasing net charge, indicative of an expanding hydrodynamic radius (rH). The degree of expansion agreed quantitatively with what has been seen from comparisons of IDPs with different charge content, yielding the general trend that every 0.1 increase in relative charge (|Q|/res) produces a roughly 5% increase in rH. While γ‐CT pH titration data followed this trend nearly perfectly, there were substantially larger deviations for the database of different IDP sequences. This suggests that other aspects of an IDP's primary amino acid sequence beyond net charge influence the sensitivity of rH to electrostatic interactions. [ABSTRACT FROM AUTHOR]

Background and objective Maintenance of genomic integrity is essential to ensure normal organismal development and to prevent diseases such as cancer. PR-Set7 (also known as Set8) is a cell cycle regulated enzyme that catalyses monomethylation of histone 4 at Lys20 (H4K20me1) to promote chromosome condensation and prevent DNA damage. Recent studies show that CRL4CDT2-mediated ubiquitylation of PR-Set7 leads to its degradation during S phase and after DNA damage. This might occur to ensure appropriate changes in chromosome structure during the cell cycle or to preserve genome integrity after DNA damage. Methods We developed a new model of lung tumor development in mice harboring a conditionally expressed allele of Cul4A. We have therefore used a mouse model to demonstrate for the first time that Cul4A is oncogenic in vivo. With this model, staining of PR-Set7 in the preneoplastic and tumor lesions in AdenoCre-induced mouse lungs was performed. Meanwhile we identified higher protein level changes of γ-tubulin and pericentrin by IHC. Results The level of PR-Set7 down-regulated in the preneoplastic and adenocarcinomous lesions following over-expression of Cul4A. We also identified higher levels of the proteins pericentrin and γ-tubulin in Cul4A mouse lungs induced by AdenoCre. Conclusion PR-Set7 is a direct target of Cul4A for degradation and involved in the formation of lung tumors in the conditional Cul4A transgenic mouse model.

Key message: After initial up-regulation, expression of TUBG1 and TUBG2 is significantly down-regulated in mature syncytia, but lack of expression of either of γ-tubulin genes reduces numbers of nematode infections and developing females.Abstract: Infective second stage juveniles of sedentary plant parasitic nematode Heterodera schachtii invade the root vascular tissue and induce a feeding site, named syncytium, formed as a result of cell hypertrophy and partial cell wall dissolution leading to a multinucleate state. Syncytium formation and maintenance involves a molecular interplay between the plant host and the developing juveniles leading to rearrangements and fragmentation of the plant cytoskeleton. In this study, we investigated the role of two Arabidopsis γ-tubulin genes (TUBG1 and TUBG2), involved in MTs nucleation during syncytium development. Expression analysis revealed that both γ-tubulin’s transcript levels changed during syncytium development and after initial up-regulation (1-3 dpi) they were significantly down-regulated in 7, 10 and 15 dpi syncytia. Moreover, TUBG1 and TUBG2 showed distinct immunolocalization patterns in uninfected roots and syncytia. Although no severe changes in syncytium anatomy and ultrastructure in tubg1-1 and tubg2-1 mutants were observed compared to syncytia induced in wild-type plants, nematode infection assays revealed reduced numbers of infecting juveniles and developed female nematodes in mutant lines. Our results indicate that the expression of both TUBG1 and TUBG2 genes, although generally down-regulated in mature syncytia, is essential for successful root infection, development of functional syncytium and nematode maturation. [ABSTRACT FROM AUTHOR]

Abstract: The orientation of the mitotic spindle (MS) is tightly regulated, but the molecular mechanisms are incompletely understood. Here we report a novel role for the multifunctional adaptor protein ALG‐2‐interacting protein X (ALIX) in regulating MS orientation in addition to its well‐established role in cytokinesis. We show that ALIX is recruited to the pericentriolar material (PCM) of the centrosomes and promotes correct orientation of the MS in asymmetrically dividing Drosophila stem cells and epithelial cells, and symmetrically dividing Drosophila and human epithelial cells. ALIX‐deprived cells display defective formation of astral microtubules (MTs), which results in abnormal MS orientation. Specifically, ALIX is recruited to the PCM via Drosophila Spindle defective 2 (DSpd‐2)/Cep192, where ALIX promotes accumulation of γ‐tubulin and thus facilitates efficient nucleation of astral MTs. In addition, ALIX promotes MT stability by recruiting microtubule‐associated protein 1S (MAP1S), which stabilizes newly formed MTs. Altogether, our results demonstrate a novel evolutionarily conserved role of ALIX in providing robustness to the orientation of the MS by promoting astral MT formation during asymmetric and symmetric cell division. [ABSTRACT FROM AUTHOR]

Microtubule organization has a crucial role in regulating cell architecture. The geometry of microtubule arrays strongly depends on the distribution of sites responsible for microtubule nucleation and minus-end attachment. In cycling animal cells, the centrosome often represents a dominant microtubule-organizing center (MTOC). However, even in cells with a radial microtubule system, many microtubules are not anchored at the centrosome, but are instead linked to the Golgi apparatus or other structures. Non-centrosomal microtubules predominate in many types of differentiated cell and in mitotic spindles. In this review, we discuss recent advances in understanding how the organization of centrosomal and non-centrosomal microtubule networks is controlled by proteins involved in microtubule nucleation and specific factors that recognize free microtubule minus ends and regulate their localization and dynamics. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

We have investigated a possible role for the inner nuclear membrane protein Samp1 (also known as TMEM201) in the mitotic machinery. Live-cell imaging showed that Samp1a-YFP (Samp1a is the short isoform of Samp1) distributed as filamentous structures in the mitotic spindle, partially colocalising with β-tubulin. Samp1 depletion resulted in an increased frequency of cells with signs of chromosomal missegregation and prolonged metaphase, indicating problems with spindle assembly and/or chromosomal alignment. Consistent with this, mitotic spindles in Samp1-depleted cells contained significantly lower levels of β-tubulin and γ-tubulin, phenotypes that were rescued by overexpression of Samp1a-YFP. We found that Samp1 can bind directly to γ-tubulin and that Samp1 co-precipitated with γ-tubulin and the HAUS6 subunit of the Augmin complex in live cells. The levels of HAUS6, in the mitotic spindle also decreased after Samp1 depletion. We show that Samp1 is involved in the recruitment of HAUS6 and γ-tubulin to the mitotic spindle. Samp1 is the first inner nuclear membrane protein shown to have a function in mitotic spindle assembly. [ABSTRACT FROM AUTHOR]

Abstract: Tubulins are an ancient family of eukaryotic proteins characterized by an amino‐terminal globular domain and disordered carboxyl terminus. These carboxyl termini play important roles in modulating the behavior of microtubules in living cells. However, the atomic‐level basis of their function is not well understood. These regions contain multiple acidic residues and their overall charges are modulated in vivo by post‐translational modifications, for example, phosphorylation. In this study, we describe an application of NMR and computer Monte Carlo simulations to investigate how the modification of local charge alters the conformational sampling of the γ‐tubulin carboxyl terminus. We compared the dynamics of two 39‐residue polypeptides corresponding to the carboxyl‐terminus of yeast γ‐tubulin. One polypeptide comprised the wild‐type amino acid sequence while the second contained a Y > D mutation at Y11 in the polypeptide (Y445 in the full protein). This mutation introduces additional negative charge at a site that is phosphorylated in vivo and produces a phenotype with perturbed microtubule function. NMR relaxation measurements show that the Y11D mutation produces dramatic changes in the millisecond‐timescale motions of the entire polypeptide. This observation is supported by Monte Carlo simulations that—similar to NMR—predict the WT γ‐CT is largely unstructured and that the substitution of Tyr 11 with Asp causes the sampling of extended conformations that are unique to the Y11D polypeptide. [ABSTRACT FROM AUTHOR]

Overexpression of γ-tubulin leads to the formation of filaments, but nothing is known about such filaments with regard to possible presence in cells, structure and probable dynamics. Here, we used mammalian cell lines to investigate the ability of γ-tubulin to form filaments. We found that γ-tubulin produces fibers called γ-tubules in a GTP-dependent manner and that γ-tubules are made up of pericentrin and the γ-tubulin complex proteins 2, 3, 5 and 6. Furthermore, we noted that the number of cells with cytosolic γ-tubules is increased in non-dividing cells. Our experiments showed that γ-tubules are polar structures that have a low regrowth rate compared to microtubules. Also, we observed that γ-tubules were disassembled by treatment with cold, colcemid, citral dimethyl acetal, dimethyl fumarate or mutation of γ-tubulin GTPase domain, but were increased in number by treatment with taxol or by stable expression of the γ-tubulin 1–333 GTPase domain. Our results demonstrate that γ-tubulin forms filaments, and such assembly is facilitated by the GTPase domain of γ-tubulin. [ABSTRACT FROM AUTHOR]

The formation of the nuclear envelope and the subsequent compartmentalization of the genome is a defining feature of eukaryotes. Traditionally, the nuclear envelope was purely viewed as a physical barrier to preserve genetic material in eukaryotic cells. However, in the last few decades, it has been revealed to be a critical cellular component in controlling gene expression and has been implicated in several human diseases. In cancer, the relevance of the cell nucleus was first reported in the mid-1800s when an altered nuclear morphology was observed in tumor cells. This review aims to give a current and comprehensive view of the role of the nuclear envelope on cancer first by recapitulating the changes of the nuclear envelope during cell division, second, by reviewing the role of the nuclear envelope in cell cycle regulation, signaling, and the regulation of the genome, and finally, by addressing the nuclear envelope link to cell migration and metastasis and its use in cancer prognosis.

Gamma-tubulin (γ-tubulin) is known to be a key component of the complexes responsible for nucleation and the provision of spatial and temporal control over the functioning of the microtubule cytoskeleton. However, the effect of heavy metal stress on the distribution of γ-tubulin containing complexes has not yet been elucidated. In this study, we present the effect of cadmium (Cd) on γ-tubulin distribution in plant cells. The localization and level of γ-tubulin was assessed in the root cells of soybean seedlings exposed to moderate (85 μM) and high (170 μM) Cd concentrations, using various immunological approaches. In response to Cd, the fluorescence signal for γ-tubulin was significantly weaker. This decrease was most pronounced in the neighborhood of the nuclei envelope and plasmalemma of interphase cells and in the mitotic arrays of dividing cells. This finding was in line with a biochemical analysis which revealed that Cd leads to a decrease in the level of some γ-tubulin isoforms and the appearance of low molecular γ-tubulin-related proteins. Interestingly, at the same time, exposure to Cd at high concentration resulted in the up-regulation of γ-tubulin encoding genes. The results indicate that Cd stress might provoke dysfunctionality in γ-tubulin complexes, which contribute to a disturbed MT cytoskeleton structure. Additionally, ultrastructural observations confirmed considerable Cd-dependent subcellular changes in the root cells of soybean seedlings. The most prominent of these included vacuolization of the cytoplasm, occurrence of autophagic bodies and electron dense deposits in the vacuole, changes in the nucleolus structure and callose deposition. The observed changes are discussed as a response to stress conditions. [ABSTRACT FROM AUTHOR]

The nearest plant homologues of animal protein kinase BRSK were identified using the methods of classical and structural bioinformatics. The selection was performed based on the sequence comparison, results of phylogenetic clustering, and analysis of domain architecture. Spatial structures of human BRSK1 and KIN10 from A. thaliana were compared. The relationship between KIN10 and the regulation of primary microtubule nucleation centers in A. thaliana was revealed. Obvious homology of plant KIN10 and mammalian BRSK1 evidence to suggest that this plant protein kinase is associated with the regulation of the structure and function of primary microtubule nucleation centers and is able to phosphorylate γ-tubulin from Arabidopsis (TUBG1 and TUBG2) at Ser131, affecting the γTuSC monomer structure as well as the γTuRC complex assembly. The effect of the modification on the TUBG1-GACP3 interaction was suggested. [ABSTRACT FROM AUTHOR]

Mitotic-spindle organizing protein associated with a ring of γ-tubulin 1 (MOZART1) is an 8.5 kDa protein linked to regulation of γ-tubulin ring complexes (γTuRCs), which are involved in nucleation of microtubules. Despite its small size, MOZART1 represents a challenging target for detailed characterization in vitro. We described herein a protocol for efficient production of recombinant human MOZART1 in Escherichia coli and assessed the properties of the purified protein using a combination of size exclusion chromatography coupled with multiangle light scattering (SEC-MALS), dynamic light scattering (DLS), and nuclear magnetic resonance (NMR) experiments. MOZART1 forms heterogeneous oligomers in solution. We identified optimal detergent and buffer conditions for recording well resolved NMR experiments allowing nearly full protein assignment and identification of three distinct alpha-helical structured regions. Finally, using NMR, we showed that MOZART1 interacts with the N-terminus (residues 1-250) of GCP3 (γ-tubulin complex protein 3). Our data illustrate the capacity of MOZART1 to form oligomers, promoting multiple contacts with a subset of protein partners in the context of microtubule nucleation. [ABSTRACT FROM AUTHOR]

The organization of microtubule networks is crucial for controlling chromosome segregation during cell division, for positioning and transport of different organelles, and for cell polarity and morphogenesis. The geometry of microtubule arrays strongly depends on the localization and activity of the sites where microtubules are nucleated and where their minus ends are anchored. Such sites are often clustered into structures known as microtubule-organizing centers, which include the centrosomes in animals and spindle pole bodies in fungi. In addition, other microtubules, as well as membrane compartments such as the cell nucleus, the Golgi apparatus, and the cell cortex, can nucleate, stabilize, and tether microtubule minus ends. These activities depend on microtubule-nucleating factors, such as γ-tubulin-containing complexes and their activators and receptors, and microtubule minus end-stabilizing proteins with their binding partners. Here, we provide an overview of the current knowledge on how such factors work together to control microtubule organization in different systems. [ABSTRACT FROM AUTHOR]

This study aimed to investigate the assembly characteristics of ciliature and cortical microtubules and the localization of tubulins in different depths and regions of the cortex. The hypotrichous ciliates have closely arranged cilia and a highly complex microtubular system. Direct fluorescence and immunofluorescence labeling were used to observe ciliary organelles and cortical microtubular cytoskeleton in Euplotes eurystomus. An immunofluorescence analysis demonstrated that α-tubulin localized to the ventral and dorsal ciliary organelles and their associated microtubules, while γ-tubulin localized to the basal bodies of ciliary organelles, macronuclear membrane, and excretory pore of a contractile vacuole in the interphase. A direct fluorescence analysis showed that the ciliature and cortical microtubules in the deep cortex were more clearly marked by fluorescent taxoid (FLUTAX). Interestingly, α- and γ-tubulins also colocalized to the ringlike ciliary base-associated microtubules of dorsal kineties. The short microtubular bundles between the bases of transverse cirri could be marked by FLUTAX and γ-tubulin rather than α-tubulin, suggesting that tubulins in cortical microtubules in E. eurystomus varied, and the differentiation of cortical microtubules in the hypotrichous ciliate tended to be versatile. Also, during morphogenesis, γ-tubulin also localized to the base of ciliary primordium, where new basal bodies were formed, suggesting that the endocellular position of γ-tubulin in ciliates was related to basal bodies and regulated by the cell cycle. This study might help understand the assembly characteristics and tubulin composition of microtubules in different depths and regions of the cortex in hypotrichous ciliate E. eurystomus. [ABSTRACT FROM AUTHOR]

Regulation of the γ-tubulin ring complex (γTuRC) through targeting and activation restricts nucleation of microtubules to microtubuleorganizing centers (MTOCs), aiding in the assembly of ordered microtubule arrays. However, the mechanistic basis of this important regulation remains poorly understood. Here, we show that, in human cells, γTuRC integrity, determined by the presence of γ-tubulin complex proteins (GCPs; also known as TUBGCPs) 2-6, is a prerequisite for interaction with the targeting factor NEDD1, impacting on essentially all γ-tubulin-dependent functions. Recognition of γTuRC integrity is mediated by MZT1, which binds not only to the GCP3 subunit as previously shown, but cooperatively also to other GCPsthrough a conserved hydrophobic motif present in the N-termini of GCP2, GCP3, GCP5 and GCP6. MZT1 knockdown causes severe cellular defects under conditions that leave γTuRC intact, suggesting that the essential function of MZT1 is not in γTuRC assembly. Instead, MZT1 specifically binds fully assembled γTuRC to enable interaction with NEDD1 for targeting, and with the CM1 domain of CDK5RAP2 for stimulating nucleation activity. Thus, MZT1 is a 'priming factor' for γTuRC that allows spatial regulation of nucleation. [ABSTRACT FROM AUTHOR]

Summary The generation of cellular microtubules is initiated at specific sites such as the centrosome and the Golgi apparatus that contain nucleation complexes rich in γ-tubulin. The microtubule growing plus-ends are stabilized by plus-end tracking proteins (+TIPs), mainly EB1 and associated proteins. Myomegalin was identified as a centrosome/Golgi protein associated with cyclic nucleotide phosphodiesterase. We show here that Myomegalin exists as several isoforms. We characterize two of them. One isoform, CM-MMG, harbors a conserved domain (CM1), recently described as a nucleation activator, and is related to a family of γ-tubulin binding proteins, which includes Drosophila centrosomin. It localizes at the centrosome and at the cis-Golgi in an AKAP450-dependent manner. It recruits γ-tubulin nucleating complexes and promotes microtubule nucleation. The second isoform, EB-MMG, is devoid of CM1 domain and has a unique N-terminus with potential EB1-binding sites. It localizes at the cis-Golgi and can localize to microtubule plus-ends. EB-MMG binds EB1 and affects its loading on microtubules and microtubule growth. Depletion of Myomegalin by small interfering RNA delays microtubule growth from the centrosome and Golgi apparatus, and decreases directional migration of RPE1 cells. In conclusion, the Myomegalin gene encodes different isoforms that regulate microtubules. At least two of these have different roles, demonstrating a previously unknown mechanism to control microtubules in vertebrate cells.