Gamma-tubulin Complex Proteins Research Articles

γ-tubulin complex controls the nucleation of tubulin-based structures in Apicomplexa.

Apicomplexan parasites rely on tubulin structures throughout their cell and life cycles, particularly in the polymerization of spindle microtubules to separate the replicated nucleus into daughter cells. Additionally, tubulin structures, including conoid and subpellicular microtubules, provide the necessary rigidity and structure for dissemination and host cell invasion. However, it is unclear whether these tubulin structures are nucleated via a highly conserved γ-tubulin complex or through a specific process unique to apicomplexans. This study demonstrates that Toxoplasma γ-tubulin is responsible for nucleating spindle microtubules, akin to higher eukaryotes, facilitating nucleus division in newly formed parasites. Interestingly, γ-tubulin colocalizes with nascent conoid and subpellicular microtubules during division, potentially nucleating these structures as well. Loss of γ-tubulin results in significant morphological defects due to impaired nucleus scission and the loss of conoid and subpellicular microtubule nucleation, crucial for parasite shape and rigidity. Additionally, the nucleation process of tubulin structures involves a concerted action of γ-tubulin and Gamma Tubulin Complex proteins (GCPs), recapitulating the localization and phenotype of γ-tubulin. This study also introduces new molecular markers for cytoskeletal structures and applies iterative expansion microscopy to reveal microtubule-based architecture in Cryptosporidium parvum sporozoites, further demonstrating the conserved localization and probable function of γ-tubulin in Cryptosporidium.

A stable sub-complex between GCP4, GCP5 and GCP6 promotes the assembly of γ-tubulin ring complexes.

γ-Tubulin is the main protein involved in the nucleation of microtubules in all eukaryotes. It forms two different complexes with proteins of the GCP family (γ-tubulin complex proteins): γ-tubulin small complexes (γTuSCs) that contain γ-tubulin, and GCPs 2 and 3; and γ-tubulin ring complexes (γTuRCs) that contain multiple γTuSCs in addition to GCPs 4, 5 and 6. Whereas the structure and assembly properties of γTuSCs have been intensively studied, little is known about the assembly of γTuRCs and the specific roles of GCPs 4, 5 and 6. Here, we demonstrate that two copies of GCP4 and one copy each of GCP5 and GCP6 form a salt (KCl)-resistant sub-complex within the γTuRC that assembles independently of the presence of γTuSCs. Incubation of this sub-complex with cytoplasmic extracts containing γTuSCs leads to the reconstitution of γTuRCs that are competent to nucleate microtubules. In addition, we investigate sequence extensions and insertions that are specifically found at the N-terminus of GCP6, and between the GCP6 grip1 and grip2 motifs. We also demonstrate that these are involved in the assembly or stabilization of the γTuRC.

Mechanical Shielding in Plant Nuclei.

In animal single cells in culture, nuclear geometry and stiffness can be affected by mechanical cues, with important consequences for chromatin status and gene expression. This calls for additional investigation into the corresponding physiological relevance in a multicellular context and in different mechanical environments. Using the Arabidopsis root as a model system, and combining morphometry and micro-rheometry, we found that hyperosmotic stress decreases nuclear circularity and size and increases nuclear stiffness in meristematic cells. These changes were accompanied by enhanced expression of touch response genes. The nuclear response to hyperosmotic stress was rescued upon return to iso-osmotic conditions and could even lead to opposite trends upon hypo-osmotic stress. Interestingly, nuclei in a mutant impaired in the functions of the gamma-tubulin complex protein 3 (GCP3) interacting protein (GIP)/MZT1 proteins at the nuclear envelope were almost insensitive to such osmotic changes. The gip1gip2 mutant exhibited constitutive hyperosmotic stress response with stiffer and deformed nuclei, as well as touch response gene induction. The mutant was also resistant to lethal hyperosmoticconditions. Altogether, we unravel a stereotypical geometric, mechanical, and genetic nuclear response to hyperosmotic stress in plants. Our data also suggest that chromatin acts as a gel that stiffens in hyperosmotic conditions and that the nuclear-envelope-associated protein GIPs act as negative regulators of this response.

Bi-allelic Pathogenic Variants in TUBGCP2 Cause Microcephaly and Lissencephaly Spectrum Disorders

Lissencephaly comprises a spectrum of malformations of cortical development. This spectrum includes agyria, pachygyria, and subcortical band heterotopia; each represents anatomical malformations of brain cortical development caused by neuronal migration defects. The molecular etiologies of neuronal migration anomalies are highly enriched for genes encoding microtubules and microtubule-associated proteins, and this enrichment highlights the critical role for these genes in cortical growth and gyrification. Using exome sequencing and family based rare variant analyses, we identified a homozygous variant (c.997C>T [p.Arg333Cys]) in TUBGCP2, encoding gamma-tubulin complex protein 2 (GCP2), in two individuals from a consanguineous family; both individuals presented with microcephaly and developmental delay. GCP2 forms the multiprotein γ-tubulin ring complex (γ-TuRC) together with γ-tubulin and other GCPs to regulate the assembly of microtubules. By querying clinical exome sequencing cases and through GeneMatcher-facilitated collaborations, we found three additional families with bi-allelic variation and similarly affected phenotypes including a homozygous variant (c.1843G>C [p.Ala615Pro]) in two families and compound heterozygous variants consisting of one missense variant (c.889C>T [p.Arg297Cys]) and one splice variant (c.2025-2A>G) in another family. Brain imaging from all five affected individuals revealed varying degrees of cortical malformations including pachygyria and subcortical band heterotopia, presumably caused by disruption of neuronal migration. Our data demonstrate that pathogenic variants in TUBGCP2 cause an autosomal recessive neurodevelopmental trait consisting of a neuronal migration disorder, and our data implicate GCP2 as a core component of γ-TuRC in neuronal migrating cells.

Haploinsufficiency of GCP4 induces autophagy and leads to photoreceptor degeneration due to defective spindle assembly in retina

Retinopathy, owing to damage to the retina, often causes vision impairment, and the underlying molecular mechanisms are largely unknown. Using a gene targeting strategy, we generated mice with the essential gene Tubgcp4 knocked out. Homozygous mutation of Tubgcp4 resulted in early embryonic lethality due to abnormal spindle assembly caused by GCP4 (gamma-tubulin complex protein 4, encoded by Tubgcp4) depletion. Heterozygotes were viable through dosage compensation of one wild-type allele. However, haploinsufficiency of GCP4 affected the assembly of γ-TuRCs (γ-tubulin ring complexes) and disrupted autophagy homeostasis in retina, thus leading to photoreceptor degeneration and retinopathy. Notably, GCP4 exerted autophagy inhibition by competing with ATG3 for interaction with ATG7, thus interfering with lipidation of LC3B. Our findings justify dosage effects of essential genes that compensate for null alleles in viability of mutant mice and uncover dosage-dependent roles of GCP4 in embryo development and retinal homeostasis. These data have also clinical implications in genetic counseling on embryonic lethality and in development of potential therapeutic targets associated with retinopathy.

MGO3 and GIP1 act synergistically for the maintenance of centromeric cohesion

ABSTRACTThe control of genomic maintenance during S phase is crucial in eukaryotes. It involves the establishment of sister chromatid cohesion, ensuring faithful chromosome segregation, as well as proper DNA replication and repair to preserve genetic information. In animals, nuclear periphery proteins - including inner nuclear membrane proteins and nuclear pore-associated components - are key factors which regulate DNA integrity. Corresponding functional homologues are not so well known in plants which may have developed specific mechanisms due to their sessile life. We have already characterized the Gamma-tubulin Complex Protein 3-interacting proteins (GIPs) as essential regulators of centromeric cohesion at the nuclear periphery. GIPs were also shown to interact with TSA1, first described as a partner of the epigenetic regulator MGOUN3 (MGO3)/BRUSHY1 (BRU1)/TONSOKU (TSK) involved in genomic maintenance. Here, using genetic analyses, we show that the mgo3gip1 mutants display an impaired and pleiotropic development including fasciation. We also provide evidence for the contribution of both MGO3 and GIP1 to the regulation of centromeric cohesion in Arabidopsis.

γ-Tubulin complex in Trypanosoma brucei: molecular composition, subunit interdependence and requirement for axonemal central pair protein assembly.

γ-Tubulin complex constitutes a key component of the microtubule-organizing center and nucleates microtubule assembly. This complex differs in complexity in different organisms: the budding yeast contains the γ-tubulin small complex (γTuSC) composed of γ-tubulin, gamma-tubulin complex protein (GCP)2 and GCP3, whereas animals contain the γ-tubulin ring complex (γTuRC) composed of γTuSC and three additional proteins, GCP4, GCP5 and GCP6. In Trypanosoma brucei, the composition of the γ-tubulin complex remains elusive, and it is not known whether it also regulates assembly of the subpellicular microtubules and the spindle microtubules. Here we report that the γ-tubulin complex in T. brucei is composed of γ-tubulin and three GCP proteins, GCP2-GCP4, and is primarily localized in the basal body throughout the cell cycle. Depletion of GCP2 and GCP3, but not GCP4, disrupted the axonemal central pair microtubules, but not the subpellicular microtubules and the spindle microtubules. Furthermore, we showed that the γTuSC is required for assembly of two central pair proteins and that γTuSC subunits are mutually required for stability. Together, these results identified an unusual γ-tubulin complex in T. brucei, uncovered an essential role of γTuSC in central pair protein assembly, and demonstrated the interdependence of individual γTuSC components for maintaining a stable complex.

Microtubule initiation from the nuclear surface controls cortical microtubule growth polarity and orientation in Arabidopsis thaliana.

The nuclear envelope in plant cells has long been known to be a microtubule organizing center (MTOC), but its influence on microtubule organization in the cell cortex has been unclear. Here we show that nuclear MTOC activity favors the formation of longitudinal cortical microtubule (CMT) arrays. We used green fluorescent protein (GFP)-tagged gamma tubulin-complex protein 2 (GCP2) to identify nuclear MTOC activity and GFP-tagged End-Binding Protein 1b (EB1b) to track microtubule growth directions. We found that microtubules initiate from nuclei and enter the cortex in two directions along the long axis of the cell, creating bipolar longitudinal CMT arrays. Such arrays were observed in all cell types showing nuclear MTOC activity, including root hairs, recently divided cells in root tips, and the leaf epidermis. In order to confirm the causal nature of nuclei in bipolar array formation, we displaced nuclei by centrifugation, which generated a corresponding shift in the bipolarity split point. We also found that bipolar CMT arrays were associated with bidirectional trafficking of vesicular components to cell ends. Together, these findings reveal a conserved function of plant nuclear MTOCs and centrosomes/spindle pole bodies in animals and fungi, wherein all structures serve to establish polarities in microtubule growth.

Involvement of YODA and mitogen activated protein kinase 6 in Arabidopsis post-embryogenic root development through auxin up-regulation and cell division plane orientation.

The role of YODA MITOGEN ACTIVATED PROTEIN KINASE KINASE KINASE 4 (MAPKKK4) upstream of MITOGEN ACTIVATED PROTEIN KINASE 6 (MPK6) was studied during post-embryonic root development of Arabidopsis thaliana. Loss- and gain-of-function mutants of YODA (yda1 and ΔNyda1) were characterized in terms of root patterning, endogenous auxin content and global proteomes. We surveyed morphological and cellular phenotypes of yda1 and ΔNyda1 mutants suggesting possible involvement of auxin. Endogenous indole-3-acetic acid (IAA) levels were up-regulated in both mutants. Proteomic analysis revealed up-regulation of auxin biosynthetic enzymes tryptophan synthase and nitrilases in these mutants. The expression, abundance and phosphorylation of MPK3, MPK6 and MICROTUBULE ASSOCIATED PROTEIN 65-1 (MAP65-1) were characterized by quantitative polymerase chain reaction (PCR) and western blot analyses and interactions between MAP65-1, microtubules and MPK6 were resolved by quantitative co-localization studies and co-immunoprecipitations. yda1 and ΔNyda1 mutants showed disoriented cell divisions in primary and lateral roots, abortive cytokinesis, and differential subcellular localization of MPK6 and MAP65-1. They also showed deregulated expression of TANGLED1 (TAN1), PHRAGMOPLAST ORIENTING KINESIN 1 (POK1), and GAMMA TUBULIN COMPLEX PROTEIN 4 (GCP4). The findings that MPK6 localized to preprophase bands (PPBs) and phragmoplasts while the mpk6-4 mutant transformed with MPK6AEF (alanine (A)-glutamic acid (E)-phenylanine (F)) showed a root phenotype similar to that of yda1 demonstrated that MPK6 is an important player downstream of YODA. These data indicate that YODA and MPK6 are involved in post-embryonic root development through an auxin-dependent mechanism regulating cell division and mitotic microtubule (PPB and phragmoplast) organization.

Axin localizes to the centrosome and is involved in microtubule nucleation

Axin is known to have an important role in the degradation of beta-catenin in the Wnt pathway. Here, we reveal a new function of Axin at the centrosome. Axin was localized to the centrosome in various cell lines and formed a complex with gamma-tubulin. Knockdown of Axin reduced the localization of gamma-tubulin and gamma-tubulin complex protein 2-components of the gamma-tubulin ring complex-to the centrosome and the centrosomal microtubule nucleation activity after treatment with nocodazole. These phenotypes could not be rescued by the reduction in the levels of beta-catenin. Although the expression of Axin rescued these phenotypes in Axin-knockdown cells, overexpression of Axin2, which is highly homologous to Axin, could not. Axin2 was also localized to the centrosome, but it did not form a complex with gamma-tubulin. These results suggest that Axin, but not Axin2, is involved in microtubule nucleation by forming a complex with gamma-tubulin at the centrosome.

Identification of a novel small Arabidopsis protein interacting with gamma‐tubulin complex protein 3

In higher plants, microtubules (MTs) show dynamic structural changes during cell cycle and development progression. A precise control of MT nucleation at dispersed sites is one way used to regulate the cytoskeletal organization. Some gamma-tubulin complex proteins (GCPs) were previously identified in Arabidopsis thaliana (At). They are directly involved in the nucleation process. Nevertheless, no additional player which may anchor the nucleating complex or regulate the nucleation activity has been found in plant cells so far. Therefore, our aim was the identification of Arabidopsis proteins interacting with MT nucleating complexes and particularly with AtGCP3. Performing a yeast two-hybrid screen, we discovered a new protein which we called AtGCP3 Interacting Protein 1 (AtGIP1). The possible role of this protein during the nucleation process is discussed.

Arabidopsis GCP2 and GCP3 are part of a soluble γ‐tubulin complex and have nuclear envelope targeting domains

In higher plants, microtubules (MTs) are assembled in distinctive arrays in the absence of a defined organizing center. Three MT nucleation sites have been described: the nuclear surface, the cell cortex and cortical MT branch points. The Arabidopsis thaliana (At) genome contains putative orthologues encoding all the components of characterized mammalian nucleation complexes: gamma-tubulin and gamma-tubulin complex proteins GCP2 to GCP6. We have cloned the cDNA encoding AtGCP2, and show that gamma-tubulin, AtGCP2 and AtGCP3 are part of the same tandem affinity-purified complex and are present in a large membrane-associated complex. In addition, small soluble gamma-tubulin complexes of the size expected for a gamma-tubulin core complex are recruited to isolated nuclei. Using immunogold labelling, AtGCP3 is localized to both the nuclear envelope (NE) and the plasma membrane. To identify domains that could play a role in targeting complexes to these nucleation sites, truncated AtGCP2- and AtGCP3-green fluorescent protein fusion proteins were expressed in BY-2 cells. Several domains from AtGCP2 and AtGCP3 are capable of targeting fusions to the NE. We propose that regulated recruitment of soluble gamma-tubulin-containing complexes is responsible for nucleation at dispersed sites in plant cells and contributes to the formation and organization of the various MT arrays.

Quick Guide: γ-Tubulin

What is it?γ-Tubulin is a member of the tubulin superfamily and is required for nucleating the polymerization of microtubules in vivo. Microtubules are dynamic cytoskeletal polymers that assemble from two other members of the tubulin superfamily, α-tubulin and β-tubulin. Microtubules make up the mitotic and meiotic spindles, and are important for establishing cell polarity and vesicle trafficking. The tubulin superfamily is still growing, and now includes tubulins δ through η (delta through eta, in case you've forgotten your Greek). α-, β- and γ-tubulin are conserved in all eukaryotic groups, whereas the other superfamily members are not.How was it found?γ-Tubulin was identified by Oakley and colleagues as a suppressor of a β-tubulin mutation in Aspergillus nidulans.Where is it?γ-Tubulin does not assemble into microtubules. In most cells it is found at the microtubule organizing center, an organelle devoted to microtubule nucleation and anchoring. In animal cells the organizing center is called the centrosome, in fungi it is called the spindle pole body, and in plants it isn't called anything because plants make do without a discrete organizing center. The centrosome consists of a pair of centrioles — microtubule structures with beautiful nine-fold symmetry – surrounded by pericentriolar material. The pericentriolar material nucleates microtubules and contains γ-tubulin. In fungi too γ-tubulin is associated with the surfaces of the spindle pole body that nucleate microtubles. Although γ-tubulin is thought to act primarily at the organizing center, in most cells the majority of γ-tubulin is in the cytoplasm.What's it good for?γ-Tubulin nucleates microtubules, allowing cells to control when and where microtubules polymerize. This has been shown both in vivo, by interfering with γ-tubulin function, and in vitro, by assaying the activity of γ-tubulin. Analysis of γ-tubulin mutants in fungi suggests that γ-tubulin might also have an independent role in cell cycle control.Does it act alone? No. When purified from animal cells, γ-tubulin is a large complex with a molecular weight of several million daltons. In addition to γ-tubulin there are at least five other proteins in this complex, all of which are related to each other and are conserved in animals and plants. We call these proteins the GCPs (gamma-tubulin complex proteins), although there are several other names in the literature. Remarkably, yeast lacks all but two of the GCPs; those GCPs and γ-tubulin form a much smaller complex than that found in animal cells.Size and shape matter! Microtubules are complex polymers, with approximately 13 protofilaments arranged into a tube. The γ-tubulin complex has a size and shape that is very similar to that of a microtubule end, suggesting that it serves as a template for microtubule assembly. The γ-tubulin complex as purified from several organisms is a ring of approximately 25 nm in diameter made up of repeating subunits, with a cap structure on one end, and is often called the γ-tubulin ring complex (γTuRC). The current model for how all the proteins fit together is that the repeating subunit in the ring is γ-tubulin plus the two GCPs that are conserved in all eukaryotes, and that the cap structure is made up of the other GCPs.Getting attached to γ-tubulin Most microtubules grow from the centrosome, so there must be a way to attach the γ-tubulin complex to the centrosome. Although it is not clear how it works, this attachment is likely to be regulated in the cell cycle. The amount of γ-tubulin at the centrosome increases dramatically at the beginning of mitosis, aiding in the formation of the mitotic spindle, then decreases at the end of mitosis. Attachment of the γ-tubulin complex to the nascent centrosome is also an important part of fertilization in most animals since the sperm supplies the centrioles and the egg supplies the γ-tubulin complex; it is only after they get together that the first centrosome is formed and development proceeds.Where can I find out more?

The plant cytoskeleton

The plant cytoskeleton is a dynamic, three dimensional array of filamentous protein polymers, consisting of microtubules (MTs) and microfilaments (MFs). These cytoskeletal components play active roles in most, if not all aspects of plant cell growth, development and interaction with the environment. Recent advances in molecular genetics, cell imaging and the exploitation of plant systems, such as Arabidopsis thaliana have resulted in substantial advances in our understanding of the protein composition, regulation, dynamics of the cytoskeleton and interactions between MTs and MFs. MTs consist of a variety of tubulin isotypes (coded for by an array of tubulin genes and subsequently altered through post-translational modifications) and a large number of MT-associated proteins (MAPs). The complexity of MT chemistry (tubulins and MAPs) has helped explain how very similarly looking MTs can accomplish the wide array of observed stabilities, dynamics and functions. Although plants lack the recognizable MT organizing centers (MTOCs) found in other eukaryotes, MT assembly and reorganization is accomplished through complexes of gamma tubulin (γ-tubulin) and gamma tubulin complex proteins (GCPs) which regulate when, where, and in which pattern MTs are formed. Once formed, MT dynamics and function is controlled by a wide-array of MAPs, many of which have comparable homologs in other eukaryotic systems while other MAPs are plant-specific. Similar to MTs, MFs in plants exhibit a variety of actin isotypes (coded by an array of actin genes) and a large number of actin-binding proteins (ABPs). Actin proteins are divided into those involved in vegetative processes and those involved in reproductive processes. The specific functions of MFs are controlled by the activities of various ABPs. Many of the process originally thought to involve only MTs, have now been shown to require the proper functioning of both MTs and MFs. All aspects of cell reproduction (establishment of division plane, mitosis and cytokinesis) involve both MTs and MFs. Cell growth and shape is also controlled by the cytoskeleton. While MTs play active roles in regulating wall patterning and subsequent shape of cells growing via a diffuse growth mechanism, MFs are the key cytoskeletal elements important in defining and controlling cell expansion and shape in tip-growing systems. While great strides have been made in unraveling the complexities of cytoskeletal function in plants, much more needs doing. MTs and MFs appear to play a much more integrative role in plant development than previously believed. It is predicted that many more MAPs and ABPs await discovery, resulting more clarity in our understanding of the role of MTs and MFs in plant growth, development and interaction with the environment.