Tubulin cofactor A gene silencing in mammalian cells induces changes in microtubule cytoskeleton, cell cycle arrest and cell death

Nolasco, Sofia; Bellido, Javier; Gonçalves, João; Zabala, Juan Carlos; Soares, Helena

doi:10.1016/j.febslet.2005.05.022

Cited by 47 publications

(46 citation statements)

References 36 publications

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“…The Mecp2 gene was not on the list because a truncated mRNA corresponding to the 3′ coding region of Mecp2, where Affymetrix probe sets hybridize, was expressed in KO neurons. Among the identified differentially expressed genes, Prefoldin 5 is involved in actin and tubulin folding and cytoskeleton formation (Hartl and Hayer-Hartl, 2002;Nolasco et al, 2005), and Syndecan 2 has been shown to be critical for synaptogenesis (Ethell and Yamaguchi, 1999;Ethell et al, 2001). We have confirmed that the expression levels of both Syndecan 2 and Prefoldin 5 mRNA were higher in KO neurons using Real time PCR analyses (Fig 5D-E).…”

Section: Altered Gene Expression Of Synaptic Proteins In Mecp2-deficisupporting

confidence: 66%

Mecp2 deficiency leads to delayed maturation and altered gene expression in hippocampal neurons

Smrt

Eaves-Egenes

Barkho

et al. 2007

Neurobiology of Disease

205

222

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It is well known that Rett Syndrome, a severe postnatal childhood neurological disorder is mostly caused by mutations in the MECP2 gene. However, how deficiencies in MeCP2 contribute to the neurological dysfunction of Rett Syndrome is not clear. We aimed to resolve the role of MeCP2 epigenetic regulation in postnatal brain development in a Mecp2-deficient mouse model. We found that, while Mecp2 was not critical for the production of immature neurons in the dentate gyrus (DG) of the hippocampus, the newly generated neurons exhibited pronounced deficits in neuronal maturation, including delayed transition into a more mature stage, altered expression of presynaptic proteins, and reduced dendritic spine density. Furthermore, analysis of gene expression profiles of isolated DG granule neurons revealed abnormal expression levels of a number of genes previously shown to be important for synaptogenesis. Our studies suggest that MeCP2 plays a central role in neuronal maturation, which might be mediated through epigenetic control of expression pathways that are instrumental in both dendritic development and synaptogenesis.

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Section: Altered Gene Expression Of Synaptic Proteins In Mecp2-deficisupporting

confidence: 66%

Mecp2 deficiency leads to delayed maturation and altered gene expression in hippocampal neurons

Smrt

Eaves-Egenes

Barkho

et al. 2007

Neurobiology of Disease

205

222

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“…S. pombe cofactor A is dispensable for cell viability (13) and is also not required for tubulin folding using an in vitro tubulin folding assay (1). In contrast, Nolasco et al (46) found that loss of cofactor A in mammalian cells led to a decrease in soluble tubulin, microtubule alterations, and a loss of cell viability.…”

Section: Discussionmentioning

confidence: 87%

Cofactor D Functions as a Centrosomal Protein and Is Required for the Recruitment of the γ-Tubulin Ring Complex at Centrosomes and Organization of the Mitotic Spindle

Cunningham

Kahn

2008

Journal of Biological Chemistry

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Microtubules are highly dynamic structures, composed of ␣/␤-tubulin heterodimers. Biosynthesis of the functional dimer involves the participation of several chaperones, termed cofactors A-E, that act on folding intermediates downstream of the cytosolic chaperonin CCT (1, 2). We show that cofactor D is also a centrosomal protein and that overexpression of either the fulllength protein or either of two centrosome localization domains leads to the loss of anchoring of the ␥-tubulin ring complex and of nucleation of microtubule growth at centrosomes. In contrast, depletion of cofactor D by short interfering RNA results in mitotic spindle defects. Because none of these changes in cofactor D activity produced a change in the levels of ␣-or ␤-tubulin, we conclude that these newly discovered functions for cofactor D are distinct from its previously described role in tubulin folding. Thus, we describe a new role for cofactor D at centrosomes that is important to its function in polymerization of tubulin and organization of the mitotic spindle.Centrosomes are the major microtubule-organizing center in animal cells and are composed of two centrioles (barrelshaped structures composed of ␣/␤-tubulin heterodimers) surrounded by a dense fibrillar network of proteins called the pericentriolar material (PCM).2 A key step in the initiation of new microtubule growth is the regulated recruitment to the PCM of ␥-tubulin ring complexes (␥-TuRCs), consisting of ␥-tubulin and ␥-complex proteins (GCPs) (3-7), that promote the polymerization of ␣/␤-tubulin heterodimers. In addition to nucleating new microtubule growth, centrosomes also organize cytosolic microtubules during interphase, spindle microtubules during mitosis, and axonemes in ciliogenesis (8, 9).Formation of ␣/␤ tubulin heterodimers is promoted by five tubulin-specific co-chaperones, termed cofactors A-E, that act on ␣-and ␤-tubulin folding intermediates in a stepwise process that generates polymerizable heterodimers (1). Demonstration of the roles for these five cofactors in folding tubulin heterodimers comes from in vitro folding assays, which also allowed purification of the five co-chaperones (1, 10). This function is consistent with genetic studies in Saccharomyces cerevisiae (11,12), Schizosaccharomyces pombe (13-15), Arabidopsis thaliana (16,17), and Caenorhabditis elegans (18) in which mutations in cofactor D (CoD) yielded defects in microtubule-dependent processes, including maintenance of chromosome number.That the tubulin-specific cofactors may play additional roles in regulating microtubule stability or dynamicity was first proposed by Tian et al. (2), who found that overexpression of bovine CoD in HeLa cells led to the loss of microtubules and decreased levels of ␣-tubulin in cells. With additional in vitro data showing that purified bovine CoD disrupts the tubulin heterodimer, the authors proposed a role for CoD in heterodimer destruction, with a secondary loss of microtubules (19,20). A regulated destructive pathway was suggested by the observations that increa...

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“…It has been found, for example, that cofactor A has a highly extended helical conformation (33), and we speculate that it could potentially interact with H11 and H12, protecting the quasi-native protein from the degradation pathway. Evidence to support this idea has recently come from the observation that depletion of cofactor A using siRNA causes a large reduction in cellular tubulin (34). The inability of either cofactor A or cofactor D to bind properly to the ␤-tubulin released from CCT might, thus, trigger its degradation.…”

Section: Discussionmentioning

confidence: 99%

Mutations Affecting β-Tubulin Folding and Degradation

Wang

Tian

Cowan

et al. 2006

Journal of Biological Chemistry

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Revertants of a colcemid-resistant Chinese hamster ovary cell line with an altered (D45Y) ␤-tubulin have allowed the identification of four cis-acting mutations (L187R, Y398C, a 12-amino acid in-frame deletion, and a C-terminal truncation) that act by destabilizing the mutant tubulin and preventing it from incorporating into microtubules. These unstable ␤-tubulins fail to form heterodimers and are predominantly found in association with the chaperonin CCT, suggesting that they cannot undergo productive folding. In agreement with these in vivo observations, we show that the defective ␤-tubulins do not stably interact with cofactors involved in the tubulin folding pathway and, hence, fail to exchange with ␤-tubulin in purified ␣␤ heterodimers. Treatment of cells with MG132 causes an accumulation of the aberrant tubulins, indicating that improperly folded ␤-tubulin is degraded by the proteasome. Rapid degradation of the mutant tubulin does not elicit compensatory changes in wild-type tubulin synthesis or assembly. Instead, loss of ␤-tubulin from the mutant allele causes a 30 -40% decrease in cellular tubulin content with no obvious effect on cell growth or survival.Microtubules are essential cytoskeletal structures responsible for much of the vesicular traffic in cells, organization of the endoplasmic reticulum and Golgi apparatus, and segregation of sister chromatids before cell division. They assemble by the endwise concatenation of ␣␤ tubulin heterodimers to form protofilaments, 13 of which associate laterally to form the walls of microtubules in most eukaryotic cells. ␣-and ␤-tubulin subunits each follow a complex folding pathway before joining to form assembly-competent heterodimers. This process involves prefoldin (1, 2), CCT 2 (cytosolic chaperonin containing TCP-1, also called TCP-1 complexes, TriC, or cytosolic chaperonin) (3-5), and the downstream participation of a series of protein cofactors (6 -9). Prefoldin binds to nascent chains and is thought to target newly synthesized tubulin to CCT (1), which then releases these polypeptides in a quasinative state. The released ␤-tubulin is captured by either cofactor A or cofactor D. In a parallel pathway, quasi-native ␣-tubulin polypeptides are captured by either cofactor B or cofactor E. Cofactor E/␣ and cofactor D/␤ complexes interact to form a supercomplex. Entry of cofactor C into this supercomplex triggers GTP hydrolysis by ␤-tubulin, a reaction that results in the release of newly formed assembly-competent ␣␤ tubulin heterodimers (6, 7).Mammalian cells express multiple tubulin genes, each of which encodes a distinct ␣-or ␤-tubulin isotype (10, 11). As far as is known, these distinct gene products follow a common folding pathway. In addition to their role in the de novo assembly of tubulin dimers, cofactors C, D, and E act in concert on native heterodimers as GTPase-activating proteins. This reaction occurs at a tubulin concentration far below that required for polymerization into microtubules and is thought to act primarily as a quality control mechanism...

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Tubulin cofactor A gene silencing in mammalian cells induces changes in microtubule cytoskeleton, cell cycle arrest and cell death

Cited by 47 publications

References 36 publications

Mecp2 deficiency leads to delayed maturation and altered gene expression in hippocampal neurons

Mecp2 deficiency leads to delayed maturation and altered gene expression in hippocampal neurons

Cofactor D Functions as a Centrosomal Protein and Is Required for the Recruitment of the γ-Tubulin Ring Complex at Centrosomes and Organization of the Mitotic Spindle

Mutations Affecting β-Tubulin Folding and Degradation

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