Drosophila Tubulin-specific chaperone E functions at neuromuscular synapses and is required for microtubule network formation

Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China.
Development (Impact Factor: 6.46). 04/2009; 136(9):1571-81. DOI: 10.1242/dev.029983
Source: PubMed


Hypoparathyroidism, mental retardation and facial dysmorphism (HRD) is a fatal developmental disease caused by mutations in tubulin-specific chaperone E (TBCE). A mouse Tbce mutation causes progressive motor neuronopathy. To dissect the functions of TBCE and the pathogenesis of HRD, we generated mutations in Drosophila tbce, and manipulated its expression in a tissue-specific manner. Drosophila tbce nulls are embryonic lethal. Tissue-specific knockdown and overexpression of tbce in neuromusculature resulted in disrupted and increased microtubules, respectively. Alterations in TBCE expression also affected neuromuscular synapses. Genetic analyses revealed an antagonistic interaction between TBCE and the microtubule-severing protein Spastin. Moreover, treatment of muscles with the microtubule-depolymerizing drug nocodazole implicated TBCE as a tubulin polymerizing protein. Taken together, our results demonstrate that TBCE is required for the normal development and function of neuromuscular synapses and that it promotes microtubule formation. As defective microtubules are implicated in many neurological and developmental diseases, our work on TBCE may offer novel insights into their basis.

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    • "The TBC proteins' functions are finely balanced: their loss or their overexpression are both lethal in most eukaryotes, stemming from a complete loss of the MT cytoskeleton (Steinborn et al., 2002; Lacefield et al., 2006; Jin et al., 2009). In budding yeast, the first identified chromosomal instability (CIN) phenotypes, showing severe mitotic spindle defects due to loss of MTs, were ultimately traced to loss of TBC proteins (Hoyt et al., 1990, 1997; Antoshechkin and Han, 2002; Steinborn et al., 2002; Lacefield et al., 2006; Jin et al., 2009). In humans, missense mutations in TBCE and TBCB are linked to hypo-parathyroidism facial dysmorphism (also termed Kenny-Caffey syndrome) and giant axonal neuropathy, in which developmental defects are observed due to impairment of MT cytoskeleton function (Parvari et al., 2002; Wang et al., 2005). "
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    ABSTRACT: Microtubule dynamics and polarity stem from the polymerization of αß-tubulin heterodimers. Five conserved tubulin cofactors/chaperones and the Arl2 GTPase regulate α- and β-tubulin assembly into heterodimers and maintain the soluble tubulin pool in the cytoplasm, but their physical mechanisms are unknown. Here, we reconstitute a core tubulin chaperone consisting of tubulin cofactors TBCD, TBCE and Arl2, and reveal a cage-like structure for regulating αβ-tubulin. Biochemical assays and electron microscopy structures of multiple intermediates show the sequential binding of αβ-tubulin dimer followed by tubulin cofactor TBCC onto this chaperone, forming a ternary complex in which Arl2 GTP hydrolysis is activated to alter αβ-tubulin conformation. A GTP-state locked Arl2 mutant inhibits ternary complex dissociation in vitro and causes severe defects in microtubule dynamics in vivo. Our studies suggest a revised paradigm for tubulin cofactors and Arl2 functions as a catalytic chaperone that regulates soluble αβ-tubulin assembly and maintenance to support microtubule dynamics.
    eLife Sciences 07/2015; 4. DOI:10.7554/eLife.08811 · 9.32 Impact Factor
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    • "The MT cytoskeleton of a cell is subject to both stabilizing and destabilizing factors that act coordinately to maintain the MT architecture. For example, an antagonistic relationship in maintaining the MT network has been demonstrated between tubulin-specific chaperone E (TBCE), required for the formation of tubulin heterodimers, and the microtubule severing protein spastin [74]. It is possible that stai acts downstream of TBCE, and its removal results in a diminished MT network, as observed in TBCE mutant larvae, suggesting an antagonistic relationship with MT severing proteins spastin and/or katanin. "
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    ABSTRACT: Axonal transport, a form of long-distance, bi-directional intracellular transport that occurs between the cell body and synaptic terminal, is critical in maintaining the function and viability of neurons. We have identified a requirement for the stathmin (stai) gene in the maintenance of axonal microtubules and regulation of axonal transport in Drosophila. The stai gene encodes a cytosolic phosphoprotein that regulates microtubule dynamics by partitioning tubulin dimers between pools of soluble tubulin and polymerized microtubules, and by directly binding to microtubules and promoting depolymerization. Analysis of stai function in Drosophila, which has a single stai gene, circumvents potential complications with studies performed in vertebrate systems in which mutant phenotypes may be compensated by genetic redundancy of other members of the stai gene family. This has allowed us to identify an essential function for stai in the maintenance of the integrity of axonal microtubules. In addition to the severe disruption in the abundance and architecture of microtubules in the axons of stai mutant Drosophila, we also observe additional neurological phenotypes associated with loss of stai function including a posterior paralysis and tail-flip phenotype in third instar larvae, aberrant accumulation of transported membranous organelles in stai deficient axons, a progressive bang-sensitive response to mechanical stimulation reminiscent of the class of Drosophila mutants used to model human epileptic seizures, and a reduced adult lifespan. Reductions in the levels of Kinesin-1, the primary anterograde motor in axonal transport, enhance these phenotypes. Collectively, our results indicate that stai has an important role in neuronal function, likely through the maintenance of microtubule integrity in the axons of nerves of the peripheral nervous system necessary to support and sustain long-distance axonal transport.
    PLoS ONE 06/2013; 8(6):e68324. DOI:10.1371/journal.pone.0068324 · 3.23 Impact Factor
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    • "TBCs, by modulating the concentration of tubulin dimers available for MT polymerization, are possible candidates for regulating specific cellular functions during oogenesis, as well as other developmental processes. Indeed, dTBCE, the only tubulin cofactor studied in flies, was shown to be required for the normal development of neuromuscular synapses (Jin et al., 2009). The Drosophila genome contains a single TBCB orthologue, annotated as CG11242 (Tweedie et al., 2009). "
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    ABSTRACT: Microtubules (MTs) are essential for cell division, shape, intracellular transport, and polarity. MT stability is regulated by many factors, including MT-associated proteins and proteins controlling the amount of free tubulin heterodimers available for polymerization. Tubulin-binding cofactors are potential key regulators of free tubulin concentration, since they are required for α-β-tubulin dimerization in vitro. In this paper, we show that mutation of the Drosophila tubulin-binding cofactor B (dTBCB) affects the levels of both α- and β-tubulins and dramatically destabilizes the MT network in different fly tissues. However, we find that dTBCB is dispensable for the early MT-dependent steps of oogenesis, including cell division, and that dTBCB is not required for mitosis in several tissues. In striking contrast, the absence of dTBCB during later stages of oogenesis causes major defects in cell polarity. We show that dTBCB is required for the polarized localization of the axis-determining mRNAs within the oocyte and for the apico-basal polarity of the surrounding follicle cells. These results establish a developmental function for the dTBCB gene that is essential for viability and MT-dependent cell polarity, but not cell division.
    Molecular biology of the cell 08/2012; 23(18):3591-601. DOI:10.1091/mbc.E11-07-0633 · 4.47 Impact Factor
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