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.
    Full-text · Article · Jul 2015 · eLife Sciences
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    • "Misregulated MTs lead to abnormal NMJ synapses in Drosophila (Jin et al., 2009; Nahm et al., 2013; Roos et al., 2000; Ruiz-Canada et al., 2004; Sherwood et al., 2004; Trotta et al., 2004; Wang et al., 2007; Yao et al., 2011; Zhang et al., 2001). To determine whether Katanin 60 plays a role in synapse development, we co-stained larval NMJs with anti-horseradish peroxidase (HRP) to reveal the neuronal membrane, and with anti-cysteine string protein (CSP) to label synaptic vesicles. "
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    ABSTRACT: Microtubules (MTs) are crucial for diverse biological processes including cell division, cell growth and motility, intracellular transport and the maintenance of cell shape. MT abnormalities are associated with neurodevelopmental and neurodegenerative diseases such as hereditary spastic paraplegia. Among many MT regulators, katanin was the first identified MT-severing protein, but its neuronal functions have not yet been examined in a multicellular organism. Katanin consists of two subunits; the catalytic subunit katanin 60 contains an AAA (ATPases associated with a variety of cellular activities) domain and breaks MT fibers while hydrolyzing ATP, whereas katanin 80 is a targeting and regulatory subunit. To dissect the in vivo functions of Katanin, we generated mutations in Drosophila Katanin 60 and manipulated its expression in a tissue-specific manner. Null mutants of Katanin 60 are pupal lethal, demonstrating that it is essential for viability. Loss-of-function mutants of Katanin 60 showed excess satellite boutons, reduced neurotransmission efficacy, and more enlarged cisternae at neuromuscular junctions. In peripheral sensory neurons, loss of Katanin 60 led to increased elaboration of dendrites, whereas overexpression of Katanin 60 resulted in the opposite. Genetic interaction analyses indicated that increased levels of MT acetylation increase its susceptibility to Katanin-mediated severing in neuronal and non-neuronal systems. Taken together, our results demonstrate for the first time that Katanin 60 is required for the normal development of neuromuscular synapses and dendrites.
    Preview · Article · Mar 2014 · Development
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    • "Electrophysiology. Excitatory junctional potentials (EJPs) and spontaneous miniature EJPs (mEJPs) at NMJs were recorded using intracellular electrodes (Jin et al., 2009; Wang et al., 2010). "
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    ABSTRACT: The precise regulation of synaptic growth is critical for the proper formation and plasticity of functional neural circuits. Identification and characterization of factors that regulate synaptic growth and function have been under intensive investigation. Here we report that brain tumor (brat), which was identified as a translational repressor in multiple biological processes, plays a crucial role at Drosophila neuromuscular junction (NMJ) synapses. Immunohistochemical analysis demonstrated that brat mutants exhibited synaptic overgrowth characterized by excess satellite boutons at NMJ terminals, whereas electron microscopy revealed increased synaptic vesicle size but reduced density at active zones compared with wild-types. Spontaneous miniature excitatory junctional potential amplitudes were larger and evoked quantal content was lower at brat mutant NMJs. In agreement with the morphological and physiological phenotypes, loss of Brat resulted in reduced FM1-43 uptake at the NMJ terminals, indicating that brat regulates synaptic endocytosis. Genetic analysis revealed that the actions of Brat at synapses are mediated through mothers against decapentaplegic (Mad), the signal transduction effector of the bone morphogenetic protein (BMP) signaling pathway. Furthermore, biochemical analyses showed upregulated levels of Mad protein but normal mRNA levels in the larval brains of brat mutants, suggesting that Brat suppresses Mad translation. Consistently, knockdown of brat by RNA interference in Drosophila S2 cells also increased Mad protein level. These results together reveal an important and previously unidentified role for Brat in synaptic development and endocytosis mediated by suppression of BMP signaling.
    Full-text · Article · Jul 2013 · The Journal of Neuroscience : The Official Journal of the Society for Neuroscience
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