Tubulin-specific chaperone E: Biological Overview | References
Gene name - Tubulin-specific chaperone E
Synonyms - CG7861
Cytological map position - 42A8-42A8
Function - tubulin chaperone
Symbol - Tbce
FlyBase ID: FBgn0033055
Genetic map position - 2R:1,930,160..1,932,475 [+]
Classification - CAP-Gly domain, Leucine-rich repeats, ubiquitin-like (UBL) domain
Cellular location - cytoplasmic
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, mutations were generated in Drosophila tbce, and its expression was manipulated in a tissue-specific manner. Drosophila tbce nulls are embryonic lethal. Tissue-specific knockdown and overexpression of tbce in the 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, these 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, this work on TBCE may offer novel insights into their basis (Jin, 2009).
Microtubules (MTs), one of the major building blocks of cells, play a crucial role in a diverse array of biological functions including cell division, cell growth and motility, intracellular transport and the maintenance of cell shape. As MTs are important in all eukaryotes, it is not surprising that defects in MTs are associated with a number of severe human diseases, including Fragile X mental retardation and autosomal dominant hereditary spastic paraplegia (AD-HSP). MTs are formed by polymerization of tubulin heterodimers consisting of one α- and one β-tubulin polypeptide. The formation of α-β tubulin heterodimers is mediated by a group of five tubulin chaperones, TBCA-TBCE (Tian, 1996) (for reviews, see Lewis, 1997; Nogales, 2000; see Domain analysis of the tubulin cofactor system: a model for tubulin folding and dimerization). TBCA and TBCD assist in the folding of β-tubulin, whereas TBCB and TBCE facilitate the folding of α-tubulin (Lewis, 1997; Tian, 2006; Jin, 2009 and references therein),
A group of rare, recessive and fatal congenital diseases, collectively called hypoparathyroidism, mental retardation and facial dysmorphism (HRD), is caused by mutations in the gene encoding TBCE (Parvari, 2002). TBCE contains three functional domains: a glycine-rich cytoskeleton-associated protein domain (CAP-Gly) that binds α-tubulin, a series of leucine-rich repeats (LRR), and an ubiquitin-like (UBL) domain; the latter two mediate protein-protein interactions (Bartolini, 2005; Grynberg, 2003; Parvari, 2002). Identification of the HRD disease gene revealed a 12 bp deletion in TBCE that leads to the expression of a mutated TBCE protein lacking four amino acids in the CAP-Gly domain (Parvari, 2002). The mutation causes lower MT density at the MT organizing center, perturbed MT polarity and decreased precipitable MT, while total tubulin remains unchanged (Parvari, 2002). Remarkably, overexpression of TBCE in cultured cells also results in disrupted MTs (Bhamidipati, 2000; Sellin, 2008; Tian, 2006). Thus, both loss-of-function mutations and overexpression of TBCE disrupt the MT network in mammalian systems (Jin, 2009).
Two independent studies have demonstrated that a Trp524Gly substitution at the last residue of mouse TBCE results in progressive motor neuronopathy (PMN), which has been widely used as a model for human motor neuron diseases. Similar to what has been reported for cells from human HRD patients, the point mutation in mouse Tbce leads to a reduced number of MTs in axons (Bommel, 2002). Isolated motor neurons from mutant mice exhibit shorter axons and irregular axonal swellings (Martin, 2002). More specifically, axonal MTs are lost progressively from distal to proximal, which correlates with dying-back axonal degeneration in mutant mice (Schaefer, 2007). This demonstrates a mechanistic link between TBCE-mediated tubulin polymerization and neurodegeneration (Jin, 2009).
TBCE is well conserved across species, from yeast to human. Genetic analyses of the TBCE homolog in S. pombe, Sto1P, show that it is essential for viability and that it plays a crucial role in the formation of cytoplasmic MTs and in the assembly of mitotic spindles (Grishchuk, 1999; Radcliffe, 1999). S. cerevisiae mutants of the TBCE homolog PAC2 show increased sensitivity to the MT-depolymerizing agent benomyl (Hoyt, 1997). Similarly, tbce mutants of Arabidopsis have defective MTs, leading to embryonic lethality (Steinborn, 2002; Jin, 2009 and references therein).
The Drosophila genome contains a TBCE ortholog, listed as CG7861 in FlyBase, but no studies of it have been reported. To gain a mechanistic insight into the in vivo functions of TBCE, different mutations were introduced into Drosophila tbce. Drosophila tbce nulls are embryonic lethal, indicating that it is an essential gene. The developmental, physiological and pharmacological consequences with regard to neuromuscular synapses and MT formation were examined when the expression of TBCE was altered specifically in neurons or muscles using the UAS-Gal4 system. It was found that TBCE is required for the normal development and function of neuromuscular synapses and that it promotes MT formation in vivo (Jin, 2009).
How MTs functions at synapses is poorly understood. Futsch, a MT-associated protein, stabilizes MTs in presynaptic neurons, and futsch mutants show reduced bouton number and increased bouton size, whereas spastin mutants have the opposite phenotype of increased bouton number but decreased bouton size. This analyses show that TBCE plays a role at synapses. Except for the presynaptic overexpression of tbce, all other manipulations of tbce at either side of the NMJ synapse caused increased branching number, increased bouton number and decreased bouton size, demonstrating that tbce is required for normal NMJ synapse development. Given the dramatic MT alterations on both pre- and postsynaptic sides, the NMJ phenotypes appear subtle. The seemingly conflicting result that both overexpression and knockdown of tbce on the postsynaptic side led to similar phenotypes in synapse development supports an existing hypothesis that abnormal synaptic growth results from the disruption of MT dynamics, rather than from an alteration in the absolute quantity of MTs (Jin, 2009).
Increased neurotransmission, reflected in both EJP and mEJP amplitude, was observed upon presynaptic alteration of tbce expression, whereas postsynaptic manipulations of tbce showed normal neurotransmission. This suggests that synaptic neurotransmission is sensitive to pre-but not postsynaptic MT alteration, although postsynaptic alterations of tbce had a significant effect on synapse development. Interestingly, both overexpression and knockdown of tbce on the presynaptic side led to a similar increase in both EJP and mEJP amplitude. The increased EJP amplitude observed upon presynaptic alterations of TBCE might be accounted for by increased mEJP amplitude. The increase in mEJP amplitude could be caused by an increase in presynaptic vesicle size, an increase in the concentration of vesicular glutamate, or an increase in postsynaptic glutamate receptor sensitivity. It is interesting to note that the mEJP is also increased in both Fmr1-null and Fmr1-overexpression NMJ synapses. However, the exact mechanism by which TBCE, and other MT regulators, affect neurotransmission remains to be elucidated (Jin, 2009).
Genetic analyses revealed an antagonistic interaction between TBCE and Spastin. TBCE promotes MT formation, whereas Spastin severs MTs. Autosomal dominant hereditary spastic paraplegia (AD-HSP) is a heterogeneous group of neurodegenerative disorders characterized by progressive and bilateral spasticity of the lower limbs, with specific degeneration of the longest axons in the CNS. Forty to fifty percent of all AD-HSP cases are caused by mutations in spastin. However, the MT-related pathology of human patients with spastin mutation has not been documented (Jin, 2009).
Overexpression of spastin in Drosophila neuromusculature and in cultured cells causes dramatically fragmented and reduced MTs. Surprisingly, morphologically normal muscles are present in patients with spastin mutations, although large-scale disruption of MT pathways was detected at the molecular level. No MT defects were reported in a mouse model in which the endogenous spastin is truncated. Similarly, spastin-null mutants of Drosophila show no dramatic change in MT appearance in muscles, suggesting that Spastin plays a fine-tuning role in MT dynamics. Indeed, spastin nulls are late pupal lethal with a few adult escapers, further confirming a subtle role for Spastin in MT regulation. By comparison, tbce nulls are embryonic lethal, whereas knockdown of tbce leads to a dramatically reduced MT network in Drosophila neuromusculature. Thus, in contrast to the nuanced role of endogenous Spastin, TBCE plays a crucial role in MT formation (Jin, 2009).
Although Drosophila possesses a TBCE ortholog, no previous studies of it have been reported. This work shows that tbce is essential for early neuromuscular development in Drosophila. In vivo evidence is provided demonstrating that Drosophila TBCE is both required and sufficient for MT formation, supporting early in vitro biochemical studies that showed that TBCE assists in α-β-tubulin heterodimer formation (Tian, 1996; Tian, 1997; Jin, 2009 and references therein).
Overexpression of tbce produced increased MTs. This is the first report of increased MT formation when a tubulin chaperone is overexpressed, and is contrary to reports in other systems. Overexpression of human TBCE in cultured cells leads to complete disruption of MTs (Bhamidipati, 2000; Sellin, 2008; Tian, 2006), as does overexpression of a TBCE-like protein (Bartolini, 2005; Keller, 2005; Sellin, 2008). It was further hypothesized that the UBL domains present in TBCE and the TBCE-like protein might contribute to the degradation of tubulin via the proteasomal pathway (Bartonili). In addition, the overexpression of other tubulin chaperones, such as TBCD, results in a similar disruption of MTs (Bhamidipati, 2000; Martin, 2000). These in vivo data are consistent with the early in vitro observation that TBCD or TBCE in excess destroys tubulin heterodimers by sequestering the bound tubulin subunit, leading to the destabilization of the freed partner subunit (Tian, 1997). It is thus believed that in addition to assisting in the folding pathway, TBCE also interacts with native tubulins to disrupt α-β-tubulin heterodimers (Bhamidipati, 2000). The discrepancy between the overexpression result and findings of others could have several explanations: (1) the use of different experimental systems: transgenic animals in this work and cultured cells in other studies (Bhamidipati, 2000; Sellin, 2008; Tian, 2006); (2) different systems might have different expression levels of tbce, leading to varying effects on MTs; (3) Drosophila and human TBCE might have diverged functions. Further analyses are needed to reconcile the conflicts in the effects of TBCE overexpression in these different systems. In general, however, tbce mutant phenotypes are consistent in all species examined so far, from yeast to human, indicating that the function of TBCE in promoting MT formation has been well-conserved throughout evolution (Jin, 2009).
Search PubMed for articles about Drosophila Tbce
Bartolini, F., Tian, G., Piehl, M., Cassimeris, L., Lewis, S. A. and Cowan, N. J. (2005). Identification of a novel tubulin-destabilizing protein related to the chaperone cofactor E. J. Cell Sci. 118: 1197-1207. PubMed ID: 15728251
Bhamidipati, A., Lewis, S. A. and Cowan, N. J. (2000). ADP ribosylation factor-like protein 2 (Arl2) regulates the interaction of tubulin-folding cofactor D with native tubulin. J. Cell Biol. 149: 1087-1096. PubMed ID: 10831612
Bommel, H., Xie, G., Rossoll, W., Wiese, S., Jablonka, S., Boehm, T. and Sendtner, M. (2002). Missense mutation in the tubulin-specific chaperone E (Tbce) gene in the mouse mutant progressive motor neuronopathy, a model of human motoneuron disease. J. Cell Biol. 159: 563-569. PubMed ID: 12446740
Grishchuk, E. L. and McIntosh, J. R. (1999). Sto1p, a fission yeast protein similar to tubulin folding cofactor E, plays an essential role in mitotic microtubule assembly. J. Cell Sci. 112: 1979-1988. PubMed ID: 10341216
Grynberg, M., Jaroszewski, L. and Godzik, A. (2003). Domain analysis of the tubulin cofactor system: a model for tubulin folding and dimerization. BMC Bioinformatics 4: 46. PubMed ID: 14536023
Hoyt, M. A., Macke, J. P., Roberts, B. T. and Geiser, J. R. (1997). Saccharomyces cerevisiae PAC2 functions with CIN1, 2 and 4 in a pathway leading to normal microtubule stability. Genetics 146: 849-857. PubMed ID: 9215891
Jin, S., Pan, L., Liu, Z., Wang, Q., Xu, Z. and Zhang, Y. Q. (2009). Drosophila Tubulin-specific chaperone E functions at neuromuscular synapses and is required for microtubule network formation. Development 136(9): 1571-81. PubMed ID: 19297412
Keller, C. E. and Lauring, B. P. (2005). Possible regulation of microtubules through destabilization of tubulin. Trends Cell Biol. 15: 571-573. PubMed ID: 16202601
Lewis, S. A., Tian, G. and Cowan, N. J. (1997). The alpha- and beta-tubulin folding pathways. Trends Cell Biol. 7: 479-484. PubMed ID: 17709011
Martin, N., Jaubert, J., Gounon, P., Salido, E., Haase, G., Szatanik, M. and Guenet, J. L. (2002). A missense mutation in Tbce causes progressive motor neuronopathy in mice. Nat. Genet. 32: 443-447. PubMed ID: 12389029
Nogales, E. (2000). Structural insights into microtubule function. Annu. Rev. Biochem. 69: 277-302. PubMed ID: 10966460
Parvari, R., Hershkovitz, E., Grossman, N., Gorodischer, R., Loeys, B., Zecic, A., Mortier, G., Gregory, S., Sharony, R., Kambouris, M. et al. (2002). Mutation of TBCE causes hypoparathyroidism-retardation-dysmorphism and autosomal recessive Kenny-Caffey syndrome. Nat. Genet. 32: 448-452. PubMed ID: 12389028
Radcliffe, P. A., Hirata, D., Vardy, L. and Toda, T. (1999). Functional dissection and hierarchy of tubulin-folding cofactor homologues in fission yeast. Mol. Biol. Cell 10: 2987-3001. PubMed ID: 10473641
Schaefer, M. K., Schmalbruch, H., Buhler, E., Lopez, C., Martin, N., Guenet, J. L. and Haase, G. (2007). Progressive motor neuronopathy: a critical role of the tubulin chaperone TBCE in axonal tubulin routing from the Golgi apparatus. J. Neurosci. 27: 8779-8789. PubMed ID: 17699660
Sellin, M. E., Holmfeldt, P., Stenmark, S. and Gullberg, M. (2008). Op18/Stathmin counteracts the activity of overexpressed tubulin-disrupting proteins in a human leukemia cell line. Exp. Cell Res. 314: 1367-1377. PubMed ID: 18262179
Steinborn, K., Maulbetsch, C., Priester, B., Trautmann, S., Pacher, T., Geiges, B., Kuttner, F., Lepiniec, L., Stierhof, Y. D., Schwarz, H. et al. (2002). The Arabidopsis PILZ group genes encode tubulin-folding cofactor orthologs required for cell division but not cell growth. Genes Dev. 16: 959-971. PubMed ID: 11959844
Tian, G., Huang, Y., Rommelaere, H., Vandekerckhove, J., Ampe, C. and Cowan, N. J. (1996). Pathway leading to correctly folded beta-tubulin. Cell 86: 287-296. PubMed ID: 8706133
Tian, G., Lewis, S. A., Feierbach, B., Stearns, T., Rommelaere, H., Ampe, C. and Cowan, N. J. (1997). Tubulin subunits exist in an activated conformational state generated and maintained by protein cofactors. J. Cell Biol. 138: 821-832. PubMed ID: 9265649
Tian, G., Huang, M. C., Parvari, R., Diaz, G. A. and Cowan, N. J. (2006). Cryptic out-of-frame translational initiation of TBCE rescues tubulin formation in compound heterozygous HRD. Proc. Natl. Acad. Sci. 103: 13491-13496. PubMed ID: 16938882
date revised: 15 July 2009
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