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Self protein-protein interactions are involved in TPPP/p25 mediated microtubule bundling.

DeBonis S, Neumann E, Skoufias DA - Sci Rep (2015)

Bottom Line: However, the C-terminally truncated TPPP/p25 shows a lower Bmax for microtubule binding, suggesting that it may bind to a site of tubulin that is masked in microtubules.Bimolecular fluorescent complementation assays in cells expressing combinations of various TPPP/p25 fragments, but not that of the central folded domain, resulted in the generation of a fluorescence signal colocalized with perinuclear microtubule bundles insensitive to microtubule inhibitors.The data suggest that the central folded domain of TPPP/p25 following binding to microtubules can drive s homotypic protein-protein interactions leading to bundled microtubules.

View Article: PubMed Central - PubMed

Affiliation: Université de Grenoble Alpes, F-38044 Grenoble, France.

ABSTRACT
TPPP/p25 is a microtubule-associated protein, detected in protein inclusions associated with various neurodegenerative diseases. Deletion analysis data show that TPPP/p25 has two microtubule binding sites, both located in intrinsically disordered domains, one at the N-terminal and the other in the C-terminal domain. In copolymerization assays the full-length protein exhibits microtubule stimulation and bundling activity. In contrast, at the same ratio relative to tubulin, truncated forms of TPPP/p25 exhibit either lower or no microtubule stimulation and no bundling activity, suggesting a cooperative phenomenon which is enhanced by the presence of the two binding sites. The binding characteristics of the N- and C-terminally truncated proteins to taxol-stabilized microtubules are similar to the full-length protein. However, the C-terminally truncated TPPP/p25 shows a lower Bmax for microtubule binding, suggesting that it may bind to a site of tubulin that is masked in microtubules. Bimolecular fluorescent complementation assays in cells expressing combinations of various TPPP/p25 fragments, but not that of the central folded domain, resulted in the generation of a fluorescence signal colocalized with perinuclear microtubule bundles insensitive to microtubule inhibitors. The data suggest that the central folded domain of TPPP/p25 following binding to microtubules can drive s homotypic protein-protein interactions leading to bundled microtubules.

No MeSH data available.


Related in: MedlinePlus

TPPP/p25-TPPP/p25 interactions in cells detected by BiFC on microtubule bundles.(a) The generated BiFC signal resulting from the FLVN-FLVC, FLVN-ΔNVC, FLVN-ΔCVC complex formation colocalizes with perinuclear microtubule bundles. Note the absence of BiFC signal despite the presence of microtubule bundles due to the expression of the FLVN in cells (scale bars = 34 μm). (b) Microtubule bundles in cells transfected with the wild type TPPP/p25 are resistant to nocodazole and vinblastine treatment (scale bars = 17 μm).
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f6: TPPP/p25-TPPP/p25 interactions in cells detected by BiFC on microtubule bundles.(a) The generated BiFC signal resulting from the FLVN-FLVC, FLVN-ΔNVC, FLVN-ΔCVC complex formation colocalizes with perinuclear microtubule bundles. Note the absence of BiFC signal despite the presence of microtubule bundles due to the expression of the FLVN in cells (scale bars = 34 μm). (b) Microtubule bundles in cells transfected with the wild type TPPP/p25 are resistant to nocodazole and vinblastine treatment (scale bars = 17 μm).

Mentions: The perinuclear filamentous BiFC colocalized with dense perinuclear MT bundles (Fig. 6a). Expression of the FLVN-FLVC, FLVN-ΔNVC and FLVN-ΔCVC pairs induced the formation of the MT-bundles. Although the FL-core pair did not produce BiFC, there were still perinuclear MT bundles formed because of the bundling activity of the full-length protein overexpressed in cells. It is worth mentioning that single plasmid transfections gave the same patterns of localization of the various constructs, eliminating the possibility that the dimerization of Venus protein was the cause of the MT bundling (Fig. S5). Similar MT bundles were observed previously when GFP-tagged TPPP/p25 was expressed in cells2628. Interestingly, the perinuclear MT bundles formed due to the expression of the FLVN-FLVC pair were resistant to MT depolymerizing drugs such as nocodazole and vinblastine. In non-transfected cells the entire MT network was depolymerized in the presence of nocodazole, whereas in the BiFC positive cells the MT bundles resisted depolymerization. In vinblastine treated cells tubulin paracrystals were observed in nontransfected cells46, whereas in the BiFC positive cells there were no tubulin paracrystals formed, probably due to the lack of enough soluble tubulin since the perinuclear MT bundles resisted depolymerization.


Self protein-protein interactions are involved in TPPP/p25 mediated microtubule bundling.

DeBonis S, Neumann E, Skoufias DA - Sci Rep (2015)

TPPP/p25-TPPP/p25 interactions in cells detected by BiFC on microtubule bundles.(a) The generated BiFC signal resulting from the FLVN-FLVC, FLVN-ΔNVC, FLVN-ΔCVC complex formation colocalizes with perinuclear microtubule bundles. Note the absence of BiFC signal despite the presence of microtubule bundles due to the expression of the FLVN in cells (scale bars = 34 μm). (b) Microtubule bundles in cells transfected with the wild type TPPP/p25 are resistant to nocodazole and vinblastine treatment (scale bars = 17 μm).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4542545&req=5

f6: TPPP/p25-TPPP/p25 interactions in cells detected by BiFC on microtubule bundles.(a) The generated BiFC signal resulting from the FLVN-FLVC, FLVN-ΔNVC, FLVN-ΔCVC complex formation colocalizes with perinuclear microtubule bundles. Note the absence of BiFC signal despite the presence of microtubule bundles due to the expression of the FLVN in cells (scale bars = 34 μm). (b) Microtubule bundles in cells transfected with the wild type TPPP/p25 are resistant to nocodazole and vinblastine treatment (scale bars = 17 μm).
Mentions: The perinuclear filamentous BiFC colocalized with dense perinuclear MT bundles (Fig. 6a). Expression of the FLVN-FLVC, FLVN-ΔNVC and FLVN-ΔCVC pairs induced the formation of the MT-bundles. Although the FL-core pair did not produce BiFC, there were still perinuclear MT bundles formed because of the bundling activity of the full-length protein overexpressed in cells. It is worth mentioning that single plasmid transfections gave the same patterns of localization of the various constructs, eliminating the possibility that the dimerization of Venus protein was the cause of the MT bundling (Fig. S5). Similar MT bundles were observed previously when GFP-tagged TPPP/p25 was expressed in cells2628. Interestingly, the perinuclear MT bundles formed due to the expression of the FLVN-FLVC pair were resistant to MT depolymerizing drugs such as nocodazole and vinblastine. In non-transfected cells the entire MT network was depolymerized in the presence of nocodazole, whereas in the BiFC positive cells the MT bundles resisted depolymerization. In vinblastine treated cells tubulin paracrystals were observed in nontransfected cells46, whereas in the BiFC positive cells there were no tubulin paracrystals formed, probably due to the lack of enough soluble tubulin since the perinuclear MT bundles resisted depolymerization.

Bottom Line: However, the C-terminally truncated TPPP/p25 shows a lower Bmax for microtubule binding, suggesting that it may bind to a site of tubulin that is masked in microtubules.Bimolecular fluorescent complementation assays in cells expressing combinations of various TPPP/p25 fragments, but not that of the central folded domain, resulted in the generation of a fluorescence signal colocalized with perinuclear microtubule bundles insensitive to microtubule inhibitors.The data suggest that the central folded domain of TPPP/p25 following binding to microtubules can drive s homotypic protein-protein interactions leading to bundled microtubules.

View Article: PubMed Central - PubMed

Affiliation: Université de Grenoble Alpes, F-38044 Grenoble, France.

ABSTRACT
TPPP/p25 is a microtubule-associated protein, detected in protein inclusions associated with various neurodegenerative diseases. Deletion analysis data show that TPPP/p25 has two microtubule binding sites, both located in intrinsically disordered domains, one at the N-terminal and the other in the C-terminal domain. In copolymerization assays the full-length protein exhibits microtubule stimulation and bundling activity. In contrast, at the same ratio relative to tubulin, truncated forms of TPPP/p25 exhibit either lower or no microtubule stimulation and no bundling activity, suggesting a cooperative phenomenon which is enhanced by the presence of the two binding sites. The binding characteristics of the N- and C-terminally truncated proteins to taxol-stabilized microtubules are similar to the full-length protein. However, the C-terminally truncated TPPP/p25 shows a lower Bmax for microtubule binding, suggesting that it may bind to a site of tubulin that is masked in microtubules. Bimolecular fluorescent complementation assays in cells expressing combinations of various TPPP/p25 fragments, but not that of the central folded domain, resulted in the generation of a fluorescence signal colocalized with perinuclear microtubule bundles insensitive to microtubule inhibitors. The data suggest that the central folded domain of TPPP/p25 following binding to microtubules can drive s homotypic protein-protein interactions leading to bundled microtubules.

No MeSH data available.


Related in: MedlinePlus