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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.(a) Schematic representation of the generation of fluorescence based on the complementation between the N- and C-terminal domains of Venus fluorescent protein fused to proteins capable of interacting with each other. (b) BiFC signal generation in cells that were co-transfected with the myc-tagged FL fused to VN155(I152L), indicated as FLVN and either HA-tagged FL- or ΔN-, ΔC- and core- fused to the VC155, indicated as FLVC or ΔNVC, or ΔCVC or coreVC, respectively. Co-expression of the two chimeras was confirmed by immunofluorescence microscopy using antibodies recognizing either the myc- or the HA- tags. (c) Fluorescence intensities of Venus-based BiFC assays in cells transfected as described in panel (b). Each bar represents the mean ± SD from three independent experiments (at least 30 BiFC-positive cells measured for each count). Data were compared using the Mann Whitney test (n = 100, 113, 110 and 95 for FLVN-FLVC, FLVN-coreVC, ΔNVN-coreVC and ΔCVN-coreVC, respectively). The signal to noise (S/N) ratio was estimated by dividing the fluorescence intensity from the positive interaction (FLVN-FLVC; FLVN-ΔNVC; FLVN-ΔCVC) by that from the negative interaction (FLVN-coreVC) (scale bars = 17 μm).
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f5: TPPP/p25-TPPP/p25 interactions in cells detected by BiFC.(a) Schematic representation of the generation of fluorescence based on the complementation between the N- and C-terminal domains of Venus fluorescent protein fused to proteins capable of interacting with each other. (b) BiFC signal generation in cells that were co-transfected with the myc-tagged FL fused to VN155(I152L), indicated as FLVN and either HA-tagged FL- or ΔN-, ΔC- and core- fused to the VC155, indicated as FLVC or ΔNVC, or ΔCVC or coreVC, respectively. Co-expression of the two chimeras was confirmed by immunofluorescence microscopy using antibodies recognizing either the myc- or the HA- tags. (c) Fluorescence intensities of Venus-based BiFC assays in cells transfected as described in panel (b). Each bar represents the mean ± SD from three independent experiments (at least 30 BiFC-positive cells measured for each count). Data were compared using the Mann Whitney test (n = 100, 113, 110 and 95 for FLVN-FLVC, FLVN-coreVC, ΔNVN-coreVC and ΔCVN-coreVC, respectively). The signal to noise (S/N) ratio was estimated by dividing the fluorescence intensity from the positive interaction (FLVN-FLVC; FLVN-ΔNVC; FLVN-ΔCVC) by that from the negative interaction (FLVN-coreVC) (scale bars = 17 μm).

Mentions: A number of MAPs, have the ability to induce MT bundles under certain conditions. The bundling activity of PRC1 and its homologues Ase1 and MAP65-1 were shown to be due to dimer formation following MT-binding7. Similarly, tau has also been reported to oligomerize along MTs40. In order to investigate the MT-dependent self-association of TPPP/p25 and map the domains responsible for this interaction in cells we employed a Bimolecular Fluorescent Complementation (BiFC) assay41. For BiFC, the N-terminal and the C-terminal domains of a fluorescent protein (e.g., GFP, Venus) are each fused separately to two partners hypothesized to interact with each other42. If the two partners indeed interact, then upon interaction the two domains of the split fluorescent protein will come into close proximity and fold into the native structure, which will fluoresce upon excitation. We have chosen to use the Venus fluorescent protein since it has superior BiFC properties than GFP (Fig. 5a)43. A necessary step for the BiFC assay is to generate a negative control in order to distinguish the true BiFC signal from the non-specific BiFC signal generated by the intrinsic tendency of the two fluorescent protein domains to complement each other44. Our in vitro MT binding data indicated that the core domain is not interacting with MTs and therefore we hypothesized that in the absence of MT binding it would have a low tendency for self association and therefore should produce low BiFC signal. Furthermore, we used two different Venus N-terminal domains, one comprising the first 173 residues and the other comprising the first 155 residues carrying a I152L mutation. Both Venus N-terminal fragments are able to complement the Venus C-terminal domain starting from residue 155. The VN155(I52L) fragment has been reported to reduce the nonspecific BiFC and therefore to produce higher BiFC signal to noise ratios45.


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.(a) Schematic representation of the generation of fluorescence based on the complementation between the N- and C-terminal domains of Venus fluorescent protein fused to proteins capable of interacting with each other. (b) BiFC signal generation in cells that were co-transfected with the myc-tagged FL fused to VN155(I152L), indicated as FLVN and either HA-tagged FL- or ΔN-, ΔC- and core- fused to the VC155, indicated as FLVC or ΔNVC, or ΔCVC or coreVC, respectively. Co-expression of the two chimeras was confirmed by immunofluorescence microscopy using antibodies recognizing either the myc- or the HA- tags. (c) Fluorescence intensities of Venus-based BiFC assays in cells transfected as described in panel (b). Each bar represents the mean ± SD from three independent experiments (at least 30 BiFC-positive cells measured for each count). Data were compared using the Mann Whitney test (n = 100, 113, 110 and 95 for FLVN-FLVC, FLVN-coreVC, ΔNVN-coreVC and ΔCVN-coreVC, respectively). The signal to noise (S/N) ratio was estimated by dividing the fluorescence intensity from the positive interaction (FLVN-FLVC; FLVN-ΔNVC; FLVN-ΔCVC) by that from the negative interaction (FLVN-coreVC) (scale bars = 17 μm).
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f5: TPPP/p25-TPPP/p25 interactions in cells detected by BiFC.(a) Schematic representation of the generation of fluorescence based on the complementation between the N- and C-terminal domains of Venus fluorescent protein fused to proteins capable of interacting with each other. (b) BiFC signal generation in cells that were co-transfected with the myc-tagged FL fused to VN155(I152L), indicated as FLVN and either HA-tagged FL- or ΔN-, ΔC- and core- fused to the VC155, indicated as FLVC or ΔNVC, or ΔCVC or coreVC, respectively. Co-expression of the two chimeras was confirmed by immunofluorescence microscopy using antibodies recognizing either the myc- or the HA- tags. (c) Fluorescence intensities of Venus-based BiFC assays in cells transfected as described in panel (b). Each bar represents the mean ± SD from three independent experiments (at least 30 BiFC-positive cells measured for each count). Data were compared using the Mann Whitney test (n = 100, 113, 110 and 95 for FLVN-FLVC, FLVN-coreVC, ΔNVN-coreVC and ΔCVN-coreVC, respectively). The signal to noise (S/N) ratio was estimated by dividing the fluorescence intensity from the positive interaction (FLVN-FLVC; FLVN-ΔNVC; FLVN-ΔCVC) by that from the negative interaction (FLVN-coreVC) (scale bars = 17 μm).
Mentions: A number of MAPs, have the ability to induce MT bundles under certain conditions. The bundling activity of PRC1 and its homologues Ase1 and MAP65-1 were shown to be due to dimer formation following MT-binding7. Similarly, tau has also been reported to oligomerize along MTs40. In order to investigate the MT-dependent self-association of TPPP/p25 and map the domains responsible for this interaction in cells we employed a Bimolecular Fluorescent Complementation (BiFC) assay41. For BiFC, the N-terminal and the C-terminal domains of a fluorescent protein (e.g., GFP, Venus) are each fused separately to two partners hypothesized to interact with each other42. If the two partners indeed interact, then upon interaction the two domains of the split fluorescent protein will come into close proximity and fold into the native structure, which will fluoresce upon excitation. We have chosen to use the Venus fluorescent protein since it has superior BiFC properties than GFP (Fig. 5a)43. A necessary step for the BiFC assay is to generate a negative control in order to distinguish the true BiFC signal from the non-specific BiFC signal generated by the intrinsic tendency of the two fluorescent protein domains to complement each other44. Our in vitro MT binding data indicated that the core domain is not interacting with MTs and therefore we hypothesized that in the absence of MT binding it would have a low tendency for self association and therefore should produce low BiFC signal. Furthermore, we used two different Venus N-terminal domains, one comprising the first 173 residues and the other comprising the first 155 residues carrying a I152L mutation. Both Venus N-terminal fragments are able to complement the Venus C-terminal domain starting from residue 155. The VN155(I52L) fragment has been reported to reduce the nonspecific BiFC and therefore to produce higher BiFC signal to noise ratios45.

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