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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

Microtubule binding properties of TPPP/p25.(a) Taxol stabilized microtubules (2 μM) were incubated in the presence of 2 μM of each of the different TPPP/p25 fragments (FL, ΔN, ΔC and core) for 15 min before centrifugation. Coomassie-stained gel of microtubule bound and unbound TPPP/ p25 fragments present in the pellets and in the supernatants, respectively. (b) Bar graph showing the changes in turbidity of tubulin solutions (15 μM) in the presence of stoichiometric amounts of the different TPPP/ p25 fragments. The OD350 of control tubulin solutions at mass steady state were subtracted from the tubulin solutions assembled in the presence of the different TPPP/ p25 fragments tagged with either 6×HIS at their C-termini or GFP at the N-termini. Data were compared using the Mann Whitney test (p values for the HIS- vs the GFP- tagged fragments were 1, 0,666, 0,667 and 0,333 for FL, ΔN, ΔC, and core respectively; p values for the FL, or ΔN, or ΔC fragments compared to the core were 0,028, 0,2 and 0,57, respectively; n = 4). EM images of taxol stabilized microtubules (2 μM) incubated with bacterially expressed and then purified C-terminal His- or GFP-tagged TPPP/p25 fragments at equimolar ratios (scale bars = 50 nm). (c) His tagged TPPP/p25 competes with GFP-tagged TPPP/p25 for MT binding. Taxol stabilized MTs were first co-incubated with 2 μM of 6×HIS-FL and 2 μM GFP-FL-TPPP/p25 and then with increasing concentrations of 6×HIS-FL. Each data point represents the mean ± SD from three independent experiments.
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f3: Microtubule binding properties of TPPP/p25.(a) Taxol stabilized microtubules (2 μM) were incubated in the presence of 2 μM of each of the different TPPP/p25 fragments (FL, ΔN, ΔC and core) for 15 min before centrifugation. Coomassie-stained gel of microtubule bound and unbound TPPP/ p25 fragments present in the pellets and in the supernatants, respectively. (b) Bar graph showing the changes in turbidity of tubulin solutions (15 μM) in the presence of stoichiometric amounts of the different TPPP/ p25 fragments. The OD350 of control tubulin solutions at mass steady state were subtracted from the tubulin solutions assembled in the presence of the different TPPP/ p25 fragments tagged with either 6×HIS at their C-termini or GFP at the N-termini. Data were compared using the Mann Whitney test (p values for the HIS- vs the GFP- tagged fragments were 1, 0,666, 0,667 and 0,333 for FL, ΔN, ΔC, and core respectively; p values for the FL, or ΔN, or ΔC fragments compared to the core were 0,028, 0,2 and 0,57, respectively; n = 4). EM images of taxol stabilized microtubules (2 μM) incubated with bacterially expressed and then purified C-terminal His- or GFP-tagged TPPP/p25 fragments at equimolar ratios (scale bars = 50 nm). (c) His tagged TPPP/p25 competes with GFP-tagged TPPP/p25 for MT binding. Taxol stabilized MTs were first co-incubated with 2 μM of 6×HIS-FL and 2 μM GFP-FL-TPPP/p25 and then with increasing concentrations of 6×HIS-FL. Each data point represents the mean ± SD from three independent experiments.

Mentions: In order to analyze the MT binding affinity of TPPP/p25 and its various fragments, we exploited a MT pelleting assay in which the amount of TPPP/p25 bound to taxol MT pellets is measured. We carried out preliminary experiments with all the His-tagged fragments of TPPP/p25 at stoichiometric 1:1 ratios with taxol stabilized MTs (Fig. 3a). The MT-pelleting assays showed that indeed the full length as well as the ΔN(49) and ΔC(158) bind to MTs whereas the core had very limited MT binding activity. The binding of the tag-free FL TPPP/p25 to taxol stabilized MTs were qualitatively similar to His-tagged FL TPPP/p25 whereas the tag-free core did not exhibit any binding to taxol MTs (Fig. S3, panels C and D).


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

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

Microtubule binding properties of TPPP/p25.(a) Taxol stabilized microtubules (2 μM) were incubated in the presence of 2 μM of each of the different TPPP/p25 fragments (FL, ΔN, ΔC and core) for 15 min before centrifugation. Coomassie-stained gel of microtubule bound and unbound TPPP/ p25 fragments present in the pellets and in the supernatants, respectively. (b) Bar graph showing the changes in turbidity of tubulin solutions (15 μM) in the presence of stoichiometric amounts of the different TPPP/ p25 fragments. The OD350 of control tubulin solutions at mass steady state were subtracted from the tubulin solutions assembled in the presence of the different TPPP/ p25 fragments tagged with either 6×HIS at their C-termini or GFP at the N-termini. Data were compared using the Mann Whitney test (p values for the HIS- vs the GFP- tagged fragments were 1, 0,666, 0,667 and 0,333 for FL, ΔN, ΔC, and core respectively; p values for the FL, or ΔN, or ΔC fragments compared to the core were 0,028, 0,2 and 0,57, respectively; n = 4). EM images of taxol stabilized microtubules (2 μM) incubated with bacterially expressed and then purified C-terminal His- or GFP-tagged TPPP/p25 fragments at equimolar ratios (scale bars = 50 nm). (c) His tagged TPPP/p25 competes with GFP-tagged TPPP/p25 for MT binding. Taxol stabilized MTs were first co-incubated with 2 μM of 6×HIS-FL and 2 μM GFP-FL-TPPP/p25 and then with increasing concentrations of 6×HIS-FL. Each data point represents the mean ± SD from three independent experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4542545&req=5

f3: Microtubule binding properties of TPPP/p25.(a) Taxol stabilized microtubules (2 μM) were incubated in the presence of 2 μM of each of the different TPPP/p25 fragments (FL, ΔN, ΔC and core) for 15 min before centrifugation. Coomassie-stained gel of microtubule bound and unbound TPPP/ p25 fragments present in the pellets and in the supernatants, respectively. (b) Bar graph showing the changes in turbidity of tubulin solutions (15 μM) in the presence of stoichiometric amounts of the different TPPP/ p25 fragments. The OD350 of control tubulin solutions at mass steady state were subtracted from the tubulin solutions assembled in the presence of the different TPPP/ p25 fragments tagged with either 6×HIS at their C-termini or GFP at the N-termini. Data were compared using the Mann Whitney test (p values for the HIS- vs the GFP- tagged fragments were 1, 0,666, 0,667 and 0,333 for FL, ΔN, ΔC, and core respectively; p values for the FL, or ΔN, or ΔC fragments compared to the core were 0,028, 0,2 and 0,57, respectively; n = 4). EM images of taxol stabilized microtubules (2 μM) incubated with bacterially expressed and then purified C-terminal His- or GFP-tagged TPPP/p25 fragments at equimolar ratios (scale bars = 50 nm). (c) His tagged TPPP/p25 competes with GFP-tagged TPPP/p25 for MT binding. Taxol stabilized MTs were first co-incubated with 2 μM of 6×HIS-FL and 2 μM GFP-FL-TPPP/p25 and then with increasing concentrations of 6×HIS-FL. Each data point represents the mean ± SD from three independent experiments.
Mentions: In order to analyze the MT binding affinity of TPPP/p25 and its various fragments, we exploited a MT pelleting assay in which the amount of TPPP/p25 bound to taxol MT pellets is measured. We carried out preliminary experiments with all the His-tagged fragments of TPPP/p25 at stoichiometric 1:1 ratios with taxol stabilized MTs (Fig. 3a). The MT-pelleting assays showed that indeed the full length as well as the ΔN(49) and ΔC(158) bind to MTs whereas the core had very limited MT binding activity. The binding of the tag-free FL TPPP/p25 to taxol stabilized MTs were qualitatively similar to His-tagged FL TPPP/p25 whereas the tag-free core did not exhibit any binding to taxol MTs (Fig. S3, panels C and D).

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