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Regulation of microtubule-based transport by MAP4.

Semenova I, Ikeda K, Resaul K, Kraikivski P, Aguiar M, Gygi S, Zaliapin I, Cowan A, Rodionov V - Mol. Biol. Cell (2014)

Bottom Line: We found that aggregation signals induced phosphorylation of threonine residues in the MT-binding domain of the Xenopus MAP4 (XMAP4), thus decreasing binding of this protein to MTs.We hypothesize that binding of XMAP4 to MTs negatively regulates dynein-dependent movement of melanosomes and positively regulates kinesin-2-based movement.Phosphorylation during pigment aggregation reduces binding of XMAP4 to MTs, thus increasing dynein-dependent and decreasing kinesin-2-dependent motility of melanosomes, which stimulates their accumulation in the cell center, whereas dephosphorylation of XMAP4 during dispersion has an opposite effect.

View Article: PubMed Central - PubMed

Affiliation: R.D. Berlin Center for Cell Analysis and Modeling and Department of Cell Biology, University of Connecticut Health Center, Farmington, CT 06030.

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XMAP4 binds dynactin but not MT motor proteins. (A) Whole-cell extracts or immunoprecipitates of EGFP-XMAP4 with rabbit polyclonal antibodies against EGFP probed with mouse monoclonal antibodies specific for EGFP (EGFP), Kif3A subunit of kinesin-2 (Kif3A), dynein IC (DIC), or the p150Glued subunit of the dynactin complex (p150Glued); p150Glued but not kinesin-2 or dynein coimmunoprecipitates with EGFP-XMAP4. (B) Immunoprecipitate of the endogenous XMAP4 with antibodies against XMAP4 MBD probed with an antibody against p150Glued; p150Glued coimmunoprecipitates with endogenous XMAP4. E, whole-cell extract; IP, immuno­precipitate.
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Figure 5: XMAP4 binds dynactin but not MT motor proteins. (A) Whole-cell extracts or immunoprecipitates of EGFP-XMAP4 with rabbit polyclonal antibodies against EGFP probed with mouse monoclonal antibodies specific for EGFP (EGFP), Kif3A subunit of kinesin-2 (Kif3A), dynein IC (DIC), or the p150Glued subunit of the dynactin complex (p150Glued); p150Glued but not kinesin-2 or dynein coimmunoprecipitates with EGFP-XMAP4. (B) Immunoprecipitate of the endogenous XMAP4 with antibodies against XMAP4 MBD probed with an antibody against p150Glued; p150Glued coimmunoprecipitates with endogenous XMAP4. E, whole-cell extract; IP, immuno­precipitate.

Mentions: To investigate whether XMAP4 interacts with MT motor proteins or the dynactin complex, we immunoprecipitated XMAP4 from whole-cell extracts and probed immunoprecipitates with antibodies specific for kinesin-2, dynein, or dynactin subunits. To avoid competition of XMAP4 binding partners with antibodies during immunoprecipitation, we expressed EGFP-XMAP4 in melanophores and used rabbit antibodies specific for the EGFP tag for immunoprecipitation. To confirm the presence of EGFP-XMAP4 in the immunoprecipitates, we probed them with mouse monoclonal EGFP antibodies. We found that, as expected, immunoprecipitates contained a protein band with electrophoretic mobility consistent with the mobility of EGFP-XMAP4 (Figure 5A). We also found, however, that antibodies against Kif3A subunit of kinesin-2 or cytoplasmic dynein IC, which recognized relevant bands in whole-cell extracts, did not react with immunoprecipitates (Figure 5A), suggesting that neither kinesin-2 nor dynein bound XMAP4 with high affinity in whole-cell extracts. In contrast to antibodies specific for MT motor proteins, antibody raised against the 150Glued subunit of the dynactin complex revealed in immunoprecipitates a protein with appropriate electrophoretic mobility (Figure 5A). To examine whether endogenous MAP4 also bound p150Glued and to exclude the possibility that the presence of p150Glued in EGFP antibody immunoprecipitates could be explained by nonspecific binding of p150Glued to EGFP, we pulled down XMAP4 from extracts of nontransfected cells using the XMAP4 MBD antibodies. Once again, we detected the p150Glued band in the immunoprecipitate obtained with MBD antibodies (Figure 5B). We conclude that XMAP4 binds the p150Glued subunit of the dynactin complex but not subunits of MT motor proteins.


Regulation of microtubule-based transport by MAP4.

Semenova I, Ikeda K, Resaul K, Kraikivski P, Aguiar M, Gygi S, Zaliapin I, Cowan A, Rodionov V - Mol. Biol. Cell (2014)

XMAP4 binds dynactin but not MT motor proteins. (A) Whole-cell extracts or immunoprecipitates of EGFP-XMAP4 with rabbit polyclonal antibodies against EGFP probed with mouse monoclonal antibodies specific for EGFP (EGFP), Kif3A subunit of kinesin-2 (Kif3A), dynein IC (DIC), or the p150Glued subunit of the dynactin complex (p150Glued); p150Glued but not kinesin-2 or dynein coimmunoprecipitates with EGFP-XMAP4. (B) Immunoprecipitate of the endogenous XMAP4 with antibodies against XMAP4 MBD probed with an antibody against p150Glued; p150Glued coimmunoprecipitates with endogenous XMAP4. E, whole-cell extract; IP, immuno­precipitate.
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Related In: Results  -  Collection

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Figure 5: XMAP4 binds dynactin but not MT motor proteins. (A) Whole-cell extracts or immunoprecipitates of EGFP-XMAP4 with rabbit polyclonal antibodies against EGFP probed with mouse monoclonal antibodies specific for EGFP (EGFP), Kif3A subunit of kinesin-2 (Kif3A), dynein IC (DIC), or the p150Glued subunit of the dynactin complex (p150Glued); p150Glued but not kinesin-2 or dynein coimmunoprecipitates with EGFP-XMAP4. (B) Immunoprecipitate of the endogenous XMAP4 with antibodies against XMAP4 MBD probed with an antibody against p150Glued; p150Glued coimmunoprecipitates with endogenous XMAP4. E, whole-cell extract; IP, immuno­precipitate.
Mentions: To investigate whether XMAP4 interacts with MT motor proteins or the dynactin complex, we immunoprecipitated XMAP4 from whole-cell extracts and probed immunoprecipitates with antibodies specific for kinesin-2, dynein, or dynactin subunits. To avoid competition of XMAP4 binding partners with antibodies during immunoprecipitation, we expressed EGFP-XMAP4 in melanophores and used rabbit antibodies specific for the EGFP tag for immunoprecipitation. To confirm the presence of EGFP-XMAP4 in the immunoprecipitates, we probed them with mouse monoclonal EGFP antibodies. We found that, as expected, immunoprecipitates contained a protein band with electrophoretic mobility consistent with the mobility of EGFP-XMAP4 (Figure 5A). We also found, however, that antibodies against Kif3A subunit of kinesin-2 or cytoplasmic dynein IC, which recognized relevant bands in whole-cell extracts, did not react with immunoprecipitates (Figure 5A), suggesting that neither kinesin-2 nor dynein bound XMAP4 with high affinity in whole-cell extracts. In contrast to antibodies specific for MT motor proteins, antibody raised against the 150Glued subunit of the dynactin complex revealed in immunoprecipitates a protein with appropriate electrophoretic mobility (Figure 5A). To examine whether endogenous MAP4 also bound p150Glued and to exclude the possibility that the presence of p150Glued in EGFP antibody immunoprecipitates could be explained by nonspecific binding of p150Glued to EGFP, we pulled down XMAP4 from extracts of nontransfected cells using the XMAP4 MBD antibodies. Once again, we detected the p150Glued band in the immunoprecipitate obtained with MBD antibodies (Figure 5B). We conclude that XMAP4 binds the p150Glued subunit of the dynactin complex but not subunits of MT motor proteins.

Bottom Line: We found that aggregation signals induced phosphorylation of threonine residues in the MT-binding domain of the Xenopus MAP4 (XMAP4), thus decreasing binding of this protein to MTs.We hypothesize that binding of XMAP4 to MTs negatively regulates dynein-dependent movement of melanosomes and positively regulates kinesin-2-based movement.Phosphorylation during pigment aggregation reduces binding of XMAP4 to MTs, thus increasing dynein-dependent and decreasing kinesin-2-dependent motility of melanosomes, which stimulates their accumulation in the cell center, whereas dephosphorylation of XMAP4 during dispersion has an opposite effect.

View Article: PubMed Central - PubMed

Affiliation: R.D. Berlin Center for Cell Analysis and Modeling and Department of Cell Biology, University of Connecticut Health Center, Farmington, CT 06030.

Show MeSH