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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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Displacement of XMAP4 from MTs by injection of MBD antibodies inhibits dispersion but not aggregation of pigment granules. (A) Immunoblotting with MBD antibodies of whole-cell extracts of control nontransfected cells (left) or melanophores overexpressing EGFP-XMAP4 (right); MBD antibodies recognize the XMAP4 band in whole-cell extracts of control cells and an additional EGFP-XMAP4 band in whole-cell extracts of EGFP-XMAP4–overexpressing cells. (B) Coomassie-stained SDS gels of pelleted MTs assembled in whole-cell extracts preincubated without added IgG (left), in the presence of control nonimmune IgG (middle), or antibodies against XMAP4 MBD (right); preincubation of whole-cell extracts with MBD antibodies prevents cosedimentation of XMAP4 but not other MAPs with MTs. (C) Live images of a melanophore expressing EGFP-XMAP4 before (left) and 30 min after (right) injection of antibodies against XMAP4 MBD; scale bar, 20 μm; the antibody injection completely removes XMAP4 from the MTs. (D) Quantification of response to dispersion (left) or aggregation (right) stimuli of melanophores microinjected with nonimmune IgG or antibodies against XMAP4 MBD. Microinjection of MBD antibodies does not significantly affect pigment aggregation but markedly inhibits pigment dispersion, as evidenced by increases in the fractions of cells with aggregated or partially responded pigment granules compared with melanophores microinjected with nonimmune IgG.
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Figure 4: Displacement of XMAP4 from MTs by injection of MBD antibodies inhibits dispersion but not aggregation of pigment granules. (A) Immunoblotting with MBD antibodies of whole-cell extracts of control nontransfected cells (left) or melanophores overexpressing EGFP-XMAP4 (right); MBD antibodies recognize the XMAP4 band in whole-cell extracts of control cells and an additional EGFP-XMAP4 band in whole-cell extracts of EGFP-XMAP4–overexpressing cells. (B) Coomassie-stained SDS gels of pelleted MTs assembled in whole-cell extracts preincubated without added IgG (left), in the presence of control nonimmune IgG (middle), or antibodies against XMAP4 MBD (right); preincubation of whole-cell extracts with MBD antibodies prevents cosedimentation of XMAP4 but not other MAPs with MTs. (C) Live images of a melanophore expressing EGFP-XMAP4 before (left) and 30 min after (right) injection of antibodies against XMAP4 MBD; scale bar, 20 μm; the antibody injection completely removes XMAP4 from the MTs. (D) Quantification of response to dispersion (left) or aggregation (right) stimuli of melanophores microinjected with nonimmune IgG or antibodies against XMAP4 MBD. Microinjection of MBD antibodies does not significantly affect pigment aggregation but markedly inhibits pigment dispersion, as evidenced by increases in the fractions of cells with aggregated or partially responded pigment granules compared with melanophores microinjected with nonimmune IgG.

Mentions: To examine whether MBD antibodies were specific for XMAP4, we performed immunoblotting with whole-cell extracts. In extracts of control, nontransfected melanophores the antibodies recognized a major band with apparent molecular weight ∼250 kDa (Figure 4A, left). The additional lower–molecular weight bands bound to MBD antibodies (Figure 4A) likely represented degradation products of XMAP4, since the corresponding proteins cosedimented with MTs assembled in cell extracts (unpublished data). In extracts of melanophores overexpressing EGFP-XMAP4, MBD antibodies recognized an additional band with electrophoretic mobility characteristic of the EGFP-XMAP4 (Figure 4A, right). The apparent molecular weights of the immunoreactive proteins were higher than the values predicted for the XMAP4 or GFP-XMAP4 based on the amino acid sequence. However, previous studies showed that the mobility of mammalian MAP4 on SDS gels was also unusually slow (Aizawa et al., 1990; West et al., 1991; Chapin et al., 1995). Therefore immunoblotting experiments showed that MBD antibodies were specific for the XMAP4.


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)

Displacement of XMAP4 from MTs by injection of MBD antibodies inhibits dispersion but not aggregation of pigment granules. (A) Immunoblotting with MBD antibodies of whole-cell extracts of control nontransfected cells (left) or melanophores overexpressing EGFP-XMAP4 (right); MBD antibodies recognize the XMAP4 band in whole-cell extracts of control cells and an additional EGFP-XMAP4 band in whole-cell extracts of EGFP-XMAP4–overexpressing cells. (B) Coomassie-stained SDS gels of pelleted MTs assembled in whole-cell extracts preincubated without added IgG (left), in the presence of control nonimmune IgG (middle), or antibodies against XMAP4 MBD (right); preincubation of whole-cell extracts with MBD antibodies prevents cosedimentation of XMAP4 but not other MAPs with MTs. (C) Live images of a melanophore expressing EGFP-XMAP4 before (left) and 30 min after (right) injection of antibodies against XMAP4 MBD; scale bar, 20 μm; the antibody injection completely removes XMAP4 from the MTs. (D) Quantification of response to dispersion (left) or aggregation (right) stimuli of melanophores microinjected with nonimmune IgG or antibodies against XMAP4 MBD. Microinjection of MBD antibodies does not significantly affect pigment aggregation but markedly inhibits pigment dispersion, as evidenced by increases in the fractions of cells with aggregated or partially responded pigment granules compared with melanophores microinjected with nonimmune IgG.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 4: Displacement of XMAP4 from MTs by injection of MBD antibodies inhibits dispersion but not aggregation of pigment granules. (A) Immunoblotting with MBD antibodies of whole-cell extracts of control nontransfected cells (left) or melanophores overexpressing EGFP-XMAP4 (right); MBD antibodies recognize the XMAP4 band in whole-cell extracts of control cells and an additional EGFP-XMAP4 band in whole-cell extracts of EGFP-XMAP4–overexpressing cells. (B) Coomassie-stained SDS gels of pelleted MTs assembled in whole-cell extracts preincubated without added IgG (left), in the presence of control nonimmune IgG (middle), or antibodies against XMAP4 MBD (right); preincubation of whole-cell extracts with MBD antibodies prevents cosedimentation of XMAP4 but not other MAPs with MTs. (C) Live images of a melanophore expressing EGFP-XMAP4 before (left) and 30 min after (right) injection of antibodies against XMAP4 MBD; scale bar, 20 μm; the antibody injection completely removes XMAP4 from the MTs. (D) Quantification of response to dispersion (left) or aggregation (right) stimuli of melanophores microinjected with nonimmune IgG or antibodies against XMAP4 MBD. Microinjection of MBD antibodies does not significantly affect pigment aggregation but markedly inhibits pigment dispersion, as evidenced by increases in the fractions of cells with aggregated or partially responded pigment granules compared with melanophores microinjected with nonimmune IgG.
Mentions: To examine whether MBD antibodies were specific for XMAP4, we performed immunoblotting with whole-cell extracts. In extracts of control, nontransfected melanophores the antibodies recognized a major band with apparent molecular weight ∼250 kDa (Figure 4A, left). The additional lower–molecular weight bands bound to MBD antibodies (Figure 4A) likely represented degradation products of XMAP4, since the corresponding proteins cosedimented with MTs assembled in cell extracts (unpublished data). In extracts of melanophores overexpressing EGFP-XMAP4, MBD antibodies recognized an additional band with electrophoretic mobility characteristic of the EGFP-XMAP4 (Figure 4A, right). The apparent molecular weights of the immunoreactive proteins were higher than the values predicted for the XMAP4 or GFP-XMAP4 based on the amino acid sequence. However, previous studies showed that the mobility of mammalian MAP4 on SDS gels was also unusually slow (Aizawa et al., 1990; West et al., 1991; Chapin et al., 1995). Therefore immunoblotting experiments showed that MBD antibodies were specific for the XMAP4.

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