Semenova I et al. (2014), Regulation of microtubule-based transport by MAP4.

XB-ART-49373

Mol Biol Cell 2014 Oct 15;2520:3119-32. doi: 10.1091/mbc.E14-01-0022.

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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 .

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Microtubule (MT)-based transport of organelles driven by the opposing MT motors kinesins and dynein is tightly regulated in cells, but the underlying molecular mechanisms remain largely unknown. Here we tested the regulation of MT transport by the ubiquitous protein MAP4 using Xenopus melanophores as an experimental system. In these cells, pigment granules (melanosomes) move along MTs to the cell center (aggregation) or to the periphery (dispersion) by means of cytoplasmic dynein and kinesin-2, respectively. 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. Overexpression of XMAP4 inhibited pigment aggregation by shortening dynein-dependent MT runs of melanosomes, whereas removal of XMAP4 from MTs reduced the length of kinesin-2-dependent runs and suppressed pigment dispersion. 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.

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Species referenced: Xenopus
Genes referenced: clip1 dnai1 kif3a map4

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	FIGURE 1:. XMAP4 is phosphorylated at threonine residues located in the proline-rich region of the MT-binding domain. Top, amino acid sequence of the XMAP4 phosphopeptide identified in the phosphoproteomic screen; Thr-758 and Thr-762 are shown in red; bottom, domain organization of the XMAP4 molecule; the molecule of XMAP4 is composed of an N-terminal projection domain, which protrudes from the MT surface, and a C-terminal MT-binding domain that contains a proline-rich region and four imperfect amino acid repeats (Aizawa etÂ al., 1991; Chapin and Bulinski, 1991); Thr-758 and Thr-762 map to the proline-rich region of the MT-binding domain.
	FIGURE 2:. Expression of XMAP4 reduces the rate of pigment aggregation. Quantification of response to dispersion (A) or aggregation (B, C) stimuli applied for 15 min (A, B) or 1 h (C) to melanophores expressing EGFP-XMAP4 (left set of bars), EGFP (middle sets of bars in A and B, right set in C), or nontransfected melanophores (right sets of bars in A and B); data are expressed as the percentages of cells with aggregated (white bars), partially responded (gray bars), or dispersed (black bars) pigment granules. Expression of EGFP-XMAP4 does not affect dispersion but significantly slows aggregation of pigment granules, as evidenced by the increase in the fractions of cells with dispersed or partially responded pigment granules 15 min after the stimulation of pigment aggregation.
	FIGURE 3:. Inhibition of pigment aggregation in melanophores overexpressing XMAP4 cannot be explained by changes in MT dynamics or loss of CLIP-170 from the MT tips. (A) Comparison of kinetics of pigment aggregation measured in cells expressing EGFP-XMAP4 (black squares) or computed based on parameters of MT dynamic instability measured in EGFP-expressing (white circles) or EGFP-XMAP4âexpressing cells (white squares); changes in parameters of MT dynamic instability caused by the overexpression of EGFP-XMAP4 cannot account for inhibition of pigment aggregation in the EGFP-XMAP4âexpressing cells. (B) Profiles of CLIP-170 fluorescence at MT plus ends normalized by maximum fluorescence and averaged for melanophores expressing EGFP (black squares) or EGFP-XMAP4 (white circles); expression of EGFP-XMAP4 does not significantly change the distribution of the CLIP-170 fluorescence at the MT plus ends.
	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-XMAP4âoverexpressing 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 Î¼m; 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.
	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, immunoÂprecipitate.
	FIGURE 6:. Phosphorylation of XMAP4 reduces inhibition of pigment aggregation and decreases binding to MTs. (A) Top, quantification of the response to a pigment aggregation stimulus of melanophores overexpressing nonphosphorylatable (left set of bars) or phosphomimetic (right set of bars) mutants or XMAP4; data are expressed as the percentages of cells with aggregated (white bars), partially responded (gray bars), or dispersed (black bars) pigment granules. Bottom, comparison of the levels of expression of XMAP4 mutants based on the EGFP fluorescence. Overexpression of the phosphomimetic XMAP4 mutant has a weaker inhibitory effect compared with the nonphosphorylatable mutant, as evidenced by a smaller fraction of cells with dispersed and larger fraction of cells with aggregated pigment granules compared with melanophores overexpressing the XMAP4 nonphosphorylatable mutant; this difference cannot be explained by the reduced expression levels of the phosphomimetic mutant, given that the mutants are expressed at approximately the same levels. (B) Cosedimentation of recombinant nonphosphorylatable and phosphomimetic XMAP4 mutant proteins with MTs in vitro. Top, Coomassie-stained gel, which shows protein composition of input (XMAP4 mutant proteinâMT mixtures before centrifugation; I), supernatants (S), and MT pellets (P). Bottom, measurement of relative amounts of nonphosphorylatable (left) and phosphomimetic (right) mutant proteins in MT pellets using quantitative immunoblotting; the data represent averages for the values measured in three independent experiments; inset, representative images of the XMAP4 mutant bands. The amount of the phosphomimetic mutant protein in the MT pellets is significantly smaller, and in the supernatants larger, than the nonphosphorylatable mutant, which indicates reduced binding to MTs.
	FIGURE 7:. Hypothesis for the regulation of MT-based transport of pigment granules by XMAP4. XMAP4 negatively regulates minus endâdirected MT transport of pigment granules by blocking the movement of dynein motors along MTs and positively regulates plus endâdirected, kinesin-2âbased transport through interaction with the granule-bound p150Glued. This interaction increases processivity of kinesin-2 motors by keeping them in proximity to the MT surface. Phosphorylation during pigment aggregation reduces binding of XMAP4 to MTs, thus increasing minus endâdirected and decreasing plus endâdirected motility of pigment granules (left), which stimulates their accumulation at the cell center, whereas dephosphorylation of XMAP4 during pigment dispersion has an opposite effect (right).

References [+] :

Aizawa, Functional analyses of the domain structure of microtubule-associated protein-4 (MAP-U). 1991, Pubmed