Semenova I et al. (2017), Stimulation of microtubule-based transport by n...

XB-ART-53928

Mol Biol Cell 2017 Jun 01;2811:1418-1425. doi: 10.1091/mbc.E16-08-0571.

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Stimulation of microtubule-based transport by nucleation of microtubules on pigment granules.

Semenova I , Gupta D , Usui T , Hayakawa I , Cowan A , Rodionov V .

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Microtubule (MT)-based transport can be regulated through changes in organization of MT transport tracks, but the mechanisms that regulate these changes are poorly understood. In Xenopus melanophores, aggregation of pigment granules in the cell center involves their capture by the tips of MTs growing toward the cell periphery, and granule aggregation signals facilitate capture by increasing the number of growing MT tips. This increase could be explained by stimulation of MT nucleation either on the centrosome or on the aggregate of pigment granules that gradually forms in the cell center. We blocked movement of pigment granules to the cell center and compared the MT-nucleation activity of the centrosome in the same cells in two signaling states. We found that granule aggregation signals did not stimulate MT nucleation on the centrosome but did increase MT nucleation activity of pigment granules. Elevation of MT-nucleation activity correlated with the recruitment to pigment granules of a major component of MT-nucleation templates, γ-tubulin, and was suppressed by γ-tubulin inhibitors. We conclude that generation of new MT transport tracks by concentration of the leading pigment granules provides a positive feedback loop that enhances delivery of trailing granules to the cell center.

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Species referenced: Xenopus laevis
Genes referenced: mapre1 tubg1 tyrp1
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	FIGURE 1:. Microinjection of TYRP1 antibodies induces formation of pigment granule clusters with reduced ability to move to the cell center. (A) Design of a granule cross-linking experiment; antibodies against TYRP1 link neighboring pigment granules to each other, inducing formation of pigment granule clusters unable to efficiently move along MTs because of their large size. (B) Immunoblotting of cell extract (left) and lysate of pigment granules (right) with TYRP1 antibodies; left and right lanes of each pair correspond to silver-stained gels and immunoblots, respectively; in cell extract and lysate of pigment granules, antibodies recognize a single TYRP1 band with molecular mass of ∼60 kDa. (C) Phase contrast images of melanophores microinjected with control nonimmune IgG (top) or TYRP1 antibodies (bottom) and treated to aggregate pigment granules; in each row, images show the distribution of pigment granules before (left) or 30 min after (middle) microinjection or 20 min after treatment of microinjected cells with melatonin to induce pigment aggregation (right); microinjection of control IgG does not significantly change the distribution of pigment granules and does not prevent their aggregation in the cell center, whereas microinjection of TYRP1 antibodies induces formation of peripheral granule clusters that fail to aggregate in the cell center after stimulation with melatonin; bar, 10 μm.
	FIGURE 2:. Pigment aggregation signals do not stimulate MT nucleation on the centrosome. Fluorescence images of melanophores expressing EGFP-EB1 injected with either control nonimmune IgG (top) or TYRP1 antibodies (bottom) treated with MSH (left) or melatonin (right); circles (D = 12 μm) outline the areas used for counting EGFP-EB1 comets; in the control cell, which aggregated pigment granules, melatonin treatment induced a significant increase in the number of EGFP-EB1 comets in the centrosome area, whereas in the TYRP1 antibody–injected cell, which did not accumulate pigment granules in the cell center, the number of comets surrounding the centrosome did not change significantly after stimulation with melatonin; bar, 10 μm.
	FIGURE 3:. Pigment granules nucleate MTs in vivo and in vitro. (A) Sequential live fluorescence images of the centrosome area of an EGFP-EB1–expressing melanophore injected with TYRP1 antibodies and treated with melatonin; an asterisk indicates the position of the centrosome, P shows the location of a clump of pigment granules, and an arrowhead marks an EGFP-EB1 comet that emerges from the pigment granule clump and moves toward the centrosome; numbers indicate time in minutes. Birth of the MT tip labeled with EGFP-EB1 away from the centrosome and its growth toward the cell center suggest nucleation of the MT on a granule clump cross-linked with TYRP1 antibodies; bar, 2 μm. (B) Left and middle, phase contrast (top) and fluorescence (bottom) images of pellets of Cy3-labeled tubulin preparations incubated in the absence (left) or presence (right) of a suspension of purified pigment granules isolated from melanophores treated with melatonin to induce granule aggregation; right, overlays of boxed areas shown in the middle; tubulin pellets that were incubated in the presence of pigment granules contain short MTs that are often attached to pigment granules; bars, 10 μm (left and middle), 2 μm (right).
	FIGURE 4:. Pigment granule aggregation signals increase nucleation of MTs on pigment granules, and this increase correlates with the recruitment of γ-tubulin. (A) Fluorescence (left) and phase contrast (right) images of pellets of immunostained MTs assembled by polymerization of twice-cycled tubulin incubated alone or with different pigment granule preparations; shown are images from (Tb) alone, (PG(D)+Tb) pigment granules isolated from cells treated with MSH to induce granule dispersion, (PG(A)+Tb) pigment granules from cells treated with melatonin to trigger aggregation and preincubated with buffer only, (PG(A)+IgG+Tb) pretreated with nonimmune rabbit IgG, (PG(A)+ γ-Tb antibody+Tb) pretreated with γ-tubulin antibodies, (PG(A)+CLASP antibody+ Tb) pretreated with CLASP antibodies, or (PG(A)+gatastatin+Tb) pretreated with γ-tubulin inhibitor gatastatin. Pigment granules increased the amount of assembled MTs, and this effect was greater in the case of granules isolated from melatonin- compared with MSH-treated cells; preincubation of granules with γ-tubulin antibodies or gatastatin but not with nonimmune IgG or CLASP antibodies reduced the amount of assembled MTs; bar, 10 μm. (B) Quantification of the number of assembled MTs in samples shown in A; each bar represents the average value of 30 measurements in two independent experiments; the data are expressed as percentage of average number of MTs assembled in the presence of pigment granules isolated from melatonin-treated cells and incubated with relevant buffer, which is taken as 100%; error bars are mean ± SEM. (C) Comparison of MT nucleation activity of pigment granules isolated from melanophores treated with MSH or melatonin by immunoblotting of granule pellets with α-tubulin antibodies; blots with α-tubulin (top) or TYRP1 (bottom; loading control) antibodies of pellets from samples containing pigment granules isolated from melatonin-treated cells (PG(A)), purified tubulin (Tb), or mixtures of purified tubulin with pigment granules isolated from MSH- or melatonin-treated melanophores (PG(D)+Tb, and PG(A)+Tb, respectively); α-tubulin bands are absent from pellets of pigment granules or tubulin preparations (an indication that pigment granule preparations do not contain significant amounts of tubulin and that MTs do not assemble in the absence of pigment granules) but present in the pellets of mixtures of purified tubulin with pigment granules (an indication of MT nucleation on pigment granules); the amount of α-tubulin is significantly higher in the case of pigment granules isolated from melatonin-treated cells, suggesting stimulation of MT nucleation by the granule-aggregating signals. (D) Immunoblotting of preparations of purified pigment granules isolated from melanophores treated with MSH (left, PG(D)) or melatonin (right, PG(A)) with antibodies against γ-tubulin (left, top), CLASP (right, top) or TYRP1 (loading control; left and right, bottom); γ-tubulin and CLASP levels are increased in preparations of pigment granules isolated from melatonin-treated cells, which suggests that pigment-aggregation signals stimulate recruitment of γ-tubulin and CLASP to pigment granules. (E) Fluorescence images of melanin-free melanophores recovering from MT depolymerization in the absence (left) or presence (right) of gatastatin (30 μM); bottom, high-magnification images of boxed areas shown in the top; gatastatin partially inhibits MT outgrowth from the centrosome; bars, 10 μm (top), 2 μm (bottom). (F) Hypothesis on the stimulation of pigment aggregation by recruitment of γ-tubulin to pigment granules.

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