CLIP-170-dependent capture of membrane organelles by microtubules initiates minus-end directed transport

Abstract

Cytoplasmic microtubules (MTs) continuously grow and shorten at free plus ends. During mitosis, this dynamic behavior allows MTs to capture chromosomes to initiate their movement to the spindle poles; however, the role of MT dynamics in capturing organelles for transport in interphase cells has not been demonstrated. Here we use Xenopus melanophores to test the hypothesis that MT dynamics significantly contribute to the efficiency of MT minus-end directed transport of membrane organelles. We demonstrate that initiation of transport of membrane-bounded melanosomes (pigment granules) to the cell center involves their capture by MT plus ends, and that inhibition of MT dynamics or loss of the MT plus-end tracking protein CLIP-170 from MT tips dramatically inhibits pigment aggregation. We conclude that MT dynamics are required for the initiation of MT transport of membrane organelles in interphase cells, and that +TIPs such as CLIP-170 play an important role in this process.

Figure 1. Stabilization of microtubules with taxol inhibits aggregation of melanosomes

(A) Phase-contrast images of melanophores treated with MSH (left) or melatonin (right) to disperse or aggregate melanosomes. Bar, 20 μm. (B) Time series of images of a melanophore with fluorescently labeled MTs treated with melatonin to induce pigment aggregation; after capture by the growing MT tip (arrow) a melanosome (arrowhead) starts moving along the MT toward the cell center. Bar, 2 μm. See also Supplemental Videos 3 and 4. (C) Quantification of responses to melatonin or MSH, applied to induce pigment aggregation or dispersion, of control non-treated melanophores or melanophores treated with the MT-stabilizing drug taxol; the data are expressed as the percentages of cells with aggregated (white bars), partially dispersed (grey bars) or completely dispersed (black bars) pigment. Quantification was performed at 10 min (aggregation) or 15 min (dispersion) after stimulation.(D) Quantification of kinetics of pigment aggregation (left panel) or dispersion (right panel) in control non-treated (white squares) or taxol-treated (black squares) melanophores; data are expressed as the percentage of change with time in the grey levels within the cell outlines; 100% corresponds to the fully dispersed state.

Figure 2. Overexpression of EB3-GFP or GFP-CLIP-170 tail removes CLIP-170 and p150^Glued from the plus ends of MTs, and inhibit pigment aggregation

(A) Hierarchy of plus-end accumulation of +TIPs: p150^Glued binds MT plus ends via CLIP-170 or EB1, CLIP-170 binding is mediated by EB1, and EB1 directly binds tubulin molecules in the MT wall; CLIP-170 and p150^Glued can bind tubulin directly, and these interactions contribute to their plus-end localization, but are not sufficient to mediate it, and therefore are not shown. (B and G) Immunostaining of EB3-GFP-overexpressing (B) or GFP-CLIP-170 tail-overexpressing (G) cells with antibodies against EB1, CLIP-170, or p150^Glued; examples of low and high magnification images are shown for each +TIP (with color codes indicated above the merged images). Representative line scan analyses of protein accumulation at MT tips in the boxed regions are shown on the right. Bars, 10 μm (left columns) or 2.5 μm (middle columns). (C and H) Fractions of MT plus ends immunostained for EB1, CLIP-170, or p150^Glued in the EB3-GFP-overexpressing (C) or GFP-CLIP-170 tail-overexpressing (H) cells. (D and I) Diagrams illustrating the effect of EB3-GFP (D) or CLIP-170 tail (I) overexpression on the composition of +TIPs at MT plus ends; EB3-GFP displaces all major +TIPs (EB1, CLIP-170, and p150^Glued) from MT plus ends, whereas GFP-CLIP-170 tail displaces CLIP-170, and p150^Glued, but not EB1. (E and J) Quantification of responses of EB3-GFP (E) or GFP-CLIP-170 tail-overexpressing (J) cells to melatonin or MSH; the data is expressed as the percentages of cells with aggregated (white bars), partially dispersed (grey bars) or completely dispersed (black bars) pigment. (F and K) Quantification of kinetics of pigment aggregation in the cells overexpressing EB3-GFP (F) or GFP-CLIP-170 (K); the data is expressed as a decrease in the values the grey levels within the cell outlines with time.

Figure 3. Overexpression of the CLIP-170 head or LIS1 displace p150^Glued from MT plus ends, but does not affect pigment aggregation

(A and F) Immunostaining of cells overexpressing GFP-CLIP-170 head or GFP-LIS1 with antibodies against EB1, CLIP-170, or p150^Glued; examples of low and high magnification images are shown for each +TIP (with color codes indicated above the merged images). Representative line scan analyses of protein accumulation at MT tips are shown on the right. Bars, 10 μm (left columns) or 2.5 μm (middle columns). (B and G) Fractions of MT plus ends immunostained for EB1, CLIP-170 or p150^Glued in the GFP-CLIP-170 head- (B) or GFP-LIS1- (G) overexpressing cells. (C and H) Diagrams illustrating the effects of CLIP-170 head (C) or LIS1 (H) overexpression on the composition of +TIPs at MT plus ends; CLIP-170 head or LIS1 displace p150^Glued, but not CLIP-170 or EB1 from the MT plus ends. (D and I) Quantification of responses of GFP-CLIP-170 head (D) or GFP-LIS1- (I) overexpressing cells to melatonin or MSH; the data is expressed as the percentages of cells with aggregated (white bars), partially dispersed (grey bars) or completely dispersed (black bars) pigment. (E and J) Quantification of kinetics of pigment aggregation in the cells overexpressing GFP-CLIP-170 head (E) or GFP-LIS1 (J); the data is expressed as a decrease in the values the grey levels within the cell outlines with time.

Figure 4. Changes in the parameters of MT dynamic instability and bidirectional movement of melanosomes along MTs do not affect significantly pigment aggregation kinetics

(A) Comparison of kinetics of pigment aggregation for cells overexpressing GFP or treated with taxol determined experimentally (open symbols), or computed using the parameters of MT dynamic instability and bi-directional melanosome movement shown in Tables 1 and 2 (filled symbols). Data are expressed as the percentage of change with time in the grey levels within the cell outlines. Computational simulations accurately reproduce kinetics of pigment aggregation in the presence (circles) or absence (squares) of taxol. (B) Computed kinetics of pigment aggregation for the taxol-treated cells, or cells overexpressing GFP, EB3-GFP, GFP-CLIP-170 tail, GFP-CLIP-170 head, or GFPLIS1. Overexpression of dominant-negative constructs, which alter the composition of +TIPs at the MT plus ends (EB3-GFP, GFP-CLIP-170 tail, GFP-CLIP-170 head, or GFPLIS1), does not significantly inhibit pigment aggregation kinetics. (C) Comparison of the kinetics of pigment aggregation experimentally measured in the cells overexpressing EB3-GFP (open squares), with the kinetics computed with the assumption that the probability of capturing of melanosomes by the CLIP-170-enriched MT plus ends is 78% (open circles) or 8% (closed circles). The kinetics computed at a low capturing probability closely matches the EB3-GFP kinetics, which confirms that a decreased pigment aggregation rate seen in cells lacking CLIP-170 at the MT plus ends could be explained by a reduced capturing ability of MTs alone.

Figure 5. Binding of CLIP-170 to melanosomes

(A) Immunoblotting of cell extract (E) or melanosomes isolated from melanophores with dispersed (D) or aggregated (A) pigment with an antibody against mammalian CLIP-170 (α-CLIP-170; left lanes), or a soluble marker glyceraldehyde-3-phosphate dehydrogenase (α-GAPDH; right lanes). CLIP-170, but not GAPDH co-purifies with melanosomes. Loading of the samples were equalized to compare the amounts of CLIP-170 and GAPDH between the melanosome preparations, and between the fractions of cytosol and melanosome extract by equalizing across the samples concentrations of melanosomes estimated by measuring optical density of the melanosome suspension at 350 nm, and by adjusting the volumes of the melanosome supernatant and extract to the volume of the initial melanosome suspension . (B) Model for CLIP-170 involvement in the binding of melanosomes to MTs. CLIP-170 concentrated at the MT plus end binds an adaptor protein on the melanosome surface, and this binding facilitates the interaction of the pigment granule-bound dynein with the MT lattice.