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. 2002 Jul 22;158(2):305-19.
doi: 10.1083/jcb.200201029. Epub 2002 Jul 15.

A role for regulated binding of p150(Glued) to microtubule plus ends in organelle transport

Patricia S Vaughan  1 , Pedro Miura, Matthew Henderson, Belinda Byrne, Kevin T Vaughan
Affiliations

A role for regulated binding of p150(Glued) to microtubule plus ends in organelle transport

Patricia S Vaughan et al. J Cell Biol. .

Abstract

A subset of microtubule-associated proteins, including cytoplasmic linker protein (CLIP)-170, dynactin, EB1, adenomatous polyposis coli, cytoplasmic dynein, CLASPs, and LIS-1, has been shown recently to target to the plus ends of microtubules. The mechanisms and functions of this binding specificity are not understood, although a role in encouraging microtubule elongation has been proposed. To extend previous work on the role of dynactin in organelle transport, we analyzed p150(Glued) by live-cell imaging. Time-lapse analysis of p150(Glued) revealed targeting to the plus ends of growing microtubules, requiring the NH2-terminal cytoskeleton-associated protein-glycine rich domain, but not EB1 or CLIP-170. Effectors of protein kinase A modulated microtubule binding and suggested p150(Glued) phosphorylation as a factor in plus-end binding specificity. Using a phosphosensitive monoclonal antibody, we mapped the site of p150(Glued) phosphorylation to Ser-19. In vivo and in vitro analysis of phosphorylation site mutants revealed that p150(Glued) phosphorylation mediates dynamic binding to microtubules. To address the function of dynamic binding, we imaged GFP-p150(Glued) during the dynein-dependent transport of Golgi membranes. Live-cell analysis revealed a transient interaction between Golgi membranes and GFP-p150(Glued)-labeled microtubules just prior to transport, implicating microtubules and dynactin in a search-capture mechanism for minus-end-directed organelles.

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Figures

Figure 1.
Figure 1.
p150 Glued binds dynamically to elongating microtubules. (A–D) COS-7 cells transfected with expression constructs encoding GFP fusions with full-length p150Glued (A, C, and D) or truncated p150Glued 1–330 (B) were analyzed by live-cell imaging. Stills from time-lapse series are presented and Quick-Time movies are available at http://www.jcb.org/cgi/content/full/jcb.200201029/DC1. Time lapse images were collected at 1-s intervals (insets). Transfected cells were subjected to acetate treatment (C) or temperature-shift (D). (E) The expression levels and microtubule-binding phenotypes of transfected cells were measured and used to bin cells into four catagories: tip-specific binding, microtubule decoration, microtubule binding, and soluble cytoplasmic. Cell percentages represent the average of three experiments and error bars depict standard deviation of the mean. (F) A single microtubule was visualized during the transition from shrinkage to elongation at the cell periphery. Bars: (A–D) 10 μm; (F) 1 μm.
Figure 2.
Figure 2.
Microtubule plus-end–specific binding of p150 Glued. (A) Cells transfected with RFP-p150Glued and GFP-tubulin were subjected to live-cell imaging and analyzed as time-lapse movies. The white arrow shows the initial position of RFP-p150Glued while the yellow arrow shows the growing microtubule tip. Elapsed time of sequence is reported in lower right corner (s.ms). (B and C) Cells transfected with GFP-p150Glued 1–330 (green) were fixed with methanol and labeled by IFM for CLIP-170 (B, red) or EB1 (C, red). Insets show higher magnification of GFP-p150Glued (green arrows), CLIP-170 (red arrows), and EB1 (red arrows). Bars: (A) 5 μm; (B and C) 10 μm.
Figure 3.
Figure 3.
Modulation of p150Gluedtargeting by effectors of protein kinase. (A) COS-7 cells were either methanol fixed and stained for p150Glued (A–C) or transfected with GFP-p150Glued and imaged as live cells (D–F). Microtubule-binding of p150Glued was compared between control cells (A and D), cells treated with 20 μM forskolin (B and E), or cells treated with 56 nM H-89 (C and F) for 2 h. Bars, 10 μm. (G) GFP fluorescence of transfected cells was measured by quantitative fluorescence microscopy and cells exhibiting low levels of expression were binned as in Fig. 1. The four panels reflect analysis of wild-type p150Glued, S19A, S19E, and T20E mutants that were treated with forskolin, H-89, or left untreated. Cell percentages represent the average of three experiments and error bars depict standard deviation of the mean.
Figure 4.
Figure 4.
Differential binding of anti-p150Glued antibodies. COS-7 cells were methanol fixed and stained for endogenous p150Glued using both monoclonal (A) and polyclonal antibodies (C). (B) Overlay of A and C highlights colocalization of both antibodies on microtubules (yellow) but not on cytoplasmic structures (polyclonal in green). Bar, 10 μm. (D and E) Two-dimensional PAGE of brain extract followed by Western blot analysis with monoclonal (D) and polyclonal (E) anti-p150Glued antibodies.
Figure 5.
Figure 5.
Mapping of phosphosensitive antibody epitope. (A) Western blot analysis of bacterially expressed recombinant p150Glued fragments containing aa 1–811 and 39–1280 (I, bacterial culture induced with IPTG; U, uninduced) probed with polyclonal (left) and monoclonal (right) anti-p150Glued antibodies (B) aa 1–38 contains four consensus PKA sites (R-X1,2-S/T), and the region of highest antigenic index predicted by the Jameson-Wolf algorithm. (C) Total cell lysates of COS-7 cells expressing wild-type p150Glued and site-directed mutants at S14, S19, and T20 were probed by Western blot analysis using monoclonal (top) and polyclonal (bottom) anti-p150Glued antibodies. (D) Wild-type and S19A polypeptides were phosphorylated in vitro by PKA for the indicated times (min). Phosphorylated samples were subjected to Western blot analysis with first monoclonal and then polyclonal anti-p150Glued antibodies.
Figure 6.
Figure 6.
Site-directed mutagenesis of candidate p150Glued phosphorylation sites. (A) GFP-p150Glued 1–330 mutant constructs S14A, T20E, S19A were transfected into COS-7 cells and analyzed by live-cell imaging. Insets show higher magnification of microtubule plus ends. Bars, 10 μm. (B) Blot overlay assays comparing the binding of recombinant p150Glued 1–330 and mutants to parallel strips of brain tubulin.
Figure 7.
Figure 7.
Summary of mutation analysis. Line diagram depicting the positions of truncation and site-directed mutants as well as binding activities of antibodies and the microtubule-binding activity of p150Glued.
Figure 8.
Figure 8.
Association of Golgi-derived membranes with elongating microtubule plus ends. COS-7 cells were cotransfected with GFP-150Glued (green) and RFP-tagged NAGT (red). Live-cell imaging was performed after BFA washout and sequential dual channel images were captured at ∼1-s intervals. (A) One example where 15 membranes undergo minus-end–directed motility during 100 s. Each membrane interacts with a GFP-positive microtubule plus end just before motility (insets, arrows). (B) Stills from a time-lapse sequence reveal the sequence of events for one NAGT-positive membrane undergoing minus-end–directed motility (arrows). (C) Forskolin treatment during BFA washout. The inset shows that these NAGT-positive membranes display some plus-end– directed excursions (arrows), but fail to resolve into vesicles or reform with the Golgi complex. Quick-Time movies of these sequences are available at http://www.jcb.org/cgi/content/full/jcb.200201029/DC1. Bars: (A) 10 μm; (B) 4 μm; (C) 10 μm.
Figure 9.
Figure 9.
Proposed role for P150Glued–microtubule interactions during membrane transport. Microtubule explores the cytoplasm and encounters a membrane (t = 1). Membrane-associated dynactin stabilizes the interaction and anchors the membrane while the microtubule continues to elongate (t = 2, 3). After a brief delay, the membrane-associated dynactin recruits cytoplasmic dynein (t = 4). After p150Glued phosphorylation (t = 4), cytoplasmic dynein becomes the only mechanism of microtubule-association. This shift from dynactin- to dynein-mediated binding allows minus-end– directed motility of the membrane (t = 5).
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