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CLIP-170 tracks growing microtubule ends by dynamically recognizing composite EB1/tubulin-binding sites.

Bieling P, Kandels-Lewis S, Telley IA, van Dijk J, Janke C, Surrey T - J. Cell Biol. (2008)

Bottom Line: In contrast, CLIP-170 does not end-track by itself but requires EB1.In contrast to its fission yeast orthologue Tip1, dynamic end tracking of CLIP-170 does not require the activity of a molecular motor.Our results demonstrate evolutionary diversity of the plus end recognition mechanism of CLIP-170 family members, whereas the autonomous end-tracking mechanism of EB family members is conserved.

View Article: PubMed Central - PubMed

Affiliation: European Molecular Biology Laboratory, Cell Biology and Biophysics Unit, 69117 Heidelberg, Germany.

ABSTRACT
The microtubule cytoskeleton is crucial for the internal organization of eukaryotic cells. Several microtubule-associated proteins link microtubules to subcellular structures. A subclass of these proteins, the plus end-binding proteins (+TIPs), selectively binds to the growing plus ends of microtubules. Here, we reconstitute a vertebrate plus end tracking system composed of the most prominent +TIPs, end-binding protein 1 (EB1) and CLIP-170, in vitro and dissect their end-tracking mechanism. We find that EB1 autonomously recognizes specific binding sites present at growing microtubule ends. In contrast, CLIP-170 does not end-track by itself but requires EB1. CLIP-170 recognizes and turns over rapidly on composite binding sites constituted by end-accumulated EB1 and tyrosinated alpha-tubulin. In contrast to its fission yeast orthologue Tip1, dynamic end tracking of CLIP-170 does not require the activity of a molecular motor. Our results demonstrate evolutionary diversity of the plus end recognition mechanism of CLIP-170 family members, whereas the autonomous end-tracking mechanism of EB family members is conserved.

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CLIP-170 recognizes composite EB1/tubulin-binding sites at the microtubule end. (A) Western blot of mock-treated and detyrosinated tubulin (tubulinΔY) either Ponceau-stained or probed with an anti–Tyr-tubulin or anti–Glu-tubulin antibody. (B) Kymographs of a growing TAMRA-labeled microtubule (red) in the presence of 75 nM EB1Y→A-GFP (left, green) or EB1-GFP (right, green) in buffer. (C) The peak signal of the EB1 comets obtained from averaged intensity profiles at the indicated conditions. Error bars indicate standard error.(D) Kymographs of a growing TAMRA-labeled microtubule (red) in the presence of 35 nM H2-GFP (green) growing with either mock-treated (left) or detyrosinated tubulin (right) in the presence of unlabeled EB1 (top) or EB1Y→A (bottom). Bars, 5 μm. (E) The peak signal of the H2 comets (top) obtained from averaged intensity profiles at the indicated conditions. Signal of H2-GFP bound to the microtubule lattice (bottom) as averaged from intensity line scans. Error bars indicate the standard error (top) or the standard deviation of the mean lattice intensity from the line scans (bottom). (F) Schematic illustration of the mechanisms of end tracking by vertebrate (left) and fission yeast (right) +TIPs. See text for details.
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fig5: CLIP-170 recognizes composite EB1/tubulin-binding sites at the microtubule end. (A) Western blot of mock-treated and detyrosinated tubulin (tubulinΔY) either Ponceau-stained or probed with an anti–Tyr-tubulin or anti–Glu-tubulin antibody. (B) Kymographs of a growing TAMRA-labeled microtubule (red) in the presence of 75 nM EB1Y→A-GFP (left, green) or EB1-GFP (right, green) in buffer. (C) The peak signal of the EB1 comets obtained from averaged intensity profiles at the indicated conditions. Error bars indicate standard error.(D) Kymographs of a growing TAMRA-labeled microtubule (red) in the presence of 35 nM H2-GFP (green) growing with either mock-treated (left) or detyrosinated tubulin (right) in the presence of unlabeled EB1 (top) or EB1Y→A (bottom). Bars, 5 μm. (E) The peak signal of the H2 comets (top) obtained from averaged intensity profiles at the indicated conditions. Signal of H2-GFP bound to the microtubule lattice (bottom) as averaged from intensity line scans. Error bars indicate the standard error (top) or the standard deviation of the mean lattice intensity from the line scans (bottom). (F) Schematic illustration of the mechanisms of end tracking by vertebrate (left) and fission yeast (right) +TIPs. See text for details.

Mentions: Finally, we asked if CLIP-170 tracks growing microtubule ends by purely hitchhiking on EB1 or if it also interacts with the tubulin polymer on microtubule ends. Both α-tubulin and EB1 carry a characteristic C-terminal EEY motif, of which the tyrosine is important for plus end tracking of CLIP-170, but not of EB1, in vivo (Badin-Larcon et al., 2004; Erck et al., 2005; Peris et al., 2006). Structural studies have shown that CAP-Gly domains can bind to the C-terminal regions of EB proteins (Honnappa et al., 2005; Weisbrich et al., 2007). To determine whether CLIP-170 interacts with both tubulin and EB1 while bound to the microtubule end, we prepared detyrosinated tubulin and an EB1 mutant with a C-terminal tyrosine-to-alanine substitution (EB1Y→A; Fig. 5 A). First, we showed that EB1 and EB1Y→A bound similarly to the growing ends of microtubules, irrespective of the tyrosination state of tubulin in vitro (Fig. 5, B and C). In contrast, both lattice association and end binding of H2-GFP in the presence of EB1 (Fig. 5 E, bottom) were drastically reduced (by 94 and 87%, respectively) when microtubules assembled from detyrosinated tubulin (Fig. 5 D, top right; and Video 5, top, available at http://www.jcb.org/cgi/content/full/jcb.200809190/DC1) instead of from normal, tyrosinated tubulin (Fig. 5 D, top left). Additionally, end tracking of H2-GFP on normal microtubules was abolished in the presence of EB1Y→A, whereas lattice binding was not strongly affected (Fig. 5 D, bottom left; Fig. 5 E, bottom; and Video 5, left). Finally, the use of detyrosinated microtubules and EB1Y→A completely abolished both end tracking and lattice binding of H2-GFP (Fig. 5 D, bottom right; Fig. 5 E; and Video 5, bottom). These measurements on dynamic microtubules agree with crystallographic data that lead to the proposal that CAP-Gly domain–containing proteins act as EEY motif–recognizing proteins (Honnappa et al., 2006; Weisbrich et al., 2007). Our results suggest that end tracking of CLIP-170 requires direct interactions with composite binding sites consisting of both tyrosinated α-tubulin and, most importantly, of end-associated, tyrosinated EB1, which explains why end accumulation of CLIP-170 is dependent on tyrosinated tubulin in vivo (Peris et al., 2006).


CLIP-170 tracks growing microtubule ends by dynamically recognizing composite EB1/tubulin-binding sites.

Bieling P, Kandels-Lewis S, Telley IA, van Dijk J, Janke C, Surrey T - J. Cell Biol. (2008)

CLIP-170 recognizes composite EB1/tubulin-binding sites at the microtubule end. (A) Western blot of mock-treated and detyrosinated tubulin (tubulinΔY) either Ponceau-stained or probed with an anti–Tyr-tubulin or anti–Glu-tubulin antibody. (B) Kymographs of a growing TAMRA-labeled microtubule (red) in the presence of 75 nM EB1Y→A-GFP (left, green) or EB1-GFP (right, green) in buffer. (C) The peak signal of the EB1 comets obtained from averaged intensity profiles at the indicated conditions. Error bars indicate standard error.(D) Kymographs of a growing TAMRA-labeled microtubule (red) in the presence of 35 nM H2-GFP (green) growing with either mock-treated (left) or detyrosinated tubulin (right) in the presence of unlabeled EB1 (top) or EB1Y→A (bottom). Bars, 5 μm. (E) The peak signal of the H2 comets (top) obtained from averaged intensity profiles at the indicated conditions. Signal of H2-GFP bound to the microtubule lattice (bottom) as averaged from intensity line scans. Error bars indicate the standard error (top) or the standard deviation of the mean lattice intensity from the line scans (bottom). (F) Schematic illustration of the mechanisms of end tracking by vertebrate (left) and fission yeast (right) +TIPs. See text for details.
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fig5: CLIP-170 recognizes composite EB1/tubulin-binding sites at the microtubule end. (A) Western blot of mock-treated and detyrosinated tubulin (tubulinΔY) either Ponceau-stained or probed with an anti–Tyr-tubulin or anti–Glu-tubulin antibody. (B) Kymographs of a growing TAMRA-labeled microtubule (red) in the presence of 75 nM EB1Y→A-GFP (left, green) or EB1-GFP (right, green) in buffer. (C) The peak signal of the EB1 comets obtained from averaged intensity profiles at the indicated conditions. Error bars indicate standard error.(D) Kymographs of a growing TAMRA-labeled microtubule (red) in the presence of 35 nM H2-GFP (green) growing with either mock-treated (left) or detyrosinated tubulin (right) in the presence of unlabeled EB1 (top) or EB1Y→A (bottom). Bars, 5 μm. (E) The peak signal of the H2 comets (top) obtained from averaged intensity profiles at the indicated conditions. Signal of H2-GFP bound to the microtubule lattice (bottom) as averaged from intensity line scans. Error bars indicate the standard error (top) or the standard deviation of the mean lattice intensity from the line scans (bottom). (F) Schematic illustration of the mechanisms of end tracking by vertebrate (left) and fission yeast (right) +TIPs. See text for details.
Mentions: Finally, we asked if CLIP-170 tracks growing microtubule ends by purely hitchhiking on EB1 or if it also interacts with the tubulin polymer on microtubule ends. Both α-tubulin and EB1 carry a characteristic C-terminal EEY motif, of which the tyrosine is important for plus end tracking of CLIP-170, but not of EB1, in vivo (Badin-Larcon et al., 2004; Erck et al., 2005; Peris et al., 2006). Structural studies have shown that CAP-Gly domains can bind to the C-terminal regions of EB proteins (Honnappa et al., 2005; Weisbrich et al., 2007). To determine whether CLIP-170 interacts with both tubulin and EB1 while bound to the microtubule end, we prepared detyrosinated tubulin and an EB1 mutant with a C-terminal tyrosine-to-alanine substitution (EB1Y→A; Fig. 5 A). First, we showed that EB1 and EB1Y→A bound similarly to the growing ends of microtubules, irrespective of the tyrosination state of tubulin in vitro (Fig. 5, B and C). In contrast, both lattice association and end binding of H2-GFP in the presence of EB1 (Fig. 5 E, bottom) were drastically reduced (by 94 and 87%, respectively) when microtubules assembled from detyrosinated tubulin (Fig. 5 D, top right; and Video 5, top, available at http://www.jcb.org/cgi/content/full/jcb.200809190/DC1) instead of from normal, tyrosinated tubulin (Fig. 5 D, top left). Additionally, end tracking of H2-GFP on normal microtubules was abolished in the presence of EB1Y→A, whereas lattice binding was not strongly affected (Fig. 5 D, bottom left; Fig. 5 E, bottom; and Video 5, left). Finally, the use of detyrosinated microtubules and EB1Y→A completely abolished both end tracking and lattice binding of H2-GFP (Fig. 5 D, bottom right; Fig. 5 E; and Video 5, bottom). These measurements on dynamic microtubules agree with crystallographic data that lead to the proposal that CAP-Gly domain–containing proteins act as EEY motif–recognizing proteins (Honnappa et al., 2006; Weisbrich et al., 2007). Our results suggest that end tracking of CLIP-170 requires direct interactions with composite binding sites consisting of both tyrosinated α-tubulin and, most importantly, of end-associated, tyrosinated EB1, which explains why end accumulation of CLIP-170 is dependent on tyrosinated tubulin in vivo (Peris et al., 2006).

Bottom Line: In contrast, CLIP-170 does not end-track by itself but requires EB1.In contrast to its fission yeast orthologue Tip1, dynamic end tracking of CLIP-170 does not require the activity of a molecular motor.Our results demonstrate evolutionary diversity of the plus end recognition mechanism of CLIP-170 family members, whereas the autonomous end-tracking mechanism of EB family members is conserved.

View Article: PubMed Central - PubMed

Affiliation: European Molecular Biology Laboratory, Cell Biology and Biophysics Unit, 69117 Heidelberg, Germany.

ABSTRACT
The microtubule cytoskeleton is crucial for the internal organization of eukaryotic cells. Several microtubule-associated proteins link microtubules to subcellular structures. A subclass of these proteins, the plus end-binding proteins (+TIPs), selectively binds to the growing plus ends of microtubules. Here, we reconstitute a vertebrate plus end tracking system composed of the most prominent +TIPs, end-binding protein 1 (EB1) and CLIP-170, in vitro and dissect their end-tracking mechanism. We find that EB1 autonomously recognizes specific binding sites present at growing microtubule ends. In contrast, CLIP-170 does not end-track by itself but requires EB1. CLIP-170 recognizes and turns over rapidly on composite binding sites constituted by end-accumulated EB1 and tyrosinated alpha-tubulin. In contrast to its fission yeast orthologue Tip1, dynamic end tracking of CLIP-170 does not require the activity of a molecular motor. Our results demonstrate evolutionary diversity of the plus end recognition mechanism of CLIP-170 family members, whereas the autonomous end-tracking mechanism of EB family members is conserved.

Show MeSH