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Published online 4 October 2004. doi:10.1083/jcb.200402082
The Rockefeller University Press, 0021-9525 $8.00
JCB, Volume 166, Number 7, 1003-1014
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Article

Conformational changes in CLIP-170 regulate its binding to microtubules and dynactin localization



Gideon Lansbergen1, Yulia Komarova3,4, Mauro Modesti1, Claire Wyman1,2, Casper C. Hoogenraad1, Holly V. Goodson5, Régis P. Lemaitre6, David N. Drechsel6, Erik van Munster7, Theodorus W.J. Gadella, Jr.7, Frank Grosveld1, Niels Galjart1, Gary G. Borisy3, and Anna Akhmanova1

1 MGC Department of Cell Biology and Genetics, Erasmus Medical Center, 3000 DR Rotterdam, Netherlands
2 Department of Radiation Oncology, Erasmus Medical Center, 3000 DR Rotterdam, Netherlands
3 Department of Cell and Molecular Biology, Northwestern University Medical School, Chicago, IL 60611
4 Laboratory of Cell Motility, A.N. Belozersky Institute, Moscow State University, Moscow, 119992, Russia
5 Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556
6 Max Planck Institute of Molecular Cell Biology and Genetics, 01307 Dresden, Germany
7 Section of Molecular Cytology and Centre for Advanced Microscopy, Swammerdam Institute for Life Science, University of Amsterdam, 1098 SM Amsterdam, Netherlands

Address correspondence to Anna Akhmanova, MGC Dept. of Cell Biology and of Genetics, Erasmus Medical Center, P.O. Box 1738, 3000 DR Rotterdam, Netherlands. Tel.: 31-10-4087166. Fax: 31-10-4089468. email: anna.akhmanova{at}chello.nl

Cytoplasmic linker protein (CLIP)-170, CLIP-115, and the dynactin subunit p150Glued are structurally related proteins, which associate specifically with the ends of growing microtubules (MTs). Here, we show that down-regulation of CLIP-170 by RNA interference results in a strongly reduced accumulation of dynactin at the MT tips. The NH2 terminus of p150Glued binds directly to the COOH terminus of CLIP-170 through its second metal-binding motif. p150Glued and LIS1, a dynein-associating protein, compete for the interaction with the CLIP-170 COOH terminus, suggesting that LIS1 can act to release dynactin from the MT tips. We also show that the NH2-terminal part of CLIP-170 itself associates with the CLIP-170 COOH terminus through its first metal-binding motif. By using scanning force microscopy and fluorescence resonance energy transfer-based experiments we provide evidence for an intramolecular interaction between the NH2 and COOH termini of CLIP-170. This interaction interferes with the binding of the CLIP-170 to MTs. We propose that conformational changes in CLIP-170 are important for binding to dynactin, LIS1, and the MT tips.

Key Words: plus end–tracking proteins; motor protein; cytoplasmic dynein; LIS1; CLIP-115

C.C. Hoogenraad's present address is The Picower Center for Learning and Memory, Massachusetts Institute of Technology, Cambridge, MA 02139.

Abbreviations used in this paper: CLIP, cytoplasmic linker protein; FRET, fluorescence resonance energy transfer; HIS, 6X histidine; IP, immunoprecipitation; MT, microtubule; RNAi, RNA interference; siRNA, small interfering RNA; SFM, scanning force microscopy; +TIP, plus end–tracking protein.


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