XMAP215 is a long thin molecule that does not increase microtubule stiffness

Author:

Cassimeris Lynne123,Gard David4,Tran P. T.56,Erickson Harold P.2

Affiliation:

1. Department of Biological Sciences, Lehigh University, Bethlehem, PA, USA

2. Cell Biology Department, Duke University School of Medicine, Durham, NC,USA

3. Author for correspondence (e-mail: lc07@lehigh.edu)

4. Biology Department, University of Utah, Salt Lake City, UT, USA

5. Biology Department, University of North Carolina, Chapel Hill, NC, USA

6. Present address: Department of Microbiology, Columbia University, New York,NY, USA

Abstract

XMAP215 is a microtubule associated protein that speeds microtubule plus end growth by seven- to tenfold and protects these ends from destabilization by the Kin I kinesin, XKCM1. To understand the mechanisms responsible for these activities, it is necessary to know the structure of XMAP215. By unidirectional shadowing and electron microscopy, XMAP215 appeared as an elongate molecule of 60±18 nm, suggesting that XMAP215 could span up to seven to eight tubulin dimers along a protofilament. Most XMAP215 molecules were straight but a subset were bent suggesting that XMAP215 is flexible. Antibodies to the C terminus labeled one end of XMAP215 with no evidence for XMAP215 dimerization. Incubation of XMAP215 and tubulin at 4°C resulted in assembly of curved protofilaments, which appeared to be incomplete tubulin rings. Measurements from rotary shadowed samples showed that tubulin/XMAP215 partial rings had an average width of 8.8±1.8 nm compared with 5.6±1.1 nm for rings assembled from tubulin dimers alone, suggesting that XMAP215 adds a width of approximately 3.2 nm to the curved tubulin protofilament. XMAP215 did not change the radius of curvature of these partial tubulin rings. Measurements of microtubule flexural rigidity by thermal fluctuations showed that XMAP215 did not change microtubule rigidity. Finally,sequence analysis shows that the N-terminal half of XMAP215 contains four repeats, each composed of multiple HEAT repeats.

Publisher

The Company of Biologists

Subject

Cell Biology

Reference47 articles.

1. Becker, B. E. and Gard, D. L. (2000). Multiple isoforms of the high molecular weight microtubule associated protein XMAP215 are expressed during development in Xenopus. Cell Motil. Cytoskel.47,282-295.

2. Charrasse, S., Lorca, T., Doree, M. and Larroque, C.(2000). The Xenopus XMAP215 and its human homologue TOG proteins interact with cyclin B1 to target p34cdc2 to microtubules during mitosis. Exp. Cell Res.254,249-256.

3. Cingolani, G., Petosa, C., Weis, K. and Muller, C. W.(1999). Structure of importin-β bound to the IBB domain of importin-alpha. Nature399,221-229.

4. Cullen, C. F., Deak, P., Glover, D. M. and Ohkura, H.(1999). mini spindles: a gene encoding a conserved microtubule-associated protein required for the integrity of the mitotic spindle in Drosophila.J. Cell Biol.146;1005-1018.

5. Desai, A. and Mitchison, T. J. (1997). Microtubule polymerization dynamics. Annu. Rev. Cell Biol.13,83-117.

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