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

Lynne Cassimeris^1,2,, David Gard³, P. T. Tran^4,* and Harold P. Erickson²

¹ Department of Biological Sciences, Lehigh University, Bethlehem, PA, USA
² Cell Biology Department, Duke University School of Medicine, Durham, NC, USA
³ Biology Department, University of Utah, Salt Lake City, UT, USA
⁴ Biology Department, University of North Carolina, Chapel Hill, NC, USA
^* Present address: Department of Microbiology, Columbia University, New York, NY, USA
Author for correspondence (e-mail: lc07{at}lehigh.edu )

View larger version (90K): [in a new window]   Fig. 1. XMAP215 is a long rod-shaped molecule. (A) Gallery of uni-directional shadowed images of XMAP215 selected from two different XMAP215 preparations. Samples were sprayed onto mica and shadowed with platinum/carbon as described in the Materials and Methods. Most molecules appeared relatively straight, but a significant number were bent (e.g. bottom row), suggesting that XMAP215 is a flexible molecule. Scale bar: 100 nm. (B) Corresponding SDS-PAGE gel of the peak glycerol fraction used for shadowing. A small fraction of the sample was proteolyzed; this is commonly observed with XMAP215. The positions of Mr markers are shown. (C) Histogram of XMAP215 length distribution. The lengths of individual XMAP215 molecules were measured from digitized EM negatives of uni-directional shadowed XMAP215. The mean XMAP215 length of 60 nm is marked with an arrow.

View larger version (71K): [in a new window]   Fig. 2. XMAP215 is a monomer. (A) Immunoblot of purified XMAP215 and a Xenopus egg extract probed with an antibody raised against the C terminus of TOGp. The antibody recognizes purified XMAP215 and a 215 kDa protein in Xenopus egg extracts. Positions of Mr markers are shown. (B) XMAP215 was incubated with (top row) or without (bottom row) an antibody to its C terminus before uni-directional shadowing. In the presence of antibody, the XMAP215 rod was decorated by a single globular structure that had the size of an antibody molecule, suggesting that XMAP215 is a monomer. Scale bar: 100 nm.

View larger version (76K): [in a new window]   Fig. 3. XMAP215 binds partial tubulin protofilament rings. Tubulin (4 µM) was incubated with XMAP215 (0.4 µM) in 0.1xBRB80 and 15% glycerol for 1 hour on ice before rotary shadowing. Under these conditions, a fraction of the tubulin assembled into partial rings (top row). In the absence of XMAP215, rings were not detected and the observed molecules were approximately the size of single tubulin dimers (middle row). Tubulin could be assembled into partial rings without XMAP215 (bottom row) by increasing the glycerol concentration (30%) and incubation time (several hours). Scale bar: 100 nm.

View larger version (26K): [in a new window]   Fig. 4. XMAP215 increases the width (A) and length (B) of partial tubulin rings. By contrast, XMAP215 does not change the radius of curvature of the protofilament rings (C). (D) A partial ring and the circle overlay used to measure the radius. The radius is shown in inches on the micrograph and was then converted to nm. For A,B, the means are denoted by arrows. Histograms are presented as the percent of the total sample falling within each range to compensate for differences in sample size.

View larger version (47K): [in a new window]   Fig. 5. (A) Alignment of the four subdomains within the N-terminal half of XMAP215, labeled N1-N4 and identified specifically on the last line. Amino acids conserved in three or four of the subdomains are indicated above the alignment. The HEAT repeats identified in TOGp by Neuwald and Hirano (Neuwald and Hirano, 2000) are shown in red (we transferred the repeats from TOGp to the closely related XMAP215 sequence). This alignment shows four segments where three subdomains have HEAT repeats, suggesting that the fourth subdomain also has one, not identified by the sequence analysis. A sixth HEAT repeat may occur in the final part of each subdomain, but this is not recognizable from the sequence. (B) A diagram of the TOGp sequence showing the location of HEAT repeats as dark boxes. The positions are nearly identical in XMAP215. A 36 amino acid insert in XMAP215 (Becker and Gard, 2000) is located N-terminal to the last four identified repeats. The other XMAP215 insert is located C-terminal to the identified HEAT repeats. The dark boxes below indicate HEAT repeats identified by Neuwald and Hirano (Neuwald and Hirano, 2000), and the open boxes indicate additional repeats suggested by the alignment in A. The sub-domains N1-N4 are indicated above.

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