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First published online 9 March 2004
doi: 10.1242/jcs.01021

Journal of Cell Science 117, 1719-1726 (2004)
Published by The Company of Biologists 2004
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{alpha}B-Crystallin-coated MAP microtubule resists nocodazole and calcium-induced disassembly

Yoshinobu Fujita1,*, Eri Ohto1, Eisaku Katayama2 and Yoriko Atomi1,*,{ddagger}

1 Department of Life Sciences, The Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1, Komaba, Meguro-ku, Tokyo 153-8902, Japan
2 Division of Biomolecular Imaging, Institute of Medical Science, The University of Tokyo, 4-6-1, Shiroganedai, Minato-ku, Tokyo 108-8639, Japan



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  		Fig. 1. Characterization of anti-{alpha}B-crystallin C-terminal (C1) and N-terminal (N1) peptide antibodies. (A) Coomassie-blue-stained gels of rat soleus muscle homogenate (lane 1), total L6E9 cell lysate (lane 2), purified {alpha}B-crystallin (lane 3) and tubulin (lane 4), and immunoblots with C1 antibody (lanes 5-8; corresponding to lanes 1-4) and N1 antibody (lanes 9-12; corresponding to lanes 1-4). (B) C1 antibody recognizes {alpha}B-crystallin in L6E9 cells (bottom right) as confirmed by a loss of reaction it after preadsorption of the antibody with the C-terminal peptide (bottom left). Scale bar, 25 µm.

 


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  		Fig. 2. Association of {alpha}B-crystallin with MTs. (A) Co-localization of {alpha}B-crystallin and MTs in formaldehyde fixed L6E9 cells. (B) Co-localization of {alpha}B-crystallin and MT in methanol fixed L6E9 cells. (A,B) Merged immunofluorescence micrographs (right) of the same cells visualized for {alpha}B-crystallin (left) and tubulin (middle). Insets show an enlargement (threefold) of the boxed area in the right-hand panels. (C) Co-precipitations of {alpha}B-crystallin and reconstituted MTs from L6E9 cell lysate. SDS-PAGE of total proteins (lane 1), the supernatant (lane 2), the precipitate (lane 3) in I and their immunoblots with the C1 antibody in II. Scale bars, 10 µm.

 


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  		Fig. 3. Electron micrographs of {alpha}B-crystallin bound MAP-MTs. MTs polymerized in the presence (A; B, top) or absence of {alpha}B-crystallin (B, bottom) as observed by electron microscopy. MTP (1 mg ml–1) was polymerized in the presence of {alpha}B-crystallin (40 µM as monomer). Arrowheads indicate unbound {alpha}B-crystallin. Scale bar, 100 nm (A), 50 nm (B).

 


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  		Fig. 4. Binding of {alpha}B-crystallin to reconstituted MTs. (A) CBB-stained gel of co-precipitated {alpha}B-crystallin and MAP-MTs. All lanes contain 1 mg ml–1 MAP-MTs. The other lanes also contain: 2.5 µM {alpha}B-crystallin (lane 2); 5 µM {alpha}B-crystallin (lane 3); 10 µM {alpha}B-crystallin (lane 4); 20 µM {alpha}B-crystallin (lane 5); 40 µM {alpha}B-crystallin (lane 6); 80 µM {alpha}B-crystallin (lane 7). Arrowhead indicates {alpha}B-crystallin band. (B) CBB-stained gel of co-precipitated {alpha}B-crystallin and PCT-MT. All lanes contain 10 µM PCT-MT. The other lanes also contain: 5 µM {alpha}B-crystallin (lane 2); 10 µM {alpha}B-crystallin (lane 3); 20 µM {alpha}B-crystallin (lane 4); 40 µM {alpha}B-crystallin (lane 5) and 80 µM {alpha}B-crystallin (lane 6). (C) The amount of co-precipitated {alpha}B-crystallin was plotted for input {alpha}B-crystallin. Closed circle represented for MTP-MTs and open circle for PCT-MTs.

 


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  		Fig. 5. Instability of MT in C6AS cell. (A) Co-localization of {alpha}B-crystallin and MTs in C6 glioma cells. Merged immunofluorescence micrographs of C6 glioma cells (right), visualized for {alpha}B-crystallin (left) and tubulin (middle), permeabilized with MSB containing Triton X-100 first and fixed later. Scale bar, 10 µm. (B) The expression levels of {alpha}B-crystallin in C6 normal, C6SE and C6AS glioma cells by western blotting. (C) Comparisons of the MTs remaining at 2 minutes and 10 minutes after nocodazole treatment (33 µM) in C6 normal, C6SE and C6AS cells immunostained with anti-{alpha}-tubulin antibody. Scale bars, 10 µm. (D) The immunoblot of tubulin isolated as dimers (lanes 1-6) or polymers (lanes 7-12) from C6 (odd numbers) and C6AS (even numbers). Tubulin from untreated cells (control; lanes 1, 2, 7 and 8) and treated with nocodazole for 15 minutes (lanes 3, 4, 9 and 10) and 30 minutes (lanes 5, 6, 11, and 12) were visualized after staining with anti-{alpha}-tubulin antibody. (E) The comparisons in the polymer ratio to the amount of total tubulin, as quantified by immunoblotting of the isolated dimers and polymers with (for 15 minutes and 30 minutes) and without (control) nocodazole treatment, between C6 and C6AS cells. The mean polymer ratio and the standard deviations in six samples are shown for C6 cells (white columns) and {alpha}B-crystallin-antisense-expressing C6AS cells (black columns). **Significantly different at P<0.01.

 


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  		Fig. 6. {alpha}B-Crystallin increases MT resistance to disassembly in vitro. (A) Changes of turbidity in assembly of MT proteins, over 15 minutes and disassembly after the addition of 10 µM podophyllotoxin (indicated by arrow) with (2.5 µM, 10 µM) or without {alpha}B-crystallin. (B) Mean levels of remaining MTs and standard deviations for each of three trials calculated from the ratio of the absorbance at 44-45 minutes to the level at 14-15 minutes (at polymerized peak) with different levels of {alpha}B-crystallin. {alpha}B-Crystallin (10 µM) significantly increased the amount of remaining MT compared with that in the absence of {alpha}B-crystallin. Values are means±s.d. (n=3, *significantly different at P<0.05). (C) Changes of turbidity in assembly of MT proteins over 15 minutes and disassembly after the addition of 1 mM calcium (indicated by arrow) with (2.5 µM, 5.0 µM, 10.0 µM) or without {alpha}B-crystallin. (D) The ratio of the attained polymerization as a mean value at 14-15 minutes after 113 µM calcium addition with {alpha}B-crystallin (10 µM) was significantly higher than that without {alpha}B-crystallin. Values are means±s.d. (n=6, **significantly different at P<0.01).

 

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© The Company of Biologists Ltd 2004