Microtubule Cross-Linking Proteins from Artemia
As determined by examination of negatively stained samples with the electron microscope, microtubules assembled in cell-free Artemia extracts upon addition of taxol are cross-linked. Taxol-induced microtubules, collected by centrifugation through sucrose cushions and extracted with 1 M NaCl, yielded several non-tubulin proteins, herein termed microtubuleassociated proteins (MAP). Although their dependence on microtubule assembly for sedimentation through sucrose cushions and their extraction from taxol-stabilized pellets with salt-containing buffers are characteristics shared by MAP from other organisms, the Artemia MAP do not stimulate tubulin assembly. Assembly of the Artemia MAP with purified Artemia or neural tubulin does, however, allow reconstitution of the microtubule cross-links. Electrophoretic analysis of cross-linked microtubules demonstrated that the same non-tubulin proteins co-sedimented with both types of tubulins. Formation of cross-links between the microtubules does not appear to be influenced by ATP. Probing of western blots with polyclonal and monoclonal antibodies indicates that Artemia MAP are different from neural MAP, although some epitopes may be shared by MAP from both sources. Neural MAP1 and MAP2 cDNA clones do not hybridize to Southern blots of restriction-digested Artemia DNA. The results clearly demonstrate that Artemia contain novel protein(s) with the ability to cross-link microtubules from phylogenetically disparate organisms.