Molecular mechanisms for changes in microtubule dynamics

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An activity that severs stable microtubules has previously been detected in M phase extracts, but not in interphase extracts, of Xenopus eggs. We reported previously the identification and purification from Xenopus M phase eggs of a microtubule-severing factor. It is a homo-oligomeric protein composed of 56-kDa polypeptide subunit (p56) that can sever stable microtubules slowly. Protein microsequencing indicated that p56 is a previously unidentified protein. Recently, we found andther microtubule-severing activity from Xenopus M phase eggs and purified it to near homogeneity by sequential chromatography. The newly purified factor had a single polypeptide of 48-kDa (p48) , and severed microtubules very rapidly in an ATP-independent manner. It was found that microtubule severing induced by p48 is not accompanied by significant depolymerization of microtubules. These characteristics of p48 are clearly different from those of katanin, an ATPase that severs and disassembles stable microtubules, which has been identified in sea urchin eggs by Vale and co-workers. We produced anti-p48 antibody that reacts with p48 specifically in total cell lysates. The immunoblotting with this anti-p48 antibody revealed the universal existence of p48 in almost all tissues of Xenopus and rat. Furthermore, p48 which was purified by chromatographya on anti-p48 antibody-fixed beads exhibited a microtubule severing activity, confirming the identity of p48 as a severing factor. When purified p48 was reacted with cytoplasmic microtubule network emanating from centrosomes, rapid breaking of microtubules occurred. p48 was revealed to be EF1-alpha. These results suggest that EF1-alpha may function as a microtubule-severing factor in cells.