Decreasing oncoprotein 18/stathmin levels reduces microtubule catastrophes and increases microtubule polymer in vivo

Oncoprotein 18/stathmin (Op18) has been identified recently as a protein which destabilizes microtubules. To characterize the function of Op18 in living cells, we used microinjection of anti-Op18 antibodies or antisense oligonucleotides to block either Op18 activity or expression in interphase newt lung cells. Anti-tubulin staining of cells microinjected with anti-Op18 and fixed 1-2 hours after injection showed an increase in total microtubule polymer. In contrast, microinjection of either non-immune IgG or anti-Op18 preincubated with bacterially-expressed Op18 had little effect on microtubule polymer level. Cells treated with Op18 antisense oligonucleotides for 4 days had (greater than or equal to)50% reduced levels of Op18 with no change in the soluble tubulin level. Measurement of MT polymer level in untreated, antisense or nonsense oligonucleotide treated cells demonstrated that reduced Op18 levels resulted in a 2.5-fold increase in microtubule polymer. Next, the assembly dynamics of individual microtubules at the peripheral regions of living cells were examined using video-enhanced contrast DIC microscopy. Microinjection of antibodies against oncoprotein 18 resulted in a 2.2-fold reduction in catastrophe frequency and a slight reduction in plus end elongation velocity compared to uninjected cells or cells microinjected with non-immune IgG. Preincubation of anti-Op18 antibody with recombinant Op18 greatly diminished the effects of the antibody. Similarly, treatment of cells with antisense oligonucleotides reduced catastrophes 2.5- to 3-fold compared to nonsense oligonucleotide treated or untreated cells. The other parameters of dynamic instability were unchanged after reducing Op18 with antisense oligonucleotides. These studies are consistent with Op18 functioning to regulate microtubule catastrophes during interphase in vivo.