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

Oncoprotein 18/stathmin (Op18) has been identified recently as a protein wh ich destabilizes microtubules. To characterize the function of Op18 in livi ng cells, we used microinjection of anti-Op18 antibodies or antisense oligo nucleotides 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 po lymer. In contrast, microinjection of either non-immune IgG or anti-Op18 pr eincubated with bacterially-expressed Op18 had little effect on microtubule polymer level. Cells treated with Op18 antisense oligonucleotides for 4 da ys had greater than or equal to 50% reduced levels of Op18 with no change i n the soluble tubulin level. Measurement of MT polymer level in untreated, antisense or nonsense oligonucleotide treated cells demonstrated that reduc ed Op18 levels resulted in a 2.5-fold increase in microtubule polymer. Next , the assembly dynamics of individual microtubules at the peripheral region s of living cells were examined using video-enhanced contrast DIC microscop y. Microinjection of antibodies against oncoprotein 18 resulted in a 2.2-fo ld reduction in catastrophe frequency and a slight reduction in plus end el ongation velocity compared to uninjected cells or cells microinjected with non-immune IgG. Preincubation of anti-Op18 antibody with recombinant Op18 g reatly diminished the effects of the antibody. Similarly, treatment of cell s with antisense oligonucleotides reduced catastrophes 2.5- to 3-fold compa red to nonsense oligonucleotide treated or untreated cells. The other param eters of dynamic instability were unchanged after reducing Op18 with antise nse oligonucleotides. These studies are consistent with Op18 functioning to regulate microtubule catastrophes during interphase in vivo.