INDUCTION OF MICROTUBULE CATASTROPHE BY FORMATION OF TUBULIN - GDP AND APOTUBULIN SUBUNITS AT MICROTUBULE ENDS

The recent discovery that GTP linked to latex beads binds to microtubu le ends suggested that nucleotide interactions at this site may play a role in regulating microtubule (MT) dynamics. Evidence for this was s ought using DIC microscopy to analyze effects of the free GTP and GDP concentration on the rates of MT elongation and phase transition to ra pid shortening (catastrophe, k(c)). That nucleotide can dissociate and thereby destabilize the plus end by forming nucleotide-free (apotubul in) subunits was indicated by an increase in k(c) from 0.001 to 0.05 s (-1), when the free GTP concentration was reduced from 100 to 0.5 mu M , during assembly with 15 mu M tubulin-GTP subunits (TuT). That nucleo tide can bind to the minus end was indicated by a nearly 5-fold decrea se in the rate of elongation when the free GDP concentration was incre ased from 1.6 to 175 mu M, during assembly with a mixture of 36 mu M T uT and 54 mu M TuD. Further evidence that nucleotide can bind to both ends was provided by the observation that with a mixture of 36 mu M Tu T and 54 mu M TuD, k(c) was increased from 0.0036 to 0.05 s(-1) at the plus end, and from 0.0005 to 0.005 s(-1) at the minus end, when the f ree GDP concentration was increased from 1.6 to 175 mu M. Our evidence for destabilization of microtubules by formation of apotubulin and by nucleotide exchange to form terminal TuD subunits suggests that micro tubule dynamics can be regulated in cells by an exchange factor that g enerates apotubulin subunits, or by a GTPase activating protein that f orms TuD subunits at microtubule ends.