Incorporation of radioactive tubulin into microtubules at steady state. Experimental and theoretical analyses of diffusional and directional flux.

Abstract

Subunit flux into porcine brain microtubules at steady state has been studied using both radioactive guanine nucleotide and a radioactive tubulin dimer, [3H]ethyltubulin, which is obtained by a reductive ethylation (Zeeberg, B., Cheek, J., and Caplow, M., (1980) Anal. Biochem. 104, 321--327). We have also determined the molecular rate constants for steady state dimer loss from and addition to the microtubule. This is the first study where the rate for flux of radioactive subunits at steady state has been correlated with the measured molecular rate constants. Also, we derive a theoretical analysis of the observed flux of subunits into a polymer at steady state in pulse and chase experiments; this is used to relate the flux and molecular rate constant to a steady state model. Analysis of our results shows that, at steady state, there is only a small difference in the number of assembly and disassembly events at a given microtubule end (as compared with the total number of events at both ends) in a unit of time; predominant opposite end assembly-disassembly (Margolis, R. L., and Wilson, L. (1978) Cell 13, 1--8) is, therefore, ruled out. The almost complete absence of opposite end assembly-disassembly in the steady state provides a means for very efficient regulation of the microtubule network in vivo, since a small modulation in the relative magnitudes of the molecular rate constants can increase the subunit flux dramatically.