The kinetic polarities of spindle microtubules in vivo, in crane-fly spermatocytes. II. Kinetochore microtubules in non-treated spindles.

We determined the kinetic polarities of chromosomal spindle fibre microtubules in vivo: either the kinetochore or pole ends of chromosomal spindle fibres were irradiated with near-ultraviolet light to prevent depolymerization by colcemid. Irradiations began either just before or just after colcemid addition; cells were continually irradiated and continuously immersed in colcemid. Irradiations of kinetochore ends of chromosomal spindle fibres prevented depolymerization; irradiations of pole ends did not. Therefore, since colcemid acts by binding to the 'on' (assembly) ends of microtubules, the on ends of chromosomal spindle fibre microtubules are at the kinetochores. That is, in untreated chromosomal spindle fibres in vivo tubulin monomers add to kinetochore microtubules at the kinetochore ends. Tubulin diffused from the irradiation sites: irradiations of the cytoplasm sometimes prevented depolymerization of chromosomal spindle fibres. Prevention of chromosomal spindle fibre depolymerization was dependent on the distance of the irradiated region from the nearest chromosome; the longer the distance the less likely was it that the irradiation prevented depolymerization. On the other hand, prevention of chromosomal spindle fibre depolymerization was not dependent on the distance from the irradiated spot to the nearer pole. This analysis, too, we argue, strongly suggests that the kinetochore ends of the chromosomal spindle fibres are the on ends.