The kinetic polarities of spindle microtubules in vivo, in crane-fly spermatocytes. I. Kinetochore microtubules that re-form after treatment with colcemid

Abstract

In newly formed chromosomal spindle fibres we determined the kinetic polarities of the microtubules, that is, the ends to which tubulin monomers add. Spindles disappeared after cells were continuously immersed in colcemid; then portions of the cells were continuously irradiated with a microbeam of near-ultraviolet light to reverse locally the effect of the colcemid. From the following lines of evidence we conclude: that microtubules are organized by the chromosomes; and that tubulin monomers add to the chromosomal spindle fibres at the kinetochore. When chromosomes were irradiated chromosomal spindle fibres grew in different directions, not necessarily focussed to a common pole; this would not occur if the chromosomal spindle fibres were organized by poles. Chromosomal spindle fibres were sometimes associated with only some of the chromosomes; this would not occur if the fibres were organized by the poles. Thus, chromosomal spindle fibres are organized solely by chromosomes; these spindle fibres are functional since the associated chromosomes moved in anaphase. When chromosomes were irradiated the re-formed spindle fibres grew up to 10 microns past the edges of the irradiating spot. Experimentally, free tubulin did not diffuse more than 4–5 microns from the irradiated spot. Thus we conclude that the tubulin monomers add at the kinetochores and not at the distal ends of the fibres.