Reactivating prometaphase movement in permeabilized animal cells.

We have attempted to reactivate chromosome activity in dividing, permeabilized animal cells with the aim of analysing the physiology of chromosome movements, particularly during prometaphase. We achieved reactivation on numerous occasions, but it was limited in extent and unreliable in that many cells did not respond and spindles frequently collapsed irreversibly. Of the three cell lines used, newt lung cells gave the best examples of oscillating chromosome movement resuming upon addition of ATP to permeabilised cells. Saltatory movement, severely inhibited or stopped completely during permeabilization, was reactivated considerably by addition of ATP. Only a few of the chromosomes in any spindle moved; while this activity was an ATP-dependent reactivation, it is at present too unreliable for us to experimentally distinguish between the physiology of polar and anti-polar movement. Permeabilized metaphase LLC cells underwent some interesting transformations. Upon exposure to digitonin, many metaphase spindles partially collapsed, creating a prometaphase-like rearrangement of chromosomes; when ATP was added, the spindle in many of these cells grew and reformed until a fairly normal metaphase plate was reconstituted. Less frequently, these spindles continued to elongate, drawing the chromosomes apart into two irregular masses during 'pseudoanaphase'. While our techniques are still too unreliable to permit analysis of prometaphase at the level desired, they demonstrate that the motility systems of prometaphase can survive permeabilization, as can the intrinsic ability of spindle to shorten and elongate in a manner reminiscent of anaphase elongation. Throughout all manipulations, chromosomes seemingly maintained their attachment to spindle fibres although the pseudoanaphase transformations suggested that some kinetochore fibre connections were weakened enough to be broken by spindle regrowth. [ABSTRACT FROM AUTHOR]