The attachment of kinetochores to the pro-metaphase spindle in PtK1 cells

When late prophase PtK₁ cells are chilled to 6 ° C the nuclear envelope (NE) breaks down as in normal cells but the spindle is inhibited from forming. When these cells are subsequently warmed to 18 ° C the spindle slowly forms and pro-metaphase congression ensues. Using this approach we have been able to experimentally eliminate the influence of asynchronous NE breakdown on the formation and development of the spindle, and also to slow down (and thus increase the temporal separation of) the subsequent events which occur during the initial stages of spindle formation. Correlative light and high voltage electron microscopic studies on these cells, fixed after various times of recovery, reveal the following results: 1) the centrosomes generate microtubules (MTs) well before MTs are seen to be associated with the kinetochores; 2) as in untreated PtK₁ cells (Roos, 1973a, 1976) the order in which chromosomes attach to the forming spindle is influenced by their proximity to a centrosome-kinetochores closest to a centrosome appear stretched towards the centrosome at a time during recovery when other kinetochores, more distal to the centrosome appear unstretched and unoriented; 3) as in untreated cells (Heneen, 1970; Roos, 1976) the predominant behavior during recovery is for a chromosome to initially mono-orient and associate with the near centrosome and only later to develop a bipolar association; and 4) MTs associated with early pro-metaphase kinetochores are almost always oriented towards a centrosome. — From our results we conclude that the proximity effect and the tendency of pro-metaphase chromosomes in PtK₁ to initially mono-orient and associate with the near centrosome cannot be ascribed, as suggested by Roos (1976), to influences arising during the asynchronous breakdown of the NE. Rather, our data clearly demonstrate that a kinetochore-centrosome interaction occurs during spindle formation which cannot be attributed to transient influences. The proximity effect and the predominant tendency of PtK₁ pro-metaphase chromosomes to mono-orient to the near pole are taken to signify the existance of a centrosomal influence on the attachment and orientation of chromosomes. Two possible mechanisms for this influence, both involving a structural interaction between the centrosome and the kinetochore, are outlined.