In the absence of a centrosomal linker, microtubules position the two centrosomes of a cell relatively close together.

<p>(A) RPE1 wt and RPE1 C-Nap1 KO cells were treated with the indicated reagents for 1 h at 37°C. Fixed cells were analyzed by indirect immunofluorescence with the indicated antibodies. Size bar: 10 μm. (B) Distance between centrosomes of RPE1 C-Nap1 KO cells increases upon treatment of cells with the microtubule drugs nocodazole and taxol. Cells were incubated with the indicated drugs for 1 h at 37°C and subsequently fixed and stained with γ-tubulin antibodies. The distance between centrosomes was determined as described in Materials and Methods. N = 40–60 per condition. Error bars are SEM around the mean value. (C) Cells from (B) were grouped into “cells with separated centrosomes” when the centrosomal distance was >2 μm. Error bars are SEM between 3 independent experiments. (D) Linker morphology in RPE1 cells. RPE1 wt cells were incubated for 1 h with 5 μM nocodazole or the solvent control DMSO. Cells were fixed and stained with the indicated linker antibodies and the centrosome marker γ-tubulin. RPE1 wt cells with a centrosome distance <2 μm have a functional centrosomal linker. Centrosomes of cells with a distance >2 μm are associated with linker proteins but the connection is not established. Nocodazole treatment does not cause displacement of C-Nap1 and rootletin from centrosomes. Bar: 2 μm. (E) The effect of nocodazole on the centrosome distance of RPE1 C-Nap1 KO cells is reversible. Cells were treated for 1 h with 100 nM nocodazole. Nocodazole was washed out (t = 0) the centrosome distance in fixed cells was determined 30, 60 and 120 min after wash out. N = 50 cells per time point and cell line. Error bars are SEM.