Fig. 6. Overexpression of TY1-NRKC blocks cytokinesis. (A, top panel) Western blot of overexpressed TY1-NRKC protein (time in hours). The membrane was probed with Isa-1 and indicates the increased concentration of TY1-NRKC of protein observed in induced (+) cells. The blot was also probed with the anti-TY1 tag antibody BB2. Wild-type cells (WT), induced time 0 or non-induced (-) cells did not express the tagged protein. TY1-NRKC is observed after 24 hours of induction as illustrated in (+) lanes. The blot was probed with anti-PFRA monoclonal L8C4 as a loading control. Blots were loaded with 5x10⁶ cells/lane. (A, bottom panel) Comparative growth curves of non-induced (- TET) and induced cells (+ TET) indicate that cells overexpressing TY1-NRKC have a reduced growth rate compared with non-induced cells. (B) Cytoskeletons of TY1-NRKC cells induced for 48 hours were probed by immunofluorescence with YL1/2 (b) and M25 (c). This multinucleated cell possesses one flagellum but four basal bodies (b and d), indicating that the cell passed through the cell cycle the absence of cytokinesis. The arrowheads in c and d indicate a large kinetoplast close to the basal bodies. Bar, 5 µm. (C) Counts of cells possessing at least one large kinetoplast and more than two nuclei in non-induced (- TET) and induced cells (+ TET) from 0 to 96 hours. After 96 hours of induction, 80.8% of cells possessed at least one large kinetoplast and more than two nuclei. (D) Populations of non-induced (- TET) and induced (+ TET) cultures were assessed to identify the percentage of cells possessing two basal bodies (2 BB), four basal bodies (4 BB), more than four basal bodies (>4 BB) and clusters of non-flagellated basal bodies (Clust. BB). Assessment was done from 0 to 96 hours of TY1-NRKC induction. All values are mean ± s.e.m. of three experiments.