KKT4 localizes at spindle microtubules in addition to kinetochores. (A) Examples of cells expressing YFP-KKT4 and the kinetochore marker tdTomato-KKT2 at indicated cell-cycle stages (BAP665). Note that KKT4 has additional signals that do not colocalize with kinetochores, which are especially prominent in metaphase (arrowheads; n > 30 metaphase cells). K and N indicate the kinetoplast (mitochondrial DNA) and nucleus, respectively. These organelles have distinct replication and segregation timings and serve as good cell-cycle markers (Woodward and Gull, 1990; Siegel et al., 2008). K* denotes an elongated kinetoplast and indicates that the nucleus is in S phase. (B) Examples of cells expressing YFP-KKT4 and the spindle microtubule marker tdTomato-MAP103 showing that the nonkinetochore KKT4 signals partially colocalize with spindle microtubules (n > 20 metaphase cells; BAP943). (C) Examples of cells expressing YFP-KKT4 with tdTomato-MAP103 (top) or tdTomato-KKT2 (bottom) treated with 5 nM ansamitocin for 4 h and showing that KKT4 localizes at kinetochores even when bipolar spindle formation is perturbed (n > 20 cells in 2K1N). Bars, 2 µm.