Our findings indicate that Spindly is a novel regulator of mitotic dynein, functioning specifically to target dynein to kinetochores. Introduction The spindle assembly checkpoint (SAC) NSC 87877 is critical for preventing the onset of anaphase until all chromosomes are aligned on the metaphase plate. Scaerou, R. Karess, and T. Hays. 2001. 3:1001C1007). Using a high throughput RNA interference NSC 87877 screen in S2 cells, we have identified a new protein (Spindly) that accumulates on unattached kinetochores and is required for silencing the SAC. After the depletion of Spindly, dynein cannot target to kinetochores, and, as a result, NSC 87877 cells arrest in NSC 87877 metaphase with high levels of kinetochore-bound Mad2 and RZZ. We also identified a human homologue of Spindly that serves a similar function. However, dynein’s nonkinetochore functions are unaffected by Spindly depletion. Our findings indicate that Spindly is a novel regulator of mitotic dynein, functioning specifically to target dynein to kinetochores. Introduction NSC 87877 The spindle assembly checkpoint (SAC) is critical for preventing the onset of anaphase until all chromosomes are aligned on the metaphase plate. A single misaligned kinetochore is sufficient to generate a wait anaphase signal, thereby ensuring that all sister chromatids segregate to opposite ends of the spindle and are equally distributed to the daughter cells. Failure of the SAC can lead to premature anaphase onset and aneuploidy (Liu et al., 2003; Kops et al., 2005b; for review see Kadura and Sazer, 2005). Such defects can have consequences for a whole organism, as mice that lack a full complement of SAC genes have more frequent DNA segregation errors and are more susceptible to tumor development (Baker et al., 2005). The presence of the SAC was initially inferred from observations that cells delay in metaphase when meiotic sex chromosomes fail to pair and align or after the spindle is perturbed by either microtubule poisons or microsurgery. Molecules responsible for the SAC were later identified in yeast genetic screens and named Mad1, -2, and -3 (Mad for mitotic arrest deficient) and Bub1, -2, and -3 (Bub for budding unperturbed by benzimidazole). Subsequent work showed that these proteins together with the MPS1 kinase form distinct complexes that target to the kinetochore (for reviews see Lew and Burke, 2003; Kadura and Sazer, 2005; Malmanche et al., 2006; Musacchio and Salmon, 2007). Two additional metazoan checkpoint proteins, Zw10 and Rough Deal (Rod), were later isolated as cell cycle mutants in genes (Echard et al., 2004), we performed two screens using S2 cells (Fig. 1, bCd). The first screen measured mitotic index (the percentage of phosphohistone H3Cpositive cells in a population; see Materials and methods). In the second screen, the shape of S2 cells (spread on concanavalin A [Con A]Ccoated surfaces; Rogers et al., 2003) was evaluated by visual inspection. Open in a separate window Figure 1. RNAi of Spindly alters cell morphology and causes mitotic arrest in S2 cells. (a) Domains of the Spindly protein showing predicted coiled-coil sequences in red and repeated motifs in blue; sequence alignment of residues in the repeat motifs is shown below. The locations of two nonoverlapping dsRNAs used to deplete Spindly are shown in green. A third dsRNA to the 3 UTR was also used (not depicted). (b and c) Wild-type (wt) S2 cells show a uniformly spread morphology (b), whereas Spindly RNAi-treated cells (c) show marked defects in the actin lamellae as well as increased numbers of cells with long microtubule-rich projections. Actin, red; microtubules, PDGFB green; DNA, blue. (d) The mitotic index of S2 cells is increased after the depletion of Spindly, dynein heavy chain (DHC), or a subunit of the APC (Cdc16;.