(A) Untreated S2 cells expressing GFP-tubulin (green) were fixed and stained with -tubulin antibody (red) and Hoechst 33342 (blue). re-addition in this in vitro system (Walczak et al., 1998). Function-blocking motor antibodies have been microinjected into fly embryo or mammalian tissue culture cells as another means of inhibiting kinesin function (Sharp et al., 2000c; Levesque and Compton, 2001). Small molecule inhibitors also have been developed against mammalian Eg5, a tetrameric kinesin (Mayer et al., 1999). RNAi of a few mitotic kinesins and cytoplasmic DHC have also been performed in (Powers et al., 1998; Raich et al., 1998; Gonczy et al., 1999). The most extensive genetic analyses have been performed in the fruit fly (in this paper, we use the kinesin nomenclature followed by the most commonly used kinesin subfamily name; Warangalone see Table I for closely related motors in other organisms). Mutations of several kinesins and cytoplasmic dynein cause mitotic defects, which include spindle formation defects (Klp61F [BimC/Eg5], Heck et al., 1993; Ncd [Kin C], Endow et al., 1994; Dhc64C Warangalone [cytoplasmic DHC], Robinson et al., 1999), chromosome Warangalone missegregation (Klp38B [Unc104], Alphey et al., 1997; Molina et al., 1997; Ruden et al., 1997; CENP-meta [CENP-E], Yucel et al., 2000), or cytokinesis failure (Klp38B [Unc104], Ohkura et al., 1997; Pav [MKLP1], Adams et al., 1998). Some kinesin mutants affect specifically meiotic cell divisions (e.g., Subito [ungrouped], Giunta et al., 2002; Nod [Kid], Theurkauf and Hawley, 1992; and Klp3A [chromokinesin], Williams et al., 1995). However, functional analyses have not been reported for 12 kinesin genes, and redundancies of different kinesin genes have not been extensively tested because mutant isolation and genetic crossing are not as easy to perform as in yeast. Furthermore, the effect of loss-of-function has been investigated in different tissues for each kinesin mutant (early stage embryo, larval neuroblast, etc.). Therefore, it is difficult to build a complete picture of the involvement of kinesins and dynein in Warangalone mitosis in higher eukaryotes. Table I. Kinesin superfamily genes in S2 cell system is excellent for functional analysis of mitotic genes because they are very sensitive to double-stranded RNA (dsRNA)Cmediated gene silencing (Clemens et al., 2000). We have reported previously that S2 cells spread Rabbit Polyclonal to KAPCB on Con ACcoated surfaces and execute normal mitosis (Rogers et al., 2002). This preparation provides outstanding imaging of the mitotic spindle and enables real-time observation of mitotic events by light microscopy. In this work, we have screened all 25 kinesins and cytoplasmic dynein for mitotic phenotypes in S2 cells using RNAi methods and microscopic observation, and have also performed simultaneous RNAi of multiple kinesins to investigate functional redundancy or coordination between different kinesin genes. We find that RNAi of eight kinesins and cytoplasmic dynein causes mitotic defects, including monopolar spindle formation, chromosome misalignment, anaphase delay, and cytokinesis failure. Some of Warangalone the phenotypes are unexpected, and we also report the first live-cell imaging of several mitotic kinesin defects. This paper represents the first comprehensive analysis of microtubule-based motor function during mitosis in a single metazoan cell type. Results Kinesin superfamily genes in kinesin superfamily proteins. A BLAST search was performed on the fly database using the conserved motor domain of fly conventional kinesin (1C340 aa). 25 genes emerged as exhibiting significant (E-value 1e-15) sequence homology, one more than a previous search for kinesins in the genome (Goldstein and Gunawardena, 2000). Sequence alignments of the motor and nonmotor domains with kinesins from other organisms (unpublished data) were used to assign the kinesins to different subfamilies. This analysis identified clear subfamilies and mammalian homologues for 21 of the 25 genes (Table I). The remaining four are divergent kinesins that have no homology in their tail domains to kinesins in other organisms. Five kinesins may not be present or are expressed at very low levels in S2 cells (Table I). Nevertheless, we performed RNAi for all 25 kinesins so as not to miss a potential mitotic involvement of a low copy number kinesin. Characterization of mitosis in untreated S2 cells Before investigating RNAi-induced mitotic phenotypes, we first characterized the process of cell division in untreated S2 cells. For clear imaging of.