ARTICLE Toucan Protein Is Essential for the Assembly of Syncytial Mitotic Spindles in Drosophila Melanogaster Alain Debec, 1 Muriel Grammont, 2 Guy Berson, 2 Bernard Dastugue, 2 William Sullivan, 3 and Jean-Louis Couderc 2 * 1 Observatoire Oce ´ anologique, Laboratoire de Biologie du De ´ veloppement, Universite ´ Pierre et Marie Curie/CNRS, Villefranche-sur-Mer, France 2 Laboratoire de Biochimie, UMR 384 INSERM, Clermont-Fd, France 3 Sinsheimer Laboratories, Department of Biology, University of California, Santa Cruz, CA Received 22 October 2001; Accepted 7 November 2001 Summary: The toc gene of Drosophila melanogaster en- codes a 235-kD polypeptide with a coiled-coil domain, which is highly expressed during oogenesis (Grammont et al., 1997, 2000). We now report the localization of the Toucan protein during early embryonic development. The Toucan protein is present only during the syncytial stages and is associated with the nuclear envelope and the cytoskeletal structures of the syncytial embryo. In anaphase A, Toucan is concentrated at the spindle poles near the minus end of microtubules. This microtubule association is very dynamic during the nuclear cell cycle. Mutant embryos lacking the Toucan protein are blocked in a metaphase-like state. They display abnormal and nonfunctional spindles, characterized by broad poles, detachment of the centrosomes, and failure of migration of the chromosomes. These results strongly suggest that Toucan represents a factor essential for the assem- bly and the function of the syncytial mitotic spindles. genesis 31:167–175, 2001. © 2001 Wiley-Liss, Inc. Key words: Drosophila; embryo; mitosis; microtubules; spindle assembly; mitotic poles; centrosomes; nuclear en- velope; kinesin INTRODUCTION Understanding mitotic spindle assembly represents a ma- jor issue of cell biology. In recent years it has become obvious that this phenomenon relies on cooperative mechanisms between microtubule-based motors and their associated proteins (Heald et al., 1996, 1997; Gaglio et al., 1997; Nedelec et al., 1997; Walczak et al., 1998; Heald, 2000; Sharp et al., 2000b). The general idea (re- viewed in Karsenti et al., 1996) has emerged that chro- matin stabilizes microtubules and that self-organization of the poles is achieved by focusing free microtubule ends with minus end motors. Moreover, proteins such as NuMA (Dionne et al., 1999; Gaglio et al., 1995; Merdes et al., 1996) could belong to a complex involved in main- taining the organization of the minus ends of the spindle microtubules into a morphologic pole. The establishment of spindle bipolarity requires mul- tiple microtubule-based motor activities (Gaglio et al., 1996; Walczak and Mitchison, 1996; Walczak et al., 1998; Sharp et al., 1999b; Sharp et al., 2000a). Depend- ing on the cell type, different combinations of motors and associated proteins are probably needed to organize a mitotic spindle. The early embryo of Drosophila melanogaster repre- sents an interesting case of mitotic spindle organization. This stage is characterized by a rapid succession of 13 metasynchronous divisions occurring in a syncytium. During these divisions, the mitotic spindles are assem- bled and disassembled with extraordinary speed. A ma- jor question is whether these syncytial mitotic spindles, relying only on maternal contributions, are different from the later ones of the cellularized embryo. As fast DNA synthesis at this stage requires multiple replication origins, one can imagine that the extremely rapid assem- bly/disassembly of the syncytial spindles needs special factors such as specialized forms of kinesins or centro- somal proteins. For example, two gamma-tubulins exist in D. melanogaster, and the syncytial mitoses are driven by only one isoform of maternal origin, the Tub37CD (Tavosanis et al., 1997; Wilson and Borisy, 1998). As a candidate for a factor with a specific role in mitosis during the syncytial phase, Toucan is particularly interesting. The toucan locus has been characterized as a recessive female sterile mutation in an enhancer trap mutagenesis. From sequence analysis, toucan is pre- dicted to encode a 235-kD protein with a P-loop domain, several putative phosphorylation sites and a coiled-coil region in the C-terminal domain. Toucan is expressed and required in germline cells (Grammont et al., 1997). Using specific antibodies against Toucan protein, Gram- * Correspondence to: Jean-Louis Couderc, Laboratoire di Biochimie, UMR 384 INSERM, 28 place Henri Dunant BP 38, 63 001 Clermont-Fd Cedex, France. E-mail: jl.couderc@inserm.u-clermont1.fr © 2001 Wiley-Liss, Inc. DOI 10.1002/gene.10019 genesis 31:167–175 (2001)