J Biol Chem

J Biol Chem. and early embryos. Multiphoton microscopy of embryos produced by these worms exposed the time course of child centrosome appearance and growth and the differential behavior of centrosomes destined for germ collection and somatic blastomeres. To study the part of -tubulin in nucleation and corporation of spindle microtubules, we used RNA interference (RNAi) to deplete but is required for the normal corporation and function of kinetochore and interpolar microtubules. Intro Centrosomes, the complex and dynamic organelles that serve as microtubule-organizing centers in animal cells, have intrigued biologists for more than a century (Wilson, 1925 ). They generally contain a pair of centrioles surrounded by a meshwork of pericentriolar material (Kellogg fail to assemble spindles (Oakley egg components (Joshi impair but do not completely block the assembly of mitotic spindles (Horio early embryos. For this investigation and to advance the study of microtubule-dependent processes in general, we developed methods to express and observe -tubulin, ST271 -tubulin, and histone H2B fused to green fluorescent protein (GFP) in living embryos. The large size (30 50 m) and transparency of ST271 genome sequence (Consortium, 1998 ) and a collection of sequence-tagged cDNA clones (Y. Kohara, personal communication), makes functionCdisruption studies in the early embryo a powerful analytical Rabbit Polyclonal to ALK approach. Our results suggest that centrosomes do not require -tubulin for microtubule nucleation and growth. However, it appears that centrosomes do require -tubulin to generate microtubules capable of participating in bipolar spindle assembly and function. MATERIALS AND METHODS Worm Strains strains were maintained as explained by Brenner (1974) . N2 variety Bristol was utilized for RNAi analysis and was the wild-type parent of all GFP strains generated. Strain DG800, (III;IV), carries a deficiency that removes the gene encoding -tubulin (Furuta ST271 (Hercules, CA) MRC600 scanning confocal microscope and manipulated to generate numbers in NIH Image (version 1.62f, developed by Wayne Rasband, National Institutes of Health, and available on the Internet at http://rsb.info.nih.gov/nih-image/) and Photoshop (Adobe Systems, Palo Alto, CA). Open in a separate window Number 2 Positioning of -tubulin sequences. (A) Sequences are from (HS; GenBank accession quantity “type”:”entrez-nucleotide”,”attrs”:”text”:”AF225971″,”term_id”:”6970072″AF225971), (XL; “type”:”entrez-nucleotide”,”attrs”:”text”:”M63446″,”term_id”:”214164″M63446), (DM; “type”:”entrez-nucleotide”,”attrs”:”text”:”AJ010552″,”term_id”:”3451556″AJ010552), (AN; “type”:”entrez-nucleotide”,”attrs”:”text”:”X15479″,”term_id”:”2362″X15479), and (CE, “type”:”entrez-protein”,”attrs”:”text”:”CAA80164″,”term_id”:”3877823″CAA80164.1). Amino acids on black backgrounds are shared by three or more of the -tubulins demonstrated. The open boxes enclose the peptide sequences used to generate antibodies. (B) Grid showing the percent amino acid identities between pairs of -tubulins. Generation of ST271 Transgenic Worms Expressing GFP::Tubulins and GFP::Histone Sequences from your gene were used to construct a vector designed to communicate GFP fusion proteins in the adult germ collection and in early embryos. A PCR-based strategy, with the use of sequence info from Y49E10 (Consortium, 1998 ), was used to clone a 7.7-kb genomic fragment containing the gene (Reese open reading frame with GFP from pPD103.87, which contains the S65C mutation and 3 synthetic introns (A. Open fire, S. Xu, J. Ahnn, and G. Seydoux, personal communication). The GFP is definitely followed by a unique upstream of the DNA (linearized with (2001) was used to generate a strain comprising (Thornwood, NY) Axioplan microscope. Images were captured with the use of NIH Image (version 1.62f) and a Hamamatsu C2400-00 video camera and video controller with an Argus-10 image processor (Hamamatsu City, Japan). Positions of pronuclei and their migration rates were determined with the use of a tracking system for NIH Image written by A. Pilling (unpublished data). Observation of GFP fluorescence was carried out on a multiphoton fluorescence excitation microscope built by J. White and D. Wokosin (University or college of Wisconsin), with the use of a 60 oil immersion objective and 900-nm excitation (from a.