Home » Apoptosis Inducers » COS-1 cells were transfected with constructs expressing BICD2, mycC GFPCp50 or both proteins

COS-1 cells were transfected with constructs expressing BICD2, mycC GFPCp50 or both proteins

COS-1 cells were transfected with constructs expressing BICD2, mycC GFPCp50 or both proteins. Struhl, 1989), which is usually highly conserved from to man and has two homologues in mammals, BICD1 (Baens and Marynen, 1997) and BICD2 (KIAA0699). In is essential for the establishment of CPHPC oocyte identity, as well as for the determination of the oocyte anteriorCposterior axis and its dorsalCventral polarity (Suter et al., 1989; Wharton and Struhl, 1989; Suter and Steward, 1991; Swan and Suter, 1996). Mutations in disrupt the proper accumulation and distribution of factors important for oocyte differentiation and patterning, and affect the organization and polarization of the microtubule network during oogenesis (Suter et al., 1989; Theurkauf et al., 1993; Mach and Lehmann, 1997). Based on genetic data and the localization of Bic-D protein, it has been suggested that it constitutes a part of the microtubule-dependent mRNA transport or anchoring mechanism (Swan and Suter, 1996; Mach and Lehmann, 1997; Swan et al., 1999). One of the major components of the intracellular transport machinery is usually cytoplasmic dynein, a minus-end-directed, microtubule-based motor. It is a large protein complex, which requires the activity of another multisubunit complex, dynactin, for most of its known cellular functions (for review see Karki and Holzbaur, 1999). Dynactin consists of two structural domains: an actin-like CPHPC backbone, thought to be responsible for cargo CPHPC attachment, and a projecting shoulderCsidearm that interacts with cytoplasmic dynein as well as with microtubules. The shoulderCsidearm complex contains p150Glued, dynamitin (p50) and p24 subunits, while the actin-like backbone contains Arp1, CapZ, p62, Arp11, p27 and p25 (Eckley et al., 1999). Genetic analysis in suggests that Bic-D functions in a transport pathway that involves cytoplasmic dynein and dynactin (Swan et al., 1999). This is in line with the fact that this distribution of Bic-D at different stages of oogenesis resembles the localization of the minus ends of microtubules (Mach and Lehmann, 1997) and of cytoplasmic dynein and dynactin (Li et al., 1994; McGrail et al., 1995). Bic-D has been shown to interact with the gene product (Mach and Lehmann, 1997). In addition, yeast two-hybrid analysis has suggested an association of Bic-D with lamin Dm0 (Stuurman et al., MGC79398 1999). How these interactions relate to the proposed role of Bic-D and how Bic-D acts in the dyneinCdynactin pathway are currently unclear. Recently, we isolated mouse BICD2 in a CPHPC yeast two-hybrid screen, using the microtubule binding protein CLIP-115 as bait (C.Hoogenraad, A.Akhmanova, F.Grosveld and N.Galjart, in preparation). These data suggest that, similar to Bic-D, BICD2 could also be involved in microtubule-dependent transport. Here we demonstrate an conversation between mammalian BICD2 and the dyneinCdynactin complexes, and we show that BICD2 associates with membranous organelles. We propose that BICD proteins play a direct role in dynein-mediated transport and that this function is usually conserved from to mammals. Results Association of BICD2 with dynein and dynactin Mammalian BICD2 is usually a coiled-coil protein, which, like Bic-D (Stuurman et al., 1999), contains three segments with multiple heptad repeats (Physique?1A). The C-terminal segment 3 shows the highest degree of evolutionary conservation (Physique?1A). In order to characterize BICD2, we generated polyclonal antibodies against the N-terminal a part of BICD2 [glutathione Bic-D (accession No. “type”:”entrez-protein”,”attrs”:”text”:”P16568″,”term_id”:”12644132″,”term_text”:”P16568″P16568) is usually indicated. Antisera were directed against the N-terminal two segments of BICD2 (GSTCNBICD2, antiserum #2293, amino acids 77C637) or the C-terminal segment (GSTCCBICD2, antiserum #2298, amino acids 631C821). (B) Specificity of the BICD2 antibodies. Protein extracts, prepared from mock (C) or BICD2 (+) transfected COS-1 cells, were analysed by western blotting, using #2293 or #2298 antibodies, or antibodies pre-incubated with their corresponding antigens (lanes, marked block). Around 5-collapse much less proteins draw out was packed in the entire case from the BICD2 transfections, so the sign in the transfected lanes had not been too intense weighed against the non-transfected examples. (C) Equal proteins levels of post-nuclear components (PNS), high-speed supernatant (sup.) and pellet fractions of COS-1 cells, incubated with or without 1% Triton X-100, had been analysed by traditional western blotting with antiserum #2293. (D) Coomassie Blue-stained 8% SDSCpolyacrylamide gel of immunoprecipitates from HeLa cells, using anti-BICD2 antibody #2298 or pre-immune serum #2298 (IgG). The main proteins bands had been excised through the gel and put through mass spectrometry. Determined protein, using their accession amounts, are demonstrated on the proper. Molecular pounds markers are indicated. (E) Co-immunoprecipitation of cytoplasmic dynein and dynactin with BICD2 antisera. Lysates of HeLa.