Homologue T24F1.two, which we named SAMP-1. The mammalian putative orthologue was initially identified in a proteomic screen for integral components from the inner nuclear membrane and named NET5 (Schirmer et al., 2003). NET5 was subsequently named Samp1 and shown to play a role in positioning nuclei in polarizing NIH 3T3 cells. Nuclear migration in polarizing mouse NIH3T3 cells relies on SUN-KASH bridges to couple moving actin arrays in the cytoplasm to the nucleoskeleton (Luxton et al., 2010; Folker et al., 2011). This nuclear migration also calls for Samp1, which partially colocalizes and coimmunoprecipitates with SUN proteins in transmembrane actin-associated nuclear (TAN) lines (Borrego-Pinto et al., 2012). The homologous protein in Schizosaccharomyces pombe, Ima1, interacts in yeast two-hybrid assays with the SUN protein Sad1 and has been implicated within the maintenance of nuclear morphology (Hiraoka et al., 2011). Previously a broad bioinformatics study predicted that C. elegans EMA401 SAMP-1 will be a component from the nuclear envelope and confirmed this localization in the early embryo using a transgenic SAMP-1::GFP fusion protein (Gunsalus et al., 2005). Even so, nothing else is identified concerning the function of C. elegans SAMP-1. WeSUN amin interactions to move nucleiFIGURE 6: samp-1(RNAi) animals have a weak nuclear migration defect. (A ) Embryos had been stained for SAMP-1 localization. Lateral views, with anterior left and dorsal up. Scale bars, 10 m. For each pair of pictures, SAMP-1 immunostaining is shown in white around the left and in red on the correct when it is merged with DAPI staining of nuclei in blue. (A, B) An early wild-type embryo. (C, D) A later, pre omma-stage embryo. Arrowhead points to PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/21269259 a hyp7 precursor nucleus. (E, F) A samp-1(tm2710)-null embryo is shown to demonstrate specificity with the antibody. (G) Numbers of nuclei in the dorsal cords of wild-type or samp-1(tm2710)(+); samp-1(RNAi) L1 larvae. Each and every gray dot represents a person animal. The mean and 95 CI error bars are shown. (H, I) DIC and GFP photos showing two hyp7 nuclei abnormally inside the dorsal cord (arrows) of a samp-1(tm2710)(+); samp-1(RNAi) L1 larva. The dorsal cord is up and is demarcated by the dotted line. Scale bar, 10 m.for that reason set out to examine the role of C. elegans SAMP-1 in nuclear migration. We initial characterized the intracellular localization pattern of endogenous SAMP-1 to see regardless of whether it was plausible that SAMP-1 functions at the nuclear envelope through nuclear migration in embryonic hyp7 precursor. Antibodies were raised against the C-terminus of SAMP-1. Anti AMP-1 antibodies recognized a band with the predicted size on a Western blot. The band intensity was considerably lowered in samp-1(RNAi) extracts (Supplemental Figure S2). SAMP-1 antibodies localized strongly to a ring around 4,6-diamidino-2-phenylindole (DAPI) tained nuclei, consistent with nuclear envelope staining, in all cells of wild-type early embryos but not in samp1(tm2710) probably null embryos (Figure six and Supplemental Figure S2). Hence the antibody is specific for SAMP-1, with a localization pattern anticipated for any nuclear membrane protein. Even though we didn’t test the specific localization inside the nuclear envelope, we hypothesize that SAMP-1 is definitely an inner nuclear membrane protein according to the published localization on the mouse orthologue, Samp1 (Buch et al., 2009). In later embryos in the time of hyp7 nuclear migration, SAMP-1 localization in the nuclear envelope was much less powerful and limited.