Homologue T24F1.two, which we named SAMP-1. The mammalian putative orthologue was initially located in a proteomic screen for integral elements of your inner nuclear membrane and named NET5 (Schirmer et al., 2003). NET5 was subsequently named Samp1 and shown to play a part 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 within the cytoplasm to the nucleoskeleton (Luxton et al., 2010; Folker et al., 2011). This nuclear migration also demands 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 using 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 SAMP-1 would be a component from the nuclear envelope and confirmed this localization inside the early embryo using a transgenic SAMP-1::GFP fusion protein (Gunsalus et al., 2005). However, absolutely nothing else is recognized concerning the function of C. elegans SAMP-1. WeSUN amin interactions to move nucleiFIGURE 6: samp-1(RNAi) animals possess a weak nuclear migration defect. (A ) Embryos were stained for SAMP-1 localization. Lateral views, with anterior left and dorsal up. Scale bars, ten m. For each and every pair of images, SAMP-1 immunostaining is shown in white on the left and in red around the correct when it really 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 on the antibody. (G) Numbers of nuclei inside the dorsal cords of wild-type or samp-1(tm2710)(+); samp-1(RNAi) L1 larvae. Each gray dot represents an individual animal. The mean and 95 CI error bars are shown. (H, I) DIC and GFP photos displaying 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, ten m.as a result set out to examine the role of C. elegans SAMP-1 in nuclear migration. We very first characterized the intracellular localization pattern of endogenous SAMP-1 to see whether or not it was plausible that SAMP-1 functions in the nuclear envelope throughout nuclear migration in embryonic hyp7 precursor. Antibodies had been raised against the C-terminus of SAMP-1. Anti AMP-1 antibodies recognized a band in the predicted size on a Western blot. The band intensity was drastically decreased 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, constant with nuclear envelope staining, in all cells of wild-type early embryos but not in samp1(tm2710) Fatostatin A chemical information probably null embryos (Figure six and Supplemental Figure S2). Hence the antibody is particular for SAMP-1, having a localization pattern anticipated for any nuclear membrane protein. While we did not test the particular localization inside the nuclear envelope, we hypothesize that SAMP-1 is definitely an inner nuclear membrane protein depending on the published localization of your mouse orthologue, Samp1 (Buch et al., 2009). In later embryos at the time of hyp7 nuclear migration, SAMP-1 localization at the nuclear envelope was significantly less robust and restricted.