To a subset of cells that incorporated hyp7 precursors (Figure 6, C and D). In mammalian tissue culture, Samp1 requires lamin AC for localization to the nuclear envelope (Borrego-Pinto et al., 2012). It has also been demonstrated that C. elegans UNC-84 requires LMN-1 for nuclear envelope localization (Lee et al., 2002). Surprisingly, SAMP-1 localized towards the nuclear envelope in lmn-1(RNAi) embryos2860 C. R. Bone et al.(Figure 7). In both early embryos (Figure 7, A and B) and embryos about the time of migration (Figure 7, C and D), SAMP-1 was able to localize in lamin-knockdown animals, whereas UNC-84 was not. LMN-1 staining was applied as a handle to confirm that the lmn-1(RNAi) knockdown was effective. Just after showing that SAMP-1 localizes to the nuclear envelope in migrating nuclei, we tested the extent PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/21269315 to which SAMP-1 functions to move nuclei. Homozygous samp-1(tm2710) was embryonic lethal. We thus fed samp-1(tm2710)+ adults dsRNA against samp-1 for 482 h and examined their offspring. Nuclei abnormally positioned inside the dorsal cord were counted in 266 samp-1(tm2710)+; samp1(RNAi) L1 larvae. On average, 0.four 0.1 nuclei (imply 95 CI) were observed within the dorsal cord (Figure six, G ), which can be statistically substantially when compared with wild kind (p = 0.005 by unpaired t test with Welch’s CGA 279202 Purity & Documentation correction). Occasionally, samp-1(RNAi) L1 larvae had up to five nucleiworm that failed to migrate (Figure 6G). We for that reason concluded that samp-1 plays a little but significant role in nuclear migration.DISCUSSIONThe benefits presented here combine genetic analyses, time-lapse imaging of nuclear migration, and a yeast two-hybrid screen. With each other the data supply mechanistic insights into both the molecular interaction involving the SUN protein UNC-84 and lamin and the functional implications of disruption of this interaction through nuclear migration. We showed that alleles disrupting the N-terminal nucleoplasmic domain of UNC-84 led to an intermediate nuclearMolecular Biology on the CellFIGURE 7: SAMP-1 localizes independently of LMN-1. (A ) Embryos were stained for SAMP-1 and UNC-84 localization. Lateral views, with anterior left and dorsal up. For every row, SAMP-1 immunostaining is shown in white inside the left column and in red on the proper when all channels are merged. UNC-84 is shown in white within the second column in the left and in green when merged. DAPI staining of nuclei is shown in white in the third column and in blue when merged. (A) An early embryo fed bacteria containing the empty L4440 vector as manage. (B) An early embryo fed lmn-1(RNAi). (C) A later, pre omma-stage embryo fed bacteria containing the empty L4440 vector as control. (D) A later, pre omma-stage embryo fed lmn-1(RNAi). Arrows highlight specific nuclei to supply reference points in all 4 columns. Scale bar, ten m.migration defect. We then performed a yeast two-hybrid screen to locate candidate interacting partners of the nucleoplasmic domain of UNC-84. Of interest, we identified an interaction among UNC-84 and the C. elegans lamin protein LMN-1. Moreover, the point mutation UNC-84(P91S) that led to an intermediate nuclear migration phenotype also disrupted the interaction amongst UNC-84 and LMN-1. As predicted from these data, lmn-1(RNAi) led to a comparable nuclear migration defect. Knockdown of yet another member with the nucleoskeleton, samp-1, led to a weak nuclear migration phenotype. Nuclear migrations in unc-84(P91S) embryos had been carefully analyzed by time-lapse imaging to provi.