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The expression, lamin-dependent localization and RNAi depletion phenotype for emerin in C. elegans

Yosef Gruenbaum^1,*, Kenneth K. Lee^2,*, Jun Liu³, Merav Cohen¹ and Katherine L. Wilson^2,

¹ Department of Genetics, The Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel
² Department of Cell Biology, The Johns Hopkins University School of Medicine, 725 N. Wolfe Street, Baltimore, MD 21205, USA
³ Department of Molecular Biology and Genetics, 423 Biotechnology Building, Cornell University, Ithaca, NY 14853, USA

View larger version (87K): [in a new window]   Fig. 1. Colocalization and co-immunoprecipitation of endogenous Ce-emerin and Ce-lamin in wild-type C. elegans embryos. (A) Panels show indirect immunofluorescence staining of wild-type C. elegans embryos with antibodies against endogenous Ce-emerin (red; serum 3272), endogenous Ce-lamin (green), and the overlap of both signals (yellow). Bar, 10 µm. (B) Ce-emerin and Ce-lamin are co-immunoprecipitated from C. elegans embryonic lysates by immune (I) but not preimmune (P) antibodies raised against an N-terminal peptide (serum 3930) or a C-terminal peptide (serum 3932) of Ce-lamin. Shown is a western blot of immunoprecipitates probed with antibodies against Ce-emerin (see Materials and Methods). X indicates a blank lane.

View larger version (113K): [in a new window]   Fig. 2. Ce-emerin is expressed throughout development and is ubiquitous in adult C. elegans. Shown are samples stained for DNA and stained by indirect immunofluorescence for endogenous Ce-emerin (serum 3272) and endogenous Ce-lamin. (A) Triple-staining of early embryos (`e'), plus adjacent adult tissue including the gonad (`g'). (B) Double-staining of L1 (left) and L3 (right) larvae for endogenous Ce-lamin and Ce-emerin, as indicated; anterior regions are oriented on the left, and posterior on the right. (C) Adult gonad double-stained for DNA and endogenous Ce-emerin, showing Ce-emerin-positive eggs and embryos. Bars, 10 µm.

View larger version (83K): [in a new window]   Fig. 3. Ce-emerin is not required to localize Ce-lamin, Ce-MAN1, UNC-84 or nuclear pore complexes. The panels show double-staining by indirect immunofluorescence for endogenous Ce-lamin (right panels), plus either Ce-emerin, Ce-MAN1, UNC-84 or nuclear pore complex proteins (NPCs). (A) Staining for Ce-emerin (emerin) and Ce-lamin (lamin) in uninjected wild-type embryos (WT; top row) and in emerin(RNAi) embryos (bottom row). (B) Double staining for each indicated marker on the left, and Ce-lamin on the right, in emerin(RNAi) embryos. Bars, 10 µm.

View larger version (83K): [in a new window]   Fig. 4. RNAi-induced loss of Ce-emerin protein persists into adulthood. L1 larvae that developed from emerin (RNAi) embryos were double-stained by indirect immunofluorescence for endogenous Ce-lamin (green), endogenous Ce-emerin (red), or both (combined). The cuticle, a non-cellular structure, stained red nonspecifically. Ce-emerin was not detected in any cells or tissues examined, including the pharynx (P; upper row) and gonad (G; bottom row). The pharynx has 20 muscle cells, 20 neurons, 9 epithelial cells, and 9 specialized epithelial cells named marginal cells (White, 1988). Ce-lamin served as the positive control for antibody penetration during staining. Bar, 10 µm.

View larger version (95K): [in a new window]   Fig. 5. Nuclear envelope localization of Ce-emerin depends on Ce-lamin. Ce-lamin-deficient (lmn-1(RNAi)) embryos were stained for DNA and double-stained by indirect immunofluorescence for endogenous Ce-lamin and Ce-emerin. (Top panels) Triple staining of an embryo depleted of Ce-lamin by injecting lmn-1 dsRNA (`injection'). (Bottom panels) Triple-stained embryos were inefficiently depleted of Ce-lamin by the `feeding' method (see Materials and Methods), producing nuclei with nearly normal amounts of Ce-lamin (arrow), or reduced levels of Celamin plus mildly reduced (barred arrowhead) or severely reduced (arrowhead) levels of Ce-emerin at the nuclear envelope. Nuclear envelope rim-staining for Ce-emerin decreased in parallel with Ce-lamin. Bars, 10 µm.