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First published online 1 April 2003
doi: 10.1242/jcs.00402

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A hydrophilic lamin-binding domain from the Drosophila YA protein can target proteins to the nuclear envelope

Shobana S. Mani^*, Rithwick Rajagopal, Amanda B. Garfinkel, Xiaochun Fan and Mariana F. Wolfner

Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853-2703, USA
^* Present address: Department of Ophthalmology, SUNY Upstate Medical University, Syracuse, NY13210, USA
Present address: Skirball Institute of Biomolecular Medicine, New York University, New York City, NY 10016, USA

View larger version (22K): [in a new window]   Fig. 1. Intracellular distribution of GFP-NLS and GFP-NLS-YA (506-696) fusion in Drosophila S2 cells. Fluorescence images of S2 cells transfected with GFP-NLS or GFP-NLS-YA (506-696). A-C shows a representative cell expressing GFP-NLS. (A) DAPI staining to mark the nucleus. (B) GFP fluorescence in nucleus and cytoplasm (indicated by *). (C) Overlay of A and B. D-G shows a representative field of cells expressing GFP-NLS-YA (506-696). (D) DAPI staining of nuclei (arrowhead indicates a transfected cell). (E) GFP fluorescence from the expressed fusion. (F) Overlay of panels D and E. (G) Higher magnification view of transfected cells expressing GFP-NLS-YA (506-696) showing GFP fluorescence in the nuclear periphery surrounding DNA (red). Bar, 10 µm.

View larger version (57K): [in a new window]   Fig. 2. Nuclear envelope localization and targeting of GFP-NLS-YA constructs in transfected S2 cells. Confocal images of S2 cells that express the listed GFP-NLS-YA constructs. GFP fluorescence was visualized in fixed, permeabilized cells at 24 hours post-induction. The location of GFP fluorescence was determined relative to that of DNA (left panels, propidium iodide staining), lamin Dm0 (middle panels, stained with rabbit -lamin) or actin (to visualize the cytoplasm; right panels, stained with AlexaFluor-594-coupled phalloidin). Bar, 10 µm.

View larger version (9K): [in a new window]   Fig. 3. Summary of nuclear envelope localization and lamin interaction of C-terminal deletions of YA. Regions of the YA C-terminus (light gray box) fused in frame with the GFP coding sequence (dark gray box) and SV40 T-Ag NLS (black box) tested for nuclear periphery location (Figs 1 and 2) or in the yeast two-hybrid system (Table 1) are shown. Nuclear envelope localizations of GFP-NLS-YA fusions are: +, colocalization with lamin; –, presence of the fusion in both nucleus and cytoplasm with no preferential peripheral staining of the nucleus; `misfolded', inability of the fusion protein to enter the nucleus. Interaction of the various deletion constructs with lamin Dm0 in the yeast two-hybrid assay: +, strong interaction; (+), weak interaction; –, no detectable interaction.

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