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First published online 1 June 2004
doi: 10.1242/jcs.01152

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Impairment of SHOX nuclear localization as a cause for Léri-Weill syndrome

Institute of Human Genetics, University of Heidelberg, Im Neuenheimer Feld 366, 69120 Heidelberg, Germany

View larger version (26K): [in a new window]   Fig. 1. Deletion constructs of SHOX cDNA and their subcellular localization. (A) Schematic representation of deletion constructs generated in pcDNA4/HisMax and their subcellular localization. Nu, nuclear; Cy, cytoplasmic. (B) Fluorescent images of immunostained slides. The first and third rows of images show the cell nuclei stained with Hoechst dye (blue) and the second and fourth rows of images show green staining for the SHOX deletion constructs immunostained with anti-His (green) antibodies. Note that all the constructs containing the homeodomain are localized in the nucleus. Arrows indicate subcellular localization of the SHOX deletion constructs (green) and the nuclei (blue) in corresponding cells.

View larger version (36K): [in a new window]   Fig. 2. Deletion fusion constructs and their subcellular translocation. (A) Schematic representation of the deletion fusion constructs of the SHOX homeodomain with RedStar and their subcellular localization. Nu, nuclear; Cy, cytoplasmic. (B) Fluorescent images of cells transfected with the RedStar fusion constructs. All constructs containing the five amino acids AKCRK are localized in the nucleus (blue, Hoechst staining). Arrows indicate the subcellular localization of the RedStar deletion fusion constructs. Red/pink staining is for red fluorescent protein (RFP).

View larger version (63K): [in a new window]   Fig. 3. Analysis of the wild-type SHOX and deletion construct SHOX-NLS and the respective subcellular localization. (A) Schematic representation of the wild-type SHOX and the deletion mutant SHOX-NLS where the NLS has been deleted from the wild-type SHOX. Nu, nuclear; Cy, cytoplasmic. (B) Fluorescent images of immunostained cells. The two upper panels show the cell nuclei stained with Hoechst dye (blue) and the two lower panel shows SHOX immunostaining with rabbit anti-SHOX antibodies (green). The constructs in pcDNA4/TO were overexpressed in U2Os cells. By contrast to the wild-type SHOX, which was found in the nucleus, deletion of the nuclear localization signal from the wild-type SHOX resulted in its cytoplasmic localization. Arrows indicate subcellular localization of the SHOX constructs (green) and the nuclei (blue) in corresponding cells.

View larger version (53K): [in a new window]   Fig. 4. Analysis of the mutant SHOX R173C (C517T) with the insertion construct SHOX/C517T+NLS and the respective subcellular localization. (A) Schematic representation of the missense mutant C517T with the mutation in the NLS, and the insertion mutant SHOX/C517T+NLS where the NLS has been inserted in the mutant SHOX C517T. Nu, nuclear; Cy, cytoplasmic. (B) Fluorescent images of immunostained cells. The two upper panels show the cell nuclei stained with Hoechst dye (blue) and the two lower panels show SHOX immunostaining with rabbit anti-SHOX antibodies (green) in corresponding cells. The constructs in pcDNA4/TO were overexpressed in U2Os cells. The missense mutation is within the nuclear localization signal resulting in its cytoplasmic localization (arrows in the two left panels). Insertion of the nuclear localization signal near the mutated site restored its nuclear localization activity (arrows in the two right panels).

View larger version (50K): [in a new window]   Fig. 5. Western blot analysis using cytoplasmic and nuclear extracts from U2Os cells transiently transfected with wild-type and mutant SHOX constructs. Immunofluorescence results using wild-type and mutant SHOX constructs were confirmed and complemented by western blots using cytoplasmic (Cy) and nuclear (Nu) extracts. Pyruvate kinase isozyme M2 (M2PK) at 57 kDa was used as a cytoplasmic marker and C23/nucleoloin at 116 kDa was used as a nuclear marker to detect the presence of SHOX (33 kDa) in both cytoplasmic or nuclear extracts.

View larger version (40K): [in a new window]   Fig. 6. Interspecies heterokaryon assay using the human osteosarcoma cell line F6, stably transfected with SHOX and the mouse fibroblast cell line NIH-3T3. After induction of SHOX expression, F6 cells were fused to NIH cells to make heterokaryons that were immunostained with anti-SHOX antibodies. (A) Phase contrast image of a representative heterokaryon with two larger F6 nuclei and one smaller NIH-3T3 nucleus (arrows). (B) Hoechst staining of the F6 and NIH-3T3 nuclei. Arrows indicate different nuclear staining for the F6 and NIH-3T3 nuclei that are shown in A. (C) Immunostaining using rabbit anti-SHOX antibodies (green). In more than 100 heterokaryons analyzed, not a single mouse nucleus showed positive staining for SHOX, indicating that once inside the nucleus, SHOX shows no shuttling between the nucleus and the cytoplasm. Arrows indicate positive SHOX staining only in F6, but not in NIH-3T3 nuclei.

View larger version (37K): [in a new window]   Fig. 7. Sequence alignment of the helix III of various paired-related homeodomain proteins. Amino acids from the characterized NLS for the SHOX protein are highlighted and aligned with other paired-related homeodomain proteins (boxed). Arrows at the top indicate the amino acids that are conserved in all the paired-related homeodomain proteins.

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