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First published online 16 March 2004
doi: 10.1242/jcs.01047

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Deletion mutant of FGFR4 induces onion-like membrane structures in the nucleus

Institute for Cancer Research, The Norwegian Radium Hospital, Department of Biochemistry, Montebello, 0310 Oslo, Norway

View larger version (16K): [in a new window]   Fig. 1. FGFR4 and deletion mutants. The extracellular part of FGFR4 is indicated in gray and the intracellular part in white. Numbers refer to amino acid positions and stippled lines indicate the part of the receptor that has been deleted. GFP, green fluorescent protein; SP, signal peptide; TM, transmembrane region. Arrows and `K' indicate the position of the tyrosine kinase domain.

View larger version (64K): [in a new window]   Fig. 2. Intracellular localization of full-length and deletion mutants of FGFR4 in transfected COS-1 cells. Cells transfected with R4wt, R4Tth, R4Eag/BamHI, Ext/R4, Ext/R4/Int and Ext/R4Tth, as indicated, were fixed, labeled with anti-FGFR4 antibodies and FITC-conjugated secondary antibodies, and analysed by confocal microscopy. Two examples of cells transfected with Ext/R4Tth are shown. Scale bar, 10 µm.

View larger version (103K): [in a new window]   Fig. 3. Ext/R4 localizes with the Golgi marker GM130 in a BFA-sensitive juxtanuclear region, whereas R4wt localizes to a BFA-resistant juxtanuclear compartment. COS-1 cells transfected with Ext/R4 (first and second rows) or R4wt (third and fourth rows) were fixed after pretreatment with 2 µg ml–1 BFA for 3 hours (second and fourth rows) or without BFA (first and third rows). The cells were double labeled with anti-FGFR4 antibodies and secondary FITC-conjugated antibodies (first column) and with anti-GM130 antibodies and rhodamine-conjugated secondary antibodies (second column), and analysed by confocal microscopy. Yellow color in the merged images (third column) indicates co-localization. Scale bar, 10 µm.

View larger version (50K): [in a new window]   Fig. 4. Nuclear structures induced by Ext/R4Tth are found in cells at various cell cycle stages. (A,B) COS-1 cells transfected with Ext/R4Tth were incubated with BrdU for 18 hours, then fixed and labeled with anti-BrdU (red) and anti-FGFR4 (green) antibodies. Yellow color indicates overlap between red and green signal. The Ext/R4Tth-transfected cell has not incorporated BrdU in A, whereas the transfected cell in B has. (C) Nuclear spots could be observed in pair of cells, indicating that they arose by division of a single transfected cell. Scale bar, 10 µm.

View larger version (139K): [in a new window]   Fig. 5. Ext/R4Tth forms spots along the NE that stain for protein disulfide isomerase. COS-1 cells transfected with Ext/R4Tth were fixed, labeled with antibodies and examined by confocal microscopy. (A) Three sections of a cell labeled with anti-FGFR4 antibodies and FITC-conjugated secondary antibodies. (Low) Image taken close to the coverslip. (Middle) Image taken through the middle of the nucleus. (Top) Image taken at the top of the cell/nucleus. (B) Double labeling with anti-FGFR4 (green) and anti-nucleoporin p62 (red) antibodies. (C) Double labeling with anti-FGFR4 (green) and anti-PDI (red) antibodies. Yellow color indicates the overlap of red and green signal. Scale bar, 10 µm.

View larger version (55K): [in a new window]   Fig. 6. Ext/R4Tth, R4Tth and R4wt have similar stability in the ER. COS cells transfected with R4wt, R4Tth or Ext/R4Tth were treated with BFA, metabolically labeled with [35S]-methionine for 1 hour and then lysed after 0 hours, 2 hours or 4 hours, as indicated. Receptor was immunoprecipitated with anti-FGFR4 antibody, separated by SDS-PAGE and scanned using a phosphorimager.

View larger version (27K): [in a new window]   Fig. 10. COS-1 cells transfected with Ext/R4Tth were labeled with [33P]-phosphate for 1 hour in the absence or presence of the protein kinase inhibitors BIM, H-89 or staurosporine (St) as indicated. Inhibitors were used at micromolar concentrations as indicated. The cells were solubilized and the receptor was immunoprecipitated with anti-FGFR4 antibodies and analysed by SDS-PAGE and autoradiography.

View larger version (185K): [in a new window]   Fig. 7. Ext/R4Tth-induced intranuclear membranous structures. COS cells expressing R4wt (A) or Ext/R4Tth (B-F) were labeled with antibody against FGFR4 followed by protein-A/gold. (A) R4wt localized mainly to the outer membrane of the NE. (B) Ext/R4Tth localized to the NE. Labeling of both the INM and ONM can be seen. (C-F) Ext/R4Tth was found in the nucleus in onion-like membrane structures. (F) is a magnification of the central region of the image in E). Nu, nucleus. Scale bars, 200 nm.

View larger version (66K): [in a new window]   Fig. 8. The intranuclear membrane structures contain ER markers. COS cells expressing Ext/R4Tth were labeled with antibody against calreticulin and protein-A/gold (A), or double labeled for PDI (small arrowheads) and Ext/R4Tth (large arrowheads) (B). Nu, nucleus. Scale bars, 200 nm.

View larger version (127K): [in a new window]   Fig. 9. Cytosolic membrane stacks in COS-1 cells transfected with Ext/R4Tth. (A) The cytosolic membrane stacks were positive for PDI (small gold particles) and Ext/R4Tth (large gold particles). (B) Cytosolic membrane stack showing its flattened dense structure (plastic-embedded material). (C) Invaginations into the nucleus (arrowheads) were positive for Ext/R4Tth (immunogold labeling) and contained several layers of membrane. Nu, nucleus. Scale bars, 200 nm.

View larger version (64K): [in a new window]   Fig. 11. COS-1 cells transfected with Ext-GFP/R4Tth were examined live by fluorescence microscopy. A region of the nucleus and a region of the cytoplasm was photobleached and the recovery of fluorescence was recorded by time-lapse confocal microscopy. (A,B) Two examples of recovery of nuclear spots after bleaching showing slow recovery of immobile nuclear spots (A) or faster movement of mobile spots from the juxtanuclear region into the nucleus (B).

View larger version (33K): [in a new window]   Fig. 12. Hypothetical models for formation of intranuclear membrane stacks. The cytoplasmic region of Ext/R4Tth (black dots) is assumed to mediate interactions between opposing membrane layers. (A) Ext/R4Tth localized to the INM induces invagination of the INM and further extension and folding of the invaginated membrane into whorled structures. (B) Interactions between cytoplasmic tails of Ext/R4Tth induces tight packing and adherence of ER membrane stacks and also interaction between ER and ONM. Interactions between ER and ONM and between opposing regions of the INM causes incorporation of the membrane stacks into the nucleus. Nu, nucleoplasm.

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