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First published online 28 October 2008
doi: 10.1242/jcs.038950

Journal of Cell Science 121, 3778-3785 (2008)
Published by The Company of Biologists 2008
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Structural determinants of the cellular localization and shuttling of TDP-43

Youhna M. Ayala1,*, Paola Zago1,*, Andrea D'Ambrogio1, Ya-Fei Xu2, Leonard Petrucelli2, Emanuele Buratti1 and Francisco E. Baralle1,{ddagger}

1 International Centre for Genetic Engineering and Biotechnology (ICGEB), 34012 Trieste, Italy
2 Department of Neuroscience, Mayo Clinic College of Medicine, Jacksonville, FL 32224, USA


Figure 1
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  		Fig. 1. Cellular distribution of endogenous and wt FLAG-TDP-43. (A) Diagram of wt TDP-43 showing the mutations introduced in the nuclear localization signal (NLS1 and NLS2). Other distinguishing features of TDP-43 are the two RRM domains (RRM-1 and RRM-2) and the putative nuclear export signal (NES, light green). (B) Distribution of wt TDP-43 and mutant TDP-43 in the nucleus (N) and cytoplasm (C), in non-transfected (endogenous) HeLa cells and HeLa cells and transiently transfected with FLAG-tagged wt and Myc-tagged mutant TDP-43. The detection of p84 and tubulin was carried out as control for nuclear and/or cytoplasmic contamination of the two fractions. (C) Indirect immunofluorescence of U2OS cells non-transfected (endogenous) and transiently transfected with FLAG-TDP-43. wtTDP-43 showed nuclear localization with foci formation, whereas – as expected – disruption of either NLS resulted in cytoplasmic accumulation of the mutants. Similar results were obtained in HeLa cells. The endogenous protein was visualized using anti-TDP-43 polyclonal antibody (ProteinTech) and FITC-conjugated secondary antibody; transfected TDP-43 was detected using either anti-FLAG or anti-Myc monoclonal antibodies and Texas-Red-conjugated secondary antibody. The nuclear membrane was seen using either anti-lamin A/C polyclonal antibody or anti-LAP2β polyclonal antibody, and Texas-Red- or FITC-conjugated secondary antibodies. Scale bars: 10 µm.

 

Figure 2
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  		Fig. 2. TDP-43 nuclear-cytoplasmic shuttling seen by interspecies heterokaryon assays. Transiently transfected U2OS cells were fused with mouse NIH-3T3 cells upon cycloheximide treatment. Arrows indicate mouse nuclei of the fused cell, distinguished from human nuclei using DAPI staining. (A,B) GFP-tagged as well as FLAG-tagged TDP-43 were analyzed in transfected U2OS or HeLa cells. (C,D) FLAG-tagged hnRNP A1 and hnRNP C1/C2 were used as a controls for protein shuttling. In the case of hnRNP C1/C2, GFP-TDP-43 and FLAG-hnRNP C1/C2 were co-transfected to observe specific TDP-43 shuttling in the same fused cell. Scale bars: 10 µm.

 

Figure 3
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  		Fig. 3. Effect of mutations in RRM-1. (A) Positions of the F147L/F149L and {Delta}RRM-1 mutations. (B) Nuclear-cytoplasmic distribution of the mutants as determined by western blotting. Detection of p84 and tubulin was carried out to check for nuclear (p84) or cytoplasmic (tubulin) contamination of the two fractions. (C) Localization of both mutants detected by immunofluorescence microscopy in U2OS cells using anti-FLAG monoclonal antibody. The nuclear membrane was visualized using anti-LAP2β polyclonal antibody. (D) Typical nuclei of transfected cells are shown in detail, similar results were seen in HeLa cells. Scale bars: 10 µm.

 

Figure 4
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  		Fig. 4. Chromatin fractionation of cells expressing endogenous and wt TDP-43, {Delta}RRM-1 and F147L/F149L. Nuclei from transfected cells were digested with micrococcal nuclease and separated into fractions S1, S2 (supernatant) and P (pellet). The distribution of endogenous and wt TDP-43, F147L/F149L and {Delta}RRM1 TDP-43 into the different nuclear fractions was detected by western blotting using antibodies against endogenous TDP-43 and FLAG-tagged transfected proteins. Histone H1 detection was used as control for the different nuclear fractions.

 

Figure 5
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  		Fig. 5. Biochemical fractionation of the C-terminally truncated mutants of TDP-43. (A) Diagram of the different mutants 1-366, 1-315 and {Delta}C. (B) Nuclear-cytoplasmic fractionation of HeLa cells expressing the different C-terminus mutants as detected by immunoblotting. Detection of p84 and tubulin was carried out to check for nuclear (p84) or cytoplasmic (tubulin) contamination of the two fractions.

 

Figure 6
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  		Fig. 6. Immunofluorescence images of U2OS cells transiently transfected with different C-terminal mutations of TDP-43 (1-366, 1-315 and {Delta}C). (A) Anti-FLAG monoclonal antibody and staining of the nuclear membrane were visualized with anti-LAP2β polyclonal antibody. (B) Detection of FLAG-tagged {Delta}C together with DAPI staining of the nuclei. Scale bars: 10 µm.

 

Figure 7
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  		Fig. 7. Disruption of RRM-1 increases TDP-43 binding to the chromatin and/or nuclear matrix. Under normal conditions, TDP-43 is found in the soluble nuclear fraction (S1) and bound to the nuclear matrix portion. Deletion of RRM-1 or mutation of key RNA and/or DNA-binding residues, shifts the equilibrium towards the bound fraction. RNA and/or DNA-binding mediated through RRM-1 may require protein mobility in the soluble fraction of the nucleus, whereas RRM-2 mediates recruitment to chromatin and/or the nuclear matrix.

 

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