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First published online 30 September 2008
doi: 10.1242/jcs.032847

Journal of Cell Science 121, 3413-3421 (2008)
Published by The Company of Biologists 2008
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Murine CENPF interacts with syntaxin 4 in the regulation of vesicular transport

Ryan D. Pooley1,*, Katherine L. Moynihan1,*, Victor Soukoulis1, Samyukta Reddy1, Richard Francis2, Cecilia Lo2, Li-Jun Ma3 and David M. Bader1,{ddagger}

1 Stahlman Cardiovascular Research Laboratories, Program for Developmental Biology, and Department of Medicine, Vanderbilt University Medical Center, Nashville, TN 37232-6300, USA
2 Laboratory for Developmental Biology, NIHLBI, Bethesda, MD 20892-1583, USA
3 Department of Pathology, Vanderbilt University Medical Center, Nashville, TN 37232-2561, USA


Figure 1
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  		Fig. 1. Identification of syntaxin 4 as a murine CENP-F-interacting protein. (A) A Y2H screen was conducted and described previously (Pooley et al., 2006Go). One of the interacting proteins with murine CENP-F (mCENP-F) was identified as syntaxin 4. The plasmid was isolated from a colony that survived on QDO media and subsequently sequenced. The resulting sequence was identified as the C-terminal 144 aa of syntaxin 4, labeled Y2HS4. The N-terminal 474 aa region of murine CENP-F (NTmCENP-F), was further characterized as being the region of CENP-F sufficient for syntaxin 4 interaction by Y2H. (B) Positive associations grew on QDO medium and exhibited blue color upon testing for β-Gal. As a positive control, growth was indicated by yeast transformed with pGBKT7-53 and pGADT7-T. The previously described interaction of NTmCENP-F with SNAP-25 was also used as a positive control (Pooley et al., 2006Go). The negative control with yeast co-expressing pGBTK-53 and the empty vector pGADT7 demonstrated no growth on the medium. The test interaction clearly demonstrates that NTmCENP-F does associate with the Y2HS4 portion of syntaxin 4 in yeast.

 

Figure 2
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  		Fig. 2. Transfected NTmCENPF redistributes in COS-7 cells expressing GFP-syntaxin 4. (A) COS-7 cells singly transfected with NTmCENPF show a cytoplasmic distribution of the protein (red) with a high perinuclear distribution. (B) Cells singly transfected with GFP-syntaxin 4 (green) show a significantly different distribution at defined foci throughout the cell. (C-E) When cells are cotransfected with syntaxin 4 and NTmCENPF, syntaxin 4 remains at the multiple intracellular foci, and NTmCENPF is redistributed to the same foci occupied by GFP-syntaxin 4, as seen in the merged image (E). DAPI (blue) was used to visualize the nuclei. (F) COS-7 cells were transfected with NTmCENPF-myc and GFP-syntaxin or with GFP-syntaxin 4 alone for a negative control. An immunoprecipitation was conducted with {alpha}-myc antibody, and blots were probed with {alpha}-GFP antibody. Input lanes show transfected protein expression in the lysate. GFP-syntaxin 4 was precipitated in the presence of NTmCENPF-myc. The singly transfected control shows there was no spurious binding of GFP-syntaxin 4 to the beads.

 

Figure 3
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  		Fig. 3. Endogenous CENPF and syntaxin 4 colocalize in murine cells. (A) There is significant overlap of endogenous murine CENPF (mCENPF) and syntaxin 4 expression throughout the cytoplasm in C2C12 myoblasts (merge). There is a high degree of colocalization in the perinuclear region, but CENPF has a broader distribution extending further into the cell periphery. (B) 3T3-L1 pre-adipocytes also demonstrate similar expression patterns of CENPF and syntaxin 4 to that seen in C2C12 cells. However, there is higher expression of both endogenous proteins at the cell periphery than in C2C12 myoblasts. (C) 3T3-L1 cells were differentiated as described in Materials and Methods. Both endogenous CENPF and syntaxin 4 appear to have high levels of expression distributed throughout the cells. All images are from confocal microscopy. Bar, 10 µm. (D) Murine CENPF forms an endogenous complex with syntaxin 4. Endogenous protein complexes were analyzed using C2C12 cell lysates for co-immunoprecipitation analysis with Sepharose beads alone, {alpha}-SNAP-25 antibody, syntaxin 4 antiserum, or IgG antibody alone. After precipitation, elution and western blotting, the blot was probed with {alpha}-CENPF antibody. Lane 1 demonstrates the presence of CENPF in the lysate. Lane 2 demonstrates the absence of precipitation with beads alone. Lane 3 is a positive control, showing that CENPF precipitates with SNAP-25. Lane 4 shows that CENPF precipitates with syntaxin 4. Lane 5 demonstrates the lack of precipitation with non-immune IgG.

 

Figure 4
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  		Fig. 4. N-terminal CENPF expression localizes to foci containing both TGN and recycling endosome markers. NTmCENPF was transfected into C2C12 cells and markers for SNAREs, TGN and recycling endosomes were immunolabelled. (A) Syntaxin 4 did not colocalize with NTmCENPF-myc, but there was a redistribution of the protein in cells that were transfected. (B-D) SNAP-25, golgin and VAMP2 did colocalize with NTmCENPF. NTmCENPF immunofluorescence is shown in green; endogenous markers are in red. DAPI was used to visualize the nuclei (blue). Bar, 10 µm.

 

Figure 5
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  		Fig. 5. Transiently expressed NTmCENPF and syntaxin 4 colocalize at the TGN in COS-7 cells. COS-7 cells were cotransfected with NTmCENPF and GFP-syntaxin 4, and only those cells expressing both transient proteins were analyzed. (A) NTmCENPF and GFP-syntaxin 4 colocalize with the TGN marker golgin-97. (B) VAMP2 also colocalizes to a high degree with NTmCENPF and GFP-syntaxin 4. (C) VAMP3 does not demonstrate any significant redistribution to NTmCENPF–GFP-syntaxin 4 foci. (D) Rab11a, a marker of recycling endosomes, does not demonstrate noticeable colocalization. (E) Early endosomes, stained with EEA1, also show no significant redistribution to NTmCENPF–GFP-syntaxin 4 foci. Therefore, the NTmCENPF–GFP-syntaxin 4 complex is specific for localization at the TGN. NTmCENPF staining is indicated in blue, GFP-syntaxin 4 in green and the third marker, as indicated, is in red. Bar, 10 µm.

 

Figure 6
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  		Fig. 6. NTmCENPF expression interferes with cell coupling. (A) Cultured 3T3 fibroblasts were transfected with control GFP or NTmCENPF and GFP-SNAP-25 or NTmCENPF and GFP-syntaxin 4 and living, transfected cells were loaded with sulforhodamine101 dye (dark red cell with yellow spot). The first and second tier cells are outlined to quantify transfer; first tier cells touch the injected cell and second tier cells are those dye-transferred cells not touching the injected cell. (B) Percentage of dye spread was quantified according to methods described and the cells expressing NTmCENPF showed significantly less coupling at the second tier level.

 

Figure 7
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  		Fig. 7. Depletion of murine CENPF alters GLUT4 trafficking. (A) 48 hours post-MO treatment, cells were processed according to described methods. Normalized to SC cell populations, cells with MO addition had a 50% decrease in radio-labeled glucose trafficking to the plasma membrane (measured as disintegration per minute; dpm). Data are normalized against the SC counts and are shown as means ± s.e.m. from three independent experiments. *One sample Student's t-test, P<0.01 versus SC control. (B) 3T3-L1 adipocytes were differentiated and depleted of mCENPF by addition of MO. As shown previously (Ashe et al., 2004Go; Pooley et al., 2006Go; Soukoulis et al., 2005Go), MO addition is specific to mCENPF depletion as demonstrated by western blot. MO treatment also had no effect on tubulin, syntaxin 4, GLUT4 or β-actin levels. (C) 3T3-L1 adipocytes were differentiated, treated with either standard control or mCENPF MO, and assayed for glucose uptake as noted in the text. Control (SC) and experimental (MO) treatments were conducted in the presence (+) and absence (–) of insulin. Glucose uptake values were normalized to SC+ and shown as percentages of that value. There is a statistically significant difference between SC– and SC+ populations (P<0.01; indicated by *), SC– and MO– populations (P<0.001; indicated by **), and SC+ and MO+ populations (P=0.0001; indicated by ***). There was not a statistically significant difference between the MO– and MO+ populations.

 

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© The Company of Biologists Ltd 2008