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First published online 21 March 2006
doi: 10.1242/jcs.02877

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Complexes of syndapin II with dynamin II promote vesicle formation at the trans-Golgi network

Michael M. Kessels^1,*, Jiaxin Dong^2,*,, Wibke Leibig^1,, Peter Westermann^2,¶ and Britta Qualmann^1,#,**

¹ Department of Neurochemistry and Molecular Biology, AG Membrane Trafficking and Cytoskeleton, Leibniz Institute for Neurobiology, 39118 Magdeburg, Germany
² Department of Cell Growth and Differentiation, Max-Delbrück-Centre for Molecular Medicine, 13092 Berlin-Buch, Germany

View larger version (44K): [in a new window]   Fig. 1. Co-sedimentation of dynamin II and syndapin II. Cytosolic proteins from HepG2 cells were centrifuged through a 10-30% glycerol gradient and fractions were analyzed by immunoblotting with the anti-dynamin II antibody C12 (A) and the anti-syndapin II antibody 3685 (B). The corresponding molecular masses were determined by positions of the marker protein catalase (230 kDa), aldolase (160 kDa) and bovine serum albumin (67 kDa), run on parallel gradients.

View larger version (32K): [in a new window]   Fig. 2. Syndapin II interacts with different splice variants of dynamin II through direct association between the SH3 domain and the PRD. (A) Immunoblot analyses of co-precipitations of GFP-tagged full-length and truncated dynamin (Dyn) II proteins overexpressed in HEK293 cells with immobilized GST (–) or a GST fusion protein of the SH3 domain of syndapin II (+). Lysates as well as co-precipitated proteins were analyzed by immunoblotting with anti-GFP antibodies. The three GFP-tagged dynamin II splice variants were all specifically co-precipitated by the syndapin II SH3 domain, whereas dynamin II lacking the PRD or GFP alone did not bind (right panel). (B) A S-peptide-tagged fusion protein containing the dynamin II PRD specifically binds endogenous syndapin II from HepG2 cell extracts, whereas a related fusion protein comprising the dynamin II PH domain does not. Eluates were analyzed by immunoblotting with anti-syndapin II antibody 2521. (C) In vitro reconstitution of the interaction with purified fusion proteins (GST-Syndapin II SH3 and S-Dynamin II PRD) demonstrates that the association of both proteins is direct. SDS-eluted proteins were analyzed by immunoblotting using the anti-dynamin II antibody C12 and anti-GST antibodies to detect the S-PRD and the GST-SH3 fusion proteins, respectively.

View larger version (29K): [in a new window]   Fig. 3. Co-localization of dynamin IIaa-GFP and Flag-syndapin II-l. (A-C) COS-7 cells cotransfected with dynamin IIaa-GFP and epitope-tagged syndapin II-l detected by GFP fluorescence (A) and anti-Flag immunostaining (C), respectively, show a complete spatial overlap of both proteins in the merged image (B; syndapin in red and dynamin in green; overlap appears yellow). Co-localization can be observed both at the perinuclear area and in the periphery of the cells. Insets represent twofold enlargements of boxed areas. Bar, 10 µm.

View larger version (51K): [in a new window]   Fig. 4. Dynamin II and syndapin II interact in vivo. (A) Complexes of dynamin II with syndapin II were immunoprecipitated from HepG2 cell extracts using anti-syndapin II antibodies (3685) and the anti-dynamin (Dyn) II antibody sc-6401, which recognizes an internal sequence of dynamin II, respectively. Eluates were analyzed by immunoblotting using the antibodies 3685 (anti-syndapin II) and C12 (anti-dynamin II). The antibody C12, which specifically recognizes the C-terminus of dynamin II, only immunoprecipitated dynamin II, but did not co-immunoprecipitate syndapin II. (B) The supernatants of the immunoprecipitations were precipitated with methanol and then immunoblotted with anti-dynamin II and anti-syndapin II antibodies. IPs, immunoprecipitates; CoIPs, coimmunoprecipitates.

View larger version (61K): [in a new window]   Fig. 5. Reconstitution of complexes of syndapin II with dynamin II at cellular membranes in vivo. (A-L) Mitochondrially targeted full-length syndapin II-l, detected by anti-Flag immunostaining (A,D,G,J), recruits different GFP-tagged splice variants of dynamin II (B,E,H,K) when cotransfected in COS-7 cells, as also demonstrated by merging the fluorescence channels (C,F,I,L). (A-F) Recruitment of dynamin IIaa-GFP (B,E) by mito-syndapin II-l that, in A, was additionally co-localized with MitoTracker (in blue; merge in A thus appears magenta). (G-I) In vivo reconstitution of protein complexes composed of mito-syndapin II-l and dynamin IIab-GFP. (J-L) Mito-syndapin II-l efficiently recruits dynamin IIbb-GFP. (M-O) By contrast, in cells cotransfected with mito-syndapin II-l P480L (M), no such recruitment was observable, but dynamin IIaa-GFP (N) shows a rather diffuse localization. O represents the corresponding merged image. Insets are twofold enlargements of boxed areas. Dyn, dynamin; mito-Sdp, mito-syndapin. Bars, 15 µm.

View larger version (100K): [in a new window]   Fig. 6. Syndapin II is bound to Golgi-enriched membranes of HepG2 cells and can be localized to the Golgi complex of COS-7 cells by immunofluorescence analysis. (A) After incubation of HepG2 cells at 37°C or at 20°C (Golgi exit block), syndapin II is detected in light membranes (mem.) and in Golgi-enriched membranes but not in heavy membranes sedimenting through 1.2 M sucrose. 30 µg membrane proteins were separated by SDS-PAGE, blotted and analyzed with anti-syndapin II antibody 3685. (B-J) Syndapin II (Sdp II) co-localizes with the Golgi marker syntaxin 6 in both untransfected COS-7 cells and in cells transfected with syndapin II-l, as seen well in the merged images (D-J; syndapin II in green, syntaxin 6 in red). Syndapin II was detected by the syndapin II-specific antibody P339 (B,E,H) and syntaxin 6 was detected with a monoclonal anti-syntaxin 6 antibody (C,F,I). Similar to endogenous syndapin II, overexpressed Xpress-tagged syndapin II-l also shows a perinuclear co-localization with syntaxin 6 (B-D). The two cells expressing Xpress-tagged syndapin II-l shown in B are marked by asterisks. Inserts show a shorter exposure of perinuclear region of the upper transfected cell. (E-G) High magnifications show that endogenous syndapin II (E) co-localizes very well with syntaxin 6 (F). (H-M) After incubation of COS-7 cells with brefeldin A to disrupt the TGN, syntaxin 6 immunostaining was found either to be collapsed into small perinuclear dots or to be dispersed (I,L). The immunostaining of endogenous (H) and overexpressed syndapin II (K) was mainly dispersed and showed no co-localization with the perinuclear syntaxin 6 accumulations formed upon BFA treatment (J,M). Bars, 10 µm.

View larger version (15K): [in a new window]   Fig. 7. Complex formation between syndapin II and dynamin II is required for vesicle formation from isolated Golgi membranes. (A) Vesicle formation from Golgi-enriched membranes of HepG2 cells is inhibited in a dose-dependent manner by the addition of the GST-tagged SH3 domain of the dynamin II-binding proteins syndapin II (circles) and amphiphysin II (squares). The assay contained Golgi-enriched membranes, ATP, GTP and 40 µg cytosolic proteins as described in the Materials and Methods. Vesicles formed were sedimented and the radioactivity of newly synthesized proteins that were packed into the vesicles was determined. The value obtained under standard conditions is arbitrarily set to 100. The experiments shown were performed in triplicate and s.d. are given. (B) The anti-syndapin II antiserum 3685 inhibits vesicle formation at Golgi-enriched membranes in a dose-dependent manner (open circles). When syndapin-specific antibodies were depleted from the antiserum 3685, inhibition was markedly reduced (filled squares). (C) Specificities and affinities of the original (left) and the depleted antiserum 3685 (right) are shown by immunostaining of western blots with 75 ng (lanes 1,4) and 15 ng (lanes 2,5) MBP-syndapin II antigen [MBP-Sdp II-I (305-388)] as well as with 30 ng (lanes 3,6) MBP-full-length syndapin I (MBP-Sdp I).

View larger version (37K): [in a new window]   Fig. 8. Trafficking of VSVG-GFP from the Golgi to the plasma membrane is inhibited upon interfering with interactions between dynamin II and syndapin II. (A,B) COS-7 cells transfected with a temperature-sensitive VSVG-GFP showed a strong perinuclear fluorescence after 15 minutes at 32°C (A; start of chase), which declined as fluorescent material was transported to the plasma membrane after 90 minutes at 32°C (B). Magnifications of boxed areas in A and B display the region of interest (gray circle) used for the quantitative image analyses (I). (C,D) Cells co-overexpressing Myc-tagged dynamin IIPRD (Dyn IIPRD) displayed perinuclear accumulations of VSVG-GFP at the start of chase (C) and after 90 minutes at 32°C (D). (E,F) Cells overexpressing the Xpress-tagged syndapin II SH3 domain also show an arrest of VSVG-GFP in the perinuclear region (F). (G,H) By contrast, co-overexpression of full-length syndapin II-l had no negative effect on Golgi-to-PM transport but VSVG-GFP was observed at the plasma membrane after 90 minutes (H). Images were processed by Adobe Photoshop to visualize clearly the different VSVG-GFP distributions. Co-transfected cells are marked by asterisks. Bar, 20 µm. (I) Averaged fluorescence intensities within the perinuclear region of interest after 90 minutes at 32°C expressed as percentage of start values. Fluorescence was measured as 8-bit gray values by unbiased experimenters. 255 corresponds to white, 0 to black. Background values were subtracted. Control (- -): start of chase, 275 cells; 90 minutes, 280 cells. Dynamin (Dyn) IIPRD: start, 83 cells; 90 minutes, 82 cells. Syndapin II SH3 domain (Sdp II SH3): start, 57 cells; 90 minutes, 32 cells. Syndapin II-l full-length (Sdp II-I): start, 66 cells; 90 minutes, 52 cells. Error bars represent standard deviations between independent data sets.

View larger version (32K): [in a new window]   Fig. 9. Acute interference with syndapin functions by introducing anti-syndapin antibodies blocks Golgi-to-PM transport of VSVG-GFP. (A,B) VSVG-GFP accumulated in the Golgi at the start of chase (A) and reached the plasma membrane after 90 minutes (end of chase) in cells treated with BioPorter (BP) and anti-syndapin preimmune (B). (C,D) By contrast, cells treated with BioPorter and anti-syndapin immunoreagent showed an arrest of VSVG-GFP signal in the perinuclear area. Images were processed by Adobe Photoshop to visualize clearly the different VSVG-GFP distributions. Bars, 10 µm. (E) Quantitative analyses of fluorescence signals in the perinuclear regions of interest clearly show the undisturbed Golgi-to-PM trafficking in cells treated with BioPorter (BP alone) or BioPorter plus anti-syndapin preimmune (BP anti-Sdp preimmune), and the inhibition of Golgi exit of VSVG-GFP in cells into which anti-syndapin antibodies (BP anti-Sdp) had been introduced. Error bars represent standard deviations between independent data sets.

View larger version (20K): [in a new window]   Fig. 10. The complex of dynamin II and syndapin II promotes vesicle formation at Golgi-enriched membranes in vitro. The protein fraction 6 (obtained by fractionation of cytosolic proteins of HepG2 cells on a glycerol gradient) that contains complexes of dynamin II with syndapin II, as demonstrated by co-immunoprecipitation with anti-syndapin II antibodies (3685) and immunoblotting with anti-syndapin II and anti-dynamin II antibodies (A), is sufficient for promoting vesicle formation at Golgi-enriched membranes in vitro (B, circles). The stimulatory activity can be removed from fraction 6 by immunoprecipitation with anti-syndapin II antibodies (B, squares).