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First published online 6 January 2004
doi: 10.1242/jcs.00892

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A role for the spectrin superfamily member Syne-1 and kinesin II in cytokinesis

Department of Cell Biology and Human Anatomy, School of Medicine, University of California, Davis, CA 95616, USA

View larger version (81K): [in a new window]   Fig. 7. KIF3B tail domain blocks Syne-1 localization to the midbody. (a-d) NRK cells transfected with myc-tagged KIF3B tail domain (c,d) or with full-length myc-KIF3B as a control (a,b). Cells were fixed and stained with anti-myc (a,c) to identify transfected cells, and with polyclonal SN120 to localize Syne-1 (b,d). While Syne-1 was concentrated at the midbody in cells transfected with the control full-length KIF3B (arrow) (b), it was significantly depleted from the midbody in cells transfected with KIF3B tail (arrow) (d). (e) To quantify this result, NRK cells transfected with KIF3B tail (tail) or with transfected with full-length KIF3B (full-length) were subjected to digital image analysis (see Materials and Methods). The amount of Syne-1 staining at the midbody (mean pixel density) was measured and is plotted as the ratio of midbody localized Syne-1 staining to background Syne-1 staining. Staining was measured in the cytoplasm directly adjacent to the midbody (error bars=standard deviation; * indicates the statistically significant difference from mock transfected cells by Student's t-test).

View larger version (48K): [in a new window]   Fig. 9. Dominant-negative KIF3B and Syne-1 fragments block syntaxin localization to the midbody. (A) Enriched mitotic NRK cells were either mock-transfected (a,b) or transfected with myc-tagged full-length KIF3B (c,d), myc-tagged KIF3B tail domain (e,f), or HA-tagged Syne-1 fragment GSSF1 (g,h). Cells were fixed and stained with anti-myc (a,c,e) or anti-HA (g) antibodies to identify transfected cells, and with anti-syntaxin (b,d,f,h). As observed with Syne-1 localization under these conditions (Fig. 7), syntaxin was easily detected at the midbody in mock transfected cells (b, arrows) and in cells transfected with the control full-length KIF3B (d, arrow). However, in cells transfected with KIF3B tail (f, arrow) or GSSF1 (h, arrow), syntaxin was significantly reduced from the midbody region. (B) To quantify these results, examples of mock transfected cells, cells transfected with full-length KIF3B (full-length), KIF3B tail (tail) and GSSF1 were subjected to digital image analysis as described for Fig. 7e (* indicates statistically significant difference from mock transfected cells by Student's t-test).

View larger version (52K): [in a new window]   Fig. 1. Expression of the Syne-1 fragment GSSF1 blocks cytokinesis in animal cells. (A) A map of Syne-1 protein showing the positions of the two fragments used in these studies, the kinesin binding fragment GSSF1 and the control fragment GSSF5 that does not bind kinesin. Also shown are the two Golgi binding sites identified previously (light blue), the proposed nuclear envelope binding site (yellow) and the kinesin II binding site identified in this study (dark blue). (B) When COS-7 cells were transfected with GFP-tagged GSSF1 (a) and nuclei were stained with DAPI (b), we found that a large proportion of transfected cells (a and b, arrow) were binucleate, whereas untransfected cells remained mononucleate (a and b, arrowheads). (C) COS-7 cell transfected with GSSF1 (a), stained with DAPI (b), examined by phase contrast microscopy (c) appears as a single binucleate cell (rather than two adjacent mononucleate cells). (D) Fluorescence activated cell sorting (FACS) assay for binucleate cells. HeLa cells were transfected with GFP-GSSF1 (black bar), GSSF5 (striped bar) or mock transfected (white bar). GFP positive cells were isolated by fluorescence activated cell sorting and the proportion of total GFP-positive cell population possessing twice the normal amount of DNA was quantified by propidium iodide fluorescence. Because mock transfected cells do not express GFP, the proportion of the total cell population with twice the normal amount of DNA is given in percent. Error bars indicate standard deviation of triplicate FACS experiments (* indicates the statistically significant difference compared to the control obtained by Student's t-test). (E) DAPI-stained cells transfected with dominant-negative fragment GSSF1 (a-c), the control fragment GSSF5 (d-f).

View larger version (71K): [in a new window]   Fig. 2. (A) COS-7 cells were co-transfected with recombinant, full-length kinesin II tagged with a myc epitope and either recombinant HA-tagged GSSF1 (a,b) or GSSF5 (c). Immune complexes were precipitated with an anti-HA antibody (a,c) or a control IgG (b) and western blots were stained with anti-KIF3B. The recombinant kinesin II can be detected in immunoprecipitates from cells expressing GSSF1 (lane a, *) but not GSSF5 (lane c). GSSF5 was used here as a control because it does not bind KIF3B in the two-hybrid system (not shown). Electrophoretic mobilities of molecular weight markers of 110 and 80 kDa are indicated to the left of the figure. (B) Co-localization of GSSF1 and KIF3B. COS-7 cells were transfected with myc-KIF3B (a) or co-transfected with myc-KIF3B and HA-GSSF1 (b,c) or myc-KIF3B and HA-GSSF5 (d). Fixed cells were stained with antibodies to myc-KIF3B (a,b,d) or HA-GSSF1 (c). KIF3B localizes to the Golgi (a and d, arrows) and microtubules (a and d, arrowheads) in cells expressing KIF3B alone (a) and in cells transfected with both KIF3B and the control fragment GSSF5 (d). By contrast, when KIF3B is co-transfected with GSSF1, KIF3B distribution is altered and both proteins co-localize in punctate cytoplasmic structures. (C) DAPI-stained COS-7 cells transfected with C-terminal tail domain of KIF3B. Several examples of bi- and multinucleate cells are shown (a-c).

View larger version (66K): [in a new window]   Fig. 3. Localization of Syne-1 during mitotic cell division. Mitotic cells were fixed and stained with anti-tubulin (green), polyclonal anti-Syne-1 (red) and DAPI (blue). Localization of Syne-1 during various stages of mitosis (a,c,e,g,i). Triple-staining of the same cells indicate the localization of Syne-1 relative to the mitotic spindle and chromosomes (b,d,f,h,j). At stages before and during metaphase (a-d) Syne-1 is localized at punctate structures typical for Golgi markers (a-d, arrows). After metaphase Syne-1 is localized at a band of material that bisects the central spindle (e-j). This localization persists through cytokinesis where midbody localization is observed (i and j).

View larger version (60K): [in a new window]   Fig. 4. Syne-1 localizes to central spindle and midbody, but not to the contractile ring. Mitotic NRK cells, double-stained with anti-Syne-1 (red) (a,b,d,e,g,h) and either with anti-tubulin (green) (a-c,e) or with fluorescein phalloidin (green) (f,h). (a) Conventional fluorescence microscopy reveals that Syne-1 co-localizes with spindle-microtubules (arrows) and that its distribution during anaphase is restricted along the spindle to the central region. (b) High-resolution image generated by optical sectioning microscopy of an anaphase mitotic spindle, double stained with antibodies to tubulin (green) and Syne-1 (red), demonstrates that Syne-1 associates with the central spindle microtubules in the form of discrete puncta (c-e, arrows). An optical sectioning image of NRK cells entering cytokinesis double stained with antibodies to tubulin (c) and Syne-1 (d) (e is an overlay of c and d) illustrates that Syne-1 localizes to a ring-like structure that surrounds the spindle midbody (d and e, arrows). (f-h) Fluorescein phalloidin stained actin of the contractile ring (f, arrowhead), which encircled the Syne-1 positive structure at the midbody (g, arrow). An Overlay (h) of f and g revealed that Syne-1 (h, arrow) did not localize to the contractile ring (h, arrowhead).

View larger version (110K): [in a new window]   Fig. 5. Association of Syne-1 and kinesin II with central spindle and midbody is detergent resistant. NRK cells were extracted with Triton X-100 before fixation and then stained with anti-Syne-1 antibody (red) (a,b), for KIF3B with anti-kinesin II antibody (red) (c,d) and DAPI (blue). Both Syne-1 and kinesin II localized to detergent insoluble structures associated with the central spindle in anaphase (arrow) (a,c) and the midbody during cytokinesis (arrow) (b,d).

View larger version (97K): [in a new window]   Fig. 6. Association of kinesin II and Syne-1 with the midbody is sensitive to ATP. To determine whether Syne-1 and kinesin II interactions with the mitotic spindle were coupled, distribution of Syne-1 (g,i,k) and kinesin II (a,c,e) was investigated in cells that had been detergent extracted in the presence of 10 mM MgSO4 (a,b,g,h), 10 mM MgATP (c,d,i,j) or 10mM of a non-hydrolyzable ATP anolog AMP-PNP (e,f,k,l). Cells were stained with antibodies against KIF3B (a,c,e) or Syne-1 (g,i,k) and then double-stained with anti-tubulin antibody (b,d,f,h,j,l) to visualize the mitotic spindle. Localization of both kinesin II and Syne-1 are greatly reduced in the presence of ATP (indicated by arrowheads) (c,i) and slightly enhanced in the presence of AMP-PNP (indicated by arrows) (e,k).

View larger version (96K): [in a new window]   Fig. 8. Syntaxin localizes to the central spindle and midbody during anphase and cytokinesis. NRK cells in late stages of mitosis were fixed and stained with antibodies against syntaxin (a,e,f,g) protein disulfide isomerase (PDI) (b), E-cadherin (c). Cells stained for cadherin were also double stained with anti-Syne-1 to visualize the central spindle (arrow) (d). Syntaxin, concentrates in the midbody during cytokinesis (a,e,g), similar to Syne-1 and kinesin II. By contrast, accumulation within the midbody was not observed for PDI (arrow) (b) or E-caherin (arrow) (c). (e-h) Cells extracted with TX-100 before fixation with (f) or without (e) ATP, or with non-hydrolyzable ATP analog AMP-PNP (g,h). Cells treated AMP-PNP were double stained with anti-syntaxin (g) and anti-tubulin (h) antibodies. Notice that, as we have observed with KIF3B and Syne-1, syntaxin association with the midbody is resistant to TX-100 extraction (e, arrow), greatly reduced in the presence of ATP (f, arrow), and stabilized in the presence of non-hydrolyzable ATP (g, arrow)