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. 2009 Jun 1;20(11):2684–2696. doi: 10.1091/mbc.E08-10-1051

Figure 3.

Figure 3.

Interaction between EB1 and XMAP215 is required for proper spindle assembly. (A) Representative images of fixed metaphase-arrested spindles assembled around replicated sperm in the presence of GST as a control or GST-tagged C-terminal fragment of EB1 (GST-C-EB1). Microtubules (red) are visualized by Cy3-tubulin and DNA (blue) is stained with Hoechst. Bar, 10 μm. (B) Average length of metaphase sperm-spindles in response to treatments as indicated in A. Error bars represent SD (n = 50 bipolar spindles per condition; one representative experiment). (C) Quantification of pole numbers in spindles assembled around sperm in extracts treated as described in A. Error bars represent SD (n = 100 structures per condition; average of two independent experiments). (D) Average spindle image (right) and averaged Cy3-tubulin fluorescence intensity along spindle's pole-to-pole axis (central longitudinal scan, left). Analyzed spindles were assembled as described in A. (E) Representative images of fixed metaphase-arrested spindles assembled around replicated sperm in the presence of GST as a control or GST-C-EB1 and excess of XMAP215. Microtubules (red) are visualized by Cy3-tubulin and DNA (blue) is stained with Hoechst. Bar, 10 μm. (F) Average length of metaphase sperm-spindles in response to treatments as indicated in E. Error bars represent SD (n = 50 bipolar spindles per condition; one representative experiment). (G) Representative images of fixed metaphase-arrested spindles assembled around replicated sperm in untreated extracts or in presence of C-terminal fragment of XMAP215 (C-XMAP215). Microtubules (red) are visualized by Cy3-tubulin and DNA (blue) is stained with Hoechst. Bar, 10 μm. (H) Average lengths of spindles shown in G. Error bars represent SD (n = 50 bipolar spindles per condition; one representative experiment. (I) Quantification of pole numbers in the spindles shown in G. Error bars represent SD (n = 100 structures per condition; average of two independent experiments). J) Average spindle image (right panel) and the plot of averaged Cy3-tubulin fluorescence along spindle's pole-to-pole axis (central longitudinal scan, left). Analyzed spindles were assembled as described in G. (K) Representative images of fixed metaphase-arrested spindles assembled around replicated sperm in untreated extracts or in the presence of C-XMAP215 and excess of EB1. Microtubules (red) are visualized by Cy3-tubulin and DNA (blue) is stained with Hoechst. Bar, 10 μm. (L) Average length of metaphase sperm-spindles in response to treatments indicated in K. Error bars represent SD (n = 50 bipolar spindles per condition; one representative experiment). (M) Quantification of percentage of bipolar, multipolar, and unfocused spindles among those assembled under conditions shown in K. Error bars represent SD (n = 50 structures per condition; average of three independent experiments).