Supplementary Figure 6: A moderate increase of network density does not block network dynamics or asymmetric spindle positioning.

(a) Control oocytes (Control) and oocytes in which the actin nucleators Fmn2 and Spire2 were moderately overexpressed (Fmn2/Spire2 moderately upregulated) were fixed and stained for F-actin. Scale bar, 5 μm. (b) The mean intensity of F-actin in the cytoplasm (labelled by fluorescent phalloidin) was measured in oocytes as shown in (a). Error bars display s.d. The number of analysed oocytes is specified in italics. P values were calculated with Student’s t-test. (c) Image correlation analysis of actin network dynamics in live oocytes expressing EGFP-UtrCH in control oocytes (Control) and in oocytes moderately overexpressing Fmn2-mCherry and mCherry-Spire2 (Fmn2/Spire2 moderately upregulated). Each image in a time series (time interval 1.57 s) was correlated with the first image. For details see Experimental Procedures. The number of analysed oocytes is specified in italics. Data are mean, with error bars displaying s.d. (d) The percentage of oocytes with a dynamic or static actin network is shown. An image correlation coefficient below 0.5 after 30 s was scored as dynamic and above 0.5 as static. The number of analysed oocytes is specified in italics. (e) Spindle movements (magenta, EGFP-MAP4; microtubules, merged with differential interference contrast [DIC]) in control live oocytes (Control) and oocytes moderately overexpressing Fmn2-mCherry and mCherry-Spire2. White ovals mark initial spindle positions. Scale bar, 10 μm. (f) The efficiency of asymmetric spindle positioning in control oocytes (Control) and in oocytes moderately overexpressing Fmn2-mCherry and mCherry-Spire2 (Fmn2/Spire2 moderately upregulated) is shown. The number of analysed oocytes is specified in italics (aggregation over 2 independent experiments). (g) The spindle was tracked in oocytes in 3D data sets (11 sections, every 8 μm) as shown in (e) and spindle movements were plotted. The number of analysed oocytes is specified in italics (aggregation over 2 independent experiments). Data are mean, with error bars displaying s.d. (h) The spindle speeds were determined from the plots in (g). The number of analysed oocytes is specified in italics (aggregation over 2 independent experiments). Box plot as in Fig. 1e.