Figure 6.
The membrane tethering of Ect2-based complexes is essential for NuMA exclusion from the equatorial membrane. (A) Schematic representation of AcGFP (Aequorea coerulescens GFP), and mono FLAG -tagged siRNA-resistant Ect2 full-length (referred to as AcGFP-Ect2r), and Ect2 without its membrane binding (PH and PBC) domain (referred to as AcGFP-Ect2rΔmem). (B) Immunoblot analysis of mitotically synchronized protein extracts made from HeLa Kyoto cells, Kyoto cells that were stably expressing either AcGFP-Ect2r or AcGFP- Ect2rΔmem. The resulting blot was probed with anti-GFP or anti-β-actin antibodies. (C–F) IF analysis of the HeLa Kyoto cells transfected with either control siRNA (C), siRNA against Ect2 (D), HeLa Kyoto cells stably expressing AcGFP-Ect2r and transfected with Ect2 siRNA (E), or HeLa Kyoto cells stably expressing AcGFP- Ect2rΔmem and transfected with Ect2 siRNA (F) for 48 h. Cells were fixed and stained with anti-α-tubulin (green) antibodies. DNA is shown in gray. % on each panel marks the cytokinesis failure as calculated by analyzing the occurrence of binucleated and multinucleated cells (n > 500 cells each from three independent experiments). The scale bar in the panel and following panels represent 10 μm. (G and H) Confocal live-imaging analysis of HeLa Kyoto cells stably expressing AcGFP-Ect2r (G) or AcGFP-Ect2rΔmem (H), which are transiently transfected with mCherry-NuMA and are depleted for endogenous Ect2 by siRNA. The recording was started 26 h post-transfection for control and Ect2 siRNA targeting endogenous Ect2. “0 min” time-point represents metaphase to anaphase transition. Yellow arrowheads depict NuMA localization at the equatorial membrane. (I) Schematic representation of quantification method used for analyzing equatorial membrane enrichment of mCherry-NuMA fluorescence intensity. (J) Equatorial membrane quantification for mCherry-NuMA in cells expressing either AcGFP-Ect2r or AcGFP-Ect2rΔmem and are depleted for endogenous Ect2 as described in I. ns, P > 0.05; **, P < 0.01; ***, P < 0.001 as determined by two-tailed unpaired Student’s t test. n = 10 cells; error bars: SD. Source data are available for this figure: SourceData F6.

The membrane tethering of Ect2-based complexes is essential for NuMA exclusion from the equatorial membrane. (A) Schematic representation of AcGFP (Aequorea coerulescens GFP), and mono FLAG -tagged siRNA-resistant Ect2 full-length (referred to as AcGFP-Ect2r), and Ect2 without its membrane binding (PH and PBC) domain (referred to as AcGFP-Ect2rΔmem). (B) Immunoblot analysis of mitotically synchronized protein extracts made from HeLa Kyoto cells, Kyoto cells that were stably expressing either AcGFP-Ect2r or AcGFP- Ect2rΔmem. The resulting blot was probed with anti-GFP or anti-β-actin antibodies. (C–F) IF analysis of the HeLa Kyoto cells transfected with either control siRNA (C), siRNA against Ect2 (D), HeLa Kyoto cells stably expressing AcGFP-Ect2r and transfected with Ect2 siRNA (E), or HeLa Kyoto cells stably expressing AcGFP- Ect2rΔmem and transfected with Ect2 siRNA (F) for 48 h. Cells were fixed and stained with anti-α-tubulin (green) antibodies. DNA is shown in gray. % on each panel marks the cytokinesis failure as calculated by analyzing the occurrence of binucleated and multinucleated cells (n > 500 cells each from three independent experiments). The scale bar in the panel and following panels represent 10 μm. (G and H) Confocal live-imaging analysis of HeLa Kyoto cells stably expressing AcGFP-Ect2r (G) or AcGFP-Ect2rΔmem (H), which are transiently transfected with mCherry-NuMA and are depleted for endogenous Ect2 by siRNA. The recording was started 26 h post-transfection for control and Ect2 siRNA targeting endogenous Ect2. “0 min” time-point represents metaphase to anaphase transition. Yellow arrowheads depict NuMA localization at the equatorial membrane. (I) Schematic representation of quantification method used for analyzing equatorial membrane enrichment of mCherry-NuMA fluorescence intensity. (J) Equatorial membrane quantification for mCherry-NuMA in cells expressing either AcGFP-Ect2r or AcGFP-Ect2rΔmem and are depleted for endogenous Ect2 as described in I. ns, P > 0.05; **, P < 0.01; ***, P < 0.001 as determined by two-tailed unpaired Student’s t test. n = 10 cells; error bars: SD. Source data are available for this figure: SourceData F6.

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