Figure S3.
NuMA exclusion from the equatorial membrane is not merely dependent on Ect2 interaction with the membrane phosphoinositides. (A) Schematic representation of AcGFP (Aequorea coerulescens GFP), and mono FLAG -tagged siRNA-resistant Ect2 full-length (referred to as AcGFP-Ect2r), Ect2 without its membrane binding (PH and PBC) domain (referred to as AcGFP-Ect2rΔmem), and mCherry-tagged C-terminus of Ect2 with membrane binding (PH and PBC) domains together with mutations in the DH domain (referred to as mCherry-Ect2CTGEF4A) that impair GDP/GTP exchange activity but not its membrane binding (see panel C, and Materials and methods, and Su et al., 2011). (B and C) Confocal live-imaging analysis of HeLa Kyoto cells stably expressing AcGFP-NuMA (B) or AcGFP-NuMA cells that are transiently transfected with phosphoinositides binding C-terminus of Ect2 fragment (mCherry-Ect2CTGEF4A). “0 min” time-point represents metaphase to anaphase transition. The scale bar in this panel and the following panels represent 10 µm. (D) Schematic representation of quantification method used for analyzing polar membrane enrichment of AcGFP-NuMA fluorescence intensity. (E) Polar membrane quantification for AcGFP-NuMA in untransfected control cells or in cells expressing mCherry-Ect2CTGEF4A. ns, P > 0.05 as determined by two-tailed unpaired Student’s t test. n = 10 cells; error bars: SD. (F and G) Confocal live-imaging analysis of HeLa Kyoto cells stably expressing AcGFP- Ect2r (F) or AcGFP-Ect2rΔmem (G). The recording was started 26 h post-transfection for Ect2 siRNA targeting endogenous Ect2. “0 min” time-point represents metaphase to anaphase transition. Maximum intensity projected images are shown for each time point. (H) Schematic representation of quantification method used for analyzing midzone enrichment of AcGFP fluorescence intensity in cells stably expressing AcGFP-Ect2r, or AcGFP-Ect2rΔmem and are depleted for endogenous Ect2. (I) Quantification of midzone enrichment of AcGFP signal in cells expressing either AcGFP-Ect2r, or AcGFP-Ect2rΔmem and are depleted for endogenous Ect2. “0 min” time-point represents metaphase to anaphase transition. ns, P > 0.05 as determined by two-tailed unpaired Student’s t test. n = 15 cells; error bars: SD.

NuMA exclusion from the equatorial membrane is not merely dependent on Ect2 interaction with the membrane phosphoinositides. (A) Schematic representation of AcGFP (Aequorea coerulescens GFP), and mono FLAG -tagged siRNA-resistant Ect2 full-length (referred to as AcGFP-Ect2r), Ect2 without its membrane binding (PH and PBC) domain (referred to as AcGFP-Ect2rΔmem), and mCherry-tagged C-terminus of Ect2 with membrane binding (PH and PBC) domains together with mutations in the DH domain (referred to as mCherry-Ect2CTGEF4A) that impair GDP/GTP exchange activity but not its membrane binding (see panel C, and Materials and methods, and Su et al., 2011). (B and C) Confocal live-imaging analysis of HeLa Kyoto cells stably expressing AcGFP-NuMA (B) or AcGFP-NuMA cells that are transiently transfected with phosphoinositides binding C-terminus of Ect2 fragment (mCherry-Ect2CTGEF4A). “0 min” time-point represents metaphase to anaphase transition. The scale bar in this panel and the following panels represent 10 µm. (D) Schematic representation of quantification method used for analyzing polar membrane enrichment of AcGFP-NuMA fluorescence intensity. (E) Polar membrane quantification for AcGFP-NuMA in untransfected control cells or in cells expressing mCherry-Ect2CTGEF4A. ns, P > 0.05 as determined by two-tailed unpaired Student’s t test. n = 10 cells; error bars: SD. (F and G) Confocal live-imaging analysis of HeLa Kyoto cells stably expressing AcGFP- Ect2r (F) or AcGFP-Ect2rΔmem (G). The recording was started 26 h post-transfection for Ect2 siRNA targeting endogenous Ect2. “0 min” time-point represents metaphase to anaphase transition. Maximum intensity projected images are shown for each time point. (H) Schematic representation of quantification method used for analyzing midzone enrichment of AcGFP fluorescence intensity in cells stably expressing AcGFP-Ect2r, or AcGFP-Ect2rΔmem and are depleted for endogenous Ect2. (I) Quantification of midzone enrichment of AcGFP signal in cells expressing either AcGFP-Ect2r, or AcGFP-Ect2rΔmem and are depleted for endogenous Ect2. “0 min” time-point represents metaphase to anaphase transition. ns, P > 0.05 as determined by two-tailed unpaired Student’s t test. n = 15 cells; error bars: SD.

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