Ect2/Cyk4/Mklp1-based complexes are localized to the equatorial membrane during anaphase. (A) Schematic representation of siRNA-resistant Ect2 construct with AcGFP (Aequorea coerulescens GFP)-tag and mono FLAG-tag at the N-terminus (referred to as AcGFP-Ect2^r). (B) Immunoblot analysis of mitotically synchronized protein extracts made from HeLa Kyoto cells, or Kyoto cells that were stably expressing AcGFP-Ect2^r. These extracts were prepared 60 h post-transfection with control siRNA (−) or siRNA against Ect2 (+). The resulting blot was probed with antibodies directed against Ect2 and β-actin. As mentioned, transgenic AcGFP-Ect2^r and endogenous (Endo.) Ect2 were detected on this immunoblot. In this and other panels, the molecular mass is indicated in kilodaltons (kD) and is shown on the left. (C–E) IF analysis of the Kyoto cells that are transfected with control siRNA (C), siRNA against Ect2 (D), or Kyoto cells stably expressing AcGFP-Ect2^r after transfection with siRNA against Ect2 (E) for 60 h. Cells were fixed and stained using anti-α-tubulin (green) antibodies. DNA is shown in gray. Multinucleation percentage (%) is shown for each siRNA condition. (n > 500 cells each from three independent experiments). The scale bar in the panel and the following panels represent 10 μm. (F) Experimental protocol for chemically induced anaphase onset of mitotically synchronized HeLa Kyoto (Control), or HeLa Kyoto cells that are stably expressing AcGFP-Ect2^r by acute Cdk1 inactivation using RO-3306. (G–L) IF analysis of HeLa Kyoto cells (Control) that are either treated with proteasome inhibitor MG132 (G and I) or MG132 and Cdk1 inhibitor RO-3306 (H and J) to synchronize them in the anaphase-like state as explained in F. Cells were fixed and stained using either anti-Plk1 (G and H; green) or anti-Mklp1 (I and J; red) antibodies. The experiment was repeated twice, and the representative cells are shown here. Quantification on the right represents midzone enrichment for Plk1 (K) and Mklp1 (L) in cells treated with MG132 or MG132+RO-3306. ***, P < 0.001 as determined by a two-tailed unpaired Student’s t test. n ≥ 12 cells, error bars: SD. (M) Co-immunoprecipitation (IP) by GFP-Trap in lysates from the AcGFP-Ect2^r expressing cells that are chemically induced in the anaphase-like state. The resulting blots were probed for Cyk4, Mklp1, p150^Glued, and GFP as indicated. IN (1% of total), IP: 20% of the total. Please note that for GFP detection in the IP fraction, only 3% of IP fraction was loaded. Note that AcGFP-Ect2^r interacts with Cyk4, and Mklp1 (centralspindlin complex), but not with dynein interacting dynactin subunit p150^Glued. (N) Schematic representation of quantification method used for analyzing equatorial membrane enrichment of AcGFP-Ect2^r fluorescence intensity. (O–Q) Images from confocal live-imaging analysis of HeLa Kyoto cells stably expressing AcGFP-Ect2^r (green) that are transfected with control siRNA (O) or siRNA against Cyk4 (P) or Mklp1 (Q). The signal intensity of AcGFP-Ect2^r is also specified by the pseudocolor gradient on the right side of the image. (R) Equatorial membrane quantification for AcGFP-Ect2^r for control siRNA, Cyk4 siRNA, or Mklp1 siRNA was performed as described in N. ***, P < 0.001 as determined by a two-tailed unpaired Student’s t test. n > 12 cells, error bars: SD. (S–U) The membrane localization of AcGFP (green) in HeLa Kyoto cells that are either expressing AcGFP-Ect2^r (S) or AcGFP-NuMA (T). The signal intensity of AcGFP-Ect2^r or AcGFP-NuMA is also specified by the pseudocolor gradient on the right side of the image. The linescan analysis of the membrane AcGFP intensity for the region (in orange) is depicted in (U). For such measurements, one quadrant of an anaphase cell from the equatorial cell membrane to the polar region of the cell membrane was analyzed to calculate the normalized intensity (U). Source data are available for this figure: SourceData F5.