Immunofluorescent localization of DΝΑ (blue), and phospho-RLC (green) in S2 cells. The scale bar = 5 µm.

(A) Expression of mutant RLC constructs with alternative codons allows for depletion of only the endogenous RLC by RNAi. Cells expressing RLC-GFP constructs were treated with dsRNA that targets the endogenous sequence for 5 days. Proteins in the cell lysates of treated and untreated cells were separated by SDS-PAGE and immunoblotting with an anti-RLC antibody revealed that the lower molecular weight endogenous RLC is specifically depleted while the higher molecular weight GFP chimera is still expressed. (B) Phosphorylation of the RLC is essential for cytokinesis. S2 cells were treated with dsRNAs for 5 days to deplete the endogenous RLC and the fraction of approximately 100 cells that were binucleated (mean±SE) as measured by DAPI staining was calculated. The bar graphs show fraction of binucleate cells with and without treatment with RNAi against native RLC for wild type cells, cells expressing wildtype RLCT20S21-GFP, non-phosphorylatable RLCA20A21-GFP, and phospho-mimic RLCE20E21-GFP. (C) Phosphorylation of the RLC is essential for myosin II recruitment to the cleavage furrow. S2 cells expressing RLC-GFP chimeras (green) and RFP-α-tubulin (red) were depleted of the endogenous RLC by RNAi treatment for five days, and timelapse fluorescence microscopy was used to film cells as they attempted to divide. Both RLCT20S21-GFP (Row 1) and RLCE20E21-GFP (Row 3) were recruited to the equatorial cortex during mitosis and the cells contracted. However, RLCA20A21-GFP (Row 2) was not recruited to the equatorial cortex at anaphase and the cells did not contract. The scale bar = 5 µm.

(A) S2 cells expressing RLCT20S21-GFP or RLCE20E21-GFP were treated with dsRNAs for 5 days to deplete Rho1, Rok, or Citron and then fixed and stained with DAPI. The fraction of binucleated cells (mean, ±SE for approximately 200 cells) was determined by fluorescence microscopy. Expression of RLCE20E21-GFP rescues depletion of Rok, but not Rho1 or Citron. (B) Expression of phospho-mimic RLC rescues Rok depletion. S2 cells expressing RLC-GFP chimeras (green) and RFP-α-tubulin (red) were depleted of the Rok by RNAi treatment for five days, and timelapse fluorescence microscopy was used to film cells as they attempted to divide. RLCT20S21-GFP (Row 1) was not recruited to the equatorial cortex at anaphase and the cells did not contract. RLCE20E21-GFP (Row 2) was recruited to the equatorial cortex during mitosis and the cells contracted. The scale bar = 5 µm.

(A) Immunofluorescent localization of DNΑ (blue) and phospho-RLC (green) in Citron depleted cells. Notice the membrane blebs in the telophase furrow as well as the anti-phospho-RLC antibody staining in the midbody. Scale bar = 5 µm. (B) Depletion of Citron probably affects the structure of telophase midbodies. S2 cells were depleted of Citron by treatment with RNAi for 5 days and then fixed and stained with an anti-phospho-RLC antibody. Late telophase cells were scored for antibody staining (mean, ±SE for 30 cells). While less than half of all control cells were stained, nearly all Citron depleted cells were stained.