Fig. 9

Degron reporters reveal membrane topology during abscission. a–f Time-lapse images of embryos expressing mCh::PH to label the plasma membrane and NMY-2::GFP::ZF1 to label non-muscle myosin in the cytokinetic ring. Scale bar: 10 µm. a In a control embryo, NMY-2::GFP::ZF1 between the anterior daughter cells (arrow) is protected from ZF1-mediated degradation due to its release outside cells in the midbody remnant after abscission. b NMY-2::GFP::ZF1 in the phagocytosed midbody remnant is protected from proteasomal degradation by engulfing membranes (n = 19). c In embryos depleted of the ESCRT-I subunit TSG-101, NMY-2::GFP::ZF1 localizes to the intercellular bridge normally. d NMY-2::GFP::ZF1 is degraded after tsg-101 knockdown, indicating that abscission is incomplete and NMY-2::GFP::ZF1 is accessible to the degradation machinery. Engulfment of the AB midbody is also delayed, likely due to incomplete abscission (n = 7). e, f Embryos depleted of the septin UNC-59 show rapid degradation of NMY-2::GFP::ZF1 from the intercellular bridge and delayed engulfment of the midbody remnant due to defects in abscission (n = 8). g Fluorescence intensity of the NMY-2::GFP::ZF1 reporter on the intercellular bridge between anterior daughter cells (AB midbody) drops significantly for 2 min after the onset of ZF1 degradation in the cytoplasm of control embryos (blue, n = 11, p < 0.05 using Student’s t-test with Bonferroni correction), showing that NMY-2 is able to diffuse out of the bridge. Fluorescence then persists, showing that formation of a diffusion barrier, symmetric abscission, and engulfment protect the degron-tagged reporter from degradation. In contrast, NMY-2::GFP::ZF1 fluorescence continues to drop in tsg-101 RNAi-treated embryos (red, n = 5, p < 0.05 compared to control after 8 min) and unc-59 mutants (green, n = 6, p < 0.05 compared to control after 3.5 min), showing that the ZF1 degron technique is sensitive to detecting small defects in abscission. Bars represent mean ± s.e.m. Source data are provided as a Source Data file. h In control embryos, degron reporters in the intercellular bridge are protected from proteasomal degradation due to release after abscission and encapsulation in a phagosome. i In abscission mutants, degron reporters are accessible to the cytosol, leading to their removal by proteasomal degradation