Figure 2.
Figure 2. The ESCRT machinery is required for midbody/midbody ring release. (A) Deconvolved wide-field image of an embryo stained for tubulin (cyan), Mklp1ZEN-4, and ESCRT-ITSG-101 (n = 5 embryos). (B) Central plane confocal images of embryos expressing GFP–ESCRT-IMVB-12 (n = 6 embryos). Times are relative to anaphase of the second division. Dashed yellow lines mark the cell boundaries. The white arrowhead and yellow arrow mark the focus of GFP-ESCRT-IMVB-12 before and after release from the cell–cell boundary, respectively. (C) Example of an ESCRT-Itsg-101(RNAi) embryo in which a 10-kD dextran probe was photoactivated after apparent closure (n = 4 embryos). Central plane images show the embryo before activation (−5 s), immediately after activation (5 s), and 140 s after activation (140 s). A kymograph was constructed by aligning strips (narrow rectangle) from images collected at 5-s intervals. Red arrow denotes the point of photoactivation. (D and E, top) Central plane confocal images of ESCRT-Itsg-101(RNAi) embryos expressing a fluorescently tagged plasma membrane probe along with the midbody marker mCherry-Mklp1ZEN-4 (D; n = 10 embryos) or the midbody ring marker GFP–CYK-7 (E; n = 6 embryos). Times are relative to anaphase of the second division. Released fragments marked with the plasma membrane probe (white arrowheads) and the mCherry-Mklp1ZEN-4–marked or GFP–CYK-7–marked midbody remnants are indicated (yellow arrows). Asterisks mark the new midbody/midbody rings arising from the second embryonic division. (bottom) Graphs plotting the times when the mCherry-Mklp1ZEN-4–marked midbodies or GFP–CYK-7–marked midbody rings were released in control and ESCRT-Itsg-101(RNAi) embryos. In cases in which the midbody/midbody ring was not released, the data point reflects the endpoint of the time-lapse sequence. (F) Timeline summarizes the key events during contractile ring constriction (light gray) and abscission (dark gray). MTs, microtubules. White boxes on the low magnification images in A, B, D, and E mark the location of the region shown at higher magnification in the adjacent images. Bars, 5 µm.

The ESCRT machinery is required for midbody/midbody ring release. (A) Deconvolved wide-field image of an embryo stained for tubulin (cyan), Mklp1ZEN-4, and ESCRT-ITSG-101 (n = 5 embryos). (B) Central plane confocal images of embryos expressing GFP–ESCRT-IMVB-12 (n = 6 embryos). Times are relative to anaphase of the second division. Dashed yellow lines mark the cell boundaries. The white arrowhead and yellow arrow mark the focus of GFP-ESCRT-IMVB-12 before and after release from the cell–cell boundary, respectively. (C) Example of an ESCRT-Itsg-101(RNAi) embryo in which a 10-kD dextran probe was photoactivated after apparent closure (n = 4 embryos). Central plane images show the embryo before activation (−5 s), immediately after activation (5 s), and 140 s after activation (140 s). A kymograph was constructed by aligning strips (narrow rectangle) from images collected at 5-s intervals. Red arrow denotes the point of photoactivation. (D and E, top) Central plane confocal images of ESCRT-Itsg-101(RNAi) embryos expressing a fluorescently tagged plasma membrane probe along with the midbody marker mCherry-Mklp1ZEN-4 (D; n = 10 embryos) or the midbody ring marker GFP–CYK-7 (E; n = 6 embryos). Times are relative to anaphase of the second division. Released fragments marked with the plasma membrane probe (white arrowheads) and the mCherry-Mklp1ZEN-4–marked or GFP–CYK-7–marked midbody remnants are indicated (yellow arrows). Asterisks mark the new midbody/midbody rings arising from the second embryonic division. (bottom) Graphs plotting the times when the mCherry-Mklp1ZEN-4–marked midbodies or GFP–CYK-7–marked midbody rings were released in control and ESCRT-Itsg-101(RNAi) embryos. In cases in which the midbody/midbody ring was not released, the data point reflects the endpoint of the time-lapse sequence. (F) Timeline summarizes the key events during contractile ring constriction (light gray) and abscission (dark gray). MTs, microtubules. White boxes on the low magnification images in A, B, D, and E mark the location of the region shown at higher magnification in the adjacent images. Bars, 5 µm.

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