Using MLF2 as a live-cell imaging marker to visualize the cell cycle–dependent dynamics of NE blebbing. (A) Orthogonal sections of 4TorKO cells expressing mScarlet-Sec61β and MLF2-GFP under a Dox-inducible promoter. Sections are from four time points in a 3D dataset from a 1,600− s time series. Time 0 s was defined by the onset of anaphase, and time 460 s shows the completion of NE reformation. NE herniations arise during a narrow window of early G1 phase immediately following cytokinesis (∼700 s) and persist through interphase. Data correspond to maximum intensity projections of 10 slices from a 3D image stack (z axis) shown in Video 1. Scale bar, 10 µm; inset, 2 µm. (B) Sequential orthogonal sections highlighting the presence of MLF2-GFP foci on branched membrane networks connected to the NE. Scale bar, 10 µm. (C) Representative montage of the fluorescence intensity growth for four MLF2-GFP foci from the left daughter cell shown in A and Video 1. Fluorescence intensities were measured from the initial appearance of foci to the end of image acquisition (700–1,600 s). Scale bar, 10 µm; inset, 1 µm. (D) Graphical representation depicting the increase in fluorescence intensity of MLF2 foci from C. Time 700–800 s is designated as “initiation phase,” time 801–1,200 s is designated as “growth phase,” and time 1,201–1,600 s is designated as “Static phase.” (E) Timeline displaying NE reformation (∼400–500 s) and the onset of herniation formation and growth (700+ s), which is represented by a blue gradient. The ranges of NE reformation and MLF2 foci formation were determined by observing five different cells (n = 5). RIU, relative intensity unit.