a, Live-cell imaging of GFP–tubulin and H2B–mChr in siRNA-treated cells. Images representative of two biological replicates, quantified in b. Time 0 refers to the time of complete CFI. Scale bar, 2 μm. b, Left, orthogonal view of the tubulin phenotype following LEM2 depletion co-stained for the nuclear envelope protein SUN2. Images representative of three biological replicates. Scale bar, 5 μm. Right, mean ± s.e.m. percentage of telophase cells with nuclear tubulin defects, lined with inner nuclear membrane (as assessed by immunofluorescence of lamin B2). Three biological replicates (siControl: n = 75, 35, 37; siLEM2-1: n = 72, 35, 37; siLEM2-2: n = 45, 34, 34). Two-tailed unpaired t-test, no multiple comparisons. c, Left, example images of 53BP1 localization by immunofluorescence in telophase U2OS cells following siRNA treatment. Scale bar, 5 μm. Right, mean ± s.e.m. percentage of telophase cells with five or more 53BP1 nuclear foci. Three biological replicates (siControl: n = 56, 52, 44; siLEM2-1: n = 64, 56, 26; siLEM2-2: n = 74, 50, 40). Two-tailed unpaired t-test, no multiple comparisons. Bottom, immunoblot confirming depletion of endogenous LEM2 in U2OS cells, using siRNA oligos previously validated in other human cell lines, including HeLa4,19 (immunoblot source data shown in Supplementary Fig. 1). d, Negative stain electron microscopy of indicated combinations of microtubules, LEM2 NTD-linker-WH , and CHMP7 FL . Scale bars, 25 nm. Images representative of two technical replicates. e, Example images of cells expressing the indicated siRNA-resistant constructs and treated with the indicated siRNAs. Cells were arrested in S-phase and then allowed to progress through one round of division, resulting in an interphase population of cells that had just exited mitosis. We observed an increased number of highly irregular nuclei in cells expressing either LEM2 ∆PR –mChr or LEM2 ∆WH –mChr compared to cells expressing full-length LEM2 or even those depleted of LEM2. Notably, deformed nuclei were commonly associated with microtubule disorganization and aberrant accumulation of LEM2 ∆PR –mChr and LEM2 ∆WH –mChr. Representative nuclear, tubulin, and LEM2 phenotypes and the correspondence to nuclear circularity score are shown. Nuclear borders and circularity scores annotated in tubulin channel. Scale bar, 5 μm. These findings suggest that interfering with cooperation between the microtubule-interacting and ESCRT-binding domains of LEM2 alters nuclear morphology, indicating that both activities are necessary, but neither is sufficient for nuclear envelope reformation. Moreover, the presence of one activity without the other is detrimental to nuclear morphology. f, Quantification of nuclear circularity in interphase parental HeLa cells and cells expressing the indicated siRNA-resistant LEM2 constructs, treated with the indicated siRNAs. Mean ± s.e.m. from three biological replicates (siControl/parental: n = 105, 46, 80; siLEM2-2/parental: n = 102, 116, 59; siControl/LEM2–mChr: n = 153, 53, 122; siLEM2-2/LEM2–mChr: n = 84, 81, 105; siControl/LEM2 ∆SY –mChr: n = 123, 68, 144; siLEM2-2/LEM2 ∆SY –mChr: n = 93, 95, 105; siControl/LEM2 ∆PR –mChr: n = 149, 123, 58; siLEM2-2/LEM2 ∆PR –mChr: n = 49, 31, 42; siControl/LEM2 ∆WH –mChr: n = 116, 96, 94; siLEM2-2/LEM2 ∆WH –mChr: n = 85, 32, 68). Two-tailed unpaired t-test comparing circularity scores less than 0.6 (indicated by blue) between the indicated treatments; no multiple comparisons. g, Immunoblot showing relative levels of the siRNA-resistant constructs fused with mCherry in parallel with endogenous LEM2. Representative of two technical replicates. For immunoblot source data, see Supplementary Fig. 1. Source Data