Fig 2: LEM2 promotes early nuclear compartmentalization via cooperation between its LCD and WH domains.a, Example images of cells treated with siLEM2 and expressing NLS-3GFP in combination with siRNA resistant LEM2 constructs corresponding to the quantification graphs shown in b and main text Fig. 3f. DNA was labeled using NucBlue. Time 0 refers to the time of complete cleavage furrow ingression. Scale bar 2 µm. b, Mean nuclear/cytoplasmic ratio of NLS-3GFP fluorescence over time in cells treated with the indicated siRNAs and expressing the indicated siRNA resistant constructs. Cells imaged at 15s intervals though 1min increments are plotted. Data was collected across at least 3 biological replicates and the mean +/- SD are plotted (siCon LEM2-mChr; n=26; siLEM2–2 LEM2-mChr: n=44; siCon LEM2?SY-mChr: n=20; siLEM2–2 LEM2?SY-mChr: n=24; siCon LEM2?PR-mChr: n=20; siLEM2–2 LEM2?PR-mChr: n=20; siCon LEM2?WH-mChr: n=24; siLEM2–2 LEM2?WH-mChr: n=16). Two-tailed unpaired t-test was used to determine p-values comparing deletion mutant lines to the full length LEM2 line under endogenous LEM2 depletion conditions at each timepoint. No multiple comparisons. Asterisk code graphed for clarity (*P < 0.05; **P < 0.005) while exact are p-values reported in the table shown at the bottom. c, Quantification of nuclear/cytoplasmic ratio of NLS-3GFP approximately 30 minutes after complete cleavage furrow ingression in parental HeLa cells treated with the indicated siRNAs. Data was collected across 3 biological replicates and plotted as mean +/- SD (siControl: n=11, 12, 6; siLEM2–2: n=18, 18, 14; siCHMP7: n=11, 6, 14). Two-tailed unpaired t-test was used to determine p-values. No multiple comparisons.

Fig 3: Control experiments for knockdown analysis. (A) Endogenous LEM2 detected at anaphase chromatin or disks. Spinning disk confocal microscopy of representative anaphase cells 72 h after treatment with siLEM2-1 or -2 confirmed that detection of signal at the chromatin surface is specific. Signal detected at the midzone with LEM2 antibody is nonspecific, persisting after knockdown of LEM2 with two independent oligos. (B) Widefield microscopy of representative interphase cells comparing detection of endogenous LEM2 at the nuclear rim 72 h after treatment with siControl or LEM2-specific oligos. (C) Specific depletion of LEM2 and CHMP7 with respective oligos used for RNAi is confirmed by immunoblot. # indicates a smaller protein product likely derived from CHMP7. (D) IST1/CHMP8 recruitment, assessed and graphed as described in Fig. 7B, performed in parallel with LEM2 analysis in Fig. 7F (siControl: 67 ± 10%, n = 104, 14, 40; siCHMP7-1: 0 ± 0%, n = 68, 26, 24; siCHMP7-2: 0 ± 0%, n = 24, 10, 48). (E) Widefield microscopy of representative interphase cells comparing detection of endogenous SUN1 at the nuclear rim 72 h after treatment with siControl or SUN1-directed oligos. (F) Specific depletion of SUN1 by RNAi confirmed by immunoblot. (G) IST1/CHMP8 recruitment, assessed and graphed as described in Fig. 7B, performed in parallel with CHMP2A analysis in Fig. 7D (siControl: 60 ± 8%, n = 80, 42, 48; siLEM2-1: 8.2 ± 4%, n = 80, 82, 30; siLEM2-2: 17 ± 7%, n = 120, 82, 78; siCHMP7-1: 5 ± 3%, n = 76, 50, 58; siCHMP7-2: 8 ± 4%, n = 58, 46, 48; siSUN1A: 53 ± 18%, n = 58, 18, 20; siSUN1B: 50 ± 7%, n = 78, 44, 36). All graphs plot mean ± SEM. *P < 0.05; **P < 0.01. N.S., not significant. Scale bars, 10 µm.

Fig 4: SATB2 interacts with the INM protein LEMD2 Model of a hypothetical NL-chromatin tether containing SATB2. Schematic representation of the nuclear envelope consisting of two lipid bilayers, the inner nuclear membrane (INM) and outer nuclear membrane (ONM), and the lamin polymer underlying the INM (modified after Zuleger et al, 2011). Depicted are the INM protein LEMD2, lamins and BAF, identified as SATB2 interaction partners (Cera et al, 2019).Immunoprecipitation of SATB2 from mouse neonatal cortical lysates. LEMD2 was detected by Western blotting in the SATB2 immunoprecipitate from control but not SATB2-deficient cortical lysate. The equal input of total protein from control and SATB2-deficient cortical lysates was controlled by ERK2 detection. Representative images of the immunoblots are shown; I (Input), F (Flow-through), B (Beads).Reverse immunoprecipitation with LEMD2 antibody and control IgG antibody, followed by WB detection of LEMD2 and SATB2. Lysates from primary cortical culture lysates were used.GST pull-down assays. Representative images of n = 3 independent experiments are shown. GST pull-down assay of transiently overexpressed V5-tagged LEMD2 (i), SATB2 (ii), and LEMD2?LEM (iii) from HeLa cell lysates using GST, GST-SATB2, and GST-LEMD2(413–503) hybrid proteins.Immunoprecipitations using anti-V5-tag antibody from lysates of HeLa cells transfected with expression plasmids encoding V5-tagged full-length SATB2 (Satb21–733) and EGFP as control. The equal input of total protein was controlled by GAPDH detection. Representative images of the immunoblots are shown; I (Input), F (Flow-through), B (Beads), M (Molecular weight marker).The CUT-like domain of SATB2 is required for the interaction with LEMD2. Immunoprecipitations using anti-V5-tag antibody from lysates of HeLa cells transfected with V5-tagged Satb21–247 and Satb21–157 deletion mutants. LEMD2 and BAF were detected only in immunoprecipitates from HeLa cells transfected with the deletion mutant containing the CUT-like domain (Satb21–247). The equal input of total protein was controlled by GAPDH detection. Representative images of the immunoblots are shown; I (Input), F (Flow-through), B (Beads), M (Molecular weight marker).CUT1 and CUT2 domains are required for the interaction with LEMD2. Immunoprecipitations using anti-V5-tag antibody from HeLa cells transfected with V5-tagged Satb2346–733 and Satb2616–733 deletion mutants. LEMD2 and BAF were detected only in immunoprecipitates from HeLa cells expressing the CUT1 and CUT2 containing deletion mutant (Satb2346–733). Source data are available online for this figure.

Fig 5: LEM2 WH domain is required for IST1 recruitment to the nascent NE and mediates polymer formation with CHMP7.a, Top: Quantification of robust IST1 recruitment to chromatin disks in late anaphase, as assessed by blind scoring. Mean +/− SEM determined from 3 independent experiments. (siControl parental: n=80, 58, 106; siLEM2–2 parental: n=78, 50, 58; siControl LEM2-mChr: n=46, 42, 62; siLEM2–2 LEM2-mChr: n=78, 51, 62; siControl LEM2ΔWH-mChr: n=138, 52, 42; siLEM2–2 LEM2ΔWH-mChr: n=112, 48, 51). Two-tailed unpaired t-test was used to determine p-values. No multiple comparisons. Bottom: Representative images by widefield showing localization of endogenous IST1 in late anaphase cells depleted of endogenous LEM2 and expressing the indicated siRNA resistant LEM2-mChr constructs. Scale 2 μm. b, Top: Quantification of the percent of early anaphase disks with robust IST1 recruitment, as assessed by blind scoring. Mean +/− SEM determined from 3 independent experiments (siControl parental: n=38, 16, 20; siLEM2–2 parental: n=24, 10, 25; siControl LEM2-mChr: n=50, 20, 24; siLEM2–2 LEM2-mChr: n=38, 14, 8; siControl LEM2ΔWH-mChr: n=44, 16, 20; siLEM2–2 LEM2ΔWH-mChr: n=20, 4, 14). Two-tailed unpaired t-test was used to determine p-values. No multiple comparisons. Bottom: Representative images by widefield showing localization of endogenous IST1 in early anaphase cells depleted of endogenous LEM2 and expressing the indicated siRNA resistant LEM2-mChr constructs. Scale 2 μm. c, Negative stain EM corresponding to the CHMP7 polymerization assay showing no polymerization for the control condition CHMP7+LEM2NTD. Representative of 2 technical replicates.