Journal: Nature

Article Title: LEM2 phase separation governs ESCRT-mediated nuclear envelope reformation

doi: 10.1038/s41586-020-2232-x

Figure Lengend Snippet: a , Live-cell imaging of GFP-Lamin B2 and LEM2-mChr; DNA is stained with NucBlue and tubulin detected with SiR-tubulin. Time 0 refers to complete cleavage furrow ingression. Representative of 10 or more cells imaged across at least 3 biological replicates. b , Multiple sequence alignment of LEM domains across LEM family proteins, highlighting a conserved four-amino acid sequence that, when mutated in Emerin (EMD m24 ), disrupts BAF binding . The position of an analogous mutation in LEM2 (LEM2 m21 ) is indicated. Scale bar 2 μm c , HeLa cells stably expressing LEM2-mCherry and EGFP-BAF live-imaged during anaphase. Representative of 10 or more cells imaged across at least 3 biological replicates for both fixed and live-cell imaging. Scale 10 μm. d , Top: A homology model for LEM2 1–72 -BAF-DNA complex , based on PDB:2BZF and PDB:2ODG , . Middle: Absorbance at 280 nm as a function of retention volume (mL) from analytical size exclusion chromatography. Retention volumes for major peaks (arrowheads) and predicted molecular weights for protein or protein-DNA complexes are listed. Bottom: SDS-PAGE of major peak for LEM2 1–72 +BAF+DNA sample. Representative of 3 technical replicates. For gel source data, see . e , LEM2’s LCD percent amino acid composition, and the compositions of two subregions, compared to an average amino acid composition. f , Schematic of LEM NTD with amino acid substitutions (S, T, or Y to D) relative to SY-rich (yellow) and PR-rich (rust) regions, in LEM2 NTD Mim1 and Mim2 constructs. LEM2 immunoblot assessing the migration pattern of full length, also shown in , and mutant LEM2-mChr constructs following separation by Phos-tag SDS-PAGE. Cell lysates were prepared from G1/S- and prometaphase- arrested cells expressing the indicated exogenous LEM2; lysates treated with lambda phosphatase (λ-PP) are indicated. Representative data from 2 biological replicates, with 1 and 3 technical replicates per biological replicate. For immunoblot source data, see . g , Amino acid sequence of the peptide corresponding to LEM2’s SY-rich and PR-rich regions. Concentration-dependent droplet formation by the LEM2 SY peptide, juxtaposed with similar data collected for full LEM2 NTD as in . Representative of 3 technical replicates. h, Fluorescence microscopy of purified LEM2 NTD with indicated molecular anions. Image representative of 2 technical replicates. Scale 2 μm.

Article Snippet: Primary antibodies [α-LEM2 (HPA017340; Sigma-Aldrich), α-mCherry (Novus Biologicals, NBP2–25157), α-Tubulin (ab18251; abcam)] were detected with HRP-coupled secondary antibodies (Thermo Fisher) and chemiluminescence.

Techniques: Live Cell Imaging, Staining, Sequencing, Binding Assay, Mutagenesis, Stable Transfection, Expressing, Size-exclusion Chromatography, SDS Page, Construct, Western Blot, Migration, Concentration Assay, Fluorescence, Microscopy, Purification

Journal: Nature

Article Title: LEM2 phase separation governs ESCRT-mediated nuclear envelope reformation

doi: 10.1038/s41586-020-2232-x

Figure Lengend Snippet: a , Live-cell imaging of GFP-tubulin and H2B-mChr in siRNA treated cells. Images representative of 2 biological replicates and quantified in b . Time 0 refers to the time of complete cleavage furrow ingression. Scale 2 μm. b , Left: Orthogonal view of the tubulin phenotype following LEM2 depletion co-stained for the NE protein SUN2. Images representative of 3 biological replicates. Scale 5 μm. Right: The mean percent of telophase cells with nuclear tubulin defects, lined with inner nuclear membrane (as assessed by immunofluorescence of Lamin B2). Data plotted are mean +/− SEM from 3 biological replicates (siControl: n =75, 35, 37; siLEM2–1: n =72, 35, 37; siLEM2–2: n =45, 34, 34). Two-tailed unpaired t-test was used to determine p-values. No multiple comparisons. c , Left: Example images of 53BP1 localization by immunofluorescence in telophase U2OS cells following siRNA treatment, as quantified on the right. Scale 5μm. Right: Quantification of the percent of telophase cells with ≥5 53BP1 nuclear foci. Mean +/− SEM percent of telophase cells with ≥5 53BP1 foci determined from 3 biological replicates (siControl: n =56, 52, 44; siLEM2–1: n =64, 56, 26; siLEM2–2: n =74, 50, 40). Two-tailed unpaired t-test was used to determine p-values. No multiple comparisons. Bottom: immunoblot confirming depletion of endogenous LEM2 in U2OS cells, using siRNA oligos previously validated in other human cell lines, including HeLa (immunoblot source data shown in ) , . d , Negative stain EM of indicated combinations of MT, LEM2 NTD-linker-WH , and CHMP7 FL . Scale 25 nm. Images representative of 2 technical replicates. e , Example images of cells expressing the indicated siRNA-resistant constructs and treated with the indicated siRNAs. LEM2-mChr in magenta, DAPI-stained DNA in blue, and tubulin immunofluorescence in green. Cells were arrested in S-phase and then allowed to progress through one round of division, resulting in an interphase population of cells that just exited mitosis. We observed an increased number of highly irregular nuclei in cells expressing either LEM2 ΔPR -mChr or LEM2 ΔWH -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 ΔPR -mChr and LEM2 ΔWH -mChr. Representative nuclear, tubulin, and LEM2 phenotypes and the correspondence to nuclear circularity score is shown. Nuclear borders and circularity scores annotated in tubulin channel. Scale 5μm. These findings suggest that interfering with cooperation between LEM2’s microtubule-interacting and ESCRT-binding domains alters nuclear morphology, indicating that both activities are necessary, but neither is sufficient for NE 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. Data plotted are mean +/− SEM from 3 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 ΔSY -mChr: n =123, 68, 144; siLEM2–2 LEM2 ΔSY -mChr: n =93, 95, 105; siControl LEM2 ΔPR -mChr: n =149, 123, 58; siLEM2–2 LEM2 ΔPR -mChr: n =49, 31, 42; siControl LEM2 ΔWH -mChr: n =116, 96, 94; siLEM2–2 LEM2 ΔWH -mChr: n =85, 32, 68). Two-tailed unpaired t-test was used to determine p-values 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 2 technical replicates. For immunoblot source data, see .

Article Snippet: Primary antibodies [α-LEM2 (HPA017340; Sigma-Aldrich), α-mCherry (Novus Biologicals, NBP2–25157), α-Tubulin (ab18251; abcam)] were detected with HRP-coupled secondary antibodies (Thermo Fisher) and chemiluminescence.

Techniques: Live Cell Imaging, Staining, Membrane, Immunofluorescence, Two Tailed Test, Western Blot, Expressing, Construct, Binding Assay, Activity Assay