Extended Data Fig. 1: The N terminus of LEM2 possesses a canonical BAF-binding LEM domain and an LCD.
From: LEM2 phase separation promotes ESCRT-mediated nuclear envelope reformation
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 CFI. Representative of ten or more cells imaged across at least three biological replicates. Scale bar, 2 μm. b, Multiple sequence alignment of LEM domains across LEM family proteins, highlighting a conserved four-amino-acid sequence that, when mutated in emerin (EMDm24), disrupts BAF binding20. The position of an analogous mutation in LEM2 (LEM2m21) is indicated. c, HeLa cells stably expressing LEM2–mCherry and EGFP–BAF live-imaged during anaphase. Representative of ten or more cells imaged across at least three biological replicates for both fixed and live-cell imaging. Scale bar, 10 μm. d, Top, a homology model for the LEM21–72 –BAF–DNA complex51, based on Protein Data Bank code (PDB): 2BZF and PDB: 2ODG32,51. 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 LEM21–72 + BAF + DNA sample. Representative of three technical replicates. For gel source data, see Supplementary Fig. 1. e, Percentage amino acid composition for the LEM2 LCD, and the compositions of two subregions, compared to an average amino acid composition. f, Schematic of LEMNTD with amino acid substitutions (S, T, or Y to D) relative to SY-rich (yellow) and PR-rich (rust) regions, in LEM2NTD Mim1 and Mim2 constructs. LEM2 immunoblot assessing the migration pattern of full-length (also shown in Fig. 1e) 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 λ-PP are indicated. Representative data from two biological replicates, with one and three technical replicates per biological replicate. For immunoblot source data, see Supplementary Fig. 1. g, Top, amino acid sequences of the peptides corresponding to the SY-rich and PR-rich regions of LEM2. Bottom, concentration-dependent droplet formation by the LEM2SY peptide, juxtaposed with similar data collected for full LEM2NTD as in Fig. 1d. Representative of three technical replicates. Scale bar, 2 μm. h, Fluorescence microscopy of purified LEM2NTD with indicated molecular anions. Image representative of two technical replicates. Scale bar, 2 μm.
