Fig 2: Absence of LAP2a reduces nucleoplasmic lamin A/C staining in immunofluorescence.(A) mEos3.2-Lmna WT and LAP2a KO sg1 cells were processed for immunofluorescence microscopy using antibody N18 against lamin A/C N-terminus, and DAPI to visualize DNA. Lamin A/C antibody N18 preferably stains nucleoplasmic lamin A/C. (B) mEos3.2-Lmna WT and LAP2a KO sg1 cells were co-cultured and processed for immunofluorescence microscopy as in (A) using antibodies N18 against lamin A/C and 1H11 against LAP2a, and DAPI to visualize DNA. Fluorescence intensity was determined for each cell along the dashed line in the red and green channel and is depicted in percent of maximum value in the graphs below. (C) LAP2a KO sg1 cells were processed as in (A) using lamin A/C antibody N18 without or with prior treatment with DNase I and RNase A to reverse N18 epitope masking. Bar: 10 µm. (D) Average nucleoplasmic fluorescence intensity from 3 wildtype and 3 LAP2a KO clones with and without prior DNase/RNase (D/R) treatment for N18 stainings and for mEos3.2 lamin A/C signal is depicted. Data represent averages ± S.E.M. Single data points are shown for each group. One-way ANOVA p value for N18 is 0.0020, F = 20.85; **p<0.01 (Tukey’s post-hoc test: WT vs. KO: p=0.0034; KO vs KO plus DNase/RNase: p=0.0033); p value for mEos3.2-lamin A/C is 0.3694 (non-significant), F = 1.181.

Fig 3: Lower lamin A/C mobility in the absence of LAP2a leads to a reduction in telomere movement.mEos3.2-Lmna WT or mEos3.2-Lmna fibroblasts lacking LAP2a (Lap2a KO sg1), lamin A/C (Lmna KO, see Figure 6—figure supplement 1), or both (Lmna/Lap2a DKO) were transiently transfected with a plasmid expressing DsRed-TRF1 to fluorescently label telomeres. The volume of telomere motion was then calculated based on its trajectory. (A) Box plots compare telomere motion between WT and Lap2a KO sg1 (left; ***p<0.0001 using Mann-Whitney U test, effect size d = 0.62) or between all four genotypes (right; Kruskal-Wallis test p<0.0001; post tests for WT ctrl versus each genotype are all highly significant with ***p<0.0001). The median is depicted within the first and third quartiles; whiskers: minimal and maximal datapoint excluding outliers (see also Figure 6—source data 1). (B) Mean square displacement (MSD) curves for telomere diffusion in mEos3.2-Lmna WT and LAP2a KO sg1 cells. The shaded area shows the distribution of 70% of the population. nWT = 471, nLap2aKO=562, nLmnaKO = 925, nDKO = 1298.Figure 6—source data 1.Volume of telomere movement in WT, LAP2a KO, Lmna KO and DKO cells.

Fig 4: Absence of LAP2a does not alter endogenous nucleoplasmic lamin A/C levels.(A) Schematic view of exons 1 and 2 (bars) and adjacent introns of the mouse Lmna locus (top), the targeting construct provided as homology-directed repair template after Cas9-mediated double strand break (middle), and the knock-in allele after successful integration of mEos3.2 (bottom). Very long introns are not displayed in their original length as indicated by a double slash. The second, light-colored exon 1 encodes the N-terminus of meiosis-specific lamin C2. The target sequence of Lmna-specific sgRNA1 in exon 1 is shown (blue). Protospacer-adjacent motif (PAM) is marked in red. Red arrowhead: expected Cas9 cut site. HA-L: Homology Arm-Left; HA-R: Homology Arm-Right. For further cell characterization see Figure 2—figure supplement 1. (B) mEos3.2-Lmna clone derived from wildtype (WT) mouse dermal fibroblasts (MDFs) and subclones generated after treatment with LAP2a-specific sgRNAs 1 or 3 (see Figure 2—figure supplement 2) were processed for western blotting using antibodies to the indicated antigens (anti lamin A/C E1, anti LAP2a 1H11). Positions of mEos-tagged and untagged lamins A and C are indicated. Wildtype and LAP2a knockout (KO) MDFs were added as controls. (C) mEos3.2-Lmna WT and LAP2a KO cells were processed for immunofluorescence microscopy using antibodies against LAP2a (1H11) and DAPI to visualize DNA. Confocal images are shown. Bar: 10 µm. (D) mEos3.2-Lmna WT cells were exposed to UV light (405 nm) to convert mEos3.2-lamin A/C from green to red. Cells were then tracked for 14 hr by live-cell imaging to test for stable integration of mEos3.2-lamin A/C into the peripheral lamina. No significant mobility of converted lamins in the lamina was observed over the entire imaging time. Pictures display merged Z-stacks to visualize most of the peripheral lamina. Bar: 10 µm. (E) Representative example of a LAP2a KO cell expressing mEos3.2-lamin A/C tracked by live-cell imaging throughout G1, followed by automated calculation of nucleoplasmic over peripheral lamin A/C ratio (t0: first available image after mitosis). The area corresponding to the nuclear lamina is delineated by red lines and was defined by a custom-made algorithm implemented in FIJI/ImageJ software. Average values for isogenic mEos3.2-Lmna LAP2a WT and KO clones were plotted in curves (F). The designation ‘sg1’ and ‘sg3’ refers to the sgRNA used to generate that clone (see Figure 2—figure supplement 2). Clones treated with sgRNAs, but still expressing LAP2a, were included as additional controls and are designated as ‘WT sg ctrl’. The 20th and 80th percentile are displayed for each curve (shaded area). nWT = 129, nWTsg1ctrl=159, nKOsg1 = 106, nWTsg3ctrl=111, nKOsg3 = 147 (G) Isogenic clones were fixed and DNA was stained with DAPI. Nucleoplasmic (N) to peripheral (P) signal intensity of mEos3.2-Lamin A/C was calculated for single cells (green dots) using a custom-made FIJI plugin and plotted versus DAPI fluorescence intensity, indicative of cell cycle stage (see also Figure 2—source data 1). Black line outlines number of cells versus DAPI intensity (histogram). Vertical dotted lines indicate what was classified as G1 and S-G2 in (H). (H) Single-cell N/P ratios from (G) were averaged for G1 and S/G2 and are shown in a box plot (median in red within the first and third quartiles; whiskers: minimal and maximal datapoint excluding outliers). The specificity of nucleoplasmic lamin A/C signal was demonstrated by comparison to lamin B1 N/P ratios (see Figure 2—figure supplement 3).Figure 2—source data 1.Nucleoplasmic to peripheral signal intensity ratio and DAPI fluorescence intensity of mEos3.2-Lamin A/C WT and LAP2a KO cells.