Attenuated Nuclear Tension Regulates Progerin-Induced Mechanosensitive Nuclear Wrinkling and Chromatin Remodeling

Abstract

Hutchinson-Gilford progeria syndrome, caused by a mutation in the LMNA gene, leads to increased levels of truncated prelamin A, progerin, in the nuclear membrane. The accumulation of progerin results in defective nuclear morphology and is associated with altered expression of linker of the nucleoskeleton and cytoskeleton complex proteins, which are critical for nuclear signal transduction via molecular coupling between the extranuclear cytoskeleton and lamin-associated nuclear envelope. However, the molecular mechanisms underlying progerin accumulation-induced nuclear deformation and its effects on intranuclear chromosomal organization remain unclear. Here, the spatiotemporal evolution of nuclear wrinkles is analyzed in response to variations in substrate stiffness using a doxycycline-inducible progerin expression system. It is found that cytoskeletal tension regulates the onset of progerin-induced nuclear envelope wrinkling and that the molecular interaction between SUN1 and LMNA controls the actomyosin-dependent attenuation of nuclear tension. Genome-wide analysis of chromatin accessibility and gene expression further suggests that an imbalance in force between the intra- and extranuclear spaces induces nuclear deformation, which specifically regulates progeria-associated gene expression via modification of mechanosensitive signaling pathways. The findings highlight the crucial role of nuclear lamin-cytoskeletal connectivity in bridging nuclear mechanotransduction and the biological aging process.

Keywords: Hutchinson–Gilford progeria syndrome; LINC complex; SUN1; actomyosin contractility; chromatin remodeling; heterochromatin; mechanosensation; nuclear deformation; nuclear tension; nuclear wrinkling; progerin.

Figure 1

Substrate stiffness‐dependent differential evolution of nuclear deformation in doxycycline‐inducible progerin‐expressing HeLa cells. A–E) Morphological alterations of doxycycline‐controlled Tet‐On HeLa cells expressing mutant lamin A protein (Δ50 LMNA/progerin). Representative confocal images depict immunofluorescence staining for progerin (red), F‐actin (green), and nuclei (DAPI, blue) in doxycycline‐untreated control cells (−Dox) or doxycycline‐treated (2 µg mL⁻¹) progerin‐expressing Tet‐On HeLa cells (+Dox). Hemispherical and cross‐sectional views of 3D‐rendered nuclei show progerin expression‐induced formation of abnormal nuclear morphology. Empty and full arrowheads indicate the absence and presence of progerin expression, respectively (A). Immunofluorescence intensity‐based quantifications of cell area (B), nuclear area (C), progerin expression (D), and the fractional occurrence of abnormal nuclear shapes (E) were performed in the absence and presence of doxycycline treatment. In panel B, 810 and 505 nuclei; in panel C, 138 and 153 nuclei; in panel D, 93 and 72 nuclei were analyzed under −Dox and +Dox conditions, respectively. For panel E, 70 to 105 nuclei were analyzed, which was independently repeated three times per each condition. Error bars indicate the standard error of the mean (S.E.M.); an unpaired t‐test was applied (***: p < 0.001, NS: not significant). F–G) Substrate stiffness‐dependent differential expression of progerin. Progerin (red), lamin B1 (green), and nuclei (DAPI, blue) of progerin‐expressing Tet‐On HeLa cells were plated on control glass substrates or polyacrylamide hydrogel (PAG) substrates with elastic moduli of 34 kPa (stiff) and 1.37 kPa (soft) (F). Control glass and stiff PAG substrates maintained doxycycline‐inducible progerin expression, while soft PAG substrates significantly reduced progerin expression (G). In panel G, >50 cells were tested per condition. Error bars indicate the S.E.M.; Student's t‐test was applied (***: p < 0.001, NS: not significant). H–J) Time‐lapse monitoring of substrate stiffness‐dependent progerin expression and nuclear deformation. Progerin intensity and nuclear envelope (NE) wrinkling were monitored every 20 min for 36 h in mCherry‐progerin‐expressing Tet‐On HeLa cells plated on control glass (H), stiff PAG (I), and soft PAG (J) substrates. Yellow dotted lines mark the nuclear boundary determined by differential interference contrast (DIC) imaging, showing nuclear spreading area. Full and empty arrowheads indicate the presence and absence of progerin (red) or NE wrinkling (blue), respectively; transparency of the full arrowheads represents the magnitude of progerin expression and NE wrinkling; oversaturated fluorescence intensity (white) indicates nuclear surface wrinkles (H–J). K–P) Quantifying differential onset of progerin expression and nuclear deformation in response to changes in substrate stiffness. Progerin expression and NE wrinkling were quantified by measuring fluorescence intensity (red curves) and the fraction of the nuclear spreading area occupied by NE wrinkling area (blue curves) after doxycycline treatment (K–M). All values were normalized using the formula (x – x _min)/(x _max – x _min) to range from 0 (min) to 1 (max). Progerin expression and NE wrinkling followed extended sigmoidal curves, with inflection points for progerin expression at 20 h and NE wrinkling at 25 h on control glass and stiff PAG substrates (K,L,N,O), which were delayed to 22 and 32 h, respectively, on soft PAG substrates (M–O). The time interval between inflection points of progerin expression and NE wrinkling extended from 5 h on control glass and stiff PAG substrates to 10 h on soft PAG substrates (P). In panels K–P, >20 cells were analyzed per condition. Error bars indicate the S.E.M.; one‐way analysis of variance (ANOVA) with Tukey's test was used for comparisons (***: p < 0.001, NS: not significant).

Figure 2

Substrate stiffness‐dependent differential NE tension. A–C) Visualization of substrate stiffness‐dependent changes in NE tension using the nesprin tension sensor. Tet‐On HeLa cells expressing fluorescence‐marker‐untagged progerin were transiently transfected with the nesprin tension sensor and plated on control glass or stiff (E ≈ 34 kPa) or soft (E ≈ 1.37 kPa) PAG substrates. NE tension was analyzed every 6 h for 36 h after doxycycline treatment. Binary layers outlining the nesprin tension sensor‐localized nuclear membrane were determined by creating polygonal hollow masks to exclude fluorescence intensity outside the nucleus (top rows). FRET signals were differentially color‐coded (bottom rows). Purple and red indicates high FRET efficiency. FRET efficiency, representing the inverse of NE tension, gradually increased in response to doxycycline‐induced progerin expression, but this rate was reduced in cells on soft PAG substrates compared to those on control glass and stiff PAG substrates (A and B vs C). D–G) Quantification of substrate stiffness‐modulated differential NE tension. Column scatter plots represent the time‐dependent increase in the FRET ratio in response to doxycycline‐induced progerin expression on control glass and stiff or soft PAG substrates. The nesprin tension sensor‐based FRET ratio increased significantly after 24 h in cells on glass (D) and stiff PAG substrates (E), but after 30 h in cells on soft PAG substrates (F). Accordingly, the FRET signal in cells on glass and stiff PAG substrates was significantly higher than in those on soft PAG substrates after 24 h (G). In panels D–G, red bars represent the mean ± S.D., and one‐way ANOVA with Tukey's test was applied for group comparisons (***: p < 0.001, **: p < 0.05, NS: not significant).

Figure 3

Mechanical model of nucleus wrinkling. A) Construction of a mechanical model mimicking progerin‐induced deformation of the nuclear surface. Experimental observations of Tet‐On‐inducible progerin expression are depicted by 3D reconstructions of confocal images (top) and the corresponding mechanical model (bottom), showing the smooth spherical shape of progerin‐absent control nuclei (left), a buckyball‐like surface configuration at the onset of progerin expression (middle), and a folded surface texture due to the progression of nuclear wrinkling (right). The color code represents displacement of the nuclear surface. B–E) Simulation of time‐dependent nucleus wrinkling in response to changes in substrate stiffness. Reduced pressure on the nucleus on compliant substrates delays surface wrinkling. The second‐order buckling (i.e., from a buckyball‐like pattern to a folded pattern) occurs at characteristic time scales of 45, 90, and 105 on stiff, medium, and soft substrates, respectively (B). (Inset) The local force balance in a membrane microelement, where membrane tension is mimicked by an equivalent internal pressure due to surface curvature and mechanical equilibrium. Time‐dependent volume changes of the nucleus on different stiffness substrates indicate that nuclei on soft substrates take the longest time, while those on stiff substrates take the shortest, based on the characteristic time to reach a specific volume change (C), where ∆V and V ₀ represent the volume change and the initial volume, respectively, and black circles mark the accelerated collapses along the nucleus triggered by the second‐order buckling. The nuclear surface tension, calculated from nuclear volume change, shows that nuclei on stiff substrates have the highest tension value and largest change rate, while nuclei on soft substrates have the lowest tension value and smallest change rate (D), where the stretching force F _e was applied to characterize the membrane tension F _memb, F ₀ represents the unit characteristic force, and the black cross indicates the breakdown of computational model due to the contact of the membrane under large deformation, respectively. Increasing internal pressure (i.e., enhanced membrane tension), corresponding to a greater 𝐾_𝑉 value, indicating a stronger resistance to external pressure reduces nuclear volume change (E), indicating that reduced nuclear tension on soft substrates delays nuclear wrinkling compared to nuclei on stiff substrates.

Figure 4

Time‐dependent alteration of gene profiling and signaling pathways in response to mechanosensitive progerin expression. A–D) Progerin‐induced, time‐dependent differential gene expression in response to changes in substrate stiffness (stiff glass substrates vs soft PAG substrates of 1.37 kPa). The similarity in gene expression profiles was assessed by Euclidean distance and complete linkage clustering, where the height of the dendrogram represents the Euclidean distance between clusters, indicating the similarity in expression profiles (A). Note that gene expression profiles remained similar between cells on stiff glass and soft PAG substrates at 18 h but clustered by substrate stiffness at 24 and 30 h (marked by red boxes), and reclustered at 36 h. The number of differentially regulated genes is displayed for each substrate stiffness by comparing with the doxycycline‐untreated control condition (glass 0 h on the left, 1.37 kPa 0 h on the right), where yellow and blue bars indicate upregulated and downregulated genes, respectively (B). Volcano plots display doxycycline treatment time‐dependent evolution of log2 fold changes in LMNA and LINC complex‐associated genes (e.g., SUN1, SUN2, SYNE1, SYNE2, and SYNE3) on stiff glass substrates (top row) or soft PAG substrates (bottom row) (C). Note that while LMNA expression increased from 6 h after doxycycline treatment on both stiff glass and soft PAG substrates, significant increases in LMNA, SYNE2, and SYNE3 were observed at 12 h on stiff substrates but at 18 h on soft substrates (marked by red dotted boxes), indicating delayed expression of nesprin on soft substrates. Gene ontology (GO) analysis was performed comparing the control glass and soft PAG substrates at different doxycycline treatment times (12, 18, 24, 30, and 36 h) for cellular components, biological processes, and molecular functions, with term sizes between 10 and 500 (D). In panel B, the criteria for significant changes in gene expression were fold change ≥ |2| and raw p‐value < 0.05. In panel C, yellow and red dots indicate specific gene expression levels corresponding to fold change ≥ |1.5|, raw p‐value < 0.5, and fold change ≤ |1.5| with raw p‐value < 0.5, respectively, with LINC complex‐associated genes colored blue. In panel D, adjusted p‐values reported from g:Profiler were derived using a one‐sided hypergeometric test and corrected by the Benjamini–Hochberg method (***: p < 0.001, **: p < 0.01, *: p < 0.05). E–H) Heatmap analysis of GO terms related to mechanosensing of substrate stiffness. Representative signaling pathways, including the Notch signaling pathway (GO:0007219, E), BMP signaling pathway (GO:0030509, F), extracellular structure organization (GO:0043062, G), and tissue homeostasis (GO:0001894, H), were visualized. Euclidean distance was used as the distance metric, and complete linkage was applied for hierarchical clustering in the analysis of each dataset. For further details, refer to the Experimental Section.

Figure 5

Time‐dependent differential epigenetic modifications in response to mechanosensitive progerin expression. A–D) Quantification of doxycycline treatment time‐dependent differential expression of H3K9me2/3 in response to changes in substrate stiffness. H3K9me2/3 expression was quantified by immunoblotting against H3K9me2/3 and GAPDH antibodies in progerin‐expressing Tet‐On HeLa cells placed on control stiff substrates (glass, A) and soft PAG substrates (1.37 kPa, B) with doxycycline treatment every 12 h for up to 36 h. (C,D) Total protein expression increased with doxycycline treatment in each condition. In panels C and D, three independently performed experiments were averaged and normalized to the values in 0 h condition. Error bars indicate the S.E.M., and one‐way ANOVA using Tukey's test was applied for comparison between groups (****: p < 0.0001, ***: p < 0.005, *: p < 0.05, NS: not significant). E–N) Spatiotemporal alterations of heterochromatic histone modifications in doxycycline‐induced progerin‐expressing cells placed on varying substrate stiffness. Tet‐On HeLa cells expressing mCherry‐tagged Δ50 LMNA (red) placed on control glass (E) and PAG substrates of 1.37 kPa (F) were immunostained for nuclei (DAPI, blue) and H3K9me2/3 (green) every 12 h after doxycycline treatment. 3D‐rendered nuclei, reconstructed from z‐stacked confocal fluorescent images, depict that doxycycline treatment time‐dependently increased progerin expression, inducing H3K9me2/3 clustering in the nuclear interior (E–N). H3K9me2/3 clusters were detected after 24 h on stiff substrates (glass, E,G–J) but appeared after 36 h on soft substrates (1.37 kPa, F,K–N). Yellow dotted lines indicate the nuclear boundary as determined by DAPI staining; white arrowheads indicate clustered H3K9me2/3 (E,F). Fluorescence intensity profiles monitored by line scanning through the maximum intensity projected nuclear images show that H3K9me2/3 clusters largely alternate with progerin staining. More intensive peaks were detected in nuclei of cells placed on stiff substrates compared to those on soft substrates (G–J vs K–N), where red and green arrowheads indicate fluorescence intensity peaks corresponding to progerin and H3K9me2/3 expression, respectively.

Figure 6

Differential chromatin accessibility in response to mechanosensitive progerin expression. A–C) Differential chromatin mobility in response to progerin expression. Time‐lapse tracking of fluorescence‐tagged chromatin was performed in TRF2 (telomeric repeat‐binding factor 2)‐transfected human dermal fibroblasts obtained from a three‐year‐old healthy control (denoted as 3 YR (Control), A) and an HGPS patient (denoted as 3 YR (HGPS), B), where nine randomly selected chromatin trajectories are displayed. (C) Quantitative analysis of the mean squared displacement (MSD) at each time lag indicates enhanced chromatin mobility in HGPS patients compared to the healthy control. D–R) ATAC sequencing‐based identification of differential key transcription factor (TF) binding motifs in response to substrate stiffness‐dependent progerin expression. Representative de novo TF binding motifs in Tet‐On HeLa cells expressing progerin were identified between cells on control stiff substrates (denoted as glass, D) and cells on soft PAG substrates (denoted as 1.37 kPa, E) at 12 h intervals after doxycycline treatment for 36 h using Homer software. The bar graph indicates the percentage of target binding motifs for CTCF, FOS::JUNB, TEAD family, KLF1, and ZNF331 (F), where each bar represents fold enrichment, defined as the percentage of target sequences with the motif divided by the percentage of background sequences with the motif. Data are normalized to doxycycline‐untreated control groups (denoted as glass 0 h, 1.37 kPa 0 h). Doxycycline treatment increased fold enrichment for TEAD (G,H), CTCF (K,L), and NFkB‐p65‐Rel (O,P), but decreased fold enrichment for JunB (I,J) and KLF1 (M,N) in control stiff substrates, with these changes diminished in soft PAG substrates. ATAC‐seq tracking of ZNF331 (Q), a known transcriptional repressor, and BMP2 (R), a component of mechanosensory pathways, was visualized using Integrative Genomics Viewer (IGV), where blue and red peaks indicate stiff glass and soft PAG substrates, respectively.

Figure 7

LINC complex‐mediated remodeling of LMNA‐associated nuclear tethering in progerin‐expressing cells. A–F) ATAC‐seq‐based identification of chromatin accessibility for LMNA‐associated LINC complex components in progerin‐expressing cells. ATAC‐seq peaks for LMNA (A,B), SUN1 (C,D), and SYNE2 (E,F) in progerin expression‐induced cells treated with doxycycline at 12 h intervals for up to 36 h on control glass (A,C,E) and soft PAG substrates (B,D,F) were visualized by Integrative Genomics Viewer (IGV). Note that as doxycycline treatment progresses, peaks for LMNA, SUN1, and SYNE2 in both control glass and soft PAG increase, indicating enhanced chromatin accessibility, but the peak height in soft substrates remains lower than that in control glass, approaching a similar level at 36 h. G–N) Differential expression of NE‐associated proteins and nuclear wrinkling in response to progerin expression. Tet‐On HeLa cells expressing mCherry‐tagged LMNA or Δ50 LMNA (progerin) were immunostained for SUN1 (green) and nucleus (DAPI, blue) (G,H) or nesprin 2 (green) and nucleus (DAPI, blue) (I,J) before (−Dox) and after (+Dox) doxycycline treatment. While doxycycline‐induced expression of mCherry‐tagged LMNA did not alter the SUN1 (G,K) and nesprin 2 contents (I,L), doxycycline‐induced expression of mCherry‐tagged Δ50 LMNA significantly increased SUN1 (H,K) and nesprin 2 (J,L). Compared to doxycycline‐induced LMNA expression, which did not induce changes in nuclear shape (G,I,M), Δ50 LMNA expression significantly increased NE wrinkling (H,J,M). SUN1 and nesprin 2 expression was more sensitive to Δ50 LMNA expression than to LMNA expression (N). In panels K–N, >50 nuclei were analyzed for each condition; error bars indicate the standard error of the mean (S.E.M.); and Student's t‐test was applied for comparison between two groups (****: p < 0.0001, ***: p < 0.001, NS: not significant). O–T) Progerin‐induced differential interaction in LMNA‐associated LINC proteins. The strength of molecular interaction between SUN1 and nesprin 2 (O,Q,R) or between SUN1 and LMNA (P,S,T) was estimated by quantifying the number and total intensity of proximity ligation assay (PLA) signals (red dots) in DAPI‐stained nuclei (blue). Hemispherical and cross‐sectional views of 3D‐rendered nuclei showed that punctate PLA signals were preferentially localized along the nuclear periphery. Note that doxycycline‐induced progerin expression significantly increased the PLA signals of SUN1 associated with LMNA (S,T), while PLA signals of SUN1 associated with nesprin 2 remained unchanged (Q,R). In panels Q, R, S, and T, >50 nuclei were analyzed for each condition; error bars indicate the S.E.M.; and an unpaired t‐test was applied to assess statistical significance (***: p < 0.001, NS: not significant).

Figure 8

Actomyosin contractility‐dependent nuclear deformation in progerin‐expressing cells. A–C) Differential actomyosin contractility in response to doxycycline‐induced progerin expression. Doxycycline‐inducible progerin‐expressing HeLa cells were immunostained for F‐actin (green), phospho‐myosin light chain 2 (pMLC2, red), and nucleus (DAPI, blue) before (−Dox) and after (+Dox) doxycycline treatment (A). (Insets) The details of pMLC2 staining along the actin stress fibers. Doxycycline‐induced progerin expression significantly increased the F‐actin (B) and pMLC2 (C) contents, which were normalized to cell area and actin stress fibers, respectively. In panels B and C, >60 cells were analyzed for each condition; error bars indicate the S.E.M.; unpaired t‐test was applied (***: p < 0.001). D–F) Differential formation of F‐actin and pMLC2 in doxycycline‐controlled progerin‐expressing Tet‐On HeLa cells in response to pharmaceutical inhibition of myosin‐dependent cytoskeletal tension. Cells were immunostained for nuclei (DAPI, blue), F‐actin (green), and pMLC2 (red) before (−Dox) and after (+Dox) doxycycline treatment, where differential concentrations of myosin‐II inhibiting blebbistatin were added (D). Compared to −Dox control cells, doxycycline‐induced progerin‐expressing cells showed significantly enhanced F‐actin, which remained unchanged in response to specific disruption of myosin activity (E). Significantly increased pMLC2 content due to doxycycline‐induced progerin expression was reversed by increasing the concentration of blebbistatin (F). Treating doxycycline‐induced progerin‐expressing cells with 15 µm blebbistatin fully restored their pMLC2 content to the level of doxycycline‐untreated progerin nonexpressing cells (F). In panels E and F, >150 cells were analyzed per condition; error bars indicate the S.E.M.; one‐way ANOVA using Tukey's test was applied (***: p < 0.001, **: p < 0.05, NS: not significant). G,H) Actomyosin contractility‐dependent differential changes of nuclear tension. Representative nesprin tension sensor‐based FRET signals along the nuclear membrane of doxycycline‐inducible progerin‐expressing cells were captured before (−Dox) and after (+Dox) doxycycline treatment in the presence of DMSO and 10 or 15 µm blebbistatin (G). Compared to doxycycline‐untreated control, doxycycline‐induced progerin expression significantly enhanced the FRET ratio, which was gradually diminished by increasing the concentration of blebbistatin and fully restored to the level of doxycycline‐untreated control condition by 15 µm blebbistatin treatment (H). I–K) Tight regulation of SUN1 expression and NE wrinkling in response to changes in actomyosin contractility. Tet‐On HeLa cells expressing mCherry‐tagged Δ50 LMNA (progerin) were treated with DMSO and 10 or 15 µm blebbistatin in the absence (−Dox) and presence (+Dox) of doxycycline before immunostaining for nucleus (DAPI, blue) and SUN1 (green) (I). Compared to the doxycycline‐untreated control, SUN1 expression and NE wrinkling were significantly increased in doxycycline‐treated progerin‐expressing cells, which was gradually diminished by increasing the concentration of blebbistatin and fully restored to the level of the control condition by 15 µm blebbistatin treatment (J,K). In panels H, J, and K, >20 cells were analyzed per condition; error bars indicate the S.E.M.; one‐way ANOVA using Tukey's test was applied for comparison between groups (***: p < 0.001, **: p < 0.05, NS: not significant).

Figure 9

SUN1‐mediated nuclear tension regulates progerin‐induced nuclear deformation. A–C) SUN1‐mediated modulation of actomyosin activity in progerin‐expressing cells. Tet‐On HeLa cells expressing progerin were immunostained for F‐actin (green), pMLC2 (red), SUN1 (orange), and nucleus (DAPI, blue) in the doxycycline‐untreated control condition (−Dox) and doxycycline‐treated conditions (+Dox) with siControl (+Dox/+siCon) or siSUN1 (+Dox/+siSUN1)‐mediated knockdown (A). Doxycycline‐induced progerin expression significantly increased the expression levels of SUN1 and pMLC2, which were maintained in siControl‐transfected cells but restored to levels similar to those observed in doxycycline‐untreated progerin nonexpressing cells after transfection with siSUN1 (B,C). In panels B and C, >150 cells were analyzed per condition; error bars indicate the S.E.M.; one‐way ANOVA using Tukey's test was applied (***: p < 0.001, NS: not significant). D–E) SUN1 expression‐dependent NE tension. Nesprin tension sensor‐based FRET signals along the nuclear membrane of doxycycline‐inducible progerin‐expressing cells transfected with siControl (+siCon) or siRNA targeting SUN1 (+siSUN1) were captured before (−Dox) and after (+Dox) doxycycline treatment (D). Doxycycline‐induced enhanced NE tension was maintained in siControl‐transfected cells but restored to the level of the doxycycline‐untreated control condition in siSUN1‐transfected cells (E). In panel E, >20 cells were analyzed per condition; error bars indicate the S.E.M.; one‐way ANOVA using Tukey's test was applied (***: p < 0.001, NS: not significant). F–H) Quantification of SUN1‐mediated NE wrinkling. mCherry‐tagged progerin‐expressing Tet‐On HeLa cells transfected with siControl (+siCon) or siRNA targeting SUN1 (+siSUN1) were immunostained for lamin B1 (green), SUN1 (yellow), and nuclear DNA (DAPI, blue) in the absence (−Dox) or presence (+Dox) of doxycycline (F). Doxycycline‐induced progerin expression significantly increased the NE wrinkling, which was maintained in siControl‐transfected cells but reduced to the level of the doxycycline‐untreated control condition in siSUN1‐transfected cells (G). The Pearson product‐moment correlation assessment applied to the merged dataset, including all conditions, showed a highly correlative relationship between SUN1 expression and NE wrinkling (r = 0.83) (H). In panels G and H, >50 cells were analyzed per condition; error bars indicate the S.E.M.; and one‐way ANOVA using Tukey's test was applied for comparison between groups (***: p < 0.001, NS: not significant). I–M) SUN1‐mediated restoration of the nuclear morphology of HGPS fibroblasts. Human dermal fibroblasts obtained from a three‐year‐old healthy control (denoted by 3 YR) and an HGPS patient were immunostained for F‐actin (green), pMLC2 (red), SUN1 (orange), and nuclei (DAPI, blue), where HGPS fibroblasts were transfected with siControl (HGPS/+siCon) or siSUN1 (HGPS/+siSUN1) (I). Full and empty arrowheads indicate the smooth and wrinkled nuclear surface, respectively. Compared to control fibroblasts, HGPS cells displayed a significantly enhanced expression of SUN1 and pMLC2, which was maintained in siControl‐transfected cells, but transfection with siSUN1 restored SUN1 and pMLC2 expression to levels similar to those observed in control cells (J,K). Nuclear wrinkles specifically featured in HGPS fibroblasts and siControl‐transfected HGPS fibroblasts were recovered in siSUN1‐transfected cells to levels comparable to those in healthy controls (L). Pearson correlation analysis applied to the merged dataset incorporating all experimental conditions showed a strong correlation between SUN1 expression and pMLC2 expression (red, r = 0.98), SUN1 expression, and NE wrinkling (blue, r = 0.99) (M). In panels J and K, >50 cells were analyzed per condition; in panel L, >20 cells were analyzed per condition; error bars indicate the S.E.M.; one‐way ANOVA using Tukey's test was applied for comparison between groups (***: p < 0.001, **: p < 0.05, NS: not significant).

Figure 10

Schematic summary depicting the functional relationship between SUN1‐mediated nuclear tension and NE wrinkling in response to progerin expression. Progerin expression accumulates LINC complex proteins SUN1 and Nesprin 2, reorganizing the actin‐binding Nesprin‐associated LINC complex at the nuclear envelope, and determining the biophysical interactions of the nuclear–cytoskeletal connection. Although the molecular linkages connecting SUN1, Nesprin 2, and F‐actin remain unchanged in response to progerin expression, nuclear tension along the SUN1–Nesprin 2–F‐actin connection is reduced by increased pMLC2. In summary, progerin‐induced morphological defects forming the surface wrinkling along the nuclear lamina are determined by the accumulation of LINC complexes proteins at the nuclear envelope and reduced nuclear tension accompanied by pMLC2 via the SUN1–Nesprin 2 bridge, regulating the expression of various genes within the nucleus. Ultimately, progerin‐induced nuclear wrinkling features increased chromatin dynamics in the heterochromatin‐rich nuclear periphery, resulting in the misregulation of mechanotransduction signal pathways in the HGPS model. Doxycycline‐induced progerin expression exhibits mechanical sensitivity to variations in substrate stiffness. Approximately 10%, 25%, and 28% of delays in onsets of progerin expression, reduction of nuclear tension, and nuclear wrinkling, respectively, on the soft substrate identifies the intracellular cytoskeletal force exerted on the nucleus as the origin of progerin‐induced nuclear wrinkling.