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Soft substrates normalize nuclear morphology and prevent nuclear rupture in fibroblasts from a laminopathy patient with compound heterozygous LMNA mutations.

Tamiello C, Kamps MA, van den Wijngaard A, Verstraeten VL, Baaijens FP, Broers JL, Bouten CC - Nucleus (2013)

Bottom Line: Consequently, tools such as mutation analysis are not adequate for predicting the course of the disease.   Here, we employ growth substrate stiffness to probe nuclear fragility in cultured dermal fibroblasts from a laminopathy patient with compound progeroid syndrome.We show that culturing of these cells on substrates with stiffness higher than 10 kPa results in malformations and even rupture of the nuclei, while culture on a soft substrate (3 kPa) protects the nuclei from morphological alterations and ruptures.Together, these data indicate that culturing of these LMNA mutated cells on substrates with a range of different stiffnesses can be used to probe the degree of nuclear fragility.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, Eindhoven University of Technology, Eindhoven, The Netherlands. c.tamiello@tue.nl

ABSTRACT
Laminopathies, mainly caused by mutations in the LMNA gene, are a group of inherited diseases with a highly variable penetrance; i.e., the disease spectrum in persons with identical LMNA mutations range from symptom-free conditions to severe cardiomyopathy and progeria, leading to early death. LMNA mutations cause nuclear abnormalities and cellular fragility in response to cellular mechanical stress, but the genotype/phenotype correlations in these diseases remain unclear. Consequently, tools such as mutation analysis are not adequate for predicting the course of the disease.   Here, we employ growth substrate stiffness to probe nuclear fragility in cultured dermal fibroblasts from a laminopathy patient with compound progeroid syndrome. We show that culturing of these cells on substrates with stiffness higher than 10 kPa results in malformations and even rupture of the nuclei, while culture on a soft substrate (3 kPa) protects the nuclei from morphological alterations and ruptures. No malformations were seen in healthy control cells at any substrate stiffness. In addition, analysis of the actin cytoskeleton organization in this laminopathy cells demonstrates that the onset of nuclear abnormalities correlates to an increase in cytoskeletal tension. Together, these data indicate that culturing of these LMNA mutated cells on substrates with a range of different stiffnesses can be used to probe the degree of nuclear fragility. This assay may be useful in predicting patient-specific phenotypic development and in investigations on the underlying mechanisms of nuclear and cellular fragility in laminopathies.

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Figure 2. Nuclear morphological abnormality regulation by substrate stiffness. (A) Immunofluorescent labeling of cell nuclei with DAPI (blue), lamin B1 (green) and overlay of the two in the most right panels allowed to distinguish between normally (upper row) and abnormally shaped nuclei (second and third row). In particular, the nucleus in the second row shows a protrusion and in the third row a bleb can be observed. Scale bars: 10 μm. (B) Frequency of abnormally shaped nuclei on increasing PA gel stiffness for LMNAmut and control NHDFα. Values represent means from at least 300 cells from two experiments. Bars represent SEM * p < 0.05, ** p < 0.01 vs NHDFα on the same substrate stiffness (C) Statistical analyses of differences in frequency of misshapen nuclei for LMNAmut and NHDFα on the different substrate stiffness’s. *, p < 0.05; no star, p > 0.05.
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Figure 2: Figure 2. Nuclear morphological abnormality regulation by substrate stiffness. (A) Immunofluorescent labeling of cell nuclei with DAPI (blue), lamin B1 (green) and overlay of the two in the most right panels allowed to distinguish between normally (upper row) and abnormally shaped nuclei (second and third row). In particular, the nucleus in the second row shows a protrusion and in the third row a bleb can be observed. Scale bars: 10 μm. (B) Frequency of abnormally shaped nuclei on increasing PA gel stiffness for LMNAmut and control NHDFα. Values represent means from at least 300 cells from two experiments. Bars represent SEM * p < 0.05, ** p < 0.01 vs NHDFα on the same substrate stiffness (C) Statistical analyses of differences in frequency of misshapen nuclei for LMNAmut and NHDFα on the different substrate stiffness’s. *, p < 0.05; no star, p > 0.05.

Mentions: Morphologically visible nuclear abnormalities are common in laminopathy cells.22 These abnormalities, seen as nuclear blebs, herniations and so-called honeycomb structures after immunostaining, seem to indicate the presence of weak spots at the nuclear membrane and/or nuclear interior. Here, we tested whether the extracellular substrate stiffness affects the frequency of these nuclear abnormalities in the LMNAmut cells. From the images of DAPI and lamin B1 immunolabeled nuclei, it became obvious that few abnormally shaped nuclei were seen in cells seeded at low stiffness after 48 h from seeding (between 3 and 4% of all nuclei) (Fig. S1). Representative images of normal and abnormally shaped nuclei are shown in Figure 2A. Further quantitative analysis of 600 nuclei per cell genotype showed that on soft substrates (3 kPa) both LMNAmut and NHDFα nuclei overall have a normal appearance (2.9 ± 0.4% NHDFα, 3.7 ± 0.4% LMNAmut, Fig. 2B). However, while in the NHDFα control fibroblasts abnormally shaped nuclei were detected in about 3.0 ± 0.7% of the cells regardless of the substrate stiffness, a significant increase of abnormally shaped LMNAmut nuclei was observed on 10, 20, 80 kPa PA gels and glass substrates (respectively 8.2 ± 0.7%, 26.9 ± 5.0%, 44.7 ± 1.7%, 22.5 ± 2.4%) (Figs. 2B and C). The fraction of misshapen nuclei in LMNAmut cells increased significantly on the 80 kPa gel (up to 44.7 ± 1.7% compared with 26.9 ± 5.0% on 20 kPa). A reason for this significant increase could be the higher cell density observed on the 80 kPa gels seeded with LMNAmut cells. As in a side experiment we observed increased nuclear aberrations with increased cell density, we therefore hypothesize that cell-cell contact played a role in the formation of nuclear abnormalities (Fig. S2). On the glass substrate results were similar to those on the 20 kPa PA gels (22.5 ± 2.4%). The findings on glass are in agreement with earlier studies, showing that 36% of all cells from this LMNAmut patient cultured on glass substrates had irregularly shaped nuclei with blebs, honeycomb figures, large and poorly defined protrusions.18


Soft substrates normalize nuclear morphology and prevent nuclear rupture in fibroblasts from a laminopathy patient with compound heterozygous LMNA mutations.

Tamiello C, Kamps MA, van den Wijngaard A, Verstraeten VL, Baaijens FP, Broers JL, Bouten CC - Nucleus (2013)

Figure 2. Nuclear morphological abnormality regulation by substrate stiffness. (A) Immunofluorescent labeling of cell nuclei with DAPI (blue), lamin B1 (green) and overlay of the two in the most right panels allowed to distinguish between normally (upper row) and abnormally shaped nuclei (second and third row). In particular, the nucleus in the second row shows a protrusion and in the third row a bleb can be observed. Scale bars: 10 μm. (B) Frequency of abnormally shaped nuclei on increasing PA gel stiffness for LMNAmut and control NHDFα. Values represent means from at least 300 cells from two experiments. Bars represent SEM * p < 0.05, ** p < 0.01 vs NHDFα on the same substrate stiffness (C) Statistical analyses of differences in frequency of misshapen nuclei for LMNAmut and NHDFα on the different substrate stiffness’s. *, p < 0.05; no star, p > 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 2: Figure 2. Nuclear morphological abnormality regulation by substrate stiffness. (A) Immunofluorescent labeling of cell nuclei with DAPI (blue), lamin B1 (green) and overlay of the two in the most right panels allowed to distinguish between normally (upper row) and abnormally shaped nuclei (second and third row). In particular, the nucleus in the second row shows a protrusion and in the third row a bleb can be observed. Scale bars: 10 μm. (B) Frequency of abnormally shaped nuclei on increasing PA gel stiffness for LMNAmut and control NHDFα. Values represent means from at least 300 cells from two experiments. Bars represent SEM * p < 0.05, ** p < 0.01 vs NHDFα on the same substrate stiffness (C) Statistical analyses of differences in frequency of misshapen nuclei for LMNAmut and NHDFα on the different substrate stiffness’s. *, p < 0.05; no star, p > 0.05.
Mentions: Morphologically visible nuclear abnormalities are common in laminopathy cells.22 These abnormalities, seen as nuclear blebs, herniations and so-called honeycomb structures after immunostaining, seem to indicate the presence of weak spots at the nuclear membrane and/or nuclear interior. Here, we tested whether the extracellular substrate stiffness affects the frequency of these nuclear abnormalities in the LMNAmut cells. From the images of DAPI and lamin B1 immunolabeled nuclei, it became obvious that few abnormally shaped nuclei were seen in cells seeded at low stiffness after 48 h from seeding (between 3 and 4% of all nuclei) (Fig. S1). Representative images of normal and abnormally shaped nuclei are shown in Figure 2A. Further quantitative analysis of 600 nuclei per cell genotype showed that on soft substrates (3 kPa) both LMNAmut and NHDFα nuclei overall have a normal appearance (2.9 ± 0.4% NHDFα, 3.7 ± 0.4% LMNAmut, Fig. 2B). However, while in the NHDFα control fibroblasts abnormally shaped nuclei were detected in about 3.0 ± 0.7% of the cells regardless of the substrate stiffness, a significant increase of abnormally shaped LMNAmut nuclei was observed on 10, 20, 80 kPa PA gels and glass substrates (respectively 8.2 ± 0.7%, 26.9 ± 5.0%, 44.7 ± 1.7%, 22.5 ± 2.4%) (Figs. 2B and C). The fraction of misshapen nuclei in LMNAmut cells increased significantly on the 80 kPa gel (up to 44.7 ± 1.7% compared with 26.9 ± 5.0% on 20 kPa). A reason for this significant increase could be the higher cell density observed on the 80 kPa gels seeded with LMNAmut cells. As in a side experiment we observed increased nuclear aberrations with increased cell density, we therefore hypothesize that cell-cell contact played a role in the formation of nuclear abnormalities (Fig. S2). On the glass substrate results were similar to those on the 20 kPa PA gels (22.5 ± 2.4%). The findings on glass are in agreement with earlier studies, showing that 36% of all cells from this LMNAmut patient cultured on glass substrates had irregularly shaped nuclei with blebs, honeycomb figures, large and poorly defined protrusions.18

Bottom Line: Consequently, tools such as mutation analysis are not adequate for predicting the course of the disease.   Here, we employ growth substrate stiffness to probe nuclear fragility in cultured dermal fibroblasts from a laminopathy patient with compound progeroid syndrome.We show that culturing of these cells on substrates with stiffness higher than 10 kPa results in malformations and even rupture of the nuclei, while culture on a soft substrate (3 kPa) protects the nuclei from morphological alterations and ruptures.Together, these data indicate that culturing of these LMNA mutated cells on substrates with a range of different stiffnesses can be used to probe the degree of nuclear fragility.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, Eindhoven University of Technology, Eindhoven, The Netherlands. c.tamiello@tue.nl

ABSTRACT
Laminopathies, mainly caused by mutations in the LMNA gene, are a group of inherited diseases with a highly variable penetrance; i.e., the disease spectrum in persons with identical LMNA mutations range from symptom-free conditions to severe cardiomyopathy and progeria, leading to early death. LMNA mutations cause nuclear abnormalities and cellular fragility in response to cellular mechanical stress, but the genotype/phenotype correlations in these diseases remain unclear. Consequently, tools such as mutation analysis are not adequate for predicting the course of the disease.   Here, we employ growth substrate stiffness to probe nuclear fragility in cultured dermal fibroblasts from a laminopathy patient with compound progeroid syndrome. We show that culturing of these cells on substrates with stiffness higher than 10 kPa results in malformations and even rupture of the nuclei, while culture on a soft substrate (3 kPa) protects the nuclei from morphological alterations and ruptures. No malformations were seen in healthy control cells at any substrate stiffness. In addition, analysis of the actin cytoskeleton organization in this laminopathy cells demonstrates that the onset of nuclear abnormalities correlates to an increase in cytoskeletal tension. Together, these data indicate that culturing of these LMNA mutated cells on substrates with a range of different stiffnesses can be used to probe the degree of nuclear fragility. This assay may be useful in predicting patient-specific phenotypic development and in investigations on the underlying mechanisms of nuclear and cellular fragility in laminopathies.

Show MeSH
Related in: MedlinePlus