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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 6. PML-NBs localization as a marker for cellular compartmentalization. (A−C) Confocal sections representative of cell nuclei were immunolabeled with Lamin B1 (red), DAPI (blue) and PML-NBs (green) to investigate the localization of PML-NBs. Nuclei were counterstained with DAPI (blue). The most right panel shows the triple overlay. Scale bars: 10 μm. (A) Nuclei showing normal morphology and internal localization of PML-NBs. Cellular compartmentalization is intact. (B) Cytoplasmic localization of PML-NBs (cytPML-NBs) around a nucleus showing an abnormal morphology (white arrowhead). Loss of cellular compartmentalization is indicated by the exit of PML-NBs to the cytoplasm. (C) CytPML-NBs could be found also in normally shaped nuclei (white arrowhead) indicating that loss of cellular compartmentalization is not directly related to nuclear morphology abnormalities. (D) Relative frequency of NHDFα and LMNAmut showing cytPML-NBs. Values represent means from at least 600 cells from 2 experiments. Bars represent SEM * p < 0.05, ** p < 0.01 vs NHDFα on the same substrate stiffness (E) Statistical analyses of differences in frequency of cytPML-NBs for LMNAmut and NHDFα on the different substrate stiffness’s. *, p < 0.05; no star, p > 0.05.
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Figure 6: Figure 6. PML-NBs localization as a marker for cellular compartmentalization. (A−C) Confocal sections representative of cell nuclei were immunolabeled with Lamin B1 (red), DAPI (blue) and PML-NBs (green) to investigate the localization of PML-NBs. Nuclei were counterstained with DAPI (blue). The most right panel shows the triple overlay. Scale bars: 10 μm. (A) Nuclei showing normal morphology and internal localization of PML-NBs. Cellular compartmentalization is intact. (B) Cytoplasmic localization of PML-NBs (cytPML-NBs) around a nucleus showing an abnormal morphology (white arrowhead). Loss of cellular compartmentalization is indicated by the exit of PML-NBs to the cytoplasm. (C) CytPML-NBs could be found also in normally shaped nuclei (white arrowhead) indicating that loss of cellular compartmentalization is not directly related to nuclear morphology abnormalities. (D) Relative frequency of NHDFα and LMNAmut showing cytPML-NBs. Values represent means from at least 600 cells from 2 experiments. Bars represent SEM * p < 0.05, ** p < 0.01 vs NHDFα on the same substrate stiffness (E) Statistical analyses of differences in frequency of cytPML-NBs for LMNAmut and NHDFα on the different substrate stiffness’s. *, p < 0.05; no star, p > 0.05.

Mentions: Given the increased presence of abnormally shaped nuclei in LMNAmut cells cultured on substrates stiffer than 3 kPa, we tested whether this was correlated with a loss of cellular compartmentalization. We chose promyeolocytic leukemia nuclear bodies (PML-NBs) as marker, as these assemblies of PML proteins are normally confined to the nuclear interior of non-proliferating cells (Fig. 6A).26 Earlier studies by De Vos et al. and Houben et al. showed that frequent loss of PML-NBs from the nucleus to the cytoplasm can be found in laminopathy cells.14,27,28 In the current experiment, approximately 600 cells for each genotype, on each substrate, were screened manually for PML-NBs localization using fluorescent microscopy. We observed cytoplasmic PML-NBs (cytPML-NBs) in cases of abnormally shaped nuclei as well as for intact nuclei (Fig. 6B and C). Therefore it is not possible to directly correlate abnormalities in the nuclear shape to the loss of cellular compartmentalization. Similarly to previous findings,14 4.4 ± 1.1% NHDFα control cells demonstrated cytPML-NBs, regardless of the substrate stiffness. On the 3kPa substrate, LMNAmut and NHDFα control cells showed no significant differences in the frequency of cells with cytPML-NBs (3.1 ± 0.5% LMNAmut and 2.0 ± 0.2% NHDFα). However, we did observe a gradual increase of LMNAmut cells with cytPML-NBs with increasing stiffness of the substrates between 3 and 20 kPa (from 3.1 ± 0.5% to 12.8 ± 1.2%), indicating increased frequency of cytPML-NBs in LMNAmut cells.


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 6. PML-NBs localization as a marker for cellular compartmentalization. (A−C) Confocal sections representative of cell nuclei were immunolabeled with Lamin B1 (red), DAPI (blue) and PML-NBs (green) to investigate the localization of PML-NBs. Nuclei were counterstained with DAPI (blue). The most right panel shows the triple overlay. Scale bars: 10 μm. (A) Nuclei showing normal morphology and internal localization of PML-NBs. Cellular compartmentalization is intact. (B) Cytoplasmic localization of PML-NBs (cytPML-NBs) around a nucleus showing an abnormal morphology (white arrowhead). Loss of cellular compartmentalization is indicated by the exit of PML-NBs to the cytoplasm. (C) CytPML-NBs could be found also in normally shaped nuclei (white arrowhead) indicating that loss of cellular compartmentalization is not directly related to nuclear morphology abnormalities. (D) Relative frequency of NHDFα and LMNAmut showing cytPML-NBs. Values represent means from at least 600 cells from 2 experiments. Bars represent SEM * p < 0.05, ** p < 0.01 vs NHDFα on the same substrate stiffness (E) Statistical analyses of differences in frequency of cytPML-NBs for LMNAmut and NHDFα on the different substrate stiffness’s. *, p < 0.05; no star, p > 0.05.
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Figure 6: Figure 6. PML-NBs localization as a marker for cellular compartmentalization. (A−C) Confocal sections representative of cell nuclei were immunolabeled with Lamin B1 (red), DAPI (blue) and PML-NBs (green) to investigate the localization of PML-NBs. Nuclei were counterstained with DAPI (blue). The most right panel shows the triple overlay. Scale bars: 10 μm. (A) Nuclei showing normal morphology and internal localization of PML-NBs. Cellular compartmentalization is intact. (B) Cytoplasmic localization of PML-NBs (cytPML-NBs) around a nucleus showing an abnormal morphology (white arrowhead). Loss of cellular compartmentalization is indicated by the exit of PML-NBs to the cytoplasm. (C) CytPML-NBs could be found also in normally shaped nuclei (white arrowhead) indicating that loss of cellular compartmentalization is not directly related to nuclear morphology abnormalities. (D) Relative frequency of NHDFα and LMNAmut showing cytPML-NBs. Values represent means from at least 600 cells from 2 experiments. Bars represent SEM * p < 0.05, ** p < 0.01 vs NHDFα on the same substrate stiffness (E) Statistical analyses of differences in frequency of cytPML-NBs for LMNAmut and NHDFα on the different substrate stiffness’s. *, p < 0.05; no star, p > 0.05.
Mentions: Given the increased presence of abnormally shaped nuclei in LMNAmut cells cultured on substrates stiffer than 3 kPa, we tested whether this was correlated with a loss of cellular compartmentalization. We chose promyeolocytic leukemia nuclear bodies (PML-NBs) as marker, as these assemblies of PML proteins are normally confined to the nuclear interior of non-proliferating cells (Fig. 6A).26 Earlier studies by De Vos et al. and Houben et al. showed that frequent loss of PML-NBs from the nucleus to the cytoplasm can be found in laminopathy cells.14,27,28 In the current experiment, approximately 600 cells for each genotype, on each substrate, were screened manually for PML-NBs localization using fluorescent microscopy. We observed cytoplasmic PML-NBs (cytPML-NBs) in cases of abnormally shaped nuclei as well as for intact nuclei (Fig. 6B and C). Therefore it is not possible to directly correlate abnormalities in the nuclear shape to the loss of cellular compartmentalization. Similarly to previous findings,14 4.4 ± 1.1% NHDFα control cells demonstrated cytPML-NBs, regardless of the substrate stiffness. On the 3kPa substrate, LMNAmut and NHDFα control cells showed no significant differences in the frequency of cells with cytPML-NBs (3.1 ± 0.5% LMNAmut and 2.0 ± 0.2% NHDFα). However, we did observe a gradual increase of LMNAmut cells with cytPML-NBs with increasing stiffness of the substrates between 3 and 20 kPa (from 3.1 ± 0.5% to 12.8 ± 1.2%), indicating increased frequency of cytPML-NBs in LMNAmut cells.

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