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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 1. Effect of substrate stiffness on cell morphology and actin cytoskeleton organization. (A) Representative bright field images of NHDFα and LMNAmut cells seeded on polyacrylamide gels with stiffness ranging from 3 kPa to 80 kPa taken 48 h after seeding. Fewer and less spread cells were present on 3 kPa polyacrylamide gels than on stiffer substrates for both cell types. Scale bar: 100 μm. (B) Actin organization in NHDFα and LMNAmut (phalloidin-TRITC, red) showed increased organization and tension on substrates stiffer than 3 kPa. Green color is given by lamin B1 staining. Scale bar: 20 μm. (C and D) Cell area and aspect ratio presented as box-and-whisker plots. The measurements of NHDFα and LMNAmut did not show significant difference, thus the values were considered as a group.
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Figure 1: Figure 1. Effect of substrate stiffness on cell morphology and actin cytoskeleton organization. (A) Representative bright field images of NHDFα and LMNAmut cells seeded on polyacrylamide gels with stiffness ranging from 3 kPa to 80 kPa taken 48 h after seeding. Fewer and less spread cells were present on 3 kPa polyacrylamide gels than on stiffer substrates for both cell types. Scale bar: 100 μm. (B) Actin organization in NHDFα and LMNAmut (phalloidin-TRITC, red) showed increased organization and tension on substrates stiffer than 3 kPa. Green color is given by lamin B1 staining. Scale bar: 20 μm. (C and D) Cell area and aspect ratio presented as box-and-whisker plots. The measurements of NHDFα and LMNAmut did not show significant difference, thus the values were considered as a group.

Mentions: We investigated the intracellular effect of increasing substrate stiffness on diseased dermal fibroblasts, isolated from a patient suffering from a progeroid syndrome due to compound heterozygous missense mutations (p.T528M and p.M540T) in the LMNA gene (LMNAmut) and we compared these findings with control human fibroblast cell line (NHDFα). For this purpose both cell types were seeded on collagen I coated polyacrylamide (PA) gels with stiffnesses ranging from 3 kPa to 80 kPa, as well as on collagen I coated glass substrates. Both cell types adhered and elongated when plated on the surface of the collagen I coated PA gels and glass substrates, except for the 3 kPa where fewer cells adhered and reduced cell spreading after attachment was observed after 48 h from seeding (Fig. 1A). Fluorescent staining (phalloidin-TRITC) of the actin cytoskeleton at 48 h after seeding suggested that both cell types could equally sense the stiffness of the substrates as their actin cytoskeleton became more stretched and organized in bundles for substrates stiffer than 3 kPa (Fig. 1B). Quantitative measurement of cell area and aspect ratio confirmed that soft substrates (3 kPa) with E = 3 kPa elicit significant lower cell spreading and elongation in both cell types (Fig. 1C-D). However, no significant differences were observed on the 10, 20, 80 kPa PA gels and glass.


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 1. Effect of substrate stiffness on cell morphology and actin cytoskeleton organization. (A) Representative bright field images of NHDFα and LMNAmut cells seeded on polyacrylamide gels with stiffness ranging from 3 kPa to 80 kPa taken 48 h after seeding. Fewer and less spread cells were present on 3 kPa polyacrylamide gels than on stiffer substrates for both cell types. Scale bar: 100 μm. (B) Actin organization in NHDFα and LMNAmut (phalloidin-TRITC, red) showed increased organization and tension on substrates stiffer than 3 kPa. Green color is given by lamin B1 staining. Scale bar: 20 μm. (C and D) Cell area and aspect ratio presented as box-and-whisker plots. The measurements of NHDFα and LMNAmut did not show significant difference, thus the values were considered as a group.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3585029&req=5

Figure 1: Figure 1. Effect of substrate stiffness on cell morphology and actin cytoskeleton organization. (A) Representative bright field images of NHDFα and LMNAmut cells seeded on polyacrylamide gels with stiffness ranging from 3 kPa to 80 kPa taken 48 h after seeding. Fewer and less spread cells were present on 3 kPa polyacrylamide gels than on stiffer substrates for both cell types. Scale bar: 100 μm. (B) Actin organization in NHDFα and LMNAmut (phalloidin-TRITC, red) showed increased organization and tension on substrates stiffer than 3 kPa. Green color is given by lamin B1 staining. Scale bar: 20 μm. (C and D) Cell area and aspect ratio presented as box-and-whisker plots. The measurements of NHDFα and LMNAmut did not show significant difference, thus the values were considered as a group.
Mentions: We investigated the intracellular effect of increasing substrate stiffness on diseased dermal fibroblasts, isolated from a patient suffering from a progeroid syndrome due to compound heterozygous missense mutations (p.T528M and p.M540T) in the LMNA gene (LMNAmut) and we compared these findings with control human fibroblast cell line (NHDFα). For this purpose both cell types were seeded on collagen I coated polyacrylamide (PA) gels with stiffnesses ranging from 3 kPa to 80 kPa, as well as on collagen I coated glass substrates. Both cell types adhered and elongated when plated on the surface of the collagen I coated PA gels and glass substrates, except for the 3 kPa where fewer cells adhered and reduced cell spreading after attachment was observed after 48 h from seeding (Fig. 1A). Fluorescent staining (phalloidin-TRITC) of the actin cytoskeleton at 48 h after seeding suggested that both cell types could equally sense the stiffness of the substrates as their actin cytoskeleton became more stretched and organized in bundles for substrates stiffer than 3 kPa (Fig. 1B). Quantitative measurement of cell area and aspect ratio confirmed that soft substrates (3 kPa) with E = 3 kPa elicit significant lower cell spreading and elongation in both cell types (Fig. 1C-D). However, no significant differences were observed on the 10, 20, 80 kPa PA gels and glass.

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