Limits...
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.

Show MeSH

Related in: MedlinePlus

Figure 3. Influence of substrate stiffness on cytoskeletal actin organization and aberrations. Confocal z-series taken from half height of the whole cell and relative orthogonal cross sections of NHDFα and LMNAmut immunocytochemical stained for F-actin in red (phalloidin-TRITC) and Lamin B1 in green at 48 h after seeding. (A) Representative fibroblast seeded on 3 kPa PA gels. It shows short and not tensed actin fibers, which are missing in the perinuclear region. An actin cap is running above the nucleus (white arrowhead). No differences could be noticed between LMNAmut and NHDFα. Thus no aberrations could be detected in the actin cytoskeleton of cells plated on soft substrates. (B) Control NHDFα on PA gel stiffer than 3 kPa, precisely on the 20 kPa PA gel. Actin stress fibers are tensed and well-structured also in the perinuclear region. The actin cap made of thick stress fibers run above the nucleus the (white arrowhead) nucleus. (C and D) Representative aberrations found in LMNAmut seeded on 20 (C) and 80 kPa (D) PA gels. Cells have a misshapen nucleus. Yellow arrowhead indicates the lack of actin fibers in the perinuclear region (D and E) and a speckled distribution of actin (E). The actin cap is running above the nucleus (white arrowhead). Scale bars: 20 μm. (E) Representative images of cells on three substrate stiffnesses. NHDFα (left panel) and LMNAmut (right panel) on 3kPa (F), 20 kPa (G) and 80 kPa (H) PA gels. Scale bar: 50 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3585029&req=5

Figure 3: Figure 3. Influence of substrate stiffness on cytoskeletal actin organization and aberrations. Confocal z-series taken from half height of the whole cell and relative orthogonal cross sections of NHDFα and LMNAmut immunocytochemical stained for F-actin in red (phalloidin-TRITC) and Lamin B1 in green at 48 h after seeding. (A) Representative fibroblast seeded on 3 kPa PA gels. It shows short and not tensed actin fibers, which are missing in the perinuclear region. An actin cap is running above the nucleus (white arrowhead). No differences could be noticed between LMNAmut and NHDFα. Thus no aberrations could be detected in the actin cytoskeleton of cells plated on soft substrates. (B) Control NHDFα on PA gel stiffer than 3 kPa, precisely on the 20 kPa PA gel. Actin stress fibers are tensed and well-structured also in the perinuclear region. The actin cap made of thick stress fibers run above the nucleus the (white arrowhead) nucleus. (C and D) Representative aberrations found in LMNAmut seeded on 20 (C) and 80 kPa (D) PA gels. Cells have a misshapen nucleus. Yellow arrowhead indicates the lack of actin fibers in the perinuclear region (D and E) and a speckled distribution of actin (E). The actin cap is running above the nucleus (white arrowhead). Scale bars: 20 μm. (E) Representative images of cells on three substrate stiffnesses. NHDFα (left panel) and LMNAmut (right panel) on 3kPa (F), 20 kPa (G) and 80 kPa (H) PA gels. Scale bar: 50 μm.

Mentions: In order to provide insight into the role of the actin cytoskeleton on the onset of nuclear abnormalities (protective mechanism of a soft environment on nuclear integrity), we investigated actin fiber organization using phalloidin-TRITC labeling. The actin cytoskeleton is known indeed to respond to substrate stiffness and affect cell shape and migration. Confocal microscopy of the phalloidin stained cells plated on 3 kPa PA gels showed a rounded morphology for both cell genotypes, with little polymerized actin formation that barely formed bundling of tensed fibers. In the perinuclear region there seemed to be no actin fibers, while we observed fibers running on top of the nucleus (actin cap) (Fig. 3A). At 10 kPa and higher stiffnesses, cells demonstrated the typical well-spread and flattened morphology with development of bundles of tense stress fibers (Figs. 1A and 3B). According to Khatau et al.,23LMNA mutant cells can lack the characteristic actin cap running above the nucleus. After analysis of confocal z-stacks of both cell genotypes in our study, we could not confirm a difference in actin cap presence. However, we did detect aberrations in actin cytoskeleton organization in about 5% of the LMNAmut cells plated on 10, 20, 80 kPa and glass, at 48 h after seeding but not on the 3 kPa. These aberrations included detachment of actin stress fibers in the perinuclear region with formation of a speckled pattern of actin which suggests actin depolymerisation in these areas (Fig. 3C−E). Similar observations were already reported for cells cultured on glass coverslips.7,17,24,25


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 3. Influence of substrate stiffness on cytoskeletal actin organization and aberrations. Confocal z-series taken from half height of the whole cell and relative orthogonal cross sections of NHDFα and LMNAmut immunocytochemical stained for F-actin in red (phalloidin-TRITC) and Lamin B1 in green at 48 h after seeding. (A) Representative fibroblast seeded on 3 kPa PA gels. It shows short and not tensed actin fibers, which are missing in the perinuclear region. An actin cap is running above the nucleus (white arrowhead). No differences could be noticed between LMNAmut and NHDFα. Thus no aberrations could be detected in the actin cytoskeleton of cells plated on soft substrates. (B) Control NHDFα on PA gel stiffer than 3 kPa, precisely on the 20 kPa PA gel. Actin stress fibers are tensed and well-structured also in the perinuclear region. The actin cap made of thick stress fibers run above the nucleus the (white arrowhead) nucleus. (C and D) Representative aberrations found in LMNAmut seeded on 20 (C) and 80 kPa (D) PA gels. Cells have a misshapen nucleus. Yellow arrowhead indicates the lack of actin fibers in the perinuclear region (D and E) and a speckled distribution of actin (E). The actin cap is running above the nucleus (white arrowhead). Scale bars: 20 μm. (E) Representative images of cells on three substrate stiffnesses. NHDFα (left panel) and LMNAmut (right panel) on 3kPa (F), 20 kPa (G) and 80 kPa (H) PA gels. Scale bar: 50 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Figure 3. Influence of substrate stiffness on cytoskeletal actin organization and aberrations. Confocal z-series taken from half height of the whole cell and relative orthogonal cross sections of NHDFα and LMNAmut immunocytochemical stained for F-actin in red (phalloidin-TRITC) and Lamin B1 in green at 48 h after seeding. (A) Representative fibroblast seeded on 3 kPa PA gels. It shows short and not tensed actin fibers, which are missing in the perinuclear region. An actin cap is running above the nucleus (white arrowhead). No differences could be noticed between LMNAmut and NHDFα. Thus no aberrations could be detected in the actin cytoskeleton of cells plated on soft substrates. (B) Control NHDFα on PA gel stiffer than 3 kPa, precisely on the 20 kPa PA gel. Actin stress fibers are tensed and well-structured also in the perinuclear region. The actin cap made of thick stress fibers run above the nucleus the (white arrowhead) nucleus. (C and D) Representative aberrations found in LMNAmut seeded on 20 (C) and 80 kPa (D) PA gels. Cells have a misshapen nucleus. Yellow arrowhead indicates the lack of actin fibers in the perinuclear region (D and E) and a speckled distribution of actin (E). The actin cap is running above the nucleus (white arrowhead). Scale bars: 20 μm. (E) Representative images of cells on three substrate stiffnesses. NHDFα (left panel) and LMNAmut (right panel) on 3kPa (F), 20 kPa (G) and 80 kPa (H) PA gels. Scale bar: 50 μm.
Mentions: In order to provide insight into the role of the actin cytoskeleton on the onset of nuclear abnormalities (protective mechanism of a soft environment on nuclear integrity), we investigated actin fiber organization using phalloidin-TRITC labeling. The actin cytoskeleton is known indeed to respond to substrate stiffness and affect cell shape and migration. Confocal microscopy of the phalloidin stained cells plated on 3 kPa PA gels showed a rounded morphology for both cell genotypes, with little polymerized actin formation that barely formed bundling of tensed fibers. In the perinuclear region there seemed to be no actin fibers, while we observed fibers running on top of the nucleus (actin cap) (Fig. 3A). At 10 kPa and higher stiffnesses, cells demonstrated the typical well-spread and flattened morphology with development of bundles of tense stress fibers (Figs. 1A and 3B). According to Khatau et al.,23LMNA mutant cells can lack the characteristic actin cap running above the nucleus. After analysis of confocal z-stacks of both cell genotypes in our study, we could not confirm a difference in actin cap presence. However, we did detect aberrations in actin cytoskeleton organization in about 5% of the LMNAmut cells plated on 10, 20, 80 kPa and glass, at 48 h after seeding but not on the 3 kPa. These aberrations included detachment of actin stress fibers in the perinuclear region with formation of a speckled pattern of actin which suggests actin depolymerisation in these areas (Fig. 3C−E). Similar observations were already reported for cells cultured on glass coverslips.7,17,24,25

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