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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 7. Spontaneous ruptures of the nuclear membrane do not occur on soft substrates. (A) Montage of selected images from a time-lapse recording of LMNAmut cell cultured on 10 kPa PA gel and transfected with EYFP-NLS, sampled at 1 min intervals for 2 h (Video S1). Nuclear membrane rupture causes the decrease in intranuclear EYFP signal and increase in cytoplasmic EYFP signal (at 0:36, yellow arrowhead). Subsequently, the nuclear signal is gradually up taken by the nucleus and the rupture appears to be restored (at 0:51, orange arrowhead) (B) Evolution in time of EYFP-NLS (mean) intensity in the nucleus of the cell shown in (A). (C) Frequency of spontaneous nuclear membrane ruptures on the different substrates for LMNAmut. Error bars represent the square root of the number of recording.
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Figure 7: Figure 7. Spontaneous ruptures of the nuclear membrane do not occur on soft substrates. (A) Montage of selected images from a time-lapse recording of LMNAmut cell cultured on 10 kPa PA gel and transfected with EYFP-NLS, sampled at 1 min intervals for 2 h (Video S1). Nuclear membrane rupture causes the decrease in intranuclear EYFP signal and increase in cytoplasmic EYFP signal (at 0:36, yellow arrowhead). Subsequently, the nuclear signal is gradually up taken by the nucleus and the rupture appears to be restored (at 0:51, orange arrowhead) (B) Evolution in time of EYFP-NLS (mean) intensity in the nucleus of the cell shown in (A). (C) Frequency of spontaneous nuclear membrane ruptures on the different substrates for LMNAmut. Error bars represent the square root of the number of recording.

Mentions: A recent study by De Vos et al.16 showed the occurrence of spontaneous nuclear ruptures in cells from laminopathy patients cultured on glass substrates. These ruptures never occurred in wild type cells (NHDFα) under the same culturing conditions. Based on these results and triggered by the finding of variable cytPML-NBs on different substrate stiffness, we hypothesized that mechanical cues provided by the extracellular environment might affect the frequency of nuclear rupture events. For this purpose we monitored living cells (about 20) for two hours at one or two minute intervals under a fluorescent microscope on 3, 10, 20, 80 kPa PA gels and glass substrate after 24−36 h from transfection with EYFP-nuclear localization signal (EYFP-NLS), which helped to check for nuclear integrity. Correct expression of EYFP-NLS was revealed by a constant intense intranuclear fluorescent signal. In NHDFα cells, as well as in LMNAmut cells on 3 kPa substrates we could not detect a nuclear rupture event in any cell examined. In contrast, for the stiffer substrates an increased frequency of nuclear rupture was detected in the LMNAmut cells, increasing from 20% (4/20) of LMNAmut with nuclear rupture on the 10 kPa substrate to 34.5% (10/29) on 80 kPa. The ruptures were visible as a sudden transient efflux of EYFP-NLS from the nucleus to the cytoplasm (Fig. 7 and Video S1). This phenomenon, which lasts about 20 min and can occur repetitively in the same cell, was followed by restoration of EYFP-NLS signal in the nucleus and was not lethal for the cells. All together, these results confirm that soft substrates do not compromise the nuclear integrity of LMNAmut cells, while stiff environments do.


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 7. Spontaneous ruptures of the nuclear membrane do not occur on soft substrates. (A) Montage of selected images from a time-lapse recording of LMNAmut cell cultured on 10 kPa PA gel and transfected with EYFP-NLS, sampled at 1 min intervals for 2 h (Video S1). Nuclear membrane rupture causes the decrease in intranuclear EYFP signal and increase in cytoplasmic EYFP signal (at 0:36, yellow arrowhead). Subsequently, the nuclear signal is gradually up taken by the nucleus and the rupture appears to be restored (at 0:51, orange arrowhead) (B) Evolution in time of EYFP-NLS (mean) intensity in the nucleus of the cell shown in (A). (C) Frequency of spontaneous nuclear membrane ruptures on the different substrates for LMNAmut. Error bars represent the square root of the number of recording.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 7: Figure 7. Spontaneous ruptures of the nuclear membrane do not occur on soft substrates. (A) Montage of selected images from a time-lapse recording of LMNAmut cell cultured on 10 kPa PA gel and transfected with EYFP-NLS, sampled at 1 min intervals for 2 h (Video S1). Nuclear membrane rupture causes the decrease in intranuclear EYFP signal and increase in cytoplasmic EYFP signal (at 0:36, yellow arrowhead). Subsequently, the nuclear signal is gradually up taken by the nucleus and the rupture appears to be restored (at 0:51, orange arrowhead) (B) Evolution in time of EYFP-NLS (mean) intensity in the nucleus of the cell shown in (A). (C) Frequency of spontaneous nuclear membrane ruptures on the different substrates for LMNAmut. Error bars represent the square root of the number of recording.
Mentions: A recent study by De Vos et al.16 showed the occurrence of spontaneous nuclear ruptures in cells from laminopathy patients cultured on glass substrates. These ruptures never occurred in wild type cells (NHDFα) under the same culturing conditions. Based on these results and triggered by the finding of variable cytPML-NBs on different substrate stiffness, we hypothesized that mechanical cues provided by the extracellular environment might affect the frequency of nuclear rupture events. For this purpose we monitored living cells (about 20) for two hours at one or two minute intervals under a fluorescent microscope on 3, 10, 20, 80 kPa PA gels and glass substrate after 24−36 h from transfection with EYFP-nuclear localization signal (EYFP-NLS), which helped to check for nuclear integrity. Correct expression of EYFP-NLS was revealed by a constant intense intranuclear fluorescent signal. In NHDFα cells, as well as in LMNAmut cells on 3 kPa substrates we could not detect a nuclear rupture event in any cell examined. In contrast, for the stiffer substrates an increased frequency of nuclear rupture was detected in the LMNAmut cells, increasing from 20% (4/20) of LMNAmut with nuclear rupture on the 10 kPa substrate to 34.5% (10/29) on 80 kPa. The ruptures were visible as a sudden transient efflux of EYFP-NLS from the nucleus to the cytoplasm (Fig. 7 and Video S1). This phenomenon, which lasts about 20 min and can occur repetitively in the same cell, was followed by restoration of EYFP-NLS signal in the nucleus and was not lethal for the cells. All together, these results confirm that soft substrates do not compromise the nuclear integrity of LMNAmut cells, while stiff environments do.

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