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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 8. Proposed mechanism for actin cytoskeleton organization and effects on nuclear abnormalities on soft and stiff substrates. Schematic representation of the cross section of a cell seeded on substrates with different stiffness. Red represents the actin cytoskeleton while green the nucleus (dark green nuclear lamina). (A) When seeded on a soft substrate NHDFα and LMNAmut cells have the same response. They develop not tensed actin stress fibers which are not directly connected to the nucleus in the perinuclear region. Fibers run below and on top of the nucleus. Since low forces are exerted on the nucleus, the onset of nuclear abnormalities is prevented. (B) NHDFα cells seeded on a stiff substrate (stiffer than 3 kPa) develop tensed actin stress fibers which are well-organized in the whole cytoplasm. Stress fibers connect to the nucleus and form also the actin cap running on top of the nucleus. (C)LMNAmut cells on top of a stiff substrate develop tensed stress fibers. These stress fibers appear to be lacking in the perinuclear region and aggregates of actin are often visible. The pushing action of the actin cap (stress fibers running on top of the nucleus) together with the uneven distribution of pushing actin in the area around the nucleus lead to the development of nuclear abnormalities, further resulting in nuclear damage.
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Figure 8: Figure 8. Proposed mechanism for actin cytoskeleton organization and effects on nuclear abnormalities on soft and stiff substrates. Schematic representation of the cross section of a cell seeded on substrates with different stiffness. Red represents the actin cytoskeleton while green the nucleus (dark green nuclear lamina). (A) When seeded on a soft substrate NHDFα and LMNAmut cells have the same response. They develop not tensed actin stress fibers which are not directly connected to the nucleus in the perinuclear region. Fibers run below and on top of the nucleus. Since low forces are exerted on the nucleus, the onset of nuclear abnormalities is prevented. (B) NHDFα cells seeded on a stiff substrate (stiffer than 3 kPa) develop tensed actin stress fibers which are well-organized in the whole cytoplasm. Stress fibers connect to the nucleus and form also the actin cap running on top of the nucleus. (C)LMNAmut cells on top of a stiff substrate develop tensed stress fibers. These stress fibers appear to be lacking in the perinuclear region and aggregates of actin are often visible. The pushing action of the actin cap (stress fibers running on top of the nucleus) together with the uneven distribution of pushing actin in the area around the nucleus lead to the development of nuclear abnormalities, further resulting in nuclear damage.

Mentions: Furthermore, disorganization of the actin cytoskeleton in the perinuclear region of LMNAmut, was observed particularly on stiffer substrates and gave indication for an abnormal distribution of forces exerted to the nucleus, enhancing nuclear morphology disturbance. In contrast to Kathau et al.,23 we observed in the LMNAmut cells the presence of the nuclear shaping actin cap. Therefore, to explain these findings, we propose that a pulling mechanism in addition to a compressive pushing mechanism might play a role in altering nuclear morphology. We suspect that, on stiff substrates, the actin cap presses tightly against the nucleus and, in addition, organization of the stress fibers around the nucleus is abnormal, enhancing the probability of disturbance and rupture in the morphology of the genetically disorganized and weakened nucleus (Fig. 8). This physical model can explain the observations on the different substrate stiffnesses. However the significant increase of misshapen nuclei on the 80 kPa PA gel is, in our opinion, due to an increase of cell-cell contact and increase in cell area and aspect ratio, which imply increase of cytoskeletal forces exerted on the nucleus. Reasons for the increase in cell area and cell aspect ratio might be found in changes in adhesive properties of the substrates. Hydrogels of increasing stiffnesses lead to increasing anchoring densities and thereby increase in cell spreading,32 while collagen absorption onto glass substrate could determine an anchoring density similar to that of the 20 kPa PA gel. While these in vitro studies cannot be directly interpolated to the in vivo situation our assay to measure nuclear weakness could well predict the development of a laminopathy phenotype in patients. A common denominator in (nearly) all laminopathies is the loss of specific tissues, seen as muscular dystrophies and/or lipodystrophies. For each of these laminopathies, its value will have to be proven.


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 8. Proposed mechanism for actin cytoskeleton organization and effects on nuclear abnormalities on soft and stiff substrates. Schematic representation of the cross section of a cell seeded on substrates with different stiffness. Red represents the actin cytoskeleton while green the nucleus (dark green nuclear lamina). (A) When seeded on a soft substrate NHDFα and LMNAmut cells have the same response. They develop not tensed actin stress fibers which are not directly connected to the nucleus in the perinuclear region. Fibers run below and on top of the nucleus. Since low forces are exerted on the nucleus, the onset of nuclear abnormalities is prevented. (B) NHDFα cells seeded on a stiff substrate (stiffer than 3 kPa) develop tensed actin stress fibers which are well-organized in the whole cytoplasm. Stress fibers connect to the nucleus and form also the actin cap running on top of the nucleus. (C)LMNAmut cells on top of a stiff substrate develop tensed stress fibers. These stress fibers appear to be lacking in the perinuclear region and aggregates of actin are often visible. The pushing action of the actin cap (stress fibers running on top of the nucleus) together with the uneven distribution of pushing actin in the area around the nucleus lead to the development of nuclear abnormalities, further resulting in nuclear damage.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 8: Figure 8. Proposed mechanism for actin cytoskeleton organization and effects on nuclear abnormalities on soft and stiff substrates. Schematic representation of the cross section of a cell seeded on substrates with different stiffness. Red represents the actin cytoskeleton while green the nucleus (dark green nuclear lamina). (A) When seeded on a soft substrate NHDFα and LMNAmut cells have the same response. They develop not tensed actin stress fibers which are not directly connected to the nucleus in the perinuclear region. Fibers run below and on top of the nucleus. Since low forces are exerted on the nucleus, the onset of nuclear abnormalities is prevented. (B) NHDFα cells seeded on a stiff substrate (stiffer than 3 kPa) develop tensed actin stress fibers which are well-organized in the whole cytoplasm. Stress fibers connect to the nucleus and form also the actin cap running on top of the nucleus. (C)LMNAmut cells on top of a stiff substrate develop tensed stress fibers. These stress fibers appear to be lacking in the perinuclear region and aggregates of actin are often visible. The pushing action of the actin cap (stress fibers running on top of the nucleus) together with the uneven distribution of pushing actin in the area around the nucleus lead to the development of nuclear abnormalities, further resulting in nuclear damage.
Mentions: Furthermore, disorganization of the actin cytoskeleton in the perinuclear region of LMNAmut, was observed particularly on stiffer substrates and gave indication for an abnormal distribution of forces exerted to the nucleus, enhancing nuclear morphology disturbance. In contrast to Kathau et al.,23 we observed in the LMNAmut cells the presence of the nuclear shaping actin cap. Therefore, to explain these findings, we propose that a pulling mechanism in addition to a compressive pushing mechanism might play a role in altering nuclear morphology. We suspect that, on stiff substrates, the actin cap presses tightly against the nucleus and, in addition, organization of the stress fibers around the nucleus is abnormal, enhancing the probability of disturbance and rupture in the morphology of the genetically disorganized and weakened nucleus (Fig. 8). This physical model can explain the observations on the different substrate stiffnesses. However the significant increase of misshapen nuclei on the 80 kPa PA gel is, in our opinion, due to an increase of cell-cell contact and increase in cell area and aspect ratio, which imply increase of cytoskeletal forces exerted on the nucleus. Reasons for the increase in cell area and cell aspect ratio might be found in changes in adhesive properties of the substrates. Hydrogels of increasing stiffnesses lead to increasing anchoring densities and thereby increase in cell spreading,32 while collagen absorption onto glass substrate could determine an anchoring density similar to that of the 20 kPa PA gel. While these in vitro studies cannot be directly interpolated to the in vivo situation our assay to measure nuclear weakness could well predict the development of a laminopathy phenotype in patients. A common denominator in (nearly) all laminopathies is the loss of specific tissues, seen as muscular dystrophies and/or lipodystrophies. For each of these laminopathies, its value will have to be proven.

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