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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 4. Effects of transient cytoD treatment on LMNAmut nuclei. Representative confocal sections of LMNAmut seeded on collagen I coated glass substrates incubated with cytoD 1μM and recovered in normal growth medium. After fixation, cells were stained with DAPI (blue) to check for nuclear abnormalities and phalloidin-TRITC (red) to check for actin organization. (A) Untreated control LMNAmut. (B) Short treatment + short recovery: LMNAmut treated for 30 min with cytoD and recovered for 1 h. (C) Long treatment + short recovery: LMNAmut treated for 3 h with cytoD and recovered for 1 h. (D) Long treatment + long recovery LMNAmut treated for 3 h with cytoD and recovered overnight. Scale bar: 20 μm. (E) Frequency of misshapen nuclei in LMNAmut upon treatment with cytoD. At least 600 cells were assessed per each group.*, p < 0.05; no star, p > 0.05
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Figure 4: Figure 4. Effects of transient cytoD treatment on LMNAmut nuclei. Representative confocal sections of LMNAmut seeded on collagen I coated glass substrates incubated with cytoD 1μM and recovered in normal growth medium. After fixation, cells were stained with DAPI (blue) to check for nuclear abnormalities and phalloidin-TRITC (red) to check for actin organization. (A) Untreated control LMNAmut. (B) Short treatment + short recovery: LMNAmut treated for 30 min with cytoD and recovered for 1 h. (C) Long treatment + short recovery: LMNAmut treated for 3 h with cytoD and recovered for 1 h. (D) Long treatment + long recovery LMNAmut treated for 3 h with cytoD and recovered overnight. Scale bar: 20 μm. (E) Frequency of misshapen nuclei in LMNAmut upon treatment with cytoD. At least 600 cells were assessed per each group.*, p < 0.05; no star, p > 0.05

Mentions: To further prove the correlation between actin cytoskeletal tension and the onset of nuclear abnormalities, LMNAmut cells grown on collagen I coated glass bottom culture dishes were incubated for different period of times with cytochalasin D (cytoD), which inhibits actin dynamics and, consequently, causes disruption of the actin-cytoskeleton (Fig. 4). Next, the drug was removed and LMNAmut cells were allowed to recover in normal growth medium for 1 h to overnight. Confocal microscopy on phalloidin-TRITC and DAPI stained LMNAmut cells showed that the short treatment (30 min) followed by 1 h recovery (short treatment + short recovery) disrupted the actin-cytoskeleton only mildly compared with untreated control LMNAmut (Fig. 4A and B). Yet, there was no difference between the frequency of misshapen nuclei in this group and in the untreated control LMNAmut (18.4 ± 2.1% vs 22.3 ± 4.0%, Fig. 4E). There was presumably not enough time for the nucleus to respond to the decrease in cytoskeletal tension or the degree of disruption did not allow any response. In contrast, a three hour treatment followed by an hour recovery (long treatment + short recovery) leads to serious disruption of the actin cytoskeleton and significantly less misshapen nuclei (11.8 ± 0.8%, Fig. 4C). Upon three hours cytoD treatment followed by overnight recovery (long treatment + long recovery), the actin cytoskeleton completely recovered from the treatment and tensed stress fibers were visible (Fig. 4D). The frequency of misshapen nuclei (19.1 ± 1.6%) was found to be comparable to that of untreated LMNAmut cells. Moreover we analyzed the changes in nuclear morphology due to cellular detachment of LMNAmut cells by trypsin, followed by re-adhesion to a glass substrate (Fig. 5). At 30 and 60 min after seeding, nuclear folding due to trypsin treatment did not yet allow a reliable analysis of nuclear shape. At this stage of attachment the actin cytoskeleton was largely disorganized, seen as absence of tense stress fibers in these cells. Starting from 2 till 8 h after seeding the frequency of misshapen nuclei was significantly lower than that at 72 h (11.0 ± 2.0%, 13.3 ± 3.8%, 14.6 ± 2.3%, 15.6 ± 2.2%, 28.3 ± 3.5% respectively at 2, 4, 8, 24 and 72 h). At these time points actin reorganization did take place in the lower regions of the cell, making contact with the glass substrate, but stress fibers were absent at close distance to the nucleus. While after 24 h of attachment the actin organization was mainly restored, showing actin fibers in close contact with the nucleus, it took even longer (up to 72 h) until cells were fully stretched, and showing the regular percentage of abnormal nuclei (Fig. 5A and B)


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 4. Effects of transient cytoD treatment on LMNAmut nuclei. Representative confocal sections of LMNAmut seeded on collagen I coated glass substrates incubated with cytoD 1μM and recovered in normal growth medium. After fixation, cells were stained with DAPI (blue) to check for nuclear abnormalities and phalloidin-TRITC (red) to check for actin organization. (A) Untreated control LMNAmut. (B) Short treatment + short recovery: LMNAmut treated for 30 min with cytoD and recovered for 1 h. (C) Long treatment + short recovery: LMNAmut treated for 3 h with cytoD and recovered for 1 h. (D) Long treatment + long recovery LMNAmut treated for 3 h with cytoD and recovered overnight. Scale bar: 20 μm. (E) Frequency of misshapen nuclei in LMNAmut upon treatment with cytoD. At least 600 cells were assessed per each group.*, p < 0.05; no star, p > 0.05
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Figure 4. Effects of transient cytoD treatment on LMNAmut nuclei. Representative confocal sections of LMNAmut seeded on collagen I coated glass substrates incubated with cytoD 1μM and recovered in normal growth medium. After fixation, cells were stained with DAPI (blue) to check for nuclear abnormalities and phalloidin-TRITC (red) to check for actin organization. (A) Untreated control LMNAmut. (B) Short treatment + short recovery: LMNAmut treated for 30 min with cytoD and recovered for 1 h. (C) Long treatment + short recovery: LMNAmut treated for 3 h with cytoD and recovered for 1 h. (D) Long treatment + long recovery LMNAmut treated for 3 h with cytoD and recovered overnight. Scale bar: 20 μm. (E) Frequency of misshapen nuclei in LMNAmut upon treatment with cytoD. At least 600 cells were assessed per each group.*, p < 0.05; no star, p > 0.05
Mentions: To further prove the correlation between actin cytoskeletal tension and the onset of nuclear abnormalities, LMNAmut cells grown on collagen I coated glass bottom culture dishes were incubated for different period of times with cytochalasin D (cytoD), which inhibits actin dynamics and, consequently, causes disruption of the actin-cytoskeleton (Fig. 4). Next, the drug was removed and LMNAmut cells were allowed to recover in normal growth medium for 1 h to overnight. Confocal microscopy on phalloidin-TRITC and DAPI stained LMNAmut cells showed that the short treatment (30 min) followed by 1 h recovery (short treatment + short recovery) disrupted the actin-cytoskeleton only mildly compared with untreated control LMNAmut (Fig. 4A and B). Yet, there was no difference between the frequency of misshapen nuclei in this group and in the untreated control LMNAmut (18.4 ± 2.1% vs 22.3 ± 4.0%, Fig. 4E). There was presumably not enough time for the nucleus to respond to the decrease in cytoskeletal tension or the degree of disruption did not allow any response. In contrast, a three hour treatment followed by an hour recovery (long treatment + short recovery) leads to serious disruption of the actin cytoskeleton and significantly less misshapen nuclei (11.8 ± 0.8%, Fig. 4C). Upon three hours cytoD treatment followed by overnight recovery (long treatment + long recovery), the actin cytoskeleton completely recovered from the treatment and tensed stress fibers were visible (Fig. 4D). The frequency of misshapen nuclei (19.1 ± 1.6%) was found to be comparable to that of untreated LMNAmut cells. Moreover we analyzed the changes in nuclear morphology due to cellular detachment of LMNAmut cells by trypsin, followed by re-adhesion to a glass substrate (Fig. 5). At 30 and 60 min after seeding, nuclear folding due to trypsin treatment did not yet allow a reliable analysis of nuclear shape. At this stage of attachment the actin cytoskeleton was largely disorganized, seen as absence of tense stress fibers in these cells. Starting from 2 till 8 h after seeding the frequency of misshapen nuclei was significantly lower than that at 72 h (11.0 ± 2.0%, 13.3 ± 3.8%, 14.6 ± 2.3%, 15.6 ± 2.2%, 28.3 ± 3.5% respectively at 2, 4, 8, 24 and 72 h). At these time points actin reorganization did take place in the lower regions of the cell, making contact with the glass substrate, but stress fibers were absent at close distance to the nucleus. While after 24 h of attachment the actin organization was mainly restored, showing actin fibers in close contact with the nucleus, it took even longer (up to 72 h) until cells were fully stretched, and showing the regular percentage of abnormal nuclei (Fig. 5A and B)

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