Cytoplasmic localization of PML particles in laminopathies

Histochem Cell Biol DOI 10.1007/s00418-012-1005-5

ORIGINAL PAPER

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Cytoplasmic localization of PML particles in laminopathies

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F. Houben • W. H. De Vos • I. P. C. Krapels • M. Coorens • G. Kierkels • M. A. F. Kamps • V. L. R. M. Verstraeten • C. L. M. Marcelis • A. van den Wijngaard F. C. S. Ramaekers • J. L. V. Broers

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Accepted: 23 July 2012 ! Springer-Verlag 2012

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Electronic supplementary material The online version of this article (doi:10.1007/s00418-012-1005-5) contains supplementary material, which is available to authorized users.

occurrence of repetitive nuclear ruptures has been described in fibroblast cultures from various laminopathy patients. Since this phenomenon was strongly correlated with disease severity, the identification of biomarkers that report on these rupture events could have diagnostic relevance. One such candidate marker is the PML nuclear body, a structure that is normally confined to the nuclear interior, but leaks out of the nucleus upon nuclear rupture. Here, we show that a variety of laminopathy cells shows the presence of these cytoplasmic PML particles, (PML CPs) and that the amount of these protein aggregates varies with severity of the disease. In addition, between clinically healthy individuals, carrying LMNA mutations, significant differences can be found. Therefore, we postulate that detection of PML CPs in patient fibroblasts could become a valuable marker for diagnosis of disease development. Keywords Lamins ! PML nuclear bodies ! Immunofluorescence ! Nuclear rupture ! Dermal fibroblasts

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Introduction

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Laminopathies are a group of diseases that result from mutations in the LMNA gene, encoding A-type lamin proteins. The different types of mutations affecting LMNA can partly explain the diversity of phenotypes. Laminopathies can be divided into five major classes: (1) striated muscle dystrophies, (2) peripheral nerve dystrophies, (3) lipodystrophies, (4) premature ageing syndromes and systemic laminopathies, and (5) heterogeneous diseases with overlapping phenotypes (Broers et al. 2006). In addition, mutations in genes encoding for lamin-associated proteins (e.g. emerin) or enzymes that are responsible for the processing of lamins (e.g. ZMPSTE24) can evoke

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Abstract There is growing evidence that laminopathies, diseases associated with mutations in the LMNA gene, are caused by a combination of mechanical and gene regulatory distortions. Strikingly, there is a large variability in disease symptoms between individual patients carrying an identical LMNA mutation. This is why classical genetic screens for mutations appear to have limited predictive value for disease development. Recently, the widespread

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F. Houben ! M. Coorens ! G. Kierkels ! M. A. F. Kamps ! F. C. S. Ramaekers ! J. L. V. Broers (&) Department of Molecular Cell Biology, CARIM, School for Cardiovascular Diseases, Maastricht University Medical Center, UNS50. Box 17, P.O. Box 616, NL-6200 MD Maastricht, The Netherlands e-mail: [email protected]

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W. H. De Vos Department of Molecular Biotechnology, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium

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W. H. De Vos NB-Photonics, Ghent University, Ghent, Belgium

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I. P. C. Krapels ! A. van den Wijngaard Department of Clinical Genetics, CARIM, School for Cardiovascular Diseases, Maastricht University Medical Center, Maastricht, The Netherlands

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V. L. R. M. Verstraeten Department of Dermatology, GROW, School for Oncology and Developmental Biology, Maastricht University Medical Center, Maastricht, The Netherlands

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C. L. M. Marcelis Department of Clinical Genetics, Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands

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Material and methods

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Patient cell cultures

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were typically devoid of characteristic PML NB components, such as DAXX and SP100 (De Vos et al. 2011), which is why we label these structures with the term cytoplasmic PML particles (PML CPs). The purpose of this study was to determine the frequency of PML CPs in different laminopathy cultures, and to examine the correlation with spontaneous nuclear membrane ruptures. In addition, we have investigated whether the frequency of PML CP containing cells in patient fibroblast cell cultures is correlated with specific laminopathy subtypes and/or progression of the disease. We found that in cell cultures from patients with LMNA mutations, having nuclear abnormalities increased cytoplasmic PML CPs can be found. However, even in patient cell cultures without overt nuclear abnormalities, the percentage of cells with PML CPs, was significantly higher, indicating that assessment of PML CPs could be a novel parameter for laminopathy detection.

We tested fibroblast cell cultures, derived from patients with laminopathy-related symptoms. Informed consent was obtained from all patients, which were seen for genetic counselling. Fibroblasts were isolated from skin biopsies and subjected to extensive genomic analysis using a specially designed cardiochip (Van den Wijngaard et al. 2009). These genomic analyses revealed mutations in LMNA, or ZMPSTE24, in some cases accompanied by mutations in other genes, potentially attributing to the disease phenotype (see below). Cultures, essentially free from epithelial cell contamination were grown in DMEM as described (Houben et al. 2009) and were passaged at least 3 times before immunocytochemical stainings were performed. The following fibroblast cultures were analysed:

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laminopathies (Broers et al. 2006). There are two major hypotheses considered to explain the resulting phenotypes, the ‘structural’ hypothesis and the ‘gene expression’ hypothesis. The first hypothesis states that absence of lamins or incorrect assembly of (mutated) lamins leads to weakening of the nuclear lamina, resulting in both nuclear and cellular weakness. As a result, especially cells, prone to mechanical stress, such as muscle cells, become damaged, resulting in tissue degeneration. The latter hypothesis (Cohen et al. 2001; Wilson et al. 2001; Vlcek and Foisner 2007; Schirmer and Foisner 2007) states that the interaction between the nuclear lamina and transcription factors is altered as a result of the mutations in the LMNA gene. Recent studies indicate that lamin A/C mutations or absence of A-type lamins can provoke a third disease mechanism, i.e. temporary decompartmentalisation due to nuclear membrane ruptures, causing inappropriate exchange between cytoplasmic and nuclear components (De Vos et al. 2011) Upon nuclear membrane rupture mobile molecules and complexes enter or leave the nucleus, causing the loss of concentration, necessary for maintaining compartment-specific function. Strikingly these ruptures are reversible and apparently non-lethal to the cells. Nuclear ruptures may explain disease development by uniting aspects of both mechanical and generegulation theories. In a previous study, we have shown that upon rupture, large protein structures such as PML nuclear bodies (PML NBs) can translocate from the nuclear compartment to the cytoplasm, and can be detected in the cytoplasm of lamin A/C deficient cells (De Vos et al. 2011). PML NBs are large intranuclear protein complexes that form functional units containing next to PML, proteins such as Sumo-1, Sp100, p53, pRB, HP1, and Daxx (Bernardi and Pandolfi 2007). Sumo-1 modification of the PML protein is necessary for deposition of PML at PML NBs, the recruitment of other associated factors such as Daxx1 and formation of the PML NBs (Sternsdorf et al. 1997; LaMorte et al. 1998; Kamitani et al. 1998; Muller et al. 1998; Ishov et al. 1999; Zhong et al. 2000; LallemandBreitenbach et al. 2001; Shen et al. 2006; Nagai et al. 2011). These bodies are implicated in a plethora of cellular functions, including chromatin organisation (Seeler and Dejean 1999; Boisvert et al. 2001), viral response (through interferon) (Ishov and Maul 1996; Regad and Chelbi-Alix 2001), DNA replication and repair (Zhong et al. 1999; Eskiw et al. 2004; Dellaire et al. 2006), transcriptional regulation (Boisvert et al. 2001; Wu et al. 2001; Wang et al. 2004), tumour suppression, induction of senescence and apoptosis (Bischof et al. 2002; Bernardi and Pandolfi 2007). Immunofluorescence staining of the PML particles that were found in the cytoplasm of lamin A/C deficient cells,

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Control fibroblast cultures from commercial suppliers or from patients with no clinical symptoms of laminopathies or LMNA mutations, with normal nuclear morphology and with normal lamin A/C immunostaining: Normal human dermal fibroblast from juvenile foreskin (NHDF-1); cat. nr C-12300 PromoCell, Heidelberg, Germany); (b) Normal human dermal fibroblast from an adult (NHDF-2) lot nr 4C0623 (17-year-old female; Cascade Biologics/GIBCO, Portland, OR, USA); (c) Normal human dermal fibroblasts (NHDF-3), kindly provided by L. Schurgers (Department of

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Histochem Cell Biol

Histochem Cell Biol

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LVNC1 is obtained from a male patient (47 years old) with Left Ventricle Non-compaction, with a heterozygous E246R mutation in the LMNA gene and an additional A161P mutation in the myosin heavy chain 7 (MYH7) gene; LVNC2 is obtained from a female patient (41 years old) with Left Ventricle Non-compaction with a heterozygous R545H mutation in the LMNA gene and an additional L396P mutation in the MYH7 gene; HCM is obtained from a male patient (30 years old) with Hypertrophic Cardiomyopathy with ventricular fibrillation, with a heterozygous R644C mutation in the LMNA gene, a heterozygous R278C mutation in the troponin T type 2 (TNNT2) gene, a heterozygous Q998R mutation in the myosin binding protein C (MYBPC3) gene and a heterozygous S126G mutation in the galactosidase alpha (GLA) gene.

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Human fibroblasts were grown on glass cover slips for at least 48 h, followed by fixation with 4 % formaldehyde in PBS (15 min at room temperature), followed by permeabilization with 0.1 % Triton X-100 in PBS, (15 min at room temperature) and immunostained as described previously (Houben et al. 2009). The following antibodies were used:

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PML (PG-M3) mouse monoclonal antibody, (Santa Cruz, Heidelberg, Germany, diluted 1:1,000), recognizing different PML isoforms; Rabbit polyclonal antibody recognizing Ki67 (Dakopatts, Glostrup, DK, diluted 1:100); Rabbit polyclonal Lamin B specific antibody, kindly provided by J.C. Courvalin (INSERM. Paris, France, diluted 1:200); Lamin C specific rabbit polyclonal antibody RalC, (MUbio Products B.V., Maastricht, The Netherlands, diluted 1:20); Lamin A/C mouse monoclonal antibody Jol2 kindly provided by Prof. C. Hutchison, University of Durham, UK, diluted 1:50).

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The following secondary antibodies were used:

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FITC conjugated goat anti-rabbit Ig (SBA/ITK Birmingham, AL, USA, diluted 1:50); Texas Red conjugated goat anti-rabbit Ig (SBA/ITK, diluted 1:80); FITC conjugated rabbit anti mouse Ig antibody (Dakopatts, Glostrup, DK, diluted 1:100).

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Cells were visualised using a Leica TCS SPE confocal laser scanning fluorescence microscope (Leica DMRBE,

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Immunofluorescence

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Compound DCM is obtained from a male patient (53 years old) with a compound mutation (R331G and a R627C) in the LMNA gene leading to Dilated Cardiomyopathy; EDMD is obtained from a female patient with Emery–Dreifuss Muscular Dystrophy with a heterozygous I531 deletion in the LMNA gene; FPLD is obtained from a patient with (Dunnigan type) Familial Partial LipoDystrophy with a heterozygous R439C mutation in the LMNA gene (Verstraeten et al. 2009); LGMD is obtained from a patient with Limb-Girdle Muscular Dystrophy with a heterozygous R644C mutation in the LMNA gene. The patient suffers from contractures, sudden loss of strength, and both hypertrophic and dilated cardiomyopathy. The age of onset was about 40 years; The compound progeroid cell culture is obtained from a 2-year-old patient with progeroid syndrome with a c.1583C.T (p.T528M) and a c.1619T.C (p.M540T) mutation in the LMNA gene (Verstraeten et al. 2006). In addition, fibroblasts from both the father (M540T) (38 years old, no clinical symptoms) and mother (33 years old, no clinical symptoms) were used. (T528 M); HGPS is obtained from a patient with Hutchinson– Gilford Progeria syndrome with a heterozygous G608G mutation in the LMNA gene (Verstraeten et al. 2006); Patient cells with a nonsense homozygous LMNA mutation (Y259X/Y259X) resulting in the absence of lamin A/C proteins in these cells. This patient, whose heterozygous family members suffer from limb-girdle muscular dystrophy, died at birth (Muchir et al. 2003); RD is obtained from a patient with Restrictive Dermopathy with homozygous c.1085_1086insT truncating mutations in the zinc metallopeptidase (STE24 homolog, S. cerevisiae, ZMPSTE24) gene (Verstraeten et al. 2006). The patient suffers from a lethal autosomal recessive skin condition characterized by abnormal facies, tight skin, sparse or absent eyelashes, and secondary joint changes;

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Biochemistry, Maastricht University, Maastricht, The Netherlands) taken from the neck of a normal subject who underwent surgery and did not show any clinical sign of connective tissue alterations (Gheduzzi et al. 2007). (d) Human dermal fibroblasts ((HDF-1) from a female patient (7 years old) with psychomotoric retardation and suspicion of peroxisomal dysfunction, but without LMNA mutation. (e) Human dermal fibroblasts (HDF-2) from a male patient (43 years old) with hypertonia and myopathy, but without a LMNA mutation.

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Dextran scrape loading followed by PML immunostaining

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Confluently growing cells were scrape loaded with 70 kDa dextran-Texas Red (dextran-TR, Molecular Probes, Oregon, USA) using a hypodermic needle as described (De Vos et al. 2011), and were allowed to regrow for different time spans (0, 4, 8, 24 h). Next, cells were fixed in 4 % formaldehyde in PBS and immunostained with the PML antibody. Confocal z-stacks were made, which allow determination of dextran uptake into the nuclear interior along with determination of the localization of PML NBs/CPs.

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Transfection and live cell imaging

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Normal as well as laminopathy fibroblasts growing in log phase were transfected with EYFP-NLS (Kremers et al. 2006) (a generous gift from Dr J. Goedhart, University of Amsterdam, The Netherlands), and/or with EYFP-PML (Wiesmeijer et al. 2002) using Genejammer (Stratagene, La Jolla, California, USA) (De Vos et al. 2011) according to the manufacturer’s instructions. Experiments were performed with cultures ranging from passage 5–23. Time-lapse recordings were made 24–48 h after transfection. Live cell imaging was performed on a Nikon A1R confocal microscope, mounted on a Nikon Ti body, equipped with a Perfect Focus System, a microscope incubator equilibrated at 36.5 "C, and a micro-chamber for humidity and CO2 control. Recordings were made using a 609/1.4 Plan Apo oil immersion lens. Alternatively, live cell imaging was performed on an inverted fluorescence microscope (Leica DIRBE, Leica Microsystems BV, Rijswijk, The Netherlands), equipped with a black and white CCD camera (CA4742-95, Hamamatsu), a polychrome II polychromator as light source for fluorescence (TILL Photonics, Martinsried, Germany) and a 209/0.7 Plan Apo lens.

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Results

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In a previous study, we have reported on the occurrence of repetitive nuclear ruptures in laminopathy cells (De Vos

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et al. 2011). Here, we extended the number of human dermal fibroblast cultures, examined and have sought for (and found) alternative markers for detection of nuclear rupture events. An overview of the different cell cultures with their specific mutations, clinical features and other characteristics is shown in Table 1. While nuclear ruptures never occurred in 149 recording of NHDF cells, this event did take place to a variable extent in fibroblast cell cultures with a LMNA mutation. The number of cells with nuclear ruptures varied considerably between cell cultures, ranging from about 5 % up to 50 % (Table 1). While the occurrence of these nuclear ruptures appears to discriminate between normal and diseased cells, alternative markers for determination of nuclear ruptures are needed. The rupture assay used, i.e. transfection of primary fibroblasts using EYFP tagged nuclear localization signal (EYFP-NLS) followed by live imaging of individual cells, is a slow and tedious assay, especially since the efficiency of fibroblast transfection is very low (below 1 %) and vital imaging of individual transfected cells is an elaborate and time-consuming protocol, with little statistical value due to the low number of cells that can be investigated. In addition, since many laboratories lack the facilities for routine transfection and live imaging, this assay would not be suitable for routine screening purposes.

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Mannheim, Germany) equipped with a 639 objective (NA 1.3) as described (De Vos et al. 2011). For quantification of PML particle distribution, the number of cells with PML CP staining was scored by counting at least 3 9 100 cells per cell culture. Counting of the average number of intranuclear PML NBs was performed by scoring at least 100 cells per culture and determining the number of PML NBs per cell by thresholding the fluorescent PML NBs within each nucleus using the spot_count macro in ImageJ (v.1.45) software (Rasband 1997–2011).

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PML NBs can be lost from the nucleus upon nuclear rupture

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In our search for additional markers for nuclear ruptures, we found that nuclear ruptures can evoke the release of PML nuclear bodies (PML NBs) from the nucleus to the cytoplasm. In Fig. 1, a selection of time lapse recordings of a representative Y259X/Y259X cell, co-transfected with EYFPNLS and EYFP-PML is shown, showing translocation of PML NBs during a rupture event. For both constructs we chose the same fluorescent tag (EYFP), since this allows the simultaneous determination of the rupture time point (NLS) along with registration of the behaviour of PML NBs, with no delay between recordings of different fluorescent channels. In addition, this reduces the amount of damaging fluorescent light needed. Pilot studies showed that EYFP-PML fluorescence is strictly confined to bright (mainly) intranuclear aggregates, easily distinguishable from the entirely diffuse intranuclear EYFP-NLS signal (data not shown). At T = 12 min a sudden complete loss of the diffuse intranuclear (NLS) signal indicates the occurrence of a nuclear rupture, followed by the efflux of at least one clearly visible PML NB from the nucleus (arrowhead). This PML NB gradually fragments (triple arrowhead) and eventually disappears completely. (see also movie 1 suppl. Material). At time point T = 1 h 14 min another PML NB

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Histochem Cell Biol Table 1 Characteristics and results of measurements of cell cultures used in this study Cell line

None

Other mutations

/

Origin/pathology

Healthy boys’ foreskin

% Nucl. ruptures EYFP-NLS (# of cells/ total #)

% Cells with PML CPs

0 (0/149)

2.5 ± 0.5 (n = 1702)

None

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Healthy female skin tissue

0 (0/23)

None

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Healthy volunteer

n.d.

(n = 3 9 100)

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% Cells with blebs and honeycombs (n = 3 9 100)

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HDF-1

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Psychomotoric retardation

n.d.

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n.d.

HDF-2

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myopathy

n.d.

2.7 ± 1.5

n.d.

Compound DCM

R331G/R627C

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Dilated cardiomyopathy

5 (1/20)

12.3 ± 2.5

15.0 ± 1.3

10 ± 2

5.7 ± 2.1

17 ± 4

23 ± 2

I531del

n.d.

Emery–Dreifuss muscular dystrophy

n.d.

R439C

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Familial partial lipodystrophy

24.6 (15/61)

LGMD

R644C/?

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Limb-girdle muscular dystrophy

7.7 (7/91)

28.0 ± 2.5

3±1

Comp.progeroid

T528M/M540T

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Progeroid

29.1 (39/134)

17.7 ± 0.6

39 ± 3

Father prog

T528 M

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None so far

Mother prog

M540T

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None so far

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EDMD FPLD

14.3 (3/21) 0 (0/23)

17.3 ± 1.7

14 ± 2

8.2 ± 3.7

9±1

G608G

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Hutchinson-Gilford Progeria Syndrome

13.6 (21/154)

16.3 ± 1.5

13 ± 1

Y259X/Y259X

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No lamin A/C protein, died shortly after birth

49.5 (93/188)

59.7 ± 5.1

63 ± 5

RD

None

ZMPSTE24: hom. c.1085_1086insT

Restrictive dermopathy

5.2 (3/58)

11.3 ± 0.6

28 ± 5

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HGPS Y259X/Y259X

LVNC1

E246R/?

MYH7:A161P

Left ventricle non-compaction

n.d.

14.7 ± 2.1

3±1

LVNC2

R545H/?

MYH7:L396P

Left ventricle non-compaction

n.d.

16.0 ± 3.6

9±2

HCM

R644C/?

TNNT2:R278C MYBPC3:Q998R GLA:S126G

Hypertrophic cardiomyopathy

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n.d. not done

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NHDF-1

Mutations in LMNA gene

escapes from the same region of the nucleus (arrow), in this case without a clearly visible decrease of the (still weak) NLS signal. In the latter case it is unclear whether the closure of the nuclear membrane did not complete yet, or another rupture event has taken place. Since also immunocytochemical staining revealed the occurrence of cytoplasmic PML particles in fixed laminopathy cells (see also (De Vos et al. 2011)), we sought to investigate the potential use of these PML CPs to serve as a proxy for the nuclear rupture events. By comparing the prevalence of PML CPs with other pathological features of the patient cells we also assessed its diagnostic potential in the detection of laminopathies.

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PML localisation in cell cultures from laminopathy and non-laminopathy patients

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In order to obtain insight into the prevalence of PML CPs, we performed immunofluorescence experiments on the fibroblast cell cultures mentioned in Table 1. A representative z-stack projection of a confocal recording of a cell of

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each cell culture, immunostained for PML, is shown in Fig. 2. We have compared control fibroblasts from different sources in order to examine the variation in number of cells with PML CPs between age, sex, and passage number. After exclusion of postmitotic cells (see below) the average number of these structures varied from 2.5 % ± 0.5 in commercially available human foreskin fibroblasts (NHDF-1) to 4.3 % ± 0.6 in cells from a healthy donor (NHDF-3, Table 1). In addition, dermal fibroblasts from patient with non-LMNA related diseases (HDF-1 and HFD2) showed similar values (Table 1). In all cell cultures from patients diagnosed with a laminopathy a significant increase in the number of cells with PML CPs was found as compared to the control cell cultures (Student’s t test, p \ 0.05 for all). In laminopathy patient cells these values ranged from 5.7 % in a case of FPLD up to 59.7 % in the cell culture from the homozygous negative LMNA patient (Y259X/Y259X) who died shortly after birth. In cell cultures from patients suffering from other mutations, next to LMNA mutations, which

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02:54 T = 12 min was followed by the escape of a PML body from the nucleus (arrowhead), which gradually disappears (triple arrowhead) Later, another PML NB escapes from the nucleus (small arrow). The complete movie (movie S1) can be viewed as supplementary material. Scale bar 10 lm

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might cause the disease phenotype, similar numbers were found, except for a cell culture from a patient with HCM, which showed only few cells (0.7 %) with PML CPs (Table 1).

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The increase in prevalence of PML CPs in laminopathy cells is not caused by cell cycle differences

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Since it is known that PML NBs relocate to the cytoplasm during mitosis [35, 36] and do not all re-enter the nucleus immediately after cytokinesis, we have investigated whether the relative abundance of PML CPs in laminopathies was partly due to differences in the cell cycle distribution between laminopathy patient cells and normal cells. To this end, we performed double immunolabelling for PML and Ki67. Ki67 shows a very characteristic, bright speckled immunostaining pattern from telophase to mid-G1 phase (Scholzen and Gerdes 2000), allowing identifying cells in which PML NBs did not re-enter the nucleus after mitosis. In addition, non-proliferating (G0) cells can be distinguished by the absence of the Ki67 staining, allowing discrimination between cycling and non-cycling cells. Figure 3 shows representative confocal acquisitions from

these immunolabelled cells. In NHDF-2 cells, PML CPs could exclusively be found in mitotic cells and in cells in early G1 phase. In addition, the number of PML CPs could be reduced to zero after serum starvation, and increased to more than 20 % after cell cycle synchronisation (data not shown). In contrast, both the compound progeroid (M540T/ T528M) and the Y259X/Y259X cells showed PML CPs in cells that were not in early post-mitotic stages, and even in non-proliferating cells, as deduced from the complete absence of Ki67 labelling (Fig. 3b, c). This latter observation is most striking in the Y259X/Y259X cells. In general, early postmitotic cells are easily detectable, even without Ki67 labelling, as pairs of cells with small, round

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Fig. 1 Loss of PML nuclear bodies upon nuclear membrane rupture. Y259X/Y259Y cells, co-transfected with EYFP-NLS (diffuse intranuclear fluorescence) and EYFP-PML (bright intranuclear spots) were studied by time-lapse microscopy. The image contrast has been stretched to visualise the pan-nuclear signal of EYFP-NLS. A rupture of the nucleus, visible as a sudden loss of diffuse intranuclear signal at

Fig. 2 a Projections of confocal z-series recordings of cells immu- c nostained with an anti-PML antibody, showing both intranuclear PML NBs as well as cytoplasmic localization of PML particles (green) in laminopathy cells, Nuclei were counterstained with DAPI (blue). Note that in all fibroblast cultures, except for NHDF and HCM, PML CPs can be found outside of the nucleus. Note also the low amount of intranuclear PML NBs in some of these cells. Scale bar 10 lm. b Graphic view of percentages of cells with PML CPs per cell culture

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Histochem Cell Biol DCM Compound

NHDF-2

EDMD

FPLD

RD

LVNC1

HGPS

Y259X/Y259X

HCM

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Compound progeroid

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LGMD

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% cells with PML CPs

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HCM

LVNC2

LVNC1

RD

Y259X/Y259X

HGPS

Comp. prog.

LGMD

FPLD

EDMD

Comp. DCM

NHDF-2

0

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Compound progeroid

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nuclei. To assess the influence of possibly undetected postmitotic cells on the numbers of PML CPs found, we have compared the counted percentages of PML CPs in different passage numbers of NHDF-1 cells. While the number of PML CPs did gradually decrease from 3.8 % (at passage 9) to 0.95 % (at passage 27), these values are well below the lowest values found in laminopathy cells (Table 1). These findings match very well with previous studies in other normal cell cultures (Jul-Larsen et al. 2009; Carracedo et al. 2011).

nuclear ruptures that has taken place in a cell population. Presuming that nuclear ruptures reduce the number of intranuclear PML NBs, an alternative method would be to count the remaining number of PML NBs. We calculated the number of PML NBs in the two most affected cell cultures (Y259X/Y259X and compound progeroid) and compared these with normal NHDF cells. Indeed, in cell cultures with high amounts of PML CPs, a larger variation in the number of PML NBs could be observed (see Fig. 4a).

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Since nuclear ruptures are temporary events, their visualisation in non-transfected cells is very difficult. Judging form our live imaging experiments, we anticipated that PML NBs, translocated to the cytoplasm upon nuclear rupture, would only remain in the cytoplasm for a limited time span, before becoming degraded. As a result, counting of PML CPs could underestimate the actual number of

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Fig. 3 Projections of confocal z-series recordings of double immunofluorescence stainings with antibodies against PML (green) and Ki67 (red) of normal NHDF cells (a), compound progeroid cells (b) and Y259X/Y259X cells (c). Note that in the control cells only

Fig. 4 Comparison of variation in number of PML nuclear bodies per c cell in normal (NHDF), compound progeroid, and Y259X/Y259X cells. a Confocal z-series projections of immunofluorescence staining with the anti-PML antibody (green in merged image), showing that the variation in number of PML NBs per cell is relatively large in compound progeroid cells compared to NHDF cells, while the Y259X/Y259X cells, show an overall decrease in number of PML NBs. Nuclei were counterstained with DAPI (blue in merged image). Note also the presence of scattered cytoplasmic PML particles in some cells of both laminopathy cell cultures and not in NHDF cells. b Graphic representation of counting of PML NBs in the three cell cultures. Note the relatively broad range of the number of PML NBs per cell in the compound progeroid cell culture

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Normally, most PML NBs are in a higher organised protein complex, tightly bound to the nuclear matrix and/or DNA (Bernardi and Pandolfi 2007). Previously, we showed that the PML structures that translocate to the cytoplasm during rupture are more mobile than the majority of the PML NBs. To verify whether there was a direct correlation between nuclear ruptures and the presence of PML CPs, we monitored the accumulative events of nuclear ruptures in cells, by loading dextran-TR in the cytoplasm of normal and laminopathy fibroblasts. In control NHDF cells, only cells that have completed mitosis after dextran-TR loading will show intranuclear dextran-TR uptake. In contrast, laminopathy cells showed an increased number of cells with intranuclear dextran-TR fluorescence due to nuclear ruptures that occurred in the time period after loading (Fig. 5). On the cell population level, a gradual increase of dextranTR containing nuclei and cells with PML CPs was observed in time (Fig. 5d, e). However, on the cellular level, there was no direct correlation between nuclear dextran-TR labelling and cytoplasmic PML body staining. While dextran-TR could be present in the nucleus, PML CPs were not always visible in the cytoplasm (cf. Fig. 5b, c). In addition, the opposite occasionally occurred, i.e. cells with PML CPs, but without intranuclear dextran-TR accumulation (data not shown).

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It is conceivable, that nuclear ruptures are more common in cells with visible nuclear aberrations (blebs, honeycombs), as these represent weak spots in the nuclear envelope, where ruptures are more prone to occur (De Vos et al. 2011). All cell cultures were immunocytochemically stained with anti-lamin (A/C) antibodies, and the number

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Based on these findings we assumed that the presence of PML CPs in the cytoplasm, is a potential novel marker for the detection of laminopathies, complementary to nuclear ruptures and structural nuclear abnormalities. In order to evaluate the potential predictive value of this assay we compared fibroblasts from a two-year old male patient with compound heterozygous mutations in the LMNA gene (Verstraeten et al. 2006) with his parents. He inherited one mutation from each parent, who is heterozygous for his or her respective mutation. The patient suffers from a progeroid syndrome, resembling symptoms of the classical Hutchinson–Gilford Progeria Syndrome, while both parents are clinically unaffected until now. The father has a

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of nuclear aberrations was determined and compared with the frequency of nuclear ruptures in NLS-EYFP transfected cells (where performed). Next, these data were compared to the frequency of PML CPs in these cultures (Fig. 6). While we expected that the presence of nuclear aberrations would predict the occurrence of nuclear ruptures in transfected cells, this was to some extent found in our experiments (linear correlation coefficient R2 = 0.71). In two cell cultures (the compound DCM cell line and the RD cell line), there was no correlation between nuclear abnormalities and ruptures. Exclusion of these two cultures from the comparison increases the correlation coefficient R2 to 0.89. When comparing the frequency of nuclear abnormalities with the percentage of cells, which have PML CPs, no correlation between these parameters could be determined in the different cell cultures (R2 = 0.52). The same holds true for a comparison between nuclear rupture events and the percentage of cells with PML CPs (R2 = 0.56). Investigations within the group of cell cultures with (additional) mutations, other than in LMNA, showed that the RD patient cell cultures displayed abnormalities as expected: due to the absence of a functional ZMPST24 protein, the cells have all characteristics of laminopathy cells, including prominent nuclear abnormalities, nuclear ruptures and PML CPs. In the two cases of LVNC prominent PML CPs are accompanied by a low (LVNC1) to moderate (LVNC1) amount of nuclear abnormalities. Whether these nuclear abnormality differences are reflected in clinical differences in the patients remains to be examined. Strikingly, the HCM patient culture shows only a slight increase in nuclear abnormalities (nuclear shape and lamina immunostaining) along with no increase in PML CPs, indicating that the nuclear barrier function is largely intact. These findings raise the question whether in this patient the LMNA mutation contributes to the disease phenotype.

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While the average number of PML NBs did not show large variations between NHDF and compound progeroid cells (12.3 ± 7.7 and 14.0 ± 9.2, respectively), the average number for Y259X/Y259X cells (9.6 ± 9.8) was lower. However, due to the huge variation in the number of PML NBs per cell, these differences were not significant. However, looking at the frequency distribution of the number of intranuclear PML NBs per cell (Fig. 4b) it becomes clear that, while in NHDF cells these counts approach a Gaussian distribution, the variation in PML NBs per cell in the compound progeroid cell culture is much larger and the curve points towards two subpopulations of cells with respect to the number of intranuclear PML NBs counted. In the Y259X/Y259X culture, relatively more cells with very low numbers (0–4) of PML NBs can be found (Fig. 4b).

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c.1583C.T (p.T528M) mutation in exon 9. This mutation is associated in literature with FPLD (Savage et al. 2004). The mother carries the c.1619T.C (p.M540T) mutation in exon 10. After immunocytochemical staining for PML, 17.7 % of the cells of the patient demonstrated PML CPs. Similarly, the father’s cells showed PML CPs in 17.3 % of the cells, while the mother’s fibroblasts only had 8.2 % PML CPs (Fig. 7). When comparing the nuclear aberrations detected by immunostaining, which lead to weak spots in the nuclear lamina, the mother’s cells also demonstrate fewer aberrations (8.7 %) compared to the father (14.3 %) or the patient (38.8 %) (Fig. 7). Closer comparison of two cultures with an identical heterozygous R644C mutation in the LMNA gene revealed striking differences. Beside this identical mutation, the cultures did not have a similar genotype or phenotype. The first cell line, LGMD, came from a male patient (about 40 years old) with limb-girdle muscular dystrophy (contractures, sudden loss of strength, and both hypertrophic and dilated cardiomyopathy) but no known mutation in other genes. The second cell line, HCM, came from a male patient (30 years old) with hypertrophic cardiomyopathy with ventricular fibrillation and with additional mutations in the TNNT2, the MYBPC3 and the GLA gene. When we compared the percentage of cells with PML CPs, the difference between the two cell cultures was dramatic (LGMD: 28.0 ± 2.0 %; HCM 0.7 ± 0.6 %), although the number of cells with nuclear aberrations in both cultures was comparable (LGMD: 3 ± 1 %; HCM 5 ± 1 %). Based on the absence of PML CPs, the absence of abnormalities in lamina A/C staining as well as the absence or nuclear shape abnormalities, we now consider that the

clinical symptoms of the HCM patient are not due to the LMNA mutation, but are due to the other gene mutations. The two cell cultures from patients with LNC both have a MYH7 mutation, next to a (different) LMNA mutation. Comparison of nuclear abnormalities showed a significant difference between the two cultures, but no differences in frequency of PML CPs were observed.

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Previously, we have documented the process of repetitive, dynamic ruptures in lamin-deficient cells, which lead to the translocation of small regulatory proteins and macromolecular complexes (De Vos et al. 2011). While live imaging of NLS-EYFP transfected cells is the most direct manner to determine whether dynamic ruptures of the nuclear membrane in living cells occur, it is too tedious for routine screening and has low statistical power. Since the duration of rupture visibility can be very short (a few minutes), it is not possible to monitor large numbers of cells simultaneously. In our attempt to search for markers which would allow alternative quantification methods for registration of nuclear ruptures we discovered that PML nuclear bodies, which, under normal growth conditions are almost completely confined to the nucleus (for a recent review see (Carracedo et al. 2011)), translocate to the cytoplasm during ruptures. While PML proteins can be present in the cytoplasm, only in case of mutations in the PML gene larger protein complexes called cytoplasmic PML bodies can form (Bellodi et al. 2006). Since hitherto nuclear ruptures have only been described in laminopathies, HIV-

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infected cells (due to lamina breakdown (de Noronha et al. 2001)) and in cancer cells that show reduction in lamin B expression (Vargas et al. 2012), registration of the translocation of PML bodies due to nuclear ruptures may be

used as a diagnostic marker for cells with deregelulated lamin expression, including laminopathies. Indeed, only a very low percentage of normal NHDF cells showed PML CPs (about 3 % in unsynchronised cultures). This

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functionally linked proteins (e.g. emerin, desmin) (Mercuri et al. 2005; Muntoni et al. 2006; Rankin et al. 2008). In these cell cultures examined, the differences in the markers for the loss of nuclear integrity may point to a negative effect of the LMNA mutation in the LGMD patient, while the negative effects on the phenotype is the HCM patient are more likely to be caused by the mutations in 3 other genes found in this patient. This indicates that testing for loss of nuclear integrity in patients and family members with this mutation could be a possible predictor for the rate of penetrance of the mutation. The exact origin of the PML CPs is currently under investigation. While vital imaging clearly showed that ruptures of the nuclear membrane could lead to the efflux of individual PML-bodies, it is premature to state that all PML CPs arise from these ruptures. Dextran-TR labelled cells occasionally showed PML CPs without signs of dextran-TR influx, indicating nuclear rupture, while also the reverse could be observed. Several factors could have a negative effect on this correlation. First, the nuclear rupture could have a short duration or could only cause a minor opening in the nuclear membrane allowing small molecules (NLS) to escape, but not the larger PML structures. Secondly, most PML NBs are normally tightly bound to the nuclear matrix, including DNA, possibly preventing the escape of most PML NBs from the nucleus. Absence of PML CPs in apparently ruptured nuclei can be explained by our observation in live imaging experiments that PML bodies seem to become degraded after relocalization to the cytoplasm (Fig. 1). However, it is also possible that other cellular processes can lead to cytoplasmic PML staining. Research from Ishov et al. (1999) and Butler et al. (2009) describe cytoplasmic PML aggregates as immature PML bodies, which lack SUMO-1, Daxx and Sp100. As the sumoylation of PML is essential for the correct formation of PML bodies (with all the associated proteins), a mutation in lamin A/C can influence the formation and localization of PML bodies and can lead to immature PML particles in the cytoplasm of cells. Vice versa, delocalization of PML to the cytoplasm may lead to desumoylation and subsequent detachment of other typical PML NB components. Lamins influence the correct localization and functioning of SUMO-1 (Sylvius et al. 2005). Certain mutations in the LMNA gene lead to an abnormal distribution of SUMO-1 and a reduction in sumoylation of proteins. However, in our laminopathy cell cultures all intranuclear PML bodies did contain SUMO-1 (data not shown). The lamin protein itself is also sumoylated, rather by SUMO-2 than by SUMO-1 (Zhang and Sarge 2008). A defect in lamin A/C sumoylation could play an important role in the underlying molecular mechanism of the familial cardiomyopathy associated laminopathies. This disturbance of sumoylation of PML-bodies, together with the

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background level represents a cell-cycle-dependent presence of cytoplasmic PML structures in G1 phase (JulLarsen et al. 2009), as confirmed by Ki67 labelling. The data from the current PML-study indicate that not only in lamin-deficient cells, but also in cell cultures from other laminopathy patients, PML CPs can be detected. This is important because lamin deficiency is an extremely rare and lethal condition and therefore clinically less relevant. While the percentage of nuclear ruptures cannot be directly correlated to the amount of cells with PML CPs, possibly due to the low number of cells in which nuclear ruptures were registered, it became evident that cell types, which demonstrated ruptures (irrespective of the frequency), had significantly more cells with PML CPs than normal cells. Since we evaluated a relative large number of normal NHDF cells (n = 149) and never found a nuclear rupture in these cells, we can state that indeed in normal cells such a rupture is extremely rare, or even absent. While we previously documented a correlation between the occurrence of nuclear rupture events and nuclear aberrations, we now compared the number of cells containing PML CPs with the number of cells having nuclear aberrations (blebs, honeycombs, and other visible weak spots of the nuclear lamina). Surprisingly no direct correlation could be observed. In addition, at the individual cell level, no such a correlation could be established: cells with apparently large numbers of nuclear deformations showed few PML CPs, while cells with apparently normal shaped nuclei showed an increased loss of PML NBs into the cytoplasm. While several explanations for this discrepancy are possible (e.g. the disintegration of PML CPs, or other routes of PML CP formation), it does not prevent PML CPs from being a potential biomarker for laminopathies. In this respect it is promising to notice that the most severe cases of laminopathies (compound progeroid, HGPS, Y259X/ Y259X) appear to show a high number of PML CPs. Additional indications come from our studies on the compound progeroid patient and his parents. The diseased child has a high number of nuclear ruptures and cytoplasmic PML bodies, significantly higher than his healthy mother, Strikingly, his symptom–free father has also relatively high numbers of ruptures and PML CPs, possibly predicting development of laminopathy-related disease symptoms in time. This age of this father (now 38 years old) is relatively young for laminopathy development in patients with this heterozygous mutation. Thus, clinical follow-up of the father is highly recommended. Another interesting case is the difference in clinical outcome in the two patients with the same R644C mutation. Previous studies show that the R644C mutation can lead to extreme phenotypic diversity and non-penetrance. (Rankin et al. 2008) The difference in clinical outcome can be explained by a second not yet identified mutation in a

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Bellodi C, Kindle K, Bernassola F, Dinsdale D, Cossarizza A, Melino G, Heery D, Salomoni P (2006) Cytoplasmic function of mutant promyelocytic leukemia (PML) and PML-retinoic acid receptoralpha. J Biol Chem 281(20):14465–14473 Bernardi R, Pandolfi PP (2007) Structure, dynamics and functions of promyelocytic leukaemia nuclear bodies. Nat Rev Mol Cell Biol 8(12):1006–1016 Bischof O, Kirsh O, Pearson M, Itahana K, Pelicci PG, Dejean A (2002) Deconstructing PML-induced premature senescence. EMBO J 21:3358–3369

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Acknowledgments The authors wish to thank Julien Cox for performing dextran experiments. We thank Dr. J. Wiegant for providing the YEFP-PML construct. We thank Dr. J. Goedhart for providing the EYFP-NLS construct.

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Boisvert FM, K MJ, Box AK, Hendzel MJ, Bazett-Jones DP (2001) The transcription coactivator CBP is a dynamic component of the promyelocytic leukemia nuclear body. J Cell Biol 152:1099–1106 Broers JL, Ramaekers FC, Bonne G, Yaou RB, Hutchison CJ (2006) Nuclear lamins: laminopathies and their role in premature ageing. Physiol Rev 86(3):967–1008 Butler JT, Hall LL, Smith KP, Lawrence JB (2009) Changing nuclear landscape and unique PML structures during early epigenetic transitions of human embryonic stem cells. J Cell Biochem 107:609–621 Carracedo A, Ito K, Pandolfi PP (2011) The nuclear bodies inside out: PML conquers the cytoplasm. Curr Opin Cell Biol 23(3): 360–366 Cohen M, Lee KK, Wilson KL, Gruenbaum Y (2001) Transcriptional repression, apoptosis, human disease and the functional evolution of the nuclear lamina. Trends Biochem Sci 26(1):41–47 de Noronha CM, Sherman MP, Lin HW, Cavrois MV, Moir RD, Goldman RD, Greene WC (2001) Dynamic disruptions in nuclear envelope architecture and integrity induced by HIV-1 Vpr. Science 294(5544):1105–1108 De Vos WH, Houben F, Kamps M, Malhas A, Verheyen F, Cox J, Manders EM, Verstraeten VL, van Steensel MA, Marcelis CL, van den Wijngaard A, Vaux DJ, Ramaekers FCS, Broers JLV (2011) Repetitive disruptions of the nuclear envelope invoke temporary loss of cellular compartmentalization in laminopathies. Hum Mol Genet 20:4175–4186 Dellaire G, Ching RW, Ahmed K, Jalali F, Tse KC, Bristow RG, Bazett-Jones DP (2006) Promyelocytic leukemia nuclear bodies behave as DNA damage sensors whose response to DNA doublestrand breaks is regulated by NBS1 and the kinases ATM, Chk2, and ATR. J Cell Biol 175:55–66 Eskiw CH, Dellaire G, Bazett-Jones DP (2004) Chromatin contributes to structural integrity of promyelocytic leukemia bodies through a SUMO-1-independent mechanism. J Biol Chem 279:9577–9585 Gheduzzi D, Boraldi F, Annovi G, DeVincenzi CP, Schurgers LJ, Vermeer C, Quaglino D, Ronchetti IP (2007) Matrix Gla protein is involved in elastic fiber calcification in the dermis of pseudoxanthoma elasticum patients. Lab Invest 87(10): 998–1008 Houben F, Willems CH, Declercq IL, Hochstenbach K, Kamps MA, Snoeckx LH, Ramaekers FC, Broers JL (2009) Disturbed nuclear orientation and cellular migration in A-type lamin deficient cells. Biochim Biophys Acta 1793(2):312–324 Ishov AM, Maul GG (1996) The periphery of nuclear domain 10 (ND10) as site of DNA virus deposition. J Cell Biol 134:815–826 Ishov AM, Sotnikov AG, Negorev D, Vladimirova OV, Neff N, Kamitani T, Yeh ET, Strauss JF III, Maul GG (1999) PML Is Critical for ND10 Formation and Recruits the PML-interacting Protein Daxx to this Nuclear Structure When Modified by SUMO-1. J Cell Biol 147(2):221–233 Jul-Larsen A, Grudic A, Bjerkvig R, Boe SO (2009) Cell-cycle regulation and dynamics of cytoplasmic compartments containing the promyelocytic leukemia protein and nucleoporins. J Cell Sci 122(Pt 8):1201–1210 Kamitani T, Kito K, Nguyen HP, Wada H, Fukada-Kamitami T, Yeh ET (1998) Identification of three major sentrinization sites in PML. J Biol Chem 273:26675–26682 Kremers GJ, Goedhart J, van Munster EB, Gadella TW Jr (2006) Cyan and yellow super fluorescent proteins with improved brightness, protein folding, and FRET Forster radius. Biochemistry 45(21):6570–6580 Lallemand-Breitenbach V, Zhu J, Puvion F (2001) Role of promyelocytic leukemia (PML) sumolation in nuclear body formation,

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phenomenon of the nuclear ruptures could thus also explain the high prevalence of PML CPs in cells from laminopathy patients. Interestingly, a direct correlation between disturbed PML organisation, increased PML mobility and the loss of lamin A/C expression has recently been reported, indicating a functional link between PML bodies and A-type lamins (Stixova et al. 2012). Currently we can only speculate on the effect of loss of (functional) PML NBs in laminopathies. While the loss is limited, as indicated by the quantification of (remaining) PML NBs, the diversity of functions, assigned to these structures allows speculating into several directions. The interaction of both lamins and PML nuclear bodies with transcription factors has long been recognized. Loss of functionality of both complexes could have a complementary effect, causing a variety of diseases. In addition, the partial loss of DNA repair capacity in laminopathies could be explained by non-functioning PML NBs, known to be important sensors of cell stress and facilitators of DNA repair and recombination (Dellaire et al. 2006). The results from the different cultures, and especially the compound progeroid patient and both his parents clearly show that the detection of PML CPs is a potential method for screening not only possible patients, but also family members of laminopathy patients, which show no clinical phenotype at first sight. This screening could be used to test whether the parents and siblings of the patient are at risk for developing clinical symptoms, or are merely subclinical and thus likely to develop symptoms over the next couple of years. In the latter case, further follow-up and preventive measures can then be provided. Taken together, these results indicate that determination of the percentage of cells with PML CPs is a potential novel diagnostic tool in detecting laminopathies. Such markers are needed in view of the poor predictive value of both classical genetic screening and of detection of nuclear aberrations (Muchir et al. 2004).

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Histochem Cell Biol

123 Journal : Large 418

Dispatch : 3-8-2012

Pages : 16

Article No. : 1005

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MS Code : HCB-2306-12-Drenckhahn

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