Laminopathy-inducing mutations reduce nuclear import of expressed prelamin A.

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Laminopathy-inducing mutations reduce nuclear import of expressed prelamin A

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T. Kiel, A. Busch, A. Meyer-Rachner, S. Hübner ∗ Julius-Maximilians-University of Würzburg, Institute of Anatomy and Cell Biology, Würzburg, Germany

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Article history: Received 11 February 2014 Received in revised form 20 May 2014 Accepted 26 May 2014 Available online xxx

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Keywords: Nuclear import NLS 16 Laminopathies 17 SSIM 18 19 Lovastatin 20 Q2 CaaX motif 14 15

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Lamins are structural components of the nuclear lamina and integral parts of the nucleoplasm. The tripartite domain structure partitions the molecule into an amino-terminal head, central rod and a carboxy-terminal tail domain. The tail domain contains a nuclear localization sequence and in most lamins an additional CaaX motif, which is necessary to post-translationally process prelamin to mature lamin. As players of nuclear and cellular integrity, lamins must possess unrestrained access to the nucleus. To study whether nuclear trafficking of lamins is compromised in laminopathies, we determined relative nuclear import activities between expressed prelamin A and selected laminopathy-inducing mutants thereof. Furthermore, the impact of inhibition of maturation on nuclear import of expressed prelamin A was examined. To perform quantitative transport measurements, import competent but lamina incorporation-deficient GFP- or DsRed-tagged prelamin A deletion mutants were used, which lacked the head and rod domain (HR-prelamin A). Nuclear accumulation of HR-prelamin A carrying the lipodystrophy and metabolic syndrome-inducing mutations R419C and L421P or progeria-causing deletions was significantly reduced, but that of the maturation-deficient mutant HR-prelamin A SSIM was significantly increased. In the case of the full length prelamin A mutants R419C and L421P altered subcellular localization and reduced lamina incorporation were detected, with the prelamin A-binding protein Narf being redistributed into R419-containing aggregates. The results suggest that impaired nuclear transport of certain prelamin A mutants may represent a contributing factor in the pathogenesis of certain laminopathies. © 2014 Elsevier Ltd. All rights reserved.

1. Introduction The lamina, a proteinaceous electron-dense layer underlying the nucleoplasmic inner leaflet of the nuclear membrane, consists of members of the intermediate filament (IF) protein family, which can be classified as either A- or B-type lamins. It was once thought that lamins were important as constituents of the mechanical integrity of the nucleus (Houben et al., 2007). However, there is an increasing number of reports showing that lamins also locate within the nucleoplasm (Bridger et al., 1993; Broers et al., 1999; Hozak et al., 1995; Moir et al., 1994), playing additional roles in many fundamental intranuclear functions (see Dechat et al., 2008, 2011; Dorner et al., 2007). Lamins contribute not only to the stability and functional integrity but also to the formation and growth

∗ Corresponding author at: Institute of Anatomy and Cell Biology, JuliusMaximilians-University, Koellikerstr. 6, D-97070 Würzburg, Germany. Tel.: +49 931 31 81311; fax: +49 931 31 82712. E-mail address: [email protected] (S. Hübner).

of the nuclear envelope (NE), and are crucial in the spatial organization of nuclear pore complexes (NPCs) (Burke and Gerace, 1986; Dabauvalle et al., 1991; Lopez-Soler et al., 2001; Prufert et al., 2004, 2005; Ralle et al., 2004). Therefore, considering the multiplicity of functions to maintain cellular homeostasis, regulated import of lamins into the nucleus is of paramount importance. Nuclear import is largely mediated by a superfamily of soluble transport receptors, which are collectively referred to as importins and involves interactions between nuclear addressing signals (nuclear localization sequences, NLSs) of karyophilic proteins and members of the importin ␣ or ␤ family. Nuclear import of lamins occurs through a Simian Virus 40 large tumour antigen (SV40 T-ag)-like NLS (Loewinger and McKeon, 1988) which locates to the tail domain. The same domain contains in most lamins a CaaX (cysteine–aliphatic–aliphatic–any residue) motif, which is a target for post-translational modifications. The modifications of the prelamin molecule include a cysteine-specific farnesylation, proteolytic cleavage of the last three C-terminal amino acids, and a carboxylmethylation of the farnesylated cysteine residue (Beck et al., 1988;

http://dx.doi.org/10.1016/j.biocel.2014.05.035 1357-2725/© 2014 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Kiel T, et al. Laminopathy-inducing mutations reduce nuclear import of expressed prelamin A. Int J Biochem Cell Biol (2014), http://dx.doi.org/10.1016/j.biocel.2014.05.035

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Bergo et al., 2002; Chelsky et al., 1989; Corrigan et al., 2005; Farnsworth et al., 1989; Pollard et al., 1990; Sinensky et al., 1994; Vorburger et al., 1989; Wolda and Glomset, 1988). In the case of prelamin A a second proteolytic step occurs which removes the now C-terminal 15 amino acids, including the farnesylcysteine methyl ester (Bergo et al., 2002; Corrigan et al., 2005; Lehner et al., 1986; Pendas et al., 2002; Weber et al., 1989). The maturation process of prelamin A to mature lamin A, which totally depends on farnesylation but less on carboxyl-methylation, is extremely efficient with prelamin A being virtually undetectable under physiological circumstances (Caron et al., 2007; Zastrow et al., 2004). The tail domain additionally contains an Ig fold (aa 428–527) (Dhe-Paganon et al., 2002; Krimm et al., 2002), which represents a binding interface for many proteins (Al-Haboubi et al., 2011; Lussi et al., 2011; Simon et al., 2010; Zastrow et al., 2004, 2006). Probably the most intriguing discovery in recent years is that mutations within A-type lamins can cause distinct heritable and de novo multisystem diseases in humans. These diseases, collectively referred to as primary laminopathies (Worman and Bonne, 2007), affect a diverse set of tissues, leading inter alia to lipodystrophies, muscular dystrophies (including cardiomyopathies) and most notably progeroid syndromes, the latter being rare genetic disorders mimicking clinical and molecular features of ageing. Two such progeroid disorders, Hutchinson–Gilford progeria syndrome (HGPS) and restrictive dermopathy (RD) result mainly through mutations, which affect the process of prelamin A maturation and culminate within in the carboxy-terminal deletion of 50 (HGPS) or 90 (RD) amino acids (De Sandre-Giovannoli et al., 2003; Eriksson et al., 2003; Navarro et al., 2004). Recent investigations indicated reduced nuclear protein import in cells expressing laminopathy-causing prelamin A mutants (Busch et al., 2009). This time, we sought to investigate effects of selected laminopathy-inducing mutations on the efficiency of nuclear import of prelamin A, using lamina incorporation incompetent but nuclear competent prelamin A molecules (i.e. prelamin A molecules deleted for the head and the rod domain). The effect of inhibition of CaaX processing on nuclear import of prelamin A was also included into our studies, using a prelamin A mutant protein unable to be farnesylated (Capell et al., 2005) and therefore to be unable to processed to mature lamin A (Beck et al., 1990; Bergo et al., 2002). Finally, changes in the subcellular distribution of (pre)lamin A-binding proteins in the presence of a selected laminopathy-causing prelamin A mutant were studied.

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2. Materials and methods

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2.1. Plasmid construction

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To generate constructs for the production of GFP-tagged human wild type and pathogenic prelamin A deletion proteins lacking the head (H) and the rod (R) domain, respective full length prelamin A expression vectors encoding wild type (WT) prelamin A and the prelamin A mutants carrying the missense mutations G465D, R482K and R527P – (Hubner et al., 2006) as templates, and cDNA encoding the NLS-containing tail domains (amino acids 382–664) were amplified applying high fidelity PCR in the presence of Gateway-compatible attB1- and attB2-specific primers. Recombination reactions were subsequently performed between attB-flanking amplicons and respective entry (pDONR207 – Invitrogen) and destination (pDEST53 – Invitrogen) vectors according to the manufacture’s instructions. The expression construct encoding the fully post-translationally processed lamin A tail (i.e. the tail of mature lamin A – amino acids 382–646) was generated as described for the HR-prelamin A mutants except that instead of the prelamin A-specific attB2 primer a lamin

A-specific attB2 primer was used. The engineered GFP fusion proteins were accordingly designated as HR-preLaAWT , HRpreLaAG465D , HR-preLaAR482L , HR-preLaAR527P and HR-LaA. In the case of the constructs coding for the HGPS- and the RDcausing HR-prelamin A mutants attB2-specific primers were used, deleting the amino acids 607–656 and 567–656, respectively, and expressed GFP fusion proteins were designated as HRpreLaAHGPS and HRpreLaARD , respectively. The GFP fusion protein representing the full length prelamin A mutant protein R419C was generated applying site-directed mutagenesis (QuickChange Mutagenesis Kit; Stratagene) to the cDNA encoding GFP-fused full length wild type prelamin A and R419C-converting forward and reversed primers. The expression vector encoding HR-prelamin AR419C was derived thereof as described above. The expression vectors encoding prelamin AR419C and prelamin AL421P , fused to monomeric DsRed, were generated by either using In-Fusion compatible primers and the respective GFP expression vector as a template and recombinational insertion of the amplicon into XhoIlinearized pEPIDsRed (R419C mutant), or by applying site-directed mutagenesis with L421P-converting forward and reversed primers to the cDNA of expression vector encoding DsRed fused to wild type prelamin A (L421P mutant). Respective fusion proteins were designated as HR-preLaAR419C (fused to GFP), preLaAR419C (fused to either GFP or DsRed) and preLaAL421P (fused to DsRed). Expression vector coding for DsRed fusion protein HRpreLaAL421P was derived from the respective full length mutant construct, but insertion of the cDNA into the EcoRI-site of pEPIDsRed by conventional ligation. The maturation-deficient prelamin A mutant-cDNA encoding DsRed-tagged HR-preLaASSIM was generated by high fidelity PCR using an SSIM-specific primer and HR-preLaAWT –cDNA as the template. All constructs were verified by sequencing. Expression vector encoding the FLAG epitopetagged prelamin A-binding protein Narf (pSVK3-FLAG-Narf) was generously provided by H.J. Worman (Columbia University, New York, USA). 2.2. Cell culture and transfection Human cervical adenocarcinoma HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated foetal calf serum, penicillin and streptomycin in a humidified 37 ◦ C incubator with 5% CO2 atmosphere. Transfection of HeLa cells was performed using Effectene (Qiagen) according to the manufacturer’s instructions. One day before transfection, cells were seeded onto 15 mm × 15 mm glass coverslips in 12-well or without coverslips in 6-well culture plates and subsequently grown for 16 h to 50–70% confluence. 2.3. Confocal laser scanning microscopy (CLSM) and image analysis Transfected cells were analyzed 16 h post transfection with equatorial imaging performed on a Zeiss LSM 510 CLSM imaging system (Carl Zeiss) equipped with a reverse 63× oil immersion objective. Acquired images were subsequently analyzed using ImageJ software 1.46r (NIH). The mean nuclear fluorescence (Fn) in relation to that in the cytoplasm (Fc), referred to as the Fn/c, was determined according to: Fn/c = (Fn − Fb)/(Fc − Fb), where Fb is the background fluorescence (i.e. regions outside the cells). Briefly, mean pixel intensities (i.e. mean grey values with a display range between 0 and 255) as a measurement of fluorescence intensity were obtained from respective regions of interest (ROI, which were well below saturation (255)). Such measurements were performed 9 times within indicated ROIs (nucleus, cytoplasm, outside the cells) and on >300 transfected cells, which were derived from at least 3 independent experiments. This approach


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allows determination of ratios of sub-compartmental fluorescence intensities in transfected cells displaying different protein expression levels. Fluorescence intensity measurements of laminaincorporated fusion proteins GFP-preLaAR419C and GFP-preLaAWT were performed on images acquired on an SP5 CLSM imaging system (Leica) in conjunction with the plot profile function of the ImageJ software (NIH). Test for significance of data was carried out using the Student’s t-test (unpaired, two-tailed), employing the InStat 2.01 software package (Graphpad Software) as previously described (Hu and Jans, 1999).

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2.4. Inhibition of isoprenoid synthesis

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CaaX processing (maturation) of endogenous prelamin A was inhibited by incubating HeLa cells for 16 h with 50 ␮M lovastatin (Sigma–Aldrich), an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase (Beck et al., 1990). In the case of ectopically produced wild type HR-preLaAWT and mutants thereof, lovastatin treatment occurred concomitant with the transfection procedure.

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2.5. Western blotting

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Western blot analysis of cellular lysates from transfected and lovastatin-treated HeLa cells was performed as follows. Cells were lysed in Laemmli sample buffer, and the resulting extracts subjected to SDS-polyacrylamide gel electrophoresis with the proteins being subsequently transferred to nitrocellulose membranes by conventional semi-dry transfer. Membranes were blocked for 1 h at room temperature in PBS containing 5% skimmed milk. Primary antibodies, (1:1000: anti-GFP, Clonech; anti-prelamin A, Santa Cruz and anti-lamin A, Imgenex) diluted in PBS/5% skimmed milk, were applied for 3 h at room temperature. Whereas antiprelamin A specifically detects prelamin A and not the mature form, lamin A, the antibody anti-lamin A recognizes both molecules, prelamin A as well as lamin A. After washing with PBS/Tween 0.5%, the membranes were incubated with secondary antibodies conjugated to horseradish peroxidase (1:600) for 1 h at room temperature. The membranes were again washed as described above and the bands detected using luminol-based enhanced chemiluminescence technique (Amersham).

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Visualization of prelamin A/Narf in transfected cells was performed using a prelamin A-/FLAG-specific antibody (Santa Cruz/Sigma–Aldrich). Cells mounted on coverslips were fixed with 4% formaldehyde in PBS for 10 min at 22 ◦ C, then permeabilized with 0.25% Triton X-100 in PBS for 5 min and incubated with the primary antibody (1:100/1000) for 20 h at 4 ◦ C. Cells were incubated with a Cy3-/Cy2-conjugated anti-goat/mouse antibody (1:600) for 1 h at 22 ◦ C. For CLSM, cells were finally mounted with 60% (w/v) glycerol in PBS containing the anti-fade agent n-propyl gallate (2%).

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3. Results

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3.1. Truncated prelamin A molecules carrying laminopathy-associated mutations As targeting of karyophilic proteins into the nucleus depends on an intact NLS, we were wondering whether laminopathy-inducing mutations within or adjacent to the NLS would affect nuclear import of expressed prelamin A. To be able to investigate transport efficiencies of prelamin A and derivatives thereof across the NPC, lamina-incorporation incompetent but nuclear import-competent GFP-fusion proteins lacking the head (H) and rod (R) domains, but

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consisting of the NLS-containing tail domain (referred to as HRpreLaAWT and respective mutants thereof), were derived from full length wild type and mutant prelamin A proteins to allow quantitative measurements of nuclear and cytoplasmic fluorescence intensity ratios. The fusion proteins used, carried the Familial Partial Lipodystrophy (Dunnigan Variety – FPLD)-causing mutations R419C, G465D and R482L (Haque et al., 2003; Shackleton et al., 2000; Speckman et al., 2000), the Emery–Dreifuss musculary dystrophy-inducing mutation R527P (Bonne et al., 1999), and the carboxy-terminal deletions accountable for progeriaassociated disease phenotypes (i.e. Hutchinson–Gilford progeria syndrome (HGPS) (De Sandre-Giovannoli et al., 2003; Eriksson et al., 2003) and restrictive dermopathy (RD) (Navarro et al., 2004)) (Fig. 1B). In addition of HR-preLaAWT, the CaaX-processed Q3 mature form HR-LaAWT was used (Fig. 1B). HR-preLaAWT was also expressed as a DsRed-fusion protein together with the HRpreLaA-mutants carrying either the metabolic syndrome-eliciting mutation L421P (Decaudain et al., 2007) or the CaaX processing mutation SSIM (Fig. 1D) (Capell et al., 2005). All GFP- and DsRedderived fusion proteins, as revealed by Western blot analysis, were correctly expressed and migrated at expected molecular weights (Fig. 1C and E). 3.2. CaaX processing of expressed truncated prelamin A mutants CaaX processing of prelamin A to mature lamin A is an extremely efficient process with prelamin A being virtually undetectable. To investigate whether deletion of the head and rod domain or overexpression in general, affect CaaX processing, Western blot analysis of crude cell extracts derived from transfected HeLa cells expressing both wild type and mutant fusion proteins and treated with or without lovastatin was performed using an antibody, detecting prelamin A sequences absent in mature lamin A (Fig. 2). Extracts from mock transfected HeLa cells, either treated with or without lovastatin, were used as controls for antibody specificity. After lovastatin treatment, two bands specific for prelamin A sequences were detected in all extracts from transfected cells, indicating the presence of endogenous non-matured prelamin A (upper bands) and of corresponding non-matured GFP-/DsRedtagged HR-prelamin A fusion proteins (lower bands) (Fig. 2A and C). In the absence of lovastatin, HR-deleted fusion proteins were efficiently CaaX-processed despite overexpression and truncation, with no or only negligible amounts of prelamin A-specific sequences being detectable (Fig. 2A and C), with the exception of the CaaX mutant HR-preLaASSIM , which is indicative for the dependency of prelamin A maturation on an intact CaaX motif (Fig. 2C). Western blot analysis of the same extracts employing a GFP-/lamin A-specific antibody to control for loading differences confirmed the presence of respective matured HR-prelamin A fusion proteins (Fig. 2B and D). 3.3. Nuclear import measurements of expressed truncated prelamin A mutants We next investigated efficiencies of nuclear accumulation of expressed wild type and mutated HR-prelamin A fusion proteins in HeLa cells, by determining ratios of fluorescence intensities in the nuclear and cytoplasmic compartments (Fn/c) 16 h post transfection. The mean Fn/c values of all fusion proteins were between 2 and 13. For comparison reasons, the Fn/c measurements of wild type HR-prelamin A (HR-preLaAWT ) were set to 100%. In relation to HR-preLaAWT (Fn/c (%) = 100 ± 7.7), we observed nuclear import efficiencies of similar magnitude for all Ig foldlocalizing mutants, i.e. HR-preLaAG465D (Fn/c (%) = 86.2 ± 6.5), HR-preLaAR482L (Fn/c (%) = 97.8 ± 7.1) and HR-preLaAL527P (Fn/c (%) = 118.4 ± 7.2). The same applied to the CaaX-processed mutant


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Fig. 1. Prelamin A and mutants derived thereof. (A) Schematic presentation of the domain structure of prelamin A. The central ␣-helical rod domain is represented by coiled-coil ␣-helices and is flanked by the amino-terminal head domain and the NLS- and Ig fold-containing tail domain. CSIM (cysteine, serine, isoleucine, methionine), NLS (nuclear localization sequence). (B and D) Head (H) and rod (R) deletion mutants derived from wild type (WT) prelamin A (HR-preLaAWT – amino acids 382–664), wild type mature lamin A (HR-LaAWT – amino acids 382–646) and the laminopathy-inducing prelamin A mutants (amino acids 382–664) with indicated mutations (circles and open triangle) fused to GFP/DsRed (see Section 3 for the laminopathy-associated phenotypes). HR-preLaASSIM is a fusion protein containing the missense mutation C661S that prevents the tail domain from CaaX processing. (C and E) Assessment of correct expression of indicated GFP/DsRed fusion proteins by Western blot analysis of crude extracts of transfected HeLa cells using a monoclonal antibody against GFP (C) or DsRed (E).

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HR-LaAWT (Fn/c (%) = 108.2 ± 8.7) (Fig. 3). Significantly reduced Fn/c values, however, were measured for the NLS mutants HRpreLaAR419C (Fn/c (%) = 54.8 ± 6.7) and HR-preLaAL421P (Fn/c (%) = 24.5 ± 2.1) and the deletion mutants HR-preLaAHGPS and HR-preLaARD (Fn/c (%) = 47.7 ± 5.4 and 32.7 ± 6.6, respectively) (Fig. 3). Our measurements additionally revealed that nuclear import of HR-LaAWT occurred with comparable efficiencies as

Fig. 2. Efficiency of CaaX processing of expressed HR prelamin A mutants. Western blot analysis of crude cells extracts obtained from transfected HeLa cells expressing head and rod deleted prelamin A mutants, with antibodies specific for prelamin A (A and C) and GFP (B) or lamin A (D). The B- and D-labelled Western Blots were used as loading controls for the Western Blots shown in A and C. The prelamin A antibody is specific to exogenous and endogenous prelamin A carboxy-termini, whereas the lamin A antibody detects all forms (matured, i.e. CaaX-processed and unmatured) of exogenously and endogenously expressed prelamin A molecules. Treatment of transfected HeLa cells with lovastatin (+) was performed to inhibit CaaX processing. Crude cell extracts from non-transfected HeLa cells, either untreated (mock) or treated with lovastatin (mock +) served as controls for the prelamin A antibody (lanes 1 and 2 in A and C). All extracts derived from lovastatin-treated cells (all +-marked fusion proteins) were positive for prelamin A-specific carboxy-termini. The Western blot results clearly indicate efficient CaaX processing of exogenously expressed fusion proteins (lanes 6–10 in A and lanes 3 and 5 in C), despite overexpression and deletion of the rod and head domains. As expected the CaaX processing incompetent fusion protein HR-preLaASSIM was positive for the prelamin A antibody (lane 7 in C). *endogenously expressed prelamin A, # exogenously expressed fusion proteins, + endogenously expressed (pre)lamin A.

HR-preLaAWT , indicating that maturation of HR-preLaAWT to HR-LaAWT makes no difference with respect to import efficiencies. Recently, we could demonstrate that expression of nuclear envelope localizing HGPS- and RD-causing prelamin A mutants negatively interfered with the nuclear import efficiencies of various artificial NLS-carrying cargo molecules in cultured cells (Busch et al., 2009). In order to rule out that HR-preLaAHGPS and HRpreLaARD retain the inhibitory characteristics of the full length proteins on global nuclear transport, we performed Fn/c measurements of DsRed-tagged HR-preLaAWT in HeLa cells co-expressing either GFP-tagged HR-preLaAWT or GFP-tagged HR-preLaARD .


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Fig. 4. Nuclear import efficiency measurements between HR-preLaAWT and CaaX processing incompetent fusion protein HR-preLaASSIM . Images acquired by CLSM were analyzed to determine the mean ratio of the nuclear to cytoplasmic fluorescence intensity (Fn/c), with the results given as relative Fn/c (Fn/c (%) ± SEM). The mean Fn/c of HR-preLaAWT was set to 100%. Compared to HR-preLaAWT mutant protein HR-preLaASSIM accumulated more efficiently in the nucleus, with mean Fn/c values between 10 and 13.

Fig. 3. Nuclear import measurements of expressed HR prelamin A mutants. (A and D) Representative CLSM images of HeLa cells transfected to express GFP-tagged (A) and DsRed-tagged (D) HR prelamin A and versions thereof, which additionally carry indicated laminopathy-inducing mutations or contain the CaaX-processed mature form of prelamin A. The images display the karyophilic nature of expressed fusion proteins, i.e. predominant fluorescence of HeLa nuclei. (B–D) Relative efficiencies of subcellular localizations of indicated proteins. Images acquired by CLSM were analyzed to determine the mean ratio of the nuclear to cytoplasmic fluorescence intensity (Fn/c), with the results given as relative Fn/c (Fn/c (%) ± SEM). The mean Fn/c of HR-preLaAWT was set to 100%. (B) Significant differences (p values 0.05), indicating that expressed HR-preLaARD does not retain the inhibitory characteristics of the full length proteins on global nuclear transport (Busch et al., 2009), rather that reduced nuclear accumulation of HR-preLaARD (and HR-preLaAHGPS – see B) is mutant-specific. All Fn/c measurements with mean values between 2 and 13 were derived from at least three separate experiments and >300 analyzed transfected cells.

We found that nuclear accumulation of DsRed-HR-preLaAWT in the presence of coexpressed GFP-tagged HR-preLaAWT occurred as efficient as DsRed-HR-preLaAWT in the presence of coexpressed GFP-tagged HR-preLaARD (Fn/c (%) = 100 ± 3.6 and 108 ± 3.6, respectively) (Fig. 3). With the cytoplasm and the nucleus being CaaX processing compartments (Barrowman et al., 2008), it is not clear whether CaaX processing precedes nuclear import or occurs thereafter and exerts an impact on the efficiency of the nuclear transport of expressed prelamin A. We therefore studied nuclear import of HR-preLaAWT under conditions of inhibition of CaaX processing. Fn/c measurements were conducted on cells expressing HR-preLaAWT in the presence and absence of lovastatin. Similar experiments with cells expressing HR-LaAWT , or the cargo molecule T-ag-NLS (Busch et al., 2009), containing an importin ␣-recognized NLS of SV40 similar to that of prelamin A, were performed to control for general effects of lovastatin on nuclear import. We found efficiency of nuclear import of HR-preLaAWT to be independent of lovastatin treatment and no negative effects of lovastatin on importin ␣-/T-ag-NLS-driven nuclear import processes under the given circumstances (data not shown). However, lovastatin-treated cells displayed a rounded cell morphology, hampering accurate Fn/c measurements on such cells. Therefore the question of effects of prelamin A processing on nuclear import efficiencies was addressed differently, using the CaaX processing-deficient mutant HRpreLaASSIM . Fn/c measurements revealed HR-preLaASSIM (Fn/c (%) = 126 ± 3.9) to be significantly more karyophilic than HRpreLaA (Fn/c (%) = 100 ± 3.8) (Fig. 4), thus accumulating within the nucleus more efficiently than its CaaX processable complement. 3.4. Subcellular localization of expressed prelamin A mutant proteins Consequences of the exogenous expression of full length prelamin A molecules carrying Ig fold-/tail-localizing mutations and subnuclear distribution of such fusion proteins have so far been investigated for proteins containing the laminopathy-inducing mutations G465D, R482L or L527P or the HGPS- and RD-causing


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Fig. 5. Subcellular localization and CaaX processing of expressed full length prelamin A NLS-mutant proteins. (A) CLSM images of HeLa cells transfected to express DsRed-preLaAR419C , DsRed-preLaAL421P or GFP-preLaAR419C , with images displaying representative subcellular localizations. (B) Quantitative analysis of subcellular localizations of indicated fusion proteins. Two populations of transfected cells were observed, with fluorescence seen at the nuclear lamina (l) exclusively or both, within the cytoplasm (c) and to different extents at the nuclear lamina (i.e. c ± l). (C) Quantification of lamina fluorescence intensities of indicated fusion proteins. The intensity of fluorescence is represented by a pseudo grey tone scale with 0 = pure black (no lamin a localization) and 1 = pure white (highest lamina localization). The scale corresponds to 255 shades, with 255 set to 1. (D) Analysis of CaaX processing of expressed GFP-preLaAR419C in HeLa cells using an antibody specific to prelamin A (anti-preLaA) in conjunction with a Cy3-conjugated anti-goat antibody. GFP-specific fluorescence is shown in the left panel, anti-prelamin A-specific fluorescence is shown on the right panel. (E) Western blot analysis of crude cell extracts obtained from HeLa cells expressing indicated fusion proteins

deletions. The mutants either formed intranuclear aggregates to different extents (Bechert et al., 2003; Hubner et al., 2006) or generated dysmorphic nuclei (Busch et al., 2009; Glynn and Glover, 2005; Goldman et al., 2004; Hubner et al., 2006; Mallampalli et al., 2005). We now conducted localization studies of full length prelamin A carrying the NLS-localizing and lipodystrophy-inducing mutation R419C or the metabolic syndrome-inducing mutation L421P in HeLa cells, either tagged with GFP (GFP-preLaAR419C ) or DsRed (DsRed-preLaAR419C , DsRed-preLaAL421P ). Similar to what has been described for the K417I and K417T mutants, which represent artificially engineered NLS mutant-containing prelamin A molecules (Loewinger and McKeon, 1988), both mutant fusion proteins were observed to be incorporated into the lamina (although at a significantly reduced level compared to the wild type form, see below). Additionally, depending on expression levels, we detected localization of the mutant proteins within dense perinuclear tubular-/dot-like structures (Fig. 5A). Quantification of subcellular localization revealed that the majority (>80%) of GFP/DsRed-preLaAR419C -expressing cells contained fluorescent protein both in cytoplasmic structures and in the lamina (Fig. 5B). Lamina incorporation of GFP-preLaAR419C compared to wild type GFPpreLaAWT was significantly reduced (>60%), a finding most likely attributable to the observed deposition of GFP-preLaAR419C in the cytoplasm (Fig. 5C). Similar quantitative measurements of lamina incorporation of the full length deletion mutants GFP-preLaAHGPS and GFP-preLaARD , were not conducted due to the dysmorphic nuclear phenotype of respective transfected cells (Busch et al., 2009; Glynn and Glover, 2005; Goldman et al., 2004; Hubner et al., 2006; Mallampalli et al., 2005). Immunostaining of GFPpreLaAR419C -expressing cells with an anti-prelamin A-antibody revealed labelling of mostly perinuclear structures and to some extent of the nuclear lamina (Fig. 5D), indicating insufficient CaaX processing of the mutant molecule. Non-transfected cells were negative for anti-prelamin A-antibody-labelling (Fig. 5D). Western blot analysis of cell extracts from GFP-preLaAR419C - and GFP-preLaAWT expressing cells, using the anti-prelamin A-antibody, confirmed presence of a prelamin A band in the former and absence of any staining in the latter, demonstrating that maturation of the mutant molecule was compromised, while GFP-preLaAWT (similar to its deletion mutant HR-preLaAWT ), despite overexpression, was efficiently matured (Fig. 5E). We further attempted to rescue the R419C-induced phenotype through cotransfection of HeLa cells with the cDNA encoding wild type prelamin A (preLaAWT fused to DsRed). Cells expressing DsRed-preLaAWT displayed predominantly lamina localization (Fig. 6A). In cells coexpressing DsRed-preLaAWT and GFP-preLaAR419C , lamina incorporation was detected for both fusion proteins but recruitment of DsRed-preLaAWT into GFP-preLaAR419C -containing cytoplasmic aggregates did not occur (Fig. 6A). Analysis of the proportion of cells localizing transfected fusion protein within the lamina or in both lamina and cytoplasmic aggregates showed that the mutant protein was found in cytoplasmic aggregates in addition to the lamina localization in 90% of transfected cells, while the wild type prelamin A was localized in both the lamina and the cytoplasm in only 7% of cells. Coexpression

with antibodies specific for prelamin A (anti-preLaA – upper Western blot) and lamin A (anti-LaA – lower Western blot). The lower Western Blot was used as a loading control for the upper Western Blot in the cases were anti-preLaA was negative for endogenous prelamin A (i.e. extracts from non-lovastatin cells shown in lane 1, 3 and 5) and exogenously expressed fusion protein GFP-preLaAWT (extracts from non-lovastatin treated cells lane 3). Crude cell extracts obtained from nontransfected HeLa cells (mock), either untreated or treated with lovastatin (+), were used as a control for the prelamin A antibody. *endogenously expressed (pre)lamin A, # exogenously expressed fusion proteins.


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Fig. 6. Subcellular localization of expressed mutant protein preLaAR419C is independent on preLaAWT expression. (A) Representative CSLM images of HeLa cells cotransfected to express indicated fusion proteins (i.e. HeLa cells expressing DsRed-preLaAWT plus GFP-preLaAR419C – upper panels, or DsRed-preLaAR419C plus GFP-preLaAR419C – lower panels). (B) Quantitative analysis of subcellular localization of indicated fusion proteins. In single transfection experiments (a) GFP-preLaAWT displayed predominant localization at the lamina (l – see also left upper panel in A), whereas localization of DsRed-preLaAR419C occurred predominantly within cytoplasmic (c) structures together with different extents of DsRed-preLaAR419C localization at the lamina (i.e. c ± l l – see also left lower panel in A). Double transfection experiments (b) revealed unaffected subcellular localization of DsRedpreLaAR419C despite coexpression of GFP-preLaAWT (i.e. c ± l l – see upper panels in A).

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of the wild type protein thus did not change the relative contribution of cytoplasmic and lamina localization of the mutant fusion protein (Fig. 6B). We finally investigated the subcellular localization of various lamin A-binding proteins in cells coexpressing preLaAR419C . We observed that cytoplasmic preLaAR419C aggregates not only failed to recruit prelamin A but were also unable to force coaggregation of the lamin A-binding proteins NUP153, SREBP1a (Fig. 7), Lap2␣ and Rb (data not shown). Singla et al. (2013) recently reported on further proteins being recruited into nuclear lamin aggregates in hepatocytes using drug- and genetic-induced porphyria models. These included transcription factors, nuclear pore proteins, ribosomal proteins and also structural proteins. In order to look for a similar scenario with respect to our cytoplasmic prelamin AR419C aggregates, we performed coexpression studies with such reported proteins, i.e. lamin B2, plakoglobin, nuclear import receptor importin ␤1, Crm1, nucleoporin 93 and ZO1. In all cases subcellular localizations of preLaAR419C -aggregates and respective proteins were mutually exclusive (data not shown). As our aggregates contained preLaAR419C , i.e. the non-matured form of lamin A, we finally looked at the prelamin A-binding protein Narf (Barton and Worman, 1999). We found the majority of preLaAR419C cytoplasmic aggregates to be positive for Narf (Fig. 7).

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In recent years identification of A-type lamin-binding proteins and expression studies of laminopathy-causing A-type lamin

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Fig. 7. Coexpression of preLaAR419C with selected lamin A-binding proteins. Representative CSLM images display HeLa cells transfected to coexpress the indicated GFP fusion proteins (i.e. GFP, GFP-NUP153 or GFP-SREBP1a) or FLAG epitope-tagged Narf (left panels) together with DsRed-preLaAR419C (right panels). In all transfected HeLa cells cytoplasmic deposits of DsRed-preLaAR419C did not sequester any of the coexpressed GFP fusion proteins or GFP. However, in the case of FLAG-Narf (which has been shown to be exclusively nuclear – Barton and Worman, 1999) significant relocalization into cytoplasmic preLaAR419C -aggregates could be observed. Note also the colocalization of GFP-NUP153 and DsRed-preLaAR419C within small nuclear aggregates, which is consistent with previous studies (Al-Haboubi et al., 2011; Hubner et al., 2006).

mutations have greatly broaden our understanding of the roles these proteins play within the nuclear compartment. The data obtained so far propose a model in which lamins act as a structural/functional scaffold for many nuclear structures and molecules (e.g. nuclear envelope, NPC, chromatin and chromatin-associated proteins). Therefore, it is quite plausible that unrestrained nuclear import of lamins is of utmost importance. In this context nuclear import measurements of expressed prelamin A head and roddomain lacking mutants were performed quantitatively and found to be significantly reduced in the case of the NLS mutants HR-preLaAR419C and HR-preLaAL421P . Although this result is not surprising, as R419C and L421P locate within the NLS, it should be taken into account that both mutations are not of artificial origin but are associated with disease-causing circumstances. More surprising was the observation that the deletion mutants HR-preLaAHGPS and HR-preLaARD displayed significantly reduced nuclear accumulation. All other mutants (i.e. HR-preLaAG465D , HR-preLaAR482L and HR-preLaAR527P ) had nuclear accumulation efficiencies similar to that of HR-preLaAWT . While reduced NLS recognition probably accounts for reduced import of HR-preLaAR419C and HR-preLaAL421P into the nucleus, it was surprising to see the HR-preLaAHGPS and HR-preLaARD mutants to accumulate within the nucleus with reduced efficiency. As it can be ruled out that the reduced efficiency of nuclear accumulation of the deletion mutants HR-preLaAHGPS and HRpreLaARD occurs due to inhibitory effects on global nuclear import (as it has previously been shown for the full length proteins – i.e. preLaAHGPS and preLaARD (Busch et al., 2009)), it can be assumed that the 50 or 90 amino acids encompassing deletions


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induce conformational changes/destabilizations of the tail domain structure, making NLS recognition by importins less favourable. Therefore, efficient nuclear import of expressed prelamin A seems not only to depend on an intact NLS (Loewinger and McKeon, 1988) and proximate phosphorylation sites (Leukel and Jost, 1995), but also on structurally relevant protein domains adjacent to the NLS (Cowan et al., 2010 and this study). In this context, it would be worthwhile to study nuclear import of the head and rod deleted prelamin A fusion protein carrying the EDMD-inducing mutation R401C (Hanisch et al., 2002; Muchir et al., 2004; Vytopil et al., 2002) quantitatively. Mutations within flanking sequences of a phosphorylation motif for PKC and Akt (amino acids 403 and 404 – Leukel and Jost, 1995; Cenni et al., 2008) could therefore possibly alter the phosphorylation-dependent nuclear import characteristics of the R401C mutant. Similar scenarios could also apply for other NLS-adjacent mutations, such as R399C, a mutation causing FPLD2/dilated cardiomyopathy type 1A (Lanktree et al., 2007; Parks et al., 2008) or R399H, which plays a role in the genesis of metabolic syndrome (Decaudain et al., 2007). To further back up our observations, future binding assays could be performed to look for differences in the binding efficiencies between respective importin(s) and the prelamin A mutants HRpreLaAR419C/L421P/HGPS/RD (Hubner et al., 1997, 2002). When fusion proteins HR-preLaAWT and HR-LaAWT were expressed, the appearance of nuclear fluorescence occurred with similar efficiency. There is no possibility to judge from the sheer observation of translocation of fluorescent protein into the nucleus whether, in the case of HR-preLaAWT , maturation takes place in the cytoplasm and whether this maturation might be a prerequisite for efficient nuclear import. Therefore, we wanted to investigate whether inhibition of farnesylation and therefore CaaX processing, i.e. maturation, would affect nuclear import of HR-preLaAWT . Before conducting these studies, we had to exclude that the deletion of head and rod domain sequences in the respective fusion protein impaired the maturation process. Western blot experiments proved that neither overexpression nor head and rod deletion prevented efficient maturation of HR-preLaAWT . We initially performed nuclear import measurements with the cargo molecule HR-preLaAWT in the absence and presence of lovastatin, together with HR-LaAWT and T-Ag-NLS, which were used to control for general effects of lovastatin on nuclear import. In this experimental set-up we found no lovastatin-dependent differences in nuclear import efficiencies for the cargo molecules HR-LaAWT and TAg-NLS (data not shown). We also observed no lovastatin effects on the nuclear import of HR-preLaAWT , suggesting that under conditions of lovastatin-inhibited CaaX processing, the import of HR-preLaAWT occurs as efficient as that of matured HR-LaAWT , indicating that cytoplasmic maturation is not a necessary prerequisite for efficient nuclear import of HR-preLaAWT (data not shown). However, lovastatin treatment resulted in rounding of respective cells making fluorescence measurements particularly within the cytoplasm not enough credible. To circumvent this issue, quantitative nuclear import measurements were performed on cells expressing a new HR-preLaA fusion protein, carrying the mutated CaaX motif sequence SSIM (HR-preLaASSIM ). In this experimental set-up, we observed HR-preLaASSIM to accumulate more efficiently within the nucleus than HR-preLaAWT , possibly due to a favourable conformation of the tail domain and thus accessibility to receptors of the nuclear import machinery. Recent studies have shown, that changes of the primary structure of the tail domain indeed have a conformational impact on the molecule (Qin et al., 2011), giving rise to conditions which may possibly contribute to changed protein-protein (such as lamin NLS–importin interactions) and protein-DNA interactions. Therefore, it would be also of future interest to look at the mutations R654X (Cowan et al., 2010; Denecke et al., 2006; Parks et al., 2008) and T655fsX49

(Decaudain et al., 2007) to see whether the truncated (R654X) or extended (T655fsX49) carboxy termini resulting therefrom, which are unable to undergo posttranslational modifications, may also exert an impact on the nuclear import characteristics of the respective mutant proteins. Last but not least, with R644 being part of the putative cleavage recognition sequence (RSY↓LLG) for the prelamin A endoprotease, Zmpste24 (Agarwal et al., 2003; Kilic et al., 1997), the pleiotropic tail mutation R644C (Genschel et al., 2001), which may inhibit the activity of Zmpste24, is another laminopathyinducing mutation that could effect maturation and hence nuclear import kinetics of the mutant protein. We next investigated subcellular localization of artificially expressed full length prelamin A containing either the NLSmutation R419C or L421P. We observed two phenotypes, cytoplasmic deposition and lamina incorporation of the mutants to occur at different extents. Compared to expressed prelamin A, lamina incorporation in the case of the R419C mutant was reduced by more than 60%. We assume that reduced lamina incorporation was attributable to compromised nuclear import resulting in increased cytoplasmic levels of dimerization competent molecules, a constellation possibly contributing to premature interaction and therefore cytoplasmic deposition of the R419C/L421P mutants in the form of tubular-/dot-like structures. The attempt to rescue the R419C-induced cellular phenotype through coexpression of wild type prelamin A failed, although such an approach has successfully been performed with the prelamin A mutant K417I (Loewinger and McKeon, 1988), as neither coexpressed preLaAWT reduced the number of cytoplasmic preLaAR419C -aggregates nor did preLaAR419C aggregates sequester preLaAWT . With regard to reduced nuclear import of the HRpreLaAHGPS/RD mutants, cytoplasmic deposition of the full length versions (i.e. preLaAHGPS/RD ) was not detected, pointing to structural differences between the tail domains with different mutations (i.e. preLaAR419C/L421P vs. preLaAHGPS/RD ), which keep the HGPS/RD-causing deletion mutants more soluble. Applying immunofluorescence studies to GFP-preLaAR419C expressing cells, to investigate efficiency of CaaX processing, we found mostly cytoplasmic aggregates to be positive for antiprelamin A (possibly due to the fact that the deposited R419C mutant represents a less CaaX processable substrate), while no antiprelamin A positive signals were detectable in HeLa cells expressing the wild type form (data not shown). Western blot analyses of respective HeLa cell extracts confirmed these results. That full length preLaAR419C is indeed a less CaaX processable substrate due to its cytoplasmic aggregation is further supported by the observation that deletion of the head and rod domains (which mediate lamin dimerization and head-to-tail polymer assembly – Stuurman et al., 1998) keeps the respective mutant (i.e. HR-preLaAR419C ) in solution (even at high expression levels), thus making it accessible to CaaX processable enzymes. It is difficult to explain from the sheer observations the impact of reduced nuclear import of truncated prelamin A mutants on the pathogenesis of the laminopathies. However, certain circumstances with respect to reduced nuclear/increased cytoplasmic levels of A-type lamins have been reported, e.g. in the context of early apoptotic events, which have been shown to be distinguished by a nuclear-to-cytoplasm shift of lamin A, possibly indicating a functional role of cytoplasmic lamin A in apoptotic processes. This is therefore quite intriguing as many primary laminopathy fibroblast cultures display increased levels of apoptosis (Bridger and Kill, 2004; Meaburn et al., 2007). Cytoplasmic relocalization of lamin A has also been described in transformed cells of colonic and gastric adenocarcinomas (Moss et al., 1999) and small cell lung cancers (Broers et al., 1993). Nevertheless, the functional significance of the cytoplasmic appearance of A-type lamins under such pathological circumstances has not yet been elucidated.


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Impaired nuclear import naturally implies a reduction of lamins in the nucleus and therefore a reduced availability for nucleoplasmic processes. In this context down-regulation of nuclear A-type lamin levels has been shown to be a prominent phenomenon in cancer cells (Capo-chichi et al., 2011). Various studies reported on the recruitment of proteins into wild type or mutant lamin A nuclear aggregates (Boudreau et al., 2012; Dreuillet et al., 2008; Hubner et al., 2006; Singla et al., 2013). In search for such proteins, coexpression studies were conducted in our preLaAR419C -expressing cells. Among established A-type lamin-binding proteins known to coaggregate with mutant lamin A molecules within the nucleus such as NUP153 (see also Fig. 7), SREBP1, Lap2␣ and Rb (Bechert et al., 2003; Hubner et al., 2006) and recently identified proteins without any relationship to A-type lamin biology (Plakoglobin, Zonula occludens protein 1, importin ␤1, Crm1, nucleoporin 93 – Singla et al., 2013) which were detected within wild type lamin A deposits, only the prelamin A-binding protein Narf was found to significantly relocalize into cytoplasmic preLaAR419C -aggregates. Although we are unable to mechanistically explain the impact of reduced nuclear import of the investigated progeria-causing prelamin A mutants on laminopathies relocalization/reduced nuclear disposability of A-type lamins in the above-reported observations clearly speak in favour of our hypothesis that unrestrained nuclear import and thus physiological subcellular distribution is required for the many lamin A-dependent processes and that perturbation of this process could represent another relevant factor contributing to the diverse disease phenotypes seen in certain laminopathies. The dyslocalization of a prelamin A-binding protein such as Narf into preLaAR419C deposits further emphasizes the importance of the undisturbed karyophilic characteristics of lamin A. Acknowledgments We thank H. Arthen, T. Reimer and A. Cubukova for skilled technical assistance and E. Asan for reading and commenting on the manuscript. We are also grateful to H.J. Worman for the human prelamin A and Narf cDNA constructs. References Agarwal AK, Fryns JP, Auchus RJ, Garg A. Zinc metalloproteinase, ZMPSTE24, is mutated in mandibuloacral dysplasia. Hum Mol Genet 2003;12:1995–2001. Al-Haboubi T, Shumaker DK, Koser J, Wehnert M, Fahrenkrog B. Distinct association of the nuclear pore protein Nup153 with A- and B-type lamins. Nucleus 2011;2:500–9. Barrowman J, Hamblet C, George CM, Michaelis S. Analysis of prelamin A biogenesis reveals the nucleus to be a CaaX processing compartment. Mol Biol Cell 2008;19:5398–408. Barton RM, Worman HJ. Prenylated prelamin A interacts with Narf, a novel nuclear protein. J Biol Chem 1999;274:30008–18. Bechert K, Lagos-Quintana M, Harborth J, Weber K, Osborn M. Effects of expressing lamin A mutant protein causing Emery–Dreifuss muscular dystrophy and familial partial lipodystrophy in HeLa cells. Exp Cell Res 2003;286:75–86. Beck LA, Hosick TJ, Sinensky M. Incorporation of a product of mevalonic acid metabolism into proteins of Chinese hamster ovary cell nuclei. J Cell Biol 1988;107:1307–16. Beck LA, Hosick TJ, Sinensky M. Isoprenylation is required for the processing of the lamin A precursor. J Cell Biol 1990;110:1489–99. Bergo MO, Gavino B, Ross J, Schmidt WK, Hong C, Kendall LV, et al. Zmpste24 deficiency in mice causes spontaneous bone fractures, muscle weakness, and a prelamin A processing defect. Proc Natl Acad Sci USA 2002;99:13049–54. Bonne G, Di Barletta MR, Varnous S, Becane HM, Hammouda EH, Merlini L, et al. Mutations in the gene encoding lamin A/C cause autosomal dominant Emery–Dreifuss muscular dystrophy. Nat Genet 1999;21:285–8. Boudreau E, Labib S, Bertrand AT, Decostre V, Bolongo PM, Sylvius N, et al. Lamin A/C mutants disturb sumo1 localization and sumoylation in vitro and in vivo. PLoS ONE 2012;7:e45918. Bridger JM, Kill IR. Aging of Hutchinson–Gilford progeria syndrome fibroblasts is characterised by hyperproliferation and increased apoptosis. Exp Gerontol 2004;39:717–24.

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Bridger JM, Kill IR, O’Farrell M, Hutchison CJ. Internal lamin structures within G1 nuclei of human dermal fibroblasts. J Cell Sci 1993;104(Pt 2):297–306. Broers JL, Machiels BM, van Eys GJ, Kuijpers HJ, Manders EM, van Driel R, et al. Dynamics of the nuclear lamina as monitored by GFP-tagged A-type lamins. J Cell Sci 1999;112(Pt 20):3463–75. Broers JL, Raymond Y, Rot MK, Kuijpers H, Wagenaar SS, Ramaekers FC. Nuclear A-type lamins are differentially expressed in human lung cancer subtypes. Am J Pathol 1993;143:211–20. Burke B, Gerace L. A cell free system to study reassembly of the nuclear envelope at the end of mitosis. Cell 1986;44:639–52. Busch A, Kiel T, Heupel WM, Wehnert M, Hubner S. Nuclear protein import is reduced in cells expressing nuclear envelopathy-causing lamin A mutants. Exp Q4 Cell Res 2009. Capell BC, Erdos MR, Madigan JP, Fiordalisi JJ, Varga R, Conneely KN, et al. Inhibiting farnesylation of progerin prevents the characteristic nuclear blebbing of Hutchinson–Gilford progeria syndrome. Proc Natl Acad Sci USA 2005;102:12879–84. Capo-chichi CD, Cai KQ, Smedberg J, Ganjei-Azar P, Godwin AK, Xu XX. Loss of Atype lamin expression compromises nuclear envelope integrity in breast cancer. Chin J Cancer 2011;30:415–25. Caron M, Auclair M, Donadille B, Bereziat V, Guerci B, Laville M, et al. Human lipodystrophies linked to mutations in A-type lamins and to HIV protease inhibitor therapy are both associated with prelamin A accumulation, oxidative stress and premature cellular senescence. Cell Death Differ 2007;14:1759–67. Cenni V, Bertacchini J, Beretti F, Lattanzi G, Bavelloni A, Riccio M, et al. Lamin A Ser404 is a nuclear target of Akt phosphorylation in C2C12 cells. J Proteome Res 2008;7:4727–35. Chelsky D, Sobotka C, O’Neill CL. Lamin B methylation and assembly into the nuclear envelope. J Biol Chem 1989;264:7637–43. Corrigan DP, Kuszczak D, Rusinol AE, Thewke DP, Hrycyna CA, Michaelis S, et al. Prelamin A endoproteolytic processing in vitro by recombinant Zmpste24. Biochem J 2005;387:129–38. Cowan J, Li D, Gonzalez-Quintana J, Morales A, Hershberger RE. Morphological analysis of 13 LMNA variants identified in a cohort of 324 unrelated patients with idiopathic or familial dilated cardiomyopathy. Circ Cardiovasc Genet 2010;3:6–14. Dabauvalle MC, Loos K, Merkert H, Scheer U. Spontaneous assembly of pore complex-containing membranes (annulate lamellae) in Xenopus egg extract in the absence of chromatin. J Cell Biol 1991;112:1073–82. De Sandre-Giovannoli A, Bernard R, Cau P, Navarro C, Amiel J, Boccaccio I, et al. Lamin a truncation in Hutchinson–Gilford progeria. Science 2003;300: 2055. Decaudain A, Vantyghem MC, Guerci B, Hecart AC, Auclair M, Reznik Y, et al. New metabolic phenotypes in laminopathies: LMNA mutations in patients with severe metabolic syndrome. J Clin Endocrinol Metab 2007;92:4835–44. Dechat T, Gesson K, Foisner R. Lamina-independent lamins in the nuclear interior serve important functions. Cold Spring Harb Symp Quant Biol 2011. Dechat T, Pfleghaar K, Sengupta K, Shimi T, Shumaker DK, Solimando L, et al. Nuclear lamins: major factors in the structural organization and function of the nucleus and chromatin. Genes Dev 2008;22:832–53. Denecke J, Brune T, Feldhaus T, Robenek H, Kranz C, Auchus RJ, et al. A homozygous ZMPSTE24 null mutation in combination with a heterozygous mutation in the LMNA gene causes Hutchinson–Gilford progeria syndrome (HGPS): insights into the pathophysiology of HGPS. Hum Mutat 2006;27:524–31. Dhe-Paganon S, Werner ED, Chi YI, Shoelson SE. Structure of the globular tail of nuclear lamin. J Biol Chem 2002;277:17381–4. Dorner D, Gotzmann J, Foisner R. Nucleoplasmic lamins and their interaction partners, LAP2alpha, Rb, and BAF, in transcriptional regulation. FEBS J 2007;274:1362–73. Dreuillet C, Harper M, Tillit J, Kress M, Ernoult-Lange M. Mislocalization of human transcription factor MOK2 in the presence of pathogenic mutations of lamin A/C. Biol Cell 2008;100:51–61. Eriksson M, Brown WT, Gordon LB, Glynn MW, Singer J, Scott L, et al. Recurrent de novo point mutations in lamin A cause Hutchinson–Gilford progeria syndrome. Nature 2003;423:293–8. Farnsworth CC, Wolda SL, Gelb MH, Glomset JA. Human lamin B contains a farnesylated cysteine residue. J Biol Chem 1989;264:20422–9. Genschel J, Bochow B, Kuepferling S, Ewert R, Hetzer R, Lochs H, et al. A R644C mutation within lamin A extends the mutations causing dilated cardiomyopathy. Hum Mutat 2001;17:154. Glynn MW, Glover TW. Incomplete processing of mutant lamin A in Hutchinson–Gilford progeria leads to nuclear abnormalities, which are reversed by farnesyltransferase inhibition. Hum Mol Genet 2005;14:2959–69. Goldman RD, Shumaker DK, Erdos MR, Eriksson M, Goldman AE, Gordon LB, et al. Accumulation of mutant lamin A causes progressive changes in nuclear architecture in Hutchinson–Gilford progeria syndrome. Proc Natl Acad Sci USA 2004;101:8963–8. Hanisch F, Neudecker S, Wehnert M, Zierz S. Hauptmann-Thannhauser muscular dystrophy and differential diagnosis of myopathies associated with contractures. Der Nervenarzt 2002;73:1004–11. Haque WA, Oral EA, Dietz K, Bowcock AM, Agarwal AK, Garg A. Risk factors for diabetes in familial partial lipodystrophy, Dunnigan variety. Diabetes Care 2003;26:1350–5. Houben F, Ramaekers FC, Snoeckx LH, Broers JL. Role of nuclear lamina–cytoskeleton interactions in the maintenance of cellular strength. Biochim Biophys Acta 2007;1773:675–86.


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G Model BC 4352 1–10 10 750 751 752 753 754 755 756 757 758 759 760 761 762 763 764 765 766 767 768 769 770 771 772 773 774 775 776 777 778 779 780 781 782 783 784 785 786 787 788 789 790 791 792 793 794 795 796 797 798 799 800 801 802


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Nuclear Import of Yeast Proteasomes.

Nucleoplasmic localization of prelamin A: implications for prenylation-dependent lamin A assembly into the nuclear lamina.

Active Nuclear Import of Membrane Proteins Revisited.

Stimulated nuclear import by β-like importins.

Computational identification of post-translational modification-based nuclear import regulations by characterizing nuclear localization signal-import receptor interaction.

Detection of mesenchymal stem cells senescence by prelamin A accumulation at the nuclear level.

A mutant nuclear protein with similarity to RNA binding proteins interferes with nuclear import in yeast.

A conserved phosphoprotein that specifically binds nuclear localization sequences is involved in nuclear import.

The nuclear import of ribosomal proteins is regulated by mTOR.

Nuclear import-export: in search of signals and mechanisms.

Nuclear import of APOBEC3F-labeled HIV-1 preintegration complexes.

Nuclear Import of Hepatitis B Virus Capsids and Genome.

Import of simian virus 40 virions through nuclear pore complexes.

Nuclear import of human histone lysine-specific demethylase LSD1.

Characterization of the nuclear import pathway for BLM protein.

Large scale RNAi reveals the requirement of nuclear envelope breakdown for nuclear import of human papillomaviruses.

Identification of the nuclear localisation signal of O-GlcNAc transferase and its nuclear import regulation.

Dynamics and regulation of nuclear import and nuclear movements of HIV-1 complexes.

CELL BIOLOGY. Disrupted nuclear import-export in neurodegeneration.

Intracellular calcium levels can regulate Importin-dependent nuclear import.

Bipartite Nuclear Localization Signal Controls Nuclear Import and DNA-Binding Activity of IFN Regulatory Factor 3.

Yeast cell-free nuclear protein import requires ATP hydrolysis.

Bending-Twisting Motions and Main Interactions in Nucleoplasmin Nuclear Import.

Transportin-1 and Transportin-2: protein nuclear import and beyond.