Journal of Cell Science 112, 1709-1719 (1999) 1709 Printed in Great Britain © The Company of Biologists Limited 1999 JCS0370

Intracellular trafficking of emerin, the Emery-Dreifuss muscular dystrophy

Cecilia Östlund1, Jan Ellenberg2, Einar Hallberg3, Jennifer Lippincott-Schwartz2 and Howard J. Worman1,* 1Departments of Medicine and of Anatomy and Cell Biology, College of Physicians and Surgeons, Columbia University, New York, New York 10032, USA 2Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA 3Center for Biotechnology, NOVUM, Karolinska Institute, 141 57 Huddinge, Sweden *Author for correspondence (E-mail: [email protected])

Accepted 18 March; published on WWW 11 May 1999

SUMMARY

Emerin is an integral protein of the inner nuclear diffusional mobility (D) of emerin is decreased in the inner membrane that is mutated or not expressed in patients nuclear membrane (D=0.10±0.01 µm2/second) compared to with Emery-Dreifuss muscular dystrophy. Confocal the ER membrane (D=0.32±0.01 µm2/second). This is in immunofluorescence microscopy studies of the agreement with a model where integral reach the intracellular targeting of truncated forms of emerin, some inner nuclear membrane by lateral diffusion and are of which are found in patients with Emery-Dreifuss retained there by association with nucleoplasmic muscular dystrophy, show that the nucleoplasmic, amino- components. Some overexpressed emerin-green fluorescent terminal domain is necessary and sufficient for nuclear protein also reaches the plasma membrane of transfected retention. When this domain is fused to a transmembrane cells, where its diffusion is similar to that in the inner segment of an integral membrane protein of the ER/plasma nuclear membrane, suggesting that emerin may also membrane, the chimeric protein is localized in the inner associate with non-nuclear structures. nuclear membrane. The transmembrane segment of emerin is not targeted to the inner nuclear membrane. Fluorescence photobleaching experiments of emerin fused Key words: , Membrane protein, Protein targeting, to green fluorescent protein demonstrate that the Fluorescence photobleaching, Muscle disease

INTRODUCTION and Leshner, 1986; Emery, 1989). Recently, mutations in the Lamin A/C have been found in individuals with this form Emery-Dreifuss muscular dystrophy (EDMD; OMIM # of EDMD (Bonne et al., 1999). 310300) is an X-linked disease characterized by early The inner nuclear membrane is one of three distinct contractures of the elbows, Achilles’ tendons and posterior membrane domains of the interphase nuclear envelope. On its neck, slow progressive muscle wasting and cardiomyopathy nucleoplasmic face it is associated with the , an with atrioventricular conduction block (Emery and Dreifuss, intermediate filament meshwork composed of proteins called 1966; Rowland et al., 1979; Emery, 1989). Positional cloning lamins (Aebi et al., 1986; Fisher et al., 1986; McKeon et al., has identified a gene on Xq28 that is mutated in 1986). Five integral membrane proteins, some with multiple individuals with EDMD (Bione et al., 1994). The protein isoforms generated by alternative RNA splicing, have so far encoded by this gene, called emerin, is a type II integral been identified as specifically localized to the inner nuclear membrane protein of 254 amino acids, with a 219 membrane in interphase. These five proteins are emerin amino-terminal domain followed by a 21 amino acid (Manilal et al., 1996; Nagano et al., 1996), lamin B receptor transmembrane segment 11 residues from the carboxyl (LBR) (Worman et al., 1988, 1990; Ye and Worman, 1994), terminus (Bione et al., 1994). Emerin, which when mutated or lamina associated polypeptide (LAP)1 (Senior and Gerace, absent is associated with a skeletal- and heart-muscle specific 1988; Martin et al., 1995), LAP2/thymopoietin (Foisner and phenotype, is expressed in the inner nuclear membrane of Gerace, 1993; Harris et al., 1994; Furukawa et al., 1995) and virtually all somatic cells and absent from this location in most MAN1 (F. Lin, D. L. Blake, I. Callebaut, I. S. Skerjanc, M. W. individuals with EDMD (Manilal et al., 1996; Nagano et al., McBurney, M. Paulin-Levasseur and H. J. Worman, 1996). A phenotypically similar disorder(s) with cardiac unpublished). The precise functions of these proteins remain condition abnormalities as a prominent feature is inherited in to be determined, but LBR, LAP1 isoform C and an autosomal dominant manner (Miller et al., 1985; Gilchrist LAP2/thymopoietin-β have been shown to bind lamins and 1710 C. Östlund and others

LBR and LAP2/thymopoietin-β have also been shown to bind obtained from American Type Culture Collection (Manassas, VA) was chromatin (Worman et al., 1988; Foisner and Gerace, 1993; Ye used as a template. In most instances, a SmaI site was engineered and Worman, 1994, 1996; Ye et al., 1997). LBR also shows within the sense PCR primer and a ClaI site in the antisense PCR homology with sterol reductases (Schuler et al., 1994; Holmer primer. PCR products were digested with these restriction et al., 1998). endonucleases and ligated into plasmid pBFT4, which also was Only a few studies have addressed how integral proteins are digested with SmaI and ClaI. pBFT4 is a pBluescript II KS (Stratagene, La Jolla, CA)-based plasmid containing a Kozak targeted to the various nuclear envelope membrane domains sequence, ATG and the FLAG-tag coding sequence 5′ to the multiple (Wozniak et al., 1992; Smith and Blobel, 1993; Soullam and cloning site. The resulting plasmids, which contained an SpeI site 5′ Worman, 1993, 1995; Furukawa et al., 1995; Ellenberg et al., to the Kozak sequence and a XhoI site 3′ to the cloned insert, were 1997; Söderqvist et al., 1997; Cartegni et al., 1997; Ellis et al., then digested with SpeI and XhoI and the isolated cassette ligated into 1998). In contrast, there has been major progress in pSVK3 that was digested with XbaI (an isoschizomer of SpeI) and understanding the targeting of soluble proteins to the nucleus XhoI. in recent years (for reviews see Görlich and Mattaj, 1996; To construct the plasmid expressing an emerin-chicken hepatic Pemberton et al., 1998). Signals capable of targeting soluble lectin (CHL) fusion protein, cDNA encoding a FLAG-tag followed by proteins through the nuclear pores cannot target integral amino acids (aa) 3-219 of emerin was ligated into pSVK3 as described membrane proteins to the inner nuclear membrane (Soullam above, using an antisense primer containing both a BspEI site and a ClaI site for PCR amplification. This plasmid was digested with BspEI and Worman, 1995). These proteins likely reach their final and XhoI and ligated with cDNA encoding aa 24-131 of CHL isolated localization in the inner nuclear membrane by lateral diffusion from plasmid LMBR-CHL (Soullam and Worman, 1993). in the endoplasmic reticulum (ER), outer nuclear and pore To express a protein with aa 117-170 of emerin fused to CHL, a membranes after synthesis on the ER (Soullam and Worman, cDNA cassette isolated from plasmid LMBR-CHL (Soullam and 1993, 1995; Furukawa et al., 1995; Ellenberg et al., 1997). Worman, 1993) by digestion with EcoRI and XhoI was ligated into According to this model, integral proteins are then retained in the EcoRI and XhoI sites of pBFT4. The ligation product was then the inner nuclear membrane by binding to structures in the digested with SmaI (which has a site in the pBFT4 multiple cloning nucleus, such as the lamina, chromatin or other nuclear site), and BspEI (which has a site in the CHL cDNA codon for aa 24). proteins. In vivo studies of LBR fused to green fluorescent A PCR product encoding aa 117-170 of emerin, amplified using a protein (GFP) have shown that LBR is relatively immobile in sense primer with a SmaI site and an antisense primer with a BspEI site, was ligated into the CHL-containing vector, yielding a cDNA the nuclear envelope, while it diffuses more freely in the ER, encoding a FLAG-tagged chimeric protein with aa 117-170 of emerin supporting this diffusion/retention model (Ellenberg et al., fused to aa 24-131 of CHL. This cDNA cassette was isolated by 1997). digestion with SpeI and XhoI and ligated into pSVK3 as described The molecular mechanism by which aberrant or no above. expression of the ubiquitously expressed inner nuclear For construction of emerin-chicken muscle pyruvate kinase membrane protein emerin can cause muscular dystrophy is not (CMPK), a cDNA fragment encoding aa 17-476 of CMPK was understood. Cartegni et al. (1997) reported the localization of isolated from plasmid p3PK (Frangioni and Neel, 1993) by digestion a protein recognized by anti-emerin antibodies to desmosomes with HindIII and ClaI. This fragment was ligated into pBFT4 that was and fasciae adherentes of intercalated discs, which are situated digested with HindIII and ClaI. The resulting plasmid was then at the site of attachment of cells to their digested with BglII and SmaI and a PCR product encoding aa 3-219 of emerin, generated using a sense primer with an engineered SmaI neighbors, and suggested that this accounts for the heart site and an antisense primer with an engineered BamHI site, was abnormalities in patients with EDMD. These data were ligated into it. The resulting chimeric cDNA was then subcloned into recently contradicted in a study by Manilal et al. (1999), where pSVK3 as described above. antibodies did not recognize the intercalated discs after affinity For construct 3-169(208), PCR amplification was performed using purification. Even if emerin is present in the intercalated discs a sense primer containing a SmaI site. The antisense primer encoded it does not, however, explain the abnormalities a modified stretch of emerin including a two-base-pair deletion at in EDMD. Furthermore, it is not clear how emerin can reach nucleotides 1564 and 1565 found in patient LB1520 described by two distinct intracellular locations in cardiac muscle, the inner Bione et al. (1994) and a SacI site found in the emerin gene at nuclear membrane and the intercalated discs. We therefore nucleotide 1580. The PCR product was digested with SmaI and SacI decided to investigate the intracellular trafficking of emerin and ligated into the SmaI and SacI sites of plasmid pACT2 (CLONTECH Laboratories, Inc., Palo Alto, CA). The resulting and how it is affected in mutant forms of the protein. plasmid, encoding a mutated form of the first 174 aa of emerin, was then digested with SacI and XhoI and ligated with wild-type emerin cDNA encoding aa 175-254, isolated from full-length emerin cDNA MATERIALS AND METHODS in pBFT4 digested with SacI and XhoI. The ligation product, encoding emerin with nucleotides 1564 and 1565 deleted, was digested with Plasmid construction SmaI and XhoI and ligated into pBFT4 digested with SmaI and XhoI, Constructs that expressed FLAG-tagged polypeptides were made in and then ligated into pSVK3 as above. plasmid pSVK3 (Pharmacia Biotech, Inc., Piscataway, NJ), which For studies of emerin fused to GFP, a FLAG-tagged construct contains a multiple cloning site downstream from the SV-40 early encoding aa 3-254 of emerin was fused, via its carboxyl terminus, to promoter. All cloning procedures were performed according to the F64L, S65T, H231L variant of GFP using the expression vector standard methods (Sambrook et al., 1989). Nucleotide numbering of pEGFP-N1 (CLONTECH Laboratories, Inc.). A PCR product emerin is based on its previously reported sequence (Bione et al., encoding aa 3-254 of emerin was digested at an EcoRI restriction site 1995). cDNAs for cloning were generated by polymerase chain present in the emerin cDNA at nucleotide 573 (encoding aa 64) and reaction (PCR) (Saiki et al., 1987), using the Gene Amp PCR System at a KpnI site engineered into the antisense primer. The digested DNA 2400 (Perkin-Elmer Cetus Corp., Norwalk, CT), with restriction sites was then ligated into the EcoRI and KpnI sites of pEGFP-N1. The engineered at the 5′ ends of the primers. A human emerin cDNA clone resulting plasmid, encoding emerin from aa 64-254 fused to GFP, was Intracellular trafficking of emerin 1711 then digested with SacI and EcoRI and ligated with cDNA encoding Briefly, for qualitative experiments shown in Fig. 6, the outlined box the FLAG-tag fused to aa 3-63 of emerin, that was isolated from was photobleached with a high zoom 488 nm laser scan using the full emerin-pBFT4 digested with SacI and EcoRI. output of a 75 mW Ar/Kr laser. Immediately afterwards, recovery was monitored at 9-second intervals with attenuated laser emission (1/100 Cell culture and transfection to 3/1000 of the bleach intensity) at low zoom, without further COS-7 cells were grown in minimal essential medium containing 10% photobleaching during the course of the experiment. To determine the fetal bovine serum (Life Technologies, Gaithersburg, MD). Cells were effective diffusion constant, D, additional quantitative FRAP transfected using DMRIE-C (Life Technologies), following the experiments were performed. Here a 4 µm wide strip that extended manufacturer’s instructions. The cells were overlaid with the lipid- across the cell and through its entire depth was photobleached at full DNA complexes for 10-22 hours. When transfecting cells with laser power and high zoom and recovery was followed only within the pSVK3-based constructs, the cells were allowed to grow in fresh photobleached strip with attenuated emission (1/1000 of the bleach medium for approximately 36 hours post-transfection. Cell cultures intensity) and low zoom at 3-second intervals without further were then split into chamber slides and grown for an additional 12- photobleaching of the sample. D was determined by fitting the 24 hours before preparation for immunofluorescence microscopy. For experimental data (Fig. 7) to an empirical formula for one-dimensional studies of emerin fused to GFP, cells were transfected directly on the diffusion (Ellenberg et al., 1997) and verified by a numerical computer chamber slides and prepared for microscopy 6-24 hours after simulation of free diffusion on the qualitative and quantitative data sets transfection. For brefeldin A treatment, cells transfected as above with (Sciaky et al., 1997). Fluorescence loss in photobleaching (FLIP; Cole FLAG-tagged emerin or FLAG-tagged emerin fused to GFP were et al., 1996) of emerin-GFP expressing cells (shown in Fig. 8) used incubated in medium containing 2 µg/ml brefeldin A (Epicentre cycles of photobleaching of the indicated areas at full laser illumination Technologies, Madison, WI) for 2.5, 4, 6, 8 or 10 hours. Brefeldin A and a single whole-cell image with attenuated emission (9/1000 of the was added when the lipid-DNA complex was removed, a time point bleach intensity) to monitor loss of fluorescence outside the when there still was little or no expression of emerin-GFP. The cells photobleached area. This routine was repeated every 80 seconds until were then examined by immunofluorescence microscopy using the ER fluorescence was completely lost to probe the continuity of ER and anti-FLAG M5 antibody as described below. nuclear envelope pools of emerin-GFP. Immunofluorescence microscopy Other chemicals Transfected cells were washed three times with phosphate-buffered Unless otherwise indicated, routine chemicals were obtained from saline (PBS) and then fixed with methanol for 6 minutes at −20¡C. either Fisher Scientific Co. (Pittsburgh, PA) or Sigma. Enzymes for The cells were permeabilized with 0.5% Triton X-100 in PBS for 2 DNA cloning were obtained from either Fisher Scientific Co. or New minutes at room temperature, washed three times with 0.1% Tween- England Biolabs (Beverly, MA). 20 in PBS (solution A) and incubated with the primary antibodies diluted in PBS containing 0.1% Tween-20 and 2% bovine serum albumin (BSA) (solution B) for 1 hour at 37¡C. The primary antibodies used were anti-FLAG M2 monoclonal antibody or anti- RESULTS FLAG M5 monoclonal antibody at a dilution of 1:200 (Sigma, St. Louis, MO), anti-lamin B1 polyclonal antibody (Cance et al., 1992) The amino-terminal domain of emerin is necessary at a dilution of 1:2,000 or anti-GM130 polyclonal antibody and sufficient for inner nuclear membrane targeting (Nakamura et al., 1995) at a dilution of 1:200. After four washes with and retention solution A, the cells were incubated with the secondary antibodies To determine the domain of emerin responsible for its inner diluted 1:200 as described for the primary antibodies. Secondary antibodies used were rhodamine-conjugated goat anti-rabbit IgG nuclear membrane localization, we transiently transfected (Biosource International, Camarillo, CA), Fluorescein isothiocyanate COS-7 cells with plasmids expressing full-length emerin and (FITC)-conjugated goat anti-mouse IgG (Jackson ImmunoResearch truncated forms of the protein. For detection of the expressed Labs, Inc., West Grove, PA) and lissamine-rhodamine-conjugated proteins, nucleotide sequences encoding FLAG-epitopes were goat anti-mouse IgG (Jackson ImmunoResearch Labs, Inc.). The cells fused to the 5′-ends of the constructs (Fig. 1A). Full-length were then washed four times with solution A and three times with emerin was localized to the nuclear rim, showing PBS. The slides were dipped in methanol, air dried and coverslips colocalization with lamin B1, a marker for the lamina/nuclear mounted using anti-fade mounting medium (5% propyl gallate, 0.25% envelope (Fig. 1B). A truncated emerin (residues 3-219), 1,4-diazabicyclo-(2,2,2)-octane, 0.0025% p-phenylenediamine in consisting of the amino-terminal nucleoplasmic domain but glycerol). For digitonin-permeabilization, the transfected cells were lacking the transmembrane segment, was retained within the washed three times with PBS and fixed with 2% paraformaldehyde in PBS for 30 minutes on ice. They were then washed three times with nucleus. This truncated emerin did not give a nuclear rim PBS and incubated with precooled 40 µg/ml digitonin (Calbiochem, staining but rather a diffuse labeling of the nucleus (Fig. 1B). La Jolla, CA) in PBS for 10 minutes on ice. The cells were then The carboxyl-terminal part of emerin (residues 197-254) washed and incubated with antibodies as described above, except that containing the transmembrane segment and the luminal tail did Tween-20 was excluded from the buffers and all steps were performed not accumulate at the nuclear rim but was localized to the entire on ice. Immunofluorescence microscopy was performed on a Zeiss ER and Golgi complex (Fig. 1B). These results show that the LSM 410 confocal laser scanning system attached to a Zeiss Axiovert signal/signals necessary for nuclear retention are in the amino- 100TV inverted microscope (Carl Zeiss, Inc., Thornwood, NY). terminal domain of emerin. They also show that the Images were processed using Photoshop software (Adobe Systems, hydrophobic region of emerin from aa 223-243 is a functional Inc., San Jose, CA) on a Macintosh G3 computer (Apple Computer, transmembrane segment, but that it is not retained in the inner Inc., Cupertino, CA). nuclear membrane. Fluorescence photobleaching experiments To confirm that the FLAG-tagged, full-length emerin Fluorescence recovery after photobleaching (FRAP) of live emerin- construct localized to the inner nuclear membrane, as opposed GFP expressing cells was performed on a 37¡C stage of a Zeiss LSM to the outer nuclear membrane, the plasma membranes of cells 410 (Carl Zeiss, Inc.) essentially as described (Ellenberg et al., 1997). expressing this construct were selectively permeabilized with 1712 C. Östlund and others

Fig. 1. The amino-terminal domain of emerin contains a nuclear envelope targeting signal. (A) Schematic diagrams of proteins expressed by the plasmid constructs used in experiments in which COS-7 cells were transfected. Domains of the proteins are represented by: black, FLAG-tag; white, nucleoplasmic and lumenal domains of emerin; gray, transmembrane segment of emerin. The amino terminus of each protein is at the left. (B) Cellular localization of full-length emerin (aa 3-254), the amino-terminal domain (aa 3- 219) and the carboxyl-terminal domain (aa 197-254) as determined by laser scanning confocal immunofluorescence microscopy. Antibodies used were anti-FLAG monoclonal antibodies recognized by FITC- conjugated secondary antibodies (left) and polyclonal antibodies against lamin B1 recognized by rhodamine- conjugated secondary antibodies (middle). The pictures to the right show an overlay of the FITC and rhodamine channels. Bars, 10 µm. digitonin (Adam et al., 1992). Most cells showed no staining nuclear membrane, COS-7 cells were transfected with a with the anti-FLAG antibodies; some overexpressing cells plasmid encoding a chimeric protein consisting of the amino- showing a typical ER-staining with no defined nuclear rim- terminal domain of emerin fused to the transmembrane staining (data not shown). To further confirm that the nuclear segment and a portion of the luminal region of CHL (Fig. 3A). envelope was intact and FLAG-tagged emerin in the inner CHL is a type II integral membrane protein of the ER, nuclear membrane was not accessible to anti-FLAG antibodies endosomes and plasma membrane (Chiacchia and Drickamer, in digitonin-permeabilized cells, cells expressing FLAG- 1984; Mellow et al., 1988). It has a cytoplasmic, amino- tagged emerin fused to GFP were treated with Triton X-100 or terminal domain of 23 amino acids followed by a digitonin (Fig. 2). The endogenous fluorescence from GFP was transmembrane domain and a luminal, carboxyl-terminal then visualized simultaneously to antibody labeling with anti- domain of 160 amino acids. The emerin-CHL fusion protein FLAG antibodies recognized by rhodamine-conjugated localized to the nuclear rim, similar to full-length emerin (Fig. secondary antibodies. Cells in which the nuclear envelopes 3B). That the fusion protein was localized to the inner nuclear were permeabilized with Triton X-100 showed an overlap of membrane was confirmed with immunofluorescence of cells the signals from the rhodamine channel and the GFP channel. permeabilized with digitonin (data not shown). To determine In cells permeabilized with digitonin, the nuclear rim was if the amino-terminal domain of emerin could also target a non- detected by GFP fluorescence but was not labeled with anti- membrane, cytosolic protein to the nucleus, this domain was FLAG antibodies. This experiment clearly demonstrated that fused to a truncated version of CMPK, which is a soluble, the majority of emerin-GFP was located in the inner nuclear cytosolic protein (Frangioni and Neel, 1993) (Fig. 3A). This membrane. fusion protein, of approximately 75 kDa, contrary to the To determine if the amino-terminal domain of emerin was amino-terminal domain of emerin itself, is too large to diffuse able to target other transmembrane polypeptides to the inner freely into the nucleus (Paine et al., 1975). When expressed in Intracellular trafficking of emerin 1713

Fig. 2. Emerin localizes to the inner nuclear membrane. (A) Schematic diagrams of the protein expressed by the transfected plasmid construct. Domains of the protein are represented by: black, FLAG-tag; white, nucleoplasmic and lumenal domains of emerin; gray, transmembrane segment of emerin; hatched, GFP. (B) Immunofluorescence studies of COS-7 cells that were transfected with plasmids expressing emerin tagged with a FLAG-tag and fused to GFP. The cells were permeabilized with Triton X-100 (upper panels) or digitonin (lower panels) and were then labeled with anti- FLAG monoclonal antibodies recognized by rhodamine- conjugated secondary antibodies (left panels). The middle panels show fluorescence from GFP and the panels to the right an overlay of signals from the two channels. Bar, 10 µm.

Fig. 3. The targeting signal/signals in the amino-terminal domain of emerin can direct an unrelated integral membrane protein to the inner nuclear membrane but not a non-membrane protein to the nucleoplasm. (A) Schematic diagrams of proteins expressed by the plasmid constructs used in transfection experiments. Domains of the proteins are represented by: black, FLAG-tag; white, nucleoplasmic domain of emerin; hatched, CHL; gray, CMPK. (B) Immunofluorescence images from laser scanning confocal immunofluorescence microscopy showing localization of the amino-terminal domain of emerin fused to truncated CHL (upper panels) and the amino-terminal domain of emerin fused to truncated CMPK (lower panels). Antibodies used were anti-FLAG monoclonal antibodies recognized by FITC-conjugated secondary antibodies (left) and polyclonal antibodies against lamin B1 recognized by rhodamine-conjugated secondary antibodies (middle). The pictures to the right show an overlay of the FITC and rhodamine channels. Bars, 10 µm.

COS-7 cells, the emerin-CMPK fusion protein remained in the localizing an integral membrane protein to the inner nuclear cytosol and was excluded from the nucleus (Fig. 3B). In membrane but not a soluble protein larger than 60 kDa to the conclusion, the emerin amino-terminal domain is capable of nucleus. 1714 C. Östlund and others Intracellular distribution of mutant forms of emerin found in patients with EDMD More than 70 different mutations in the emerin gene have been identified in patients with EDMD (Yates, 1997; http://www.path.cam.ac.uk/emd/mutation.html). To narrow down the region responsible for targeting of emerin, the intracellular localizations of some of these mutated forms were studied (Fig. 4). Two short, truncated forms of emerin, aa 3-41 and 3-44, were apparently unstable and virtually impossible to detect in cells by immunofluorescence microscopy (data not shown). Truncated forms of emerin, aa 3-74 and 3-109, showed a diffuse staining of the entire cell. These proteins are small enough to diffuse freely through the nuclear pores (Paine et al., 1975), but seem to lack the signal responsible for nuclear retention present in the full emerin amino-terminal domain. When a truncated emerin containing aa 3-147 was expressed, there was a clear accumulation in the nucleoplasm but also significant staining of the cytoplasm. A nuclear rim staining pattern was seen with a frame-shift mutation at aa 169 (3- 169(208)). This construct lacks the native transmembrane domain of emerin, as the frame shift generates a stop codon at aa 208. The out-of-frame region coincidentally encodes a completely unrelated hydrophobic stretch of amino acids (Cartegni et al., 1997). Digitonin-permeabilization confirmed the protein to be localized to the inner nuclear membrane (data not shown). Truncated forms of emerin, aa 3-205 and 3-228, showed a clear accumulation in the nucleus; however, not at the nuclear periphery but in a diffuse nuclear distribution as would be expected for proteins lacking a membrane-spanning segment. aa 117-170 in the amino-terminal domain of emerin is retained in the nucleus but is not sufficient to target an integral membrane protein to the inner nuclear membrane The results of the above experiments suggested that the nuclear retention signal/signals of emerin can be limited to the region between aa 109 and 169. To verify this, addtional constructs, encoding aa 3-170, 170-254 and 117-170 were made (Fig. 5A). When COS-7 cells were transfected with these constructs, immunofluorescence showed a nuclear localization of the polypeptides from aa 3-170 and 117-170 (Fig. 5B). This showed that these regions can retain a non-membrane protein in the nucleoplasm. The polypeptide from aa 170-254 was Fig. 4. Effects of mutations found in EDMD patients on cellular localization of emerin as determined by immunofluorescence localized to the entire ER (Fig. 5B), providing further evidence microscopy. (A) Schematic diagrams of proteins expressed by the for the lack of inner nuclear membrane retention signals in the plasmid constructs in transfected COS-7 cells. Domains of the carboxyl-terminal region of emerin. When aa 117-170 were proteins are represented by: black, FLAG-tag; white, nucleoplasmic fused to CHL (Fig. 5A), the polypeptide remained in the ER domain of emerin; gray, transmembrane segment of emerin. In and did not accumulate in the inner nuclear membrane (Fig. mutation 3-169(208), a frame-shift mutation at the codon for amino 5B). The retention signal between aa 117-170 of emerin, which acid 169 gives an out-of-frame segment between aa 169 and 208 is adequate for retention of a non-membrane protein in the (hatched). At aa 208, the frame shift causes a stop codon. nucleus, is therefore not sufficient for retention of an integral (B) Cellular localization of mutated forms of emerin containing aa protein in the inner nuclear membrane. Efficient inner nuclear 3-74, 3-109, 3-147, 3-208 with the segment between aa 169 and 208 membrane retention required the aa 3-170 portion of emerin. translated out-of-frame, 3-205 and 3-228 as indicated in the panels. Laser scanning confocal immunofluorescence was performed using Lateral diffusion of emerin in the ER, inner nuclear anti-FLAG antibodies recognized by FITC-conjugated secondary antibodies. Bar, 10 µm. membrane and plasma membrane To determine if emerin becomes immobilized when it reaches the inner nuclear membrane, photobleaching experiments were envelope (Fig. 6A). In many cells emerin-GFP localized partly performed on emerin with GFP fused to its carboxyl terminus. to the ER. This staining was predominantly seen in cells FRAP was performed on emerin-GFP localized to the nuclear overexpressing emerin-GFP at a relatively high level, and it is Intracellular trafficking of emerin 1715

Table 1. Diffusion constants (D) for emerin-GFP Membrane D (µm2/second) n Nuclear envelope 0.10±0.01 4 ER 0.32±0.01 4 Plasma membrane 0.09±0.02 5

t-tests show that the D value for emerin-GFP in the ER is significantly larger than the values for D in the nuclear envelope (P=5.8×10−7) and in the plasma membrane (P=7.7×10−8). The D values in the nuclear envelope and in the plasma membrane are not significantly different (P=0.58).

When emerin-GFP was analyzed by FRAP, the membrane pools of emerin in the nuclear envelope and ER exhibited different mobilities. The bleached nuclear envelope area did not regain full fluorescence intensity 260 seconds after photobleaching (Fig. 6A). After approximately 8 minutes, the fluorescence recovery was nearly complete (data not shown). In the bleached ER area, fluorescence recovered almost immediately and regained most of its original fluorescence intensity after approximately 60 seconds (Fig. 6B). When cells overexpressing emerin-GFP were examined 36 hours after transfection, another distinct pool of protein could be distinguished in the plasma membrane (Fig. 6C). When analyzed by FRAP, the recovery rate in the plasma membrane was similar to that in the nuclear envelope and distinctly slower compared to the recovery rate in the ER. Quantitative FRAP experiments were performed to determine the diffusion constants (D) of the different membrane pools (Fig. 7). D was 0.32±0.01 µm2/second for the ER pool, 0.10±0.01 µm2/second for the nuclear envelope pool µ 2 Fig. 5. Proteins containing aa 3-170 or 117-170 are retained in the and 0.09±0.02 m /second for the plasma membrane pool nucleus, while proteins containing aa 170-254 or aa 117-170 fused to (Table 1). Emerin was therefore significantly less mobile in CHL remain in the ER. (A) Schematic diagrams of proteins both the inner nuclear membrane and plasma membrane expressed by the plasmid constructs used in transfection relative to the ER. experiments. Domains of the proteins are represented by: black, We also used FLIP to examine emerin-GFP diffusion. In FLAG-tag; white, nucleoplasmic and lumenal domains of emerin; FLIP, an area of the cell is repeatedly photobleached and the gray, transmembrane segment of emerin; hatched, CHL. fluorescence loss in the entire cell is measured. When parts of (B) Immunofluorescence images from laser scanning confocal the ER of cells expressing emerin-GFP at high levels were immunofluorescence microscopy showing cellular localization of repeatedly bleached, the nuclear envelope retained its truncated forms of emerin, and a chimeric protein consisting of aa fluorescence after all fluorescence had disappeared from the 117-170 of emerin fused to truncated CHL. Antibodies used were anti-FLAG monoclonal antibodies recognized by FITC-conjugated ER (Fig. 8). The Golgi apparatus also retained its fluorescence secondary antibodies. Bars, 10 µm. during the experiment. These results show that the diffusional capacity of emerin decreases when the protein enters the inner nuclear membrane. Although there is a slow lateral movement likely that it represents proteins ‘backed up’ in the ER when of emerin within the inner nuclear membrane, there is very its binding/retention sites in the nuclear membrane are filled. little back flow of protein from the inner nuclear membrane to A quantitative analysis of the correlation between expression the ER. There is also very little flow of emerin-GFP from the levels and ER staining was not possible as the population of Golgi back to the ER. cells showed a high variablility in the degree of ER staining. FRAP performed on emerin-GFP localized to the ER is shown in Fig. 6B. In many cells overexpressing emerin-GFP, the DISCUSSION protein was also present in the Golgi apparatus, colocalizing with the cis-Golgi matrix protein GM130 (data not shown; We have shown that the nucleoplasmic, amino-terminal Nakamura et al., 1995). Transport of emerin through the Golgi domain of emerin is necessary and sufficient for targeting to apparatus is, however, not necessary for its localization to the and retention in the inner nuclear membrane while the inner nuclear membrane as transfected cells treated with transmembrane segment and luminal carboxyl-terminal brefeldin A still showed an inner nuclear membrane domain localizes to the ER when expressed by itself. This localization of emerin-GFP (data not shown). Brefeldin A finding is in disagreement with another study, which claimed blocks protein transport from the ER to the Golgi apparatus that the transmembrane segment of emerin was the main and causes the Golgi apparatus to disassemble (Misumi et al., determinant for inner nuclear membrane targeting (Cartegni et 1986; Lippincott-Schwartz et al., 1989; Sciaky et al., 1997). al., 1997). Our results show that while the transmembrane 1716 C. Östlund and others

Fig. 6. The mobility of emerin is decreased in the nuclear envelope and the plasma membrane compared to the ER. Confocal fluorescence studies of localization and mobilities of emerin-GFP in interphase cells by qualitative FRAP-experiments on cells expressing emerin fused to GFP, showing photobleach recovery in (A) nuclear envelope membranes, (B) ER membranes and (C) the plasma membrane. The fluorescence in the boxed regions was bleached and the fluorescence recovery is shown at 9 seconds (9s), 60 seconds (60s) and 260 seconds (260s) after the bleaching. Bars, 5 µm. segment gets inserted into the ER/outer nuclear envelope, it is and the nuclear pore membrane, for example LBR, LAP2, unable to retain the protein in the inner nuclear membrane. Our POM121 and gp210, contain targeting and retention signals in results using FRAP and FLIP also show that the targeting of the parts of the proteins directed towards the nucleo- emerin to the inner nuclear membrane is consistent with the cytoplasmic side of the nuclear membrane (Wozniak and ‘diffusion and retention’ model proposed based on studies of Blobel, 1992; Soullam and Worman, 1993, 1995; Furukawa et LBR (Soullam and Worman, 1995; Ellenberg et al., 1997). In al., 1995; Söderqvist et al., 1997). These domains likely this model, proteins synthesized on the ER can diffuse to the contain regions that bind to nuclear or pore complex proteins inner nuclear membrane and are retained there by binding to (or possibly nucleic acids). There are also examples where the nuclear ligands such as lamins or chromatin. hydrophobic transmembrane segments contribute to inner Most of the integral proteins of the inner nuclear membrane nuclear membrane retention, for example in gp210 and LBR Intracellular trafficking of emerin 1717

Fig. 7. Quantitative FRAP experiments to determine diffusion constants for emerin-GFP in the nuclear envelope membranes, ER membranes and the plasma membrane. Data points were taken at 3- second intervals. The curves displayed kinetics allowing for the determination of diffusion constants for emerin-GFP in the different membranes.

(Wozniak and Blobel, 1992; Smith and Blobel, 1993; Soullam and Worman, 1995). These transmembrane segments may contribute to retention by homodimerization within the plane of the membrane. It has previously been shown that the signals Fig. 8. FLIP experiment to study the continuity of nuclear envelope and ER membranes by repeated bleaching of parts of the cytoplasm. retaining integral proteins in the inner nuclear membrane differ Areas above the upper line and below the lower line were bleached from the nuclear localization signals, which target soluble every 80 seconds. The figure shows confocal micrographs taken proteins through the nuclear pores (Soullam and Worman, before the first bleach and after 12, 24, 36, 48 and 60 rounds of 1995). Our results show that the nucleoplasmic domain of bleaching. Bar, 5 µm. emerin, like the nucleoplasmic domain of LBR, is able to target CHL to the nuclear membrane. The emerin amino-terminal domain is, however, unable to target a cytosolic protein greater Cartegni et al. (1997), that this fortuitously encoded than 60 kDa to the nucleus, which lends further weight to the transmembrane segment can be an inner nuclear membrane hypothesis that the mechanisms for nuclear targeting differ targeting signal. More likely, the portion of the amino-terminal between soluble and integral membrane proteins. domain of emerin from aa 1-169 is sufficient for nuclear The amino-terminal domain of emerin likely contains at retention of a transmembrane protein. These results suggest least two, separate signals for inner nuclear membrane that the second necessary, but not sufficient, inner nuclear retention. One may be localized between aa 117 and 170, as a membrane retention signal must exist in emerin at the amino- non-membrane construct containing only this stretch of emerin terminal side of aa 117. is retained in the nucleus. It does not, however, contain Emerin shows two regions of homology with sufficient information to target the truncated form of CHL, LAP2/thymopoietin-β, one of the lamin-binding proteins of the implying the presence of an additional signal important for inner nuclear envelope (Bione et al., 1994; Furukawa et al., inner nuclear membrane targeting in another part of the amino- 1995). The first of these regions, between aa 6-44 of emerin terminal domain. One of the mutated forms of emerin and aa 114-152 of LAP2, is also conserved in another inner identified in a patient with EDMD has a mutation at aa 169, nuclear membrane protein, MAN1 (F. Lin, D. L. Blake, I. which causes a frame shift, yielding a region translated out-of- Callebaut, I. S. Skerjanc, M. W. McBurney, M. Paulin- frame between aa 169 and 208. This mutant form of emerin, Levasseur and H. J. Worman, unpublished). The second of as previously described by Cartegni et al. (1997) and these regions is between aa 221-254 of emerin and 409-442 of demonstrated in the present study, localizes to the inner nuclear LAP2. These regions of homology are not in domains membrane. The out-of-frame region of this mutant form of implicated in lamin-binding and nuclear envelope targeting (aa emerin coincidentally encodes a hydrophobic stretch of amino 298-373) or chromosome interaction (aa 1-85) of LAP2 acids, completely unrelated to the wild-type transmembrane (Furukawa et al., 1995, 1998). Therefore, emerin and LAP2 do region of emerin. It is difficult to believe, as proposed by not appear to be retained in the inner nuclear membrane by 1718 C. Östlund and others conserved signals. Some studies have suggested that emerin Biology, Columbia University) for help with the confocal microscopy. may also interact with nuclear lamins (Cartegni et al., 1997; This work was supported by a research grant from the Muscular Ellis et al., 1998; Squarzoni et al., 1998), suggesting that a Dystrophy Association (MDA) to H. J. Worman. C. Östlund received different lamin binding domain may be important in the inner support from The Swedish Institute, Sven and Lilly Lawski’s nuclear membrane retention of the protein. Foundation for Research in the Natural Sciences and Olof Ahlöf’s Using FRAP and FLIP, we have shown that emerin has a Foundation. E. Hallberg received support from Carl Tryggers’s Foundation and the Swedish Board for Laboratory Animals. H. J. decreased lateral mobility in the inner nuclear membrane Worman is also an Irma T. Hirschl Scholar, and supported in part on compared to the ER. Interestingly, the diffusion coefficient of this project by a grant from the National Institutes of Health (RO1- emerin in the inner nuclear membrane is not as low as that for CA66974). The confocal microscopy facility used for part of this LBR (Ellenberg et al., 1997). Emerin therefore is not project was established by National Institutes of Health grant 1S10- completely immobilized in the inner nuclear membrane by an RR10506 and is supported by National Institutes of Health Grant 5 irreversible binding to an immobile nuclear structure, for P30-CA13696 as part of the Herbert Irving Cancer Center at example chromatin, which is immobile in the interphase Columbia University. nucleus (Abney et al., 1997; Marshall et al., 1997). Emerin may instead bind to non-chromatin nuclear proteins (or nucleic acids) and diffuse more slowly as a complex in the inner REFERENCES nuclear membrane than in the ER. 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