We wished to investigate whether cytoskeletal abnormalities are induced by the absence of emerin in cells. To this end, human dermal fibroblasts (HDF) from healthy individuals and from X-EDMD patients (which were null for emerin) were investigated for possible abnormalities in the actin, vimentin, and tubulin cytoskeleton (). In stark contrast to findings in lamin A/C-null mouse embryonic fibroblasts, there were little or no differences in the organization of any of the cytoskeletal elements in emerin-null HDFs. Surprisingly, however, we did observe that the centrosome was not positioned next to the nucleus in emerin-null HDFs (, arrowheads).
Figure 1. Organization of the cytoskeleton in X-EDMD cells. Normal and emerin-null HDFs were fixed with methanol/acetone (1:1) and stained for β-actin, vimentin, and β-tubulin. No differences in the organization of the cytoskeleton between normal (more ...)
To confirm this finding, we used an antibody against pericentrin to specifically investigate the position of the centrosome. In normal HDF the centrosome was positioned next to or within 1.5 μm of the NE. In contrast, in four independent emerin-null HDF cell lines the centrosome was >3.0 μm distant from the NE (). To further investigate this phenomenon we looked at centrosome position in a cell line from an X-EDMD carrier, which has approximately equal numbers of emerin-positive and emerin-null cells. We found that in emerin-positive cells the centrosome was positioned next to the NE, whereas in emerin-negative cells the centrosome was >3.0 μm away from the NE. Finally, we investigated the centrosome position in a lamin A/C–null HDF line in which emerin was located entirely within the endoplasmic reticulum (Muchir et al., 2000
). In the lamin A/C–null cell line the centrosome was also >3.0 μm away from the NE, indicating that absence of emerin from the NE was the cause of the centrosome mislocalization (). To confirm that mislocalization of the centrosome was specific to absence of emerin from the NE, we investigated centrosome positions in a fibroblast cell line from a patient with Greenberg dysplasia, which were null for the INM protein lamin B receptor (LBR) (Waterham et al., 2003
). Like EDMD, Greenberg dysplasia and the related disorder Pelger-Huey anomaly are characterized by nuclear morphological defects (Hoffman et al., 2002
). However, in LBR-null fibroblasts, emerin was located at the NE and similarly the centrosome was positioned adjacent to the NE (), suggesting that centrosome mislocalization is specific to loss of emerin from the NE. To verify these results, we performed knockdown of emerin by siRNA in normal HDFs (). In HDFs transfected with the scrambled siRNA, centrosomes were found adjacent to the NE. In contrast, in HDFs that were transfected with siRNA specific for emerin, the centrosome was located at a distance of >3.0 μm away from the NE, similar to the distances observed in X-EDMD cells.
Figure 2. Distance of the centrosome from the nucleus in normal and EDMD cells. (A) The position of the centrosome relative to nucleus was determined in nine cell lines: two normal, four emerin null, one X-EDMD carrier, one AD-EDMD cell line (LMNA−/−), (more ...)
This very intriguing result raised the important question of how a protein that is localized in the INM could affect the position of the centrosome, an organelle that is localized at the ONM. To investigate whether a yet-unidentified emerin binding partner could help explain the observed phenomenon, we used recombinant emerin peptides in coprecipitation experiments to identify new emerin binding partners. The peptide was used as a bait to precipitate interacting partners from the Xenopus
egg extracts, which were in turn chosen because they store very large quantities of cytoskeleton and centriolar proteins in a soluble form (). Bands that coprecipitated with emerin were cut from the gel and identified by mass spectrometry. Interestingly, β-tubulin was identified as the most consistent emerin binding protein in this assay. To confirm that emerin is a microtubule (MT) binding protein, MT cosedimentation experiments were performed in which purified MTs were polymerized by taxol and incubated with the same emerin peptide (aa 73–180) or two different emerin peptides corresponding to its chromatin binding domain (aa 1–70) or most of the nucleoplasmic domain (aa 1–176) (). Emerin 73–180 and 1–176 efficiently cosedimented with MTs, whereas emerin 1–70 did not bind to MTs. To estimate the stoichiometry of emerin/microtubule interactions we calculated the tubulin/emerin binding ratios. Emerin 1–176 bound to tubulin at an approximate ratio of 1:8, which is close to the binding ratios of known microtubule-associated proteins (MAPs) (e.g., Enconsin; Bulinski and Bossler, 1994
). Emerin 73–180 bound to tubulin at an approximate ratio of 1:24, and this weaker interaction is likely due to misfolding of this peptide. Collectively, these data suggest that emerin is a novel MT-interacting protein.
Figure 3. Emerin–β-tubulin interaction and its implications for centrosome position. (A) A His-tagged recombinant emerin peptide corresponding to aa 73–180 was immobilized on Ni2+-beads and used to pull down binding partners from (more ...)
The interaction of emerin with β-tubulin led us to investigate whether MTs are involved in the attachment of the centrosome to the NE. To investigate this possibility, normal and X-EDMD fibroblasts were treated with nocodazole and its effects on MT organization and centrosome position were investigated (). As expected, nocodazole treatment led to the depolymerization of the MT network. Interestingly, when normal HDFs were treated with nocodazole the centrosome was observed to be located >3.0 μm away from the NE, just as was observed in emerin-null fibroblasts. As a control, normal HDFs were treated with latrunculin B to depolymerize the actin cytoskeleton. In latrunculin B–treated HDFs the centrosome was located adjacent to the NE, implying that only disruption of the tubulin cytoskeleton leads to an emerin-null phenocopy. We confirmed this finding using biochemical fractionation to determine whether centrosomes cosedimented with the nucleus in a range of HDF lines (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200702026/DC1
). In normal HDFs, nuclei and centrosomes cosedimented at a 1:1 ratio. In emerin-null HDFs or normal HDFs treated with nocodazole, nuclei and centrosomes cosedimented at a 1:0.4 ratio, again showing that centrosomes were detached from the NE.
All the above experiments provide strong evidence that emerin links centrosomes to the NE via a MT association. Given that emerin is a protein of the INM, this raised the question as to whether emerin acts via another protein that crosses the NE. We therefore investigated whether either SUN domain proteins or one of the nesprins is also mislocalized in emerin- null HDFs as a first step to determining whether these proteins might be involved in centrosome localization. We could not detect any change in the distribution of SUN1 or nesprin 1 (not depicted) or SUN2 or nesprin 2 (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200702026/DC1
) in these cells, suggesting that these proteins were not involved in centrosome localization. As a result of this finding we decided to reinvestigate the localization of emerin in normal fibroblasts. Using digitonin permeabilization, we showed that a considerable fraction of emerin was concentrated at the ONM, with a further dispersed fraction in the peripheral ER, with two independent anti-emerin antibodies, whereas lamins A/C and SUN2 were undetectable under similar conditions and therefore located exclusively at the INM (). The anti-Sun2 antibody used in this assay recognizes a luminal domain, indicating that not only is the INM intact, but also the ONM, strengthening the finding that a fraction of emerin resides at the ONM. To confirm that the protein detected at the ONM was indeed emerin, we performed siRNA knockdown of emerin on control fibroblasts and again stained the cells with anti-emerin antibodies after digitonin permeabilization. In these experiments knockdown was ~70% efficient, and whereas emerin was detected at the ONM in fibroblasts treated with scrambled siRNA, staining was eliminated in cells transfected with siRNA specific to emerin (). As a further control we also stained X-EDMD fibroblasts, (which are null for emerin) or an X-EDMD carrier in which emerin is absent from ~50% of cells. We found that staining of the ONM was undetectable in emerin-null fibroblasts, further supporting the presence of emerin at the ONM. This finding implies that emerin residing at the ONM can interact directly with centrioles via MTs. It has recently been reported that localization of emerin at the INM is dependent upon the presence of both nesprin1α and 2β (Wheeler et al., 2007
). To investigate whether nesprins might also be involved in localization of emerin to the ONM, we transfected normal HDF with a dominant-negative Sun1 mutant that causes loss of the ONM form of nesprin 2 (Crisp et al., 2006
). We found that emerin was still detected at the ONM in the presence of this mutant, suggesting that nesprin2 does not cause the localization of emerin to the ONM ().
Figure 4. Digitonin permeabilization of normal and X-EDMD HDFs. (A) Normal HDFs were permeabilized with digitonin and stained for emerin with two antibodies: a mouse monoclonal (4G5) and a rabbit polyclonal (AP8) antibody. Cells were also stained for lamin A/C (more ...)
Overall, our results show that emerin interacts directly with MTs and that emerin and MTs both are necessary for the association of the centrosome with the NE. Our findings are supported by previous work, which showed a colocalization of emerin with β-tubulin in mitotic cells (Dabauvalle et al., 1999
) and an enrichment of emerin at the kinetochores, near the spindle poles, during NE reassembly (Haraguchi et al., 2000
). Recent evidence has demonstrated how complexes involving lamins A/C, the SUN domain proteins, and the nesprins link the actin and intermediate filament cytoskeletons to the NE in mammalian cells (Wilhelmsen et al., 2005
; Crisp et al., 2006
). Our current data now reveal how the tubulin cytoskeleton via the centriole interacts with the NE in human fibroblasts.