QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Raharjo, W. H. Articles by Burke, B. Search for Related Content PubMed PubMed Citation Articles by Raharjo, W. H. Articles by Burke, B.

Nuclear envelope defects associated with LMNA mutations cause dilated cardiomyopathy and Emery-Dreifuss muscular dystrophy

Wahyu Hendrati Raharjo¹, Paul Enarson¹, Teresa Sullivan², Colin L. Stewart² and Brian Burke^1,*,

¹ Department of Cell Biology and Anatomy, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta T2N 4N1, Canada
² Laboratory of Cancer and Developmental Biology, NCI-FCRDC, PO Box B, Frederick, MD 21702-1201, USA
^* Present address: Department of Anatomy and Cell Biology, University of Florida, Box 100235, Gainesville, FL 32610, USA

View larger version (11K): [in a new window]   Fig. 1. Disease-linked point mutations of human lamins A and C expressed in HeLa cells. (A) Organization of the lamin A and C polypeptides. Both lamins share identical sequence from residues 1 to 566. The location of each point mutation is indicated in the diagram. The black bar within the lamin A and C nonhelical C-terminal domains marks the position of the nuclear localization sequence. The immunoblots in (B) demonstrate that HA-tagged wild-type and mutant lamin cDNAs encode proteins of the appropriate molecular weight. The various lamin A alleles (HA-LaA) appear as a doublet, with the lower band corresponding to mature lamin A. The upper band most likely corresponds to lamin A0, which has yet to undergo C-terminal proteolytic processing. This processing event appears slightly retarded in LaA L85R where the A0:A ratio is approximately 1.6:1. All of the other lamin A alleles exhibit an A0:A ratio of 1:1. The various lamin C alleles (HA-LaC) have identical mobilities.

View larger version (58K): [in a new window]   Fig. 2. Indirect immunofluorescence analysis of HA-tagged lamin A (LaA, A-J) and lamin C (LaC, K-O) alleles expressed in HeLa cells. In all cases the cells were labeled with the 12CA5 anti-HA antibody. F-J also include DNA staining with Höchst dye 33258. In the case of the lamin A mutants only LaA N195K (C and H) exhibits a distribution that is overtly different to wild-type (LaA WT, A and F). This protein remains largely nucleoplasmic and gives rise to intranuclear inclusions or aggregates in 25% of transfected cells (insets). These most likely correspond to those cells with the highest LaA N195K expression levels. Of the lamin C alleles, only the wild-type protein, LaC WT (K) and LaC R482W (N) are appropriately targeted to the nuclear envelope. The remainder localize exclusively to the nucleoplasm (L, M and O).

View larger version (55K): [in a new window]   Fig. 3. Indirect immunofluorescence analysis of HA-tagged lamin A alleles expressed in HeLa cells. Intranuclear aggregates induced by HA-LaA N195K (C) invariably contain lamin B2 (D, LaB2). In nontransfected cells both lamin A (A) and lamin B2 (B) are concentrated at the nuclear periphery (B). In A-D, the cells were double labeled with a rabbit antibody against lamin A and a mouse monoclonal antibody against lamin B2. In E-I the cells were labeled with the 12CA5 anti-HA antibody. The high-magnification views in E-G reveal that, in contrast to wild-type lamin A (LaA, E), LaA L85R (F) is distributed in a speckled pattern across the nuclear surface. Numerous small intranuclear aggregates formed by the LaA N195K mutant (G) is also shown. The difference in distribution between wild-type lamin A (H) and LaA L85R (I) is further demonstrated by confocal images of the equatorial region of a pair of nuclei. A more continuous labeling pattern is observed with wild-type lamin A than with LaA L85R.

View larger version (56K): [in a new window]   Fig. 4. Double indirect immunofluorescence analysis of HeLa cells expressing both wild-type (A,E) and mutant forms of lamin C (B-D and F-H). In each case the cells were labeled with the 12CA5 anti-HA antibody to reveal the HA-tagged lamin C alleles (A-D) and a rabbit antibody against lamin A to reveal the endogenous protein (E-H). Each of the mutants shown caused the relocation of endogenous lamin A to the nucleoplasm and often induced the formation of lamin A-containing aggregates (arrows). This lamin A aggregation phenotype is most pronounced with LaC N195K (C and G). Higher-magnification confocal views of lamin A aggregates (J) resulting from LaC N195K overexpression (I) are also shown. Nucleoli (identifiable by phase contrast microscopy, not shown), which are unlabeled by either antibody, are indicated by arrowheads (I and J).

View larger version (20K): [in a new window]   Fig. 5. Indirect immunofluorescence analysis of HA-tagged lamin A (LaA, A-E) and lamin C (LaC, G-K) alleles expressed in Lmna(–/–) fibroblasts. In each case the cells were labeled with the 12CA5 anti-HA antibody. Of the lamin A alleles only LaA N195K (C) fails to localize to the nuclear envelope. By contrast, none of the lamin C alleles, including wild-type lamin C (G) associate with the nuclear envelope. In about 35% of transfected cells spherical intranuclear LaC L85R aggregates could be observed (H, arrowheads). Appropriate localization of wild-type HA-lamin C (L) to the nuclear periphery (arrows) could be restored by cotransfection with wild-type lamin A (F). In this case, the nontagged lamin A was detected with the rabbit anti-lamin A-specific antibody (F).

View larger version (50K): [in a new window]   Fig. 6. Overexpression of lamin mutants causes a loss of nuclear envelope-associated emerin. Transfected HeLa cells (arrowheads) were doubled labeled with rabbit anti-emerin and monoclonal anti-HA antibodies. As shown in (A), expression of both LaA L530P and LaC L530P is associated with a decline in the levels of emerin detected in the nuclear envelope. Rather than distributing throughout the peripheral ER, mislocalized emerin was almost invariably found in cytoplasmic foci (arrows). As indicated in (B), comparison of cells expressing wild-type versus L530P mutants reveal that this effect is statistically significant (P<0.001). Similar, albeit milder effects are observed with LaA L85R and LaA/C N195K. LaC L85R and LaA/C R482W caused no significant (NS) emerin loss when compared with their wild-type counterparts.

View larger version (58K): [in a new window]   Fig. 7. Double indirect immunofluorescence analysis of Lmna(–/–) fibroblasts expressing both wild-type (A,B) and mutant forms of lamin A (E-L). Nontransfected cells are also shown (C,D). In each case the cells were labeled with the 12CA5 anti-HA antibody to reveal the HA-tagged lamin A alleles (La; A,C,E,G,I,K) and a rabbit antibody against emerin (Em; B,D,F,H,J,L). In the absence of A-type lamins, emerin in Lmna(–/–) fibroblasts is distributed largely throughout the peripheral ER, with little concentration in the nuclear envelope. Only wild-type lamin A (LaA WT; A,B) and LaA R482W (G,H) are capable of restoring the nuclear envelope localization of emerin in these cells.

View larger version (44K): [in a new window]   Fig. 8. Interaction of lamin A mutants with the emerin N-terminal domain (EmC, residues 3-221). Lamin and EmC cDNAs were transcribed and translated in vitro and yielded 35S-labeled translation products of the appropriate size (A). Single translation mixes containing identical levels of each lamin allele were subjected to immunoprecipitation with a guinea pig anti-lamin antibody coupled to Dynabeads. (B) Wild-type lamin A (WT) as well as each of the lamin mutants (LaA L85R, LaA N195K, LaA R482W and LaA L530P) were all efficiently immunoprecipitated with this antibody. Combined translation mixes (C) containing 35S-labeled EmC and unlabeled lamin alleles were similarly immunoprecipitated. EmC was recovered in comparable amounts in wild-type lamin A and LaA R482W immunoprecipitates. Less efficient EmC recovery was observed with LaA L85R and LaA N195K. With LaA L530P, EmC was barely detectable. Unlabeled lamins were employed in (C) to avoid the EmC band being obscured by possible lamin fragments or truncated translation products.