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First published online October 22, 2003
doi: 10.1242/10.1242/jcs.00764

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Bpag1 localization to actin filaments and to the nucleus is regulated by its N-terminus

Ottawa Health Research Institute, 501 Smyth Road, Ottawa, Ontario, Canada K1H 8L6 The University of Ottawa Center for Neuromuscular Disease, Ottawa, Ontario, Canada K1H 8M5 Department of Cellular and Molecular Medicine, and Department of Medicine, University of Ottawa, Ottawa, Ontario, Canada K1H 8M5

View larger version (37K): [in a new window]   Fig. 1. Bpag1 isoforms expressed in C2C12 myoblasts, and the N-terminal fusion proteins used in this study. (A) The Bpag1e and Bpag1b isoforms. Bpag1b includes the N-terminal unique region encoded by exons A or A' (Brown et al., 1995), an ABD consisting of two calponin homology domains (CH1 and CH2), a plakin domain, an intermediate filament binding domain (IFBD2), a spectrin-repeat region (SR rod), an EF-hand, and a GAS2 homology region which includes part of an MTBD. Only the plakin domain is shared by the Bpag1e isoform, which has a C-terminal half consisting of a coiled-coil rod domain (C-C rod) and an alternate intermediate filament binding domain (IFBD1). The Bpag1a isoform (not shown) differs from the Bpag1b isoform only in that it lacks the IFBD2 and surrounding region. The peptide sequence used for isoform-specific antibody production is also shown. (B) Semiquantitative RT-PCR using primers against various regions of Bpag1. The results indicate that Bpag1b is the predominant isoform expressed in C2C12 myoblasts, although a band amplified from the IFBD1 region of Bpag1e was also detected at low levels. Conversely, regions from the Bpag1b mRNA were detected at low levels in mouse skin, whereas the IFBD1 region from the Bpag1e mRNA was prominent in skin. Actin mRNA amplification served as a control for RNA amount used. (C) The various fusion proteins used in this study. FLAG and GFP western blots indicated that each fusion protein was expressed at the appropriate size. B, BglII; R, EcoRI; S, SalI; Sa, SapI.

View larger version (107K): [in a new window]   Fig. 2. An antibody specific for Bpag1 isoform 2 detects protein co-aligning with actin stress fibers. (A) Western blot analysis of lysates from Cos-1 cells expressing Bpag1 fusion proteins. GFP-Nterm1 and GFP–Nterm2 were expressed, as shown with an anti-GFP antibody, and used to test anti-Bpag1 sera. Serum containing antibodies against the Bpag1 A' region detected GFP-Nterm2 but not GFPNterm1. GFP-Nterm2 was not detected with the pre-immune serum (data not shown), and was strongly detected with the affinity-purified antibody (A' Ab). (B) Only diffuse background staining was ever observed in cells stained with the pre-absorbed A' antibody. (C,D) In C2C12 cells, the A' antibody (C) stained in a filamentous pattern co-aligning with centrally located actin stress fibers (stained with rhodamine-phalloidin in D; closed arrows in C and D). The A' staining typically did not occur along more peripheral actin filaments (open arrows in C and D), although colocalization did occur along more peripheral actin filaments in a few cells. Strong peri-nuclear staining was also observed in a few cells (arrowhead in C). Bars, 20 µm.

View larger version (124K): [in a new window]   Fig. 3. Endogenous Bpag1a/b2 stress fiber localization does not extend into focal contact sites, and most of the A' Ab staining occurs in and around the nucleus. (A-C) Observed by confocal microscopy, the A' antibody (A) stains in a pattern co-aligning perfectly with centrally-located stress fibers (stained with rhodamine-phalloidin in B; arrows in A and B). (D-F) In the same cells as in A-C, staining with the A' antibody (D) in an optical plane through the nuclei revealed strong nuclear (arrowheads) and perinuclear (arrow) staining (D). Rhodamine-phalloidin staining was absent from the nuclei (E). Hoechst staining was used to identify the nuclei (C,F). (G-I) When C2C12 cells were stained for both Bpag1a/b2 (G) and paxillin (H), and observed by confocal microscopy, the A' staining and the paxillin staining never overlapped (arrows in G and H). Shown is a region of a C2C12 cell with a large number of focal contact sites (paxillin staining) to illustrate the exclusion of Bpag1a/b2 from these sites (I is the merged image of G and H). Bars, 20 µm. Bar in A applies to A-F; bar in I applies to G-I.

View larger version (94K): [in a new window]   Fig. 4. Disruption of actin filaments, but not of microtubules, alters Bpag1a/b2 localization. (A,B) Treatment with 200 nM cytochalasin D caused the aggregation of stress fibers in many of the C2C12 cells (B). In cells with a few remaining actin stress fibers, some A' staining (A) was still co-aligned with the fibers (arrow in A and B). In cells with no intact central stress fibers, A' staining was diffuse, and no filamentous pattern was ever observed. (C,D) Treatment with 500 nM nocodazole caused almost complete depolymerization of microtubules in the majority of the C2C12 cells (D), with most of the remaining microtubule staining existing at and close to the microtubule organizing centers (asterisks in D). A' staining in these cells (C) was not disrupted, as it was still observed in a filamentous pattern (arrow in C). Bar in D, 20 µm, and applies to all four images.

View larger version (77K): [in a new window]   Fig. 5. Bpag1e localizes predominantly to the nucleus in C2C12 myoblasts. (A-C) In cells double-labeled for Bpag1e (A) and paxillin (B), overlap in the patterns of staining was observed in the center of a few cells (arrows in insets; insets are enlargements of the center region from the larger cell). However, the only staining in most cells was light staining in the nuclei (arrowhead in A). (D,E) Bpag1e staining (D) and Hoechst staining (E) showing that most of the Bpag1e staining was limited to the nuclei (arrowheads in D and E). In several mitotic cells noted, Bpag1e staining was absent from condensed chromosomes (arrows in D and E). (F,G) Bpag1e staining (F) was also identified in the nuclei (G; arrowheads in F and G) using bullous pemphigoid patient serum [Bpag1e (10-71)]. Bars, 20 µm.

View larger version (88K): [in a new window]   Fig. 6. The interaction of Bpag1 N-terminal fusion proteins with actin filaments in C2C12 myoblasts. (A-D) The FLAG-ABD protein (A) coaligned with actin stress fibers (B). A relatively stronger signal was also consistently noted at the ends of the fibers (insets in A-C; arrow in D). (E-H) The N-term1 protein (E) colocalized primarily with actin aggregates (F) in all cells. It never colocalized with normal-appearing stress fibers. (I-L) The Nterm2 fusion protein (I) co-aligned with stress fibers (J), but not at the ends (left-hand insets in I-K; arrows in L). In about half of the cells, Nterm2 colocalized with aberrantly bundled actin fibers (right-hand insets in I-K). Bar in K, 20 µm for all conventional fluorescence micrographs. Bars in confocal micrographs (D,H,L), 10 µm.

View larger version (93K): [in a new window]   Fig. 9. The Bpag1 isoform 2 N-terminus, but not the isoform 1 N-terminus, localizes to the nucleus of C2C12 cells when the plakin domain is included in the fusion protein. (A-C) Nterm1long (A), similar to the shorter Nterm1 fusion protein (Fig. 6E), localized to actin aggregates (B; arrows in A and B). This protein was never observed in the nuclei (arrowhead in C). (D-F) Nterm2long (D) localized to the nucleus in almost all cells (arrowhead in D and F), and was also found in the cytoplasm in over half of the cells, including along stress fibers (E; arrows in D and E). (G,H) In many of the cells, Nterm2long staining (G) appeared to be exclusively nuclear, in a Swiss-cheese pattern of staining reminiscent of the endogenous Bpag1 staining (see Figs 3 and 5). (I) The C-terminal half of the plakin domain (plakinCterm) localizes in a similar pattern in the nucleus in all cells expressing this fusion protein. Additional localization to the cytoplasm occurred only in a minority of the cells expressing plakinCterm. Bars, 20 µm.

View larger version (57K): [in a new window]   Fig. 7. The Bpag1 ABD, but not the isoform 2 N-terminus, localizes to focal contacts. (A,B) As observed by confocal microscopy, the FLAG-ABD fusion protein staining (green) consistently produced staining that overlapped with paxillin staining (red), indicating its localization to focal contact sites. (C,D) With the FLAG-Nterm2 fusion protein (green), only minor overlap with paxillin staining (red) was ever observed by confocal microscopy. Also, punctate staining consistently observed for paxillin and FLAG-ABD along the periphery of the cells (arrows in A and B) was never observed with the FLAG-Nterm2-expressing cells. Bar in D, 10 µm, and applies to all four images.

View larger version (52K): [in a new window]   Fig. 8. The ABD-containing N-terminal regions of Bpag1 can selfassociate but require the presence of the unique regions upstream from the ABD to do so. (A) Immunoblots showing 1/10 input of the lysates used for performing immunoprecipitations. GFP-Nterm1 and FLAG-Nterm1, GFP-Nterm2 and FLAG-Nterm2, or GFP-Nterm2 and FLAG-ABD were co-expressed in Cos-1 cells. All of the GFP- and FLAG-tagged proteins were expressed relatively evenly, with the exception of the FLAG-Nterm2, which was very highly expressed. This was typical of other results looking at the expression of these proteins (data not shown). (B) Immunoprecipitation with an anti-FLAG antibody pulls down GFP-Nterm1 (lane 2) in cells coexpressing FLAG-Nterm1 and GFP-Nterm1, and GFP-Nterm2 (lane 4) in cells co-expressing FLAG-Nterm2 and GFP-Nterm2. However, the GFP-Nterm2 was not co-immunoprecipitated with the FLAG-ABD fusion protein (lane 6). Lanes 1, 3, and 5 are immunoprecipitations corresponding to lanes 2, 4 and 6, respectively, which were performed without the anti-FLAG antibody as controls for the experiment.

View larger version (37K): [in a new window]   Fig. 10. Bpag1 contains a functional NLS in the plakin domain. (A) In both mouse and human Bpag1 proteins, the PVKRRRI/M motif in the plakin domain is conserved (identical amino acid residues have dark shading; similar amino acid residues have light shading). This motif is similar in the same region of mouse MACF, but differs in plectin (Plec1) and periplakin (Ppl). (B) The FLAG-Nterm2long plasmid was re-made with a mutation within the NLS sequence such that it would no longer resemble a classical NLS (Nterm2longNLS). The FLAG-NLS-ABD plasmid had the NLS coding sequence (coding for PVKRRRI) inserted immediately before the ABD insert. The mutant fusion proteins were of similar size to their wild-type counterparts (not shown). Labeling is the same as in Fig. 1. (C) Nterm2long fusion protein produced strong signal in the nucleus of 99% of the cells (arrowheads). The arrow in C points to some of the fusion protein co-aligning with stress fibers. (D) The Nterm2longNLS fusion protein staining never produced staining in the nucleus (arrowhead) above the background of untransfected cells. The protein was exclusively cytoplasmic, with much of it co-aligning with stress fibers (arrows). (E) Localization of the ABD fusion protein along stress fibers (arrow), but not in the nucleus (arrowhead). (F) With the NLS sequence placed before the ABD, 57% of the cells had labeling of the fusion protein within the nucleus (arrowhead), which was typically much stronger than the signal in the cytoplasm. The NLS-ABD fusion protein also co-aligned with stress fibers in many cells (arrow), and appeared no different than the ABD fusion protein in many. Bar in F, 20 µm, and also applies to CE. (G) Summary of the quantification of the localizations of the Nterm2long, Nterm2longNLS, ABD and NLS-ABD fusion proteins. Error bars indicate the standard errors from a minimum of three separate transfections.