BPAG1a and b Associate with EB1 and EB3 and Modulate Vesicular Transport, Golgi Apparatus Structure, and Cell Migration in C2.7 Myoblasts

BPAG1a and BPAG1b (BPAG1a/b) constitute two major isoforms encoded by the dystonin (Dst) gene and show homology with MACF1a and MACF1b. These proteins are members of the plakin family, giant multi-modular proteins able to connect the intermediate filament, microtubule and microfilament cytoskeletal networks with each other and to distinct cell membrane sites. They also serve as scaffolds for signaling proteins that modulate cytoskeletal dynamics. To gain better insights into the functions of BPAG1a/b, we further characterized their C-terminal region important for their interaction with microtubules and assessed the role of these isoforms in the cytoskeletal organization of C2.7 myoblast cells. Our results show that alternative splicing does not only occur at the 5′ end of Dst and Macf1 pre-mRNAs, as previously reported, but also at their 3′ end, resulting in expression of additional four mRNA variants of BPAG1 and MACF1. These isoform-specific C-tails were able to bundle microtubules and bound to both EB1 and EB3, two microtubule plus end proteins. In the C2.7 cell line, knockdown of BPAG1a/b had no major effect on the organization of the microtubule and microfilament networks, but negatively affected endocytosis and maintenance of the Golgi apparatus structure, which became dispersed. Finally, knockdown of BPAG1a/b caused a specific decrease in the directness of cell migration, but did not impair initial cell adhesion. These data provide novel insights into the complexity of alternative splicing of Dst pre-mRNAs and into the role of BPAG1a/b in vesicular transport, Golgi apparatus structure as well as in migration in C2.7 myoblasts

Schematic representation of BPAG1a and b domain organization.

<p>ABD, actin-binding domain; CH, calponin homology domain; SR, spectrin repeat (dotted ovals represent putative SRs, not previously identified as SRs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107535#pone.0107535-Jefferson3" target="_blank">[63]</a>, or predicted in mouse or human BPAG1a sequence by SMART <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107535#pone.0107535-Letunic1" target="_blank">[64]</a>; some SRs in the plakin domain were deduced from the alignment with plectin plakin domain <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107535#pone.0107535-Sonnenberg1" target="_blank">[65]</a>, SH3, src homology-3 domain (is atypical and embedded in SR5 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107535#pone.0107535-Ortega1" target="_blank">[66]</a>); PRD, plakin repeat domain; EFhs, EF hands; GAR, GAS2-related domain; GSRs, Gly-Ser-Arg repeats; EBBS, EB1/EB3-binding site containing a Ser-X-Ile-Pro motif (where is X is any residue); MTBD, microtubule-binding domain. BPAG1a (NP_598594) is 5379 res. long. BPAG1b-specific domain (2014 res. long, deduced from NP_604443; 7393 res.) is inserted in between SR10 and SR11 of BPAG1a (after res. 1548).</p

Knockdown of BPAG1a/b in C2.7 cells induces the dispersal of the Golgi apparatus.

<p>A) Cells treated with control and BPAG1 siRNA were fixed in PFA and immunolabeled with anti-GM130 (cis-Golgi marker, green) and anti-BPAG1a/b (red) antibodies. Blue: nuclei stained with DAPI. Scale bar: 20 µm. Arrows: examples of dispersed Golgi. B) Graph represents percentage of cells (200 cells per siRNA) with dispersed Golgi. Data are mean ± SEM, n = 3 independent experiments.</p

Protein sequence alignment of the C-terminus of BPAG1a/b and MACF1a/b.

<p>Identical amino acids are shaded in black with white letters. Conserved amino acids changes are shaded in grey. The GAR domain, sequences encoded by the alternatively spliced exons α and β, GSR repeats and EB1-binding site (EB1-BS) are indicated. The denomination «C-tail» corresponds to the sequence(s) after the GAR domain.</p

BPAG1a/b is enriched in pseudopodia and small areas at the plasma membrane.

<p>C2.7 cells were grown to high confluency, fixed in PFA, and stained with anti-BPAG1a/b (green) and A) anti-β-tubulin (red) antibodies or B) TRITC-phalloidin (red). Blue: nuclei stained with DAPI. Scale bar: 20 µm. Arrows indicate strong BPAG1a/b signals close to the plasma membrane and arrowheads BPAG1a/b localization in pseudopodia.</p

BPAG1a/b short filament pattern disappears upon microtubule depolymerization, but not the dotted pattern in C2.7 myoblasts.

<p>Cells were treated for 10 min with A) 0.32 µM or B) 10 µM nocodazole, then immediately fixed in PFA and immunolabeled with anti-β-tubulin and anti-BPAG1a/b antibodies. Blue: nuclei stained with DAPI. Scale bar: 20 µm. C) Negative control cells were treated with a corresponding volume of DMSO for 30 min, and processed for microscopy the same way. DMSO had no effect on the cells and BPAG1a/b pattern looked the same as in non-treated cells (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107535#pone-0107535-g004" target="_blank">Fig. 4B</a>).</p

Expression profile of BPAG1a/b and MACF1a/b 3′-variants in various mouse tissues.

<p>RT-PCRs were performed with primers specific to the various plakin variants or to GAPDH from total RNA. The amplicons were analyzed on agarose gel and stained with ethidium bromide.</p

BPAG1 knockdown reduces C2.7 myoblast wound invasion in a wound healing assay.

<p>A) Pictures of denuded areas in C2.7 cell layer were taken immediately after the scratch was applied (0 h) and 19 h later. Images are representative of three independent experiments. Scale bar: 50 µm. B) Graph represents quantification of wound areas reinvaded by cells by subtracting wound area at 19 h from wound area at 0 h. Results were expressed in comparison to myoblasts transfected with control siRNA. Data are mean ± SEM. *p<0.05 <i>versus</i> control, Student's t test, n = 3 independent experiments, 3 crosses per triplicate for each siRNA.</p

BPAG1 knockdown reduces the number of myoblasts migrating into a wound, myoblast migration velocity and directness in a wound healing assay.

<p>A) The fields of view at 0 h (red) and at 18 h (green) after scratch were overlaid to visualize the invading cells. Cell-free area at 0 h was colored in black to increase contrast on the overlay. Scale bar: 100 µm. B) Time lapse photographs were taken of control and BPAG1 knockdown myoblasts every 10 min for 18 h. The migratory paths of individual cells that did not divide during 18 h are shown from one of the three experiments from the lower wound edge (see panel A). C) Quantitative comparison of the number of cells transfected with BPAG1 siRNA that had migrated into an acellular area with control siRNA transfected cells. The mean cell velocities were measured by manual tracking using ImageJ and pooled from three independent experiments. The mean velocity of BPAG1 knockdown cells is reduced by 36 %. Cell migration directness was quantified by calculating Euclidian distance divided by the accumulated distance for each individual cell (120 cells per siRNA). Data are mean ± SEM. *p<0.05 vs control, Student's t-test, n = 3 independent experiments.</p

Nuclear cysteine cathepsin variants in thyroid carcinoma cells

The cysteine peptidase cathepsin B is important in thyroid physiology by being involved in thyroid prohormone processing initiated in the follicular lumen and completed in endo-lysosomal compartments. However, cathepsin B has also been localized to the extrafollicular space and is therefore suggested to promote invasiveness and metastasis in thyroid carcinomas through, e.g., ECM degradation. In this study, immunofluorescence and biochemical data from subcellular fractionation revealed that cathepsin B, in its single- and two-chain forms, is localized to endo-lysosomes in the papillary thyroid carcinoma cell line KTC-1 and in the anaplastic thyroid carcinoma cell lines HTh7 and HTh74. This distribution is not affected by thyroid stimulating hormone (TSH) incubation of HTh74, the only cell line that expresses a functional TSH-receptor. Immunofluorescence data disclosed an additional nuclear localization of cathepsin B immunoreactivity. This was supported by biochemical data showing a proteolytically active variant slightly smaller than the cathepsin B proform in nuclear fractions. We also demonstrate that immunoreactions specific for cathepsin V, but not cathepsin L, are localized to the nucleus in HTh74 in peri-nucleolar patterns. As deduced from co-localization studies and in vitro degradation assays, we suggest that nuclear variants of cathepsins are involved in the development of thyroid malignancies through modification of DNA-associated proteins