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

Abstract

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.

Functional and Genetic Analysis of Spectraplakins in Drosophila

“…Furthermore, patient studies suggest potential roles in neural development (Giorda et al, 2004;Vincent et al, 2008), human melanoma (Shimbo et al, 2010), and the infection process of Herpes virus (Pasdeloup, McElwee, Beilstein, Labetoulle, & Rixon, 2013). Finally, studies of dystonin mutant mice reveal further defects in glial cells (Bernier, De Repentigny, Mathieu, David, & Kothary, 1998;Saulnier, De Repentigny, Yong, & Kothary, 2002) potentially linking to multiple sclerosis (Laffitte et al, 2005), and neuromuscular junction defects associated with intrinsic muscle weakness (Boyer, Bernstein, & Boudreau-Larivière, 2010;Dalpe et al, 1999;Poliakova et al, 2014).…”

Microtubule-Actin Cross-Linking Factor 1

“…Alternative initiation of both MACF1a and MACF1b can result in additional isoforms (Bernier et al, 1996;Gong et al, 2001;Jefferson et al, 2006). Splice variants of the MACF1 C-tail have also been described (Poliakova et al, 2014).…”

“…The MTBD contains two alternatively spliced exons and a single SxIP motif that functions as a binding site for the EB1 family of MT tip-interacting proteins (TIPs). Alternative initiation of both MACF1a and MACF1b can result in additional isoforms, some lacking one or both CH parts of the ABD (Bernier et al, 1996;Gong, Besirli, & Lomax, 2001;Jefferson, Leung, & Liem, 2006;Kumar & Wittmann, 2012;Leung, Sun, Zheng, et al, 1999;Lin et al, 2005;Ortega, Buey, Sonnenberg, & de Pereda, 2011;Poliakova et al, 2014;Slep et al, 2005;Sonnenberg & Liem, 2007;Sonnenberg, Rojas, & de Pereda, 2007;Wu, Kodama, & Fuchs, 2008).…”

“…When combined, the two regions bind MTs more strongly than either one does in isolation (Sun et al, 2001;Suozzi et al, 2012). The MACF1 MTBD contains at least two alternatively spliced exons, which may play a role in modulation of the MT-binding capacity of MACF1 isoforms in different tissues (Poliakova et al, 2014). In addition, the C-tail of MACF1 contains a single SxIP motif.…”

Spectraplakin family proteins – cytoskeletal crosslinkers with versatile roles

“…Spectraplakins generate further diversity from their differential promoter usage and splicing, which results in a plethora of possible proteins that are able to selectively link and coordinate many biological functions within different cell types ( Fig. 1; Box 1) (Brown et al, 1995;Duchen et al, 1964;Green et al, 1990;Guo et al, 1995;Jefferson et al, 2006;Künzli et al, 2016;Leung et al, 2001b;Poliakova et al, 2014;Ruhrberg and Watt, 1997;Sawamura et al, 1991b;Stanley et al, 1981;Wiche et al, 1991). In this Review, we will discuss the structure and function of spectraplakin proteins and summarize recent studies that unveil their role in many physiological and pathological processes.…”