Characterization of neuronal plakins

Plakins are a growing family of giant-sized cytoskeletal binding proteins that crosslink cytoskeletal elements to each other and to specialized sites at the plasma membrane. Originally identified as intermediate-filament binding proteins important for maintaining epithelial tissue integrity, the discovery of new plakin genes and alternative splice variants has now made it clear that plakins can associate with multiple cytoskeletal networks. Although many of the newly identified proteins are expressed highly in the vertebrate nervous system, little is known of their function. We focus our studies on further characterizing the plakin proteins, bullous pemphogoid antigen 1 (BPAG1) and microtubule-actin crosslinking factor 1 (MACF1) in the mouse nervous system. We first examined the expression pattern of BPAG1 and MACF1 proteins and found that both proteins are expressed in several regions of the adult brain. To examine the function of MACF1 in neuronal cells, we used MACF1 conditional knockout (cKO) mice in which the inactivation of the MACF1 gene is neural-cell specific. In these mice, we find that conditional inactivation was specific for MACF1-a isoforms containing an N-terminal actin-binding domain (ABD). Expression of ABD-less MACF1 isoforms was still observed in these mice. Cultured neurons from wildtype and MACF1 cKO mice were morphologically indistinguishable.;BPAG1 and MACF1 are able to associate with microtubules. This interaction is mediated through their C-terminal microtubule-binding domain which is composed of a Gas-2 related domain (GAR) that binds to microtubules and a glycine-serine-arginine (GSR) repeat containing domain that bundles microtubules. To get a better handle on the function of neuronal plakins we sought to further characterize this domain. We report alternative splicing at the 3' end of BPAG1 and MACF1. This alternative splicing uses different 3' exons to yield four additional variants of the neural and muscle isoforms of BPAG1 and MACF1. We have analyzed the subcellular localization and cytoskeletal interactions of these isoforms and show that the novel sequences at the 3' end do not alter the microtubule bundling activity of these proteins. We also show that the novel sequences do not have an effect on interaction with EB1 family members.