Molecular polymorphism of the intermediate filament protein transitin

Abstract

Transitin is an avian intermediate filament protein whose transient expression in the progenitor cells of the muscle and nerve tissues is similar to that of mammalian nestin. Both proteins contain an alpha-helical core domain flanked by a short N-terminal head and a long C-terminal extremity. However, the tail region of transitin is significantly different from that of nestin in that it harbors a unique motif containing more than 50 leucine zipper-like heptad repeats which is not found in any other intermediate filament protein. Despite the absence of introns in this region of the transitin gene, it was reported that different isoforms of the protein were produced by exclusion or inclusion of a number of repeats generated by an unusual splicing mechanism recognizing consensus 5' and 3' splice sites contained within the coding sequence of the heptad repeat domain [Napier et al. (1999) J Mol Neurosci 12:11-22]. Two monoclonal antibodies (mAbs) reacting with repeated epitopes of this motif were used to monitor transitin expression during in vitro myogenesis of the quail myogenic cell line QM7. Confocal microscopy revealed that the subcellular domains decorated with mAbs A2B11 and VAP-5 were mutually exclusive: the intermediate filament network visualized with mAb VAP-5 appeared to abut on a submembranous domain defined by mAb A2B11. When QM7 cells were induced to differentiate by switching to medium containing low serum components, an early effect was the local loss of A2B11 cortical staining at the points of cell-cell contacts. The A2B11 signal also disappeared before that of VAP-5 in newly formed myotubes. Unexpectedly, the mutually exclusive staining pattern of the mAbs could not be explained by alternative splicing since both epitopes mapped to a repeated element preceding the consensus 5' splice sites of the heptad repeat domain. An alternative explanation would be that the central repeat domain of transitin is a polymorphic structure from which different conformations exist depending on the local context. This hypothesis is strengthened by the observation that in cultured neural crest cells, the A2B11 antigen is preferentially expressed by freely migrating crest cells whose intracellular pH and calcium concentrations are different from those of non-migrating cells.

Molecular cloning and characterization of chicken neuronal intermediate filament protein α‐internexin

“…During the past decade, several studies have contributed to the analysis of neural developmental processes in the chicken embryo, such as neurogenesis, migration, and patterning (Itasaki etal., 1999; Stern, 2005; Lu et al, 2009). Some neural IF genes of the chicken have been cloned and characterized (Zopf et al, 1987; Shaw, 1989; Henion et al, 2000; Darenfed et al, 2001; Wakamatsu et al, 2007). However, little is known about the sequence, structure, expression patterns, and function of the neuronal IF α‐internexin gene in avians.…”

Transitin is required for the differentiation of avian QM7 myoblasts into myotubes

“…Its expression in radial glia at apparent sites of cell-substratum adhesion and the localization of its mRNA to radial glia endfeet (Lee and Cole, 2000) are suggestive of local regulation of transitin concentration in specific distal compartments. In addition, our laboratory demonstrated that a subset of transitin molecules is concentrated in a submembranous domain of migrating neural crest cells in vitro (Darenfed et al, 2001). Immunoreactivity of this submembranous domain rapidly disappeared from points of cell-cell contacts when avian QM7 myoblasts were induced to fuse and differentiate into myotubes.…”

“…Localization of transitin mRNA in radial glia processes of the developing chick CNS and to Z-lines in developing skeletal muscles (Lee and Cole, 2000) suggests that transitin concentration may be regulated in specific distal compartments. In QM7 cells, we have previously shown that a subset of transitin molecules, defined by mAb A2B11, concentrated in a submembranous domain distinct from the pool of transitin molecules associated to the IF network (Darenfed et al, 2001). When QM7 cells were induced to differentiate by switching to DM, an early effect was the local loss of A2B11 staining at the points of cell-cell contacts.…”

“…After respective treatments, glass plates were removed with forceps, washed with PBS twice, and fixed with ice-cold methanol for 20 min at À20 C. Cells were then reacted with primary and secondary antibodies as described previously (Darenfed et al, 2001). For transitin and desmin co-detection, plates were double-labeled for 1 hr with monoclonal antibody VAP-5 and rabbit polyclonal antibody against desmin.…”

What's new in histochemistry and cell biology?

“…Transitin represents an avian intermediate filament with transient expression in progenitor cells of muscle and nerve tissues. Its expression during in vitro myogenesis of the quail myogenic cell line QM7 was studied by confocal immunofluorescence (Darenfed et al 2001).…”