Multiple antibodies to titin immunoreact with AHNAK and localize to the mitotic spindle machinery

Abstract

Recently, the large filamentous striated-muscle protein titin has been observed in non-muscle cells, and, in one instance, has been proposed to have a nuclear function as a chromosomal component contributing to structure and elasticity. In this study, we sought to further characterize the presumptive nuclear isoform of titin. Immunofluorescence microscopy with multiple titin-specific monoclonal antibodies shows localization to the nucleus in interphase cells and to the spindle machinery in mitotic cells in all cell types examined; localization to condensed chromosomes is not observed. An abundant 700-kDa phosphoprotein is the predominant species immunoprecipitated with these antibodies. Sequencing of peptide fragments of the immunopurified protein reveals identity to AHNAK, a nuclear phosphoprotein, an identification that was confirmed by Western blot analysis with antibodies to AHNAK and peptide fragmentation patterns. Sequence comparison suggests similarities between the repetitive heptad phi+/-phiP+/-phi+/- motif in AHNAK and the PEVK region of titin, potentially explaining the cross-reactivity observed between AHNAK antibodies and titin antibodies. Interestingly, although some AHNAK antibodies stain interphase nuclei, no evidence of mitotic spindle localization is seen, suggesting that the identity of the protein at the latter location is more closely related to titin than AHNAK. This concept is further supported by observations that cell lines not expressing AHNAK have similar antititin antibody localization to the mitotic spindle. We conclude that (1) multiple titin antibodies, particularly those recognizing the PEVK region, cross-react with AHNAK, and (2) the mitotic spindle staining observed with antititin antibodies is most likely due to the association of titin or a titin-like molecule with this structure.

Titin in insect spermatocyte spindle fibers associates with microtubules, actin, myosin and the matrix proteins skeletor, megator and chromator

“…Results from these control experiments indicate that the staining is due to the primary antibody and is not specific to any single fixation or lysis procedure. Thus, we are confident that the described staining patterns represent non-perturbed patterns of D-titin localization, patterns consistent with staining observed in C. elegans spindles (Zastrow et al, 2006) and in mammalian spindles (Wernyj et al, 2001). Whereas Machado et al (Machado et al, 1998) did not report staining of spindle fibers in Drosophila or mammalian cells with anti-KZ, the same anti-KZ antibody in the present study did stain spindle fibers.…”

Expression of Distinct Classes of Titin Isoforms in Striated and Smooth Muscles by Alternative Splicing, and Their Conserved Interaction with Filamins

“…Since the titin 9D10 antibody is known to cross-react with another giant protein, AHNAK, we performed additional Western blots with antibodies to AHNAK. 21 Finally, we included antibodies to Novex-3 titin, a ∼650 kDa splice isoform of titin co-expressed in striated muscles with conventional half-sarcomere spanning titins. 22 The antibodies to AHNAK and anti-Novex 3 antibodies did not recognize the 1 MDa species present in smooth muscles (Figure 4(a), right).…”

NME1 at the human maternal–fetal interface downregulates titin expression and invasiveness of trophoblast cells via MAPK pathway in early pregnancy

“…High expression levels of MMP and TIMP in trophoblasts during early pregnancy regulate their invasion (Wernyj et al 2001). TIMPs from decidua and trophoblast are the natural MMP inhibitors, and play an essential role in regulating the process of trophoblast invasion .…”