Targeting of cardiac muscle titin fragments to the Z-bands and dense bodies of living muscle and non-muscle cells

Abstract

A 6.5-kb N-terminal region of embryonic chick cardiac titin, including the region previously reported as part of the protein zeugmatin, has been sequenced, further demonstrating that zeugmatin is part of the N-terminal region of titin, and not a separate Z-band protein. This Z-band region of cardiac titin, from both 7-and 19-day embryos as well as from adult animals, was found to contain six different small motifs, termed z-repeats [Gautel et al., 1996: J. Cell Sci. 109:2747-2754, of approximately 45 amino acids each sandwiched between flanking regions containing Ig domains. Fragments of Z-band titin, linked to GFP, were expressed in cultured cardiomyocytes to determine which regions were responsible for Z-band targeting. Transfections of primary cultures of embryonic chick cardiomyocytes demonstrated that the z-repeats play the major role in targeting titin fragments to the Z-band. Similar transfections of skeletal myotubes and non-muscle cells lead to the localization of these cardiac z-repeats in the Z-bands of the myofibrils and the dense bodies of the stress fibers. Over-expression of these z-repeat constructs in either muscle or non-muscle cells lead to the loss of the myofibrils or stress fibers, respectively. The transfection experiments also indicated that small domains of a protein, 40 to 50 amino acids, can be studied for their localization properties in living cells if a suitable linker is placed between these small domains and the much larger 28 kDa GFP protein.

Translocation of a human focal adhesion LIM-only protein, FHL2, during myofibrillogenesis and identification of LIM2 as the principal determinants of FHL2 focal adhesion localization

“…During differentiation of the stably transfected myoblasts, FHL2 was colocalized with F-actin, which suggested that FHL2 maybe redistributing along actin filaments from focal adhesion plaques. Furthermore, LIM domain proteins can also be found in the Z-lines of the terminally differentiated muscle such as Enigma [Guy et al, 1999], CRP3/MLP [Arber and Caroni, 1996], titin [Ayoob et al, 2000], ZASP [Faulkner et al, 1999], and Cypher [Zhou et al, 1999]. We observed that FHL2 is yet another LIM-containing protein localized in the Z-lines of human myocardium; however, the LIM domain responsible for its localization in the Z-lines has not been determined.…”

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

“…8,10,12 In contrast to striated muscle titin, the giant protein species from smooth muscle and non-muscle tissues have been difficult to characterize due to an overall low level of their expression. Moreover, Western blot studies observing failure to react with titin antibodies may be explained by a splice isoform lacking the respective titin epitope, whereas a positive reaction might be caused by a cross-reactivity of titin to different proteins such as AHNAK.…”

“…11 Both the 700 kDa zeugmatin and 2000 kDa smitin proteins from avian smooth muscle were detected immunologically in dense bodies of smooth muscle and in stress fibers of non-muscle cells, implicating them in regulation of the actin cytoskeleton that is prominent in these structures. 8,10,12 Finally, a titin-sized protein was identified in the rapidly dividing carcinoma HeLa cell line and becomes here an integral part of the chromosome during metaphase. 13 Titin could potentially regulate both the elastic properties of the metaphase chromosome as well as their structural assembly.…”

Novel actions of cardiac ankyrin repeat protein

“…Smitin, a 700 kDa titin-like protein, is associated with the contractile apparatus in smooth muscle [27,28]. In non-muscle cell types, transfected, labeled z-repeats of titin have been localized to the dense bodies of stress fibers [29]. Overexpression of the z-repeat constructs results in the loss of stress fibers, suggesting the presence of a titin-like protein in non-muscle that acts to organize the stress fibers, similar to the sarcomere organizing function of titin in which CARP appears to play a role [25].…”