Constitutive and Variable Regions of Z-disk Titin/Connectin in Myofibril Formation: A Dominant-negative Screen.

Abstract

ABSTRACT. The giant muscle protein titin (connectin) is assumed to play a crucial role in the control of Zdisk assembly. Analysis of the Z-disk region of titin/connectin revealed a novel 45 residue repeat that is spliced in variable copy numbers. The repeat region is coexpressed in normal humanmyocardiumin size variants corresponding to between 5 and 7 repeats. Smaller isoforms can be detected in muscles with thinner Z-disks like M. psoas. Sequence analysis of chicken breast muscle titin/connectin reveals that in this tissue, where Z-disks are thin, only two Z-repeats are expressed. The greatly variable thickness of Z-disks is therefore correlated to a region of variable length in titin/connectin, constructed from a novel protein building block. Transfection experiments in myogenic cell lines demonstrate that overexpression of the entire integral Z-disk region of titin leads to a disruption of sarcomere assembly. The differentially expressed titin/connectin regions, however, show no dominant-negative effects on myofibril assembly and are not targeted to Z-disks. This supports the idea that the Z-repeat region of titin/connectin is responsible for the control Z-disk thickness as an element of highly variable length, due to extensive differential splicing.Assemblyof the sarcomere involves the coordinated, sequential integration of many proteins into filamentous and anchoring structures. The assembly of I-Z-I complexes is one of the initial events in sarcomeric assembly, and the proteins incorporated into this structure are therefore the first sarcomeric proteins detectable during myogenic differentiation (reviewed in 31). The giant muscle protein titin/connectin (18, 19, 33), mostly and in what follows called titin for its large size, is one of the first proteins appearing in the Z-disk in developing striated muscle (7,10, 16, 28, 29, 34). The integration of titin into nascent sarcomeres begins with the assembly of the aminoterminus into the Z-disk and proceeds towards the C-terminal M-band region (7). Titin/connectin is believed to play a major role in defining the positions of other sarcomeric proteins as a molecular blueprint ("molecular ruler") for the layout of the sarcomere (34, 38), and thus to control the sequential events in sarcomere assembly. Titin binding to myosin and myosin-associated proteins in the A-band supports this idea (5, 13, 20). Further evidence is supplied by the striking correlation of the modular array of immunoglobulin-like (Ig) and fibronectin-3-like (fn3) domains in the A-band with the ultrastructure of the sarcomere (3, 13, 14). However, the sequence of cardiac titin reveals a particular sequence complexity in both the regions predicted to be in the Z-disk and M-disk (8, 14, 24). The identification of sequence insertions in cardiac Z-disk titin which can be phosphorylated in vitro by SP-specific protein serine/threonine kinases (9, 24) suggests the titin N-terminus may be involved in controlling myofibril assembly or disassembly. Since the anchorage of titin in the Z-disk is importa...

Distinct roles for telethonin N‐versus C‐terminus in sarcomere assembly and maintenance

“…Is this conclusion consistent with the rest of the telethonin literature? On one hand, studies supporting the importance of the telethonin/titin interaction during de novo myofibrillogenesis include overexpression of N-terminal titin (residues 1-362), Z-disc titin (Z1-Z2 repeats, residues 1-200), telethonin, or C-terminal a-actinin missing its titin-binding domain, all of which result in severe myofibril disruption (Turnacioglu et al, 1997;Peckham et al, 1997;Gregorio et al, 1998;Lin et al, 1998). On the other hand, telethonin is incorporated into the Z-disc late during myofibrillogenesis (Wang et al, 2005;Sanger et al, 2009;Zhang et al, 2009), and once incorporated has very low mobility as assessed with FRAP measurements (Wang et al, 2005, Sanger et al, 2009).…”

The sarcomeric cytoskeleton: from molecules to motion

“…The interaction of Z-repeats with α-actinin has been studied in detail at the cellular, biochemical, biophysical and structural level, confirming and characterising the interaction of the helical Z-repeats with the α-actinin CaM domain (Atkinson et al, 2000(Atkinson et al, , 2001Ribeiro et al, 2014;Sorimachi et al, 1997;Young et al, 1998). There is a strict correlation of the number of titin Z-repeats with the number of Z-links: chicken pectoralis major muscle, a fast glycolytic muscle with thin Z-disks, expresses titin with two Z-repeats (Peckham et al, 1997), while cardiac and slow skeletal muscles in mammals with thick Z-disks express up to seven Z-repeats Sorimachi et al, 1997). However, it is currently unclear how their α-actinin-binding properties on the molecular level are translated into the assembled structure, as there appears to be a mismatch in the length of the repeat and the periodicity of the Z-disk as measured by EM (Luther and Squire, 2002).…”

Role of desmin filaments in chicken cardiac myofibrillogenesis

“…The Z-repeat region of titin is responsible for the control of Z-disk assembly [Peckham et al, 1997], and the A-band domain of titin is involved in the ordered assembly of thick filaments [Labeit et al, 1992]. As shown by an immunoelectron microscopic study, titin forms spots in the cytoplasm of embryonic cardiac muscle cells and cultured skeletal myoblasts [van der Ven et al, 1993;Fulton and Alftine, 1997;Tokuyasu and Maher, 1987a].…”

“…At the onset of myofibrillogenesis, Z-bodies are formed as punctate concentrations of ␣-actinin [Tokuyasu and Maher, 1987b;Sanger et al, 1984], and this is followed by anchorage of the N-terminus of the titin molecule into the Z-body . The subsequent alignment and fusion of Z-bodies of adjacent premyofibrils result in the formation of Z bands Turnaciogly et al, 1997;Peckham et al, 1997;Sorimachi et al, 1997;Nag and Lee, 1997]. Incorporation of anti-desmin antibodies may block the interactions between desmin and ␣-actinin and ␣-actinin and titin, which would explain the formation of titin aggregates and the fragmentation of myofibrils.…”