Dynamics of Z-band based proteins in developing skeletal muscle cells

Abstract

During myofibril formation, Z-bodies, small complexes of alpha-actinin and associated proteins, grow in size, fuse and align to produce Z-bands. To determine if there were changes in protein dynamics during the assembly process, Fluorescence Recovery after Photobleaching was used to measure the exchange of Z-body and Z-band proteins with cytoplasmic pools in cultures of quail myotubes. Myotubes were transfected with plasmids encoding Yellow, Green or Cyan Fluorescent Protein linked to the Z-band proteins: actin, alpha-actinin, cypher, FATZ, myotilin, and telethonin. Each Z-band protein showed a characteristic recovery rate and mobility. All except telethonin were localized in both Z-bodies and Z-bands. Proteins that were present both early in development in Zbodies and later in Z-bands had faster exchange rates in Z-bodies. These results suggest that during myofibrillogenesis, molecular interactions develop between the Z-band proteins that decrease their mobility and increase the stability of the Z-bands. A truncated construct of alpha-actinin, which localized in Z-bands in myotubes, and exhibited a very low rate of exchange, lead to disruption of myofibrils, suggesting the importance of dynamic, intact alpha-actinin molecules for the formation and maintenance of Z-bands. Our experiments reveal the Z-band to be a much more dynamic structure than its appearance in electron micrographs of cross-striated muscle cells might suggest.

α-Actinin/titin interaction: A dynamic and mechanically stable cluster of bonds in the muscle Z-disk

“…3C), would not change significantly, and even under those conditions the Z-disk would remain stable. The concerted effort of many bonds reconciles the apparently contradictory findings of a long-term stable Z-disk (half-life of titin ≈14 h) (32) and a fast exchange of its individual components (half-lives of α-actinin and other Z-disk proteins <1 min) (19,33), as has been measured in various fluorescence recovery after photobleaching studies.…”

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

“…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). In addition, truncation mutations in telethonin are associated with a late-onset limb-girdle muscular dystrophy (LGMD 2G), and muscle biopsies from LGMD 2G patients are immunonegative for telethonin (Moreira et al, 2000;Olivé et al, 2008).…”

“…In addition to binding titin, telethonin is also known to interact with other Z-disc proteins like FATZ , (MLP) muscle LIM-protein (Knöll et al, 2002), calsarcins 1-3 (Frey and Olson, 2002), minK, the potassium channel b subunit (Furukawa et al, 2001), and myostatin, a negative regulator of skeletal muscle growth (Nicholas et al, 2002). Incorporation of telethonin at the Z-disc via binding to titin and FATZ appears to be one of the last stages in maturation of the Z-disc (Wang et al, 2005).…”

The sarcomeric cytoskeleton: from molecules to motion

“…All major Z-disk proteins except telethonin show rapid dynamic exchange in cardiac and skeletal myocytes, with half-lives of about 25 s for α-actinin-2 (Ribeiro et al, 2014;Stout et al, 2008;Wang et al, 2005). This suggests that the fast-exchanging components may be in a dynamically regulated equilibrium between a high-affinity Z-disk-bound conformation and a low-affinity cytoplasmic conformation.…”