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 Z-bodies 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, led 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.

Figure 1

Fluorescence image of myotubes at their (A) spreading ends and in their (B) central regions previously transfected with plasmids encoding different Z-band proteins: (a) myotilin, (b) telethonin, (c) FATZ, (d) cypher-1s, and (e) cypher-2s. The transfected myotubes were fixed and stained with sarcomeric alpha-actinin antibodies (a′–e′). Myotilin, FATZ and cypher are localized in the Z-bodies at the ends of myotubes, but telethonin is not. All the proteins are co-localized with alpha-actinin in the Z-bands of mature myofibrils in the central regions of the myotubes. (Bar = 5 μm.)

Figure 2

Fluorescence recovery after photobleaching (FRAP) experiments on the Z-bands of mature myofibrils. Myotubes were transfected with YFP-myotilin (A), YFP-alpha-actinin (B) and YFP-telethonin (C). Images shown from left to right are pre-bleach, bleach, 1, 5 and 10 minutes recovery after bleach. The arrows indicate the bleached region in the bleach frame. (Bar = 5 μm.)

Figure 3

FRAP of different Z-band proteins in mature myofibrils. (A) The average FRAP recovery curves within 20 minutes of myotilin (black circles), cypher-1s (white circles), FATZ (black rectangles), cypher-2s (white rectangles), alpha-actinin (black triangles), actin in the Z-band (white triangles) and telethonin (black diamond). (B) Comparison of the fast (solid black columns) and slow mobile (stippled columns) fractions of different Z-band proteins. Values are the means ± SDs. Note that myotilin and cypher-1s were 100% mobile, and telethonin is the slowest protein recovering from photobleaching.

Figure 4

FRAP recovery rates of different Z-band proteins in Z-bodies and in Z-bands. (A) The FRAP curves of cypher-2s in the Z-bodies (black circle) and in the Z-bands (white circle). Note that the recovery rate for cypher is faster in the Z-bodies than in the Z-bands of the mature myofibrils. (B) Comparison of the fast and slow mobile fractions of different proteins in the Z-bodies and in the Z-bands of mature myofibrils. Values are the means ± SDs. Note that the fast mobile fractions for the proteins are always higher in the Z-bodies compared to the Z-bands.

Figure 5

A myotube expressing photoactivatable GFP-alpha-actinin was imaged by using low levels of 488 nm excitation before and after irradiation with high levels of 364 nm laser light. (A) Images (a) to (d) are pre-activation, activation, 10 and 20 minutes after activation. (Bar = 10 μm.) (B) The fluorescence intensity curve in the photoactivated region after irradiation. Note that 50 % of the fluorescence was lost after 20 minutes.

Figure 6

FRAP recovery rates of alpha-actinin (black circle) and alpha-actinin (N-273) fragment (white circle) in Z-band of mature myofibrils.

Figure 7

Fluorescence image of a myotube transfected with an alpha-actinin (N-273) fragment. (a) Alpha-actinin (N-273) fragment localized to the Z-bands of mature myofibrils one day post transfections just like the full construct alpha-actinin. (b) In contrast to transfections with the full construct of alpha-actinin, myotubes expressing the alpha-actinin fragment exhibited increasing numbers of disassembled myofibrils after one or more days.

Figure 8

FRAP recovery rates of two cypher skeletal muscle isoforms and their domains in the Z-bands of mature myofibrils. (A) The schematic diagrams of cypher-1s and cypher-2s indicates the shared PDZ domain (amino acids 1-84) present in both isoforms, and the three LIM domains (amino acids 404–472) present only in cypher-1s. (B) The average FRAP recovery curves of cypher-1s (black rectangles), cypher-2s (black circles), cypher-LIM domains (white circles) and cypher-PDZ domain (cross) in the Z-bands of mature myofibrils. The C-terminal region of cypher-2s decreases the dynamics of the cypher-2S PDZ domain, while cypher-1s amino acids 1-364 increase the dynamics of the LIM domains. (C) Comparison of the fast and slow mobile fractions of the cyphers, as well as their LIM and PDZ domains in the Z-bands of mature myofibrils. Values are the means ± SDs.

Figure 9

A model of the progression of Z-bodies in premyofibrils and nascent myofibrils to Z-bands of the mature myofibrils. Immunofluorescence experiments indicate that titin, not present in the Z-bodies of premyofibrils, is first detected in the Z-bodies of the nascent myofibrils (Rhee et al., 1994). The exchange of proteins would permit the remodeling of Z-bodies in the transitions needed to form Z-bands during myofibrillogenesis.

Figure 10

A growing list of Z-band proteins in the mature myofibrils in skeletal muscle cells. The diagram is arranged to indicate the central role of alpha-actinin in the Z-bands that directly and indirectly binds to the major elements of the muscle cell, i.e., thin and thick filaments, intermediate filaments, costameres, and membranous structures like the sarcolemma, transverse tubules and the sarcoplasmic reticulum.