PerspectiveMuscle

The Dynamic Z Bands of Striated Muscle Cells

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Sci. Signal.  12 Aug 2008:
Vol. 1, Issue 32, pp. pe37
DOI: 10.1126/scisignal.132pe37

Abstract

The distribution of proteins in the sarcomeres of skeletal and cardiac muscle gives rise to the striated pattern of these muscles. Sarcomeric protein localization is not, however, permanently fixed. There is dynamic exchange of proteins between a cytosolic pool and the sarcomere, addition of newly synthesized proteins during development and repair, and aberrant redistribution of proteins due to mutations. Recent studies have shown that two groups of proteins that localize in the sarcomere Z band can relocate, in one case to the A band, and in the other to the nucleus.

Myofibril design results in a precisely ordered framework for the localization of the dozens of proteins that generate contractile force and modulate and integrate its transmission in cardiac and skeletal muscle cells (Fig. 1). The contractile units of the myofibril, the sarcomeres, are distinguished by the striated distribution of their proteins, visible by light microscopy as three major bands, called A, I, and Z (Fig. 1). A bands comprise thick filaments of myosin and proteins that bind myosin. The middle part of the A band is termed "the M band" or "M line." The I band is composed of thin actin filaments and proteins that bind actin. In the middle of the I band is the Z band, also called "the Z line" or "Z disc" (Fig. 1). Our appreciation of the importance of sarcomere proteins in cardiac and skeletal muscle health has been reinforced by the growing realization that mutations in these proteins are involved in a number of myopathies in the heart and in skeletal muscle (14).

Fig. 1.

Diagram of a sarcomere, the repeating unit of a myofibril, and some proteins reported to be in or associated with the Z bands of mature myofibrils. The plasma membrane proteins are shown in purple. Proteins that are associated with the plasma membrane or are part of the costameres (the proteins associated with the peripheries of the Z bands) are indicated in green. All of the integral Z-band proteins, with the exception of actin, nebulin, titin, the two chaperones, and the two proteins that translocate to the nucleus (whose colors reflect the two different locations in which they are found), are indicated in orange. Thin filament proteins are indicated in blue, and thick filament proteins are indicated in gray. Thin filaments and the elongated titin molecules are tethered to the Z bands. Six parallel titin molecules connect each half of a thick myosin filament of the A band to the Z band. Other proteins besides desmin in the costamere and juxtamembrane cytoskeleton bind to several of the Z-band proteins, but these connections are not shown in the diagram.

The greatest number of sarcomeric proteins is in the Z band, which functions as a scaffold that links the sarcomeric contractile units in series by anchoring the actin and titin filaments of adjacent sarcomeres. The Z band also anchors the ends of myofibrils in specialized junctions, termed "intercalated discs" in the heart, and links sarcomeres laterally to the cell membrane through costameric proteins in both heart and skeletal muscle cells (5, 6). The multifunctional nature of the Z band is reflected in the variety of proteins that colocalize in this structure: channels, signaling molecules active in the cytoplasm and in the nucleus, enzymes, and cytoskeletal proteins (Fig. 1 and Table 1) (711). Many myopathies have been linked to proteins associated with the Z band (4); in these skeletal and cardiac muscle diseases, the Z bands are abnormal, the arrangements of the myofibrils are in disarray (1, 4, 12), or aggregates of protein (nemaline bodies) are present, often near the Z bands (13).

Table 1.

Sarcomeric proteins mentioned in the text.

In vitro methods such as yeast two-hybrid assays, overlay assays, immunoprecipitation, and glutathione-S-transferase (GST) pull-down assays have identified various interactions among Z-band proteins that can be depicted schematically (Fig. 1). Whereas such methods provide strong evidence for the depicted relationships, they do not directly address questions about protein-protein interactions in vivo. The highly ordered arrangement of proteins in the sarcomere, which persists even as contractile force is generated, suggests that binding interactions between Z-band proteins are strong and very stable. Clearly, the formation and stability of myofibrils must depend on the binding interactions of myofibrillar proteins with one another, yet little is known about these interactions in the living cell. Nor can these biochemical methods hint at the dynamics of Z-band proteins revealed by the quantitative biophysical approach of FRAP (fluorescence recovery after photobleaching), a technique that has shown that, superimposed on the addition of newly synthesized proteins to the Z bands, there is active exchange between proteins in a cytoplasmic pool and the same species of protein residing in the Z bands (14, 15).

Two recent papers (10, 11) show that sarcomeric localization of a myofibrillar protein is not always confined to a single sarcomeric band. Moreover, these studies have uncovered some of the factors controlling protein localization. Etard et al. (11) reported the unexpected localization of two myosin chaperones, Unc45b and Hsp90a, in the Z bands of zebrafish skeletal muscle cells. This Z-band localization is surprising because these two chaperones are necessary for the folding of the globular regions of the myosin heavy chains that will assemble into the A bands. Expression of chaperone-GFP (green fluorescent protein) fusion proteins during myofibrillogenesis showed diffuse distribution of Unc45b and Hsp90a in the cytoplasm of zebrafish skeletal muscle cells in live embryos at 24 hours postfertilization (hpf) followed by Z-band localization at later stages, 48 hpf. The same pattern was seen if embryos expressing Unc45b-GFP were fixed in a mixture of acetone and paraformaldehyde. Subjecting embryos that expressed chaperone-GFP fusion proteins to stress factors, such as cold temperatures or laser damage to the plasma membrane, induced chaperone relocalization from the Z bands to the A bands. Conversely, raising the temperature or recovery of the membranes from photodamage led to the movement of the chaperones back to the Z bands from the A bands.

Fixation of the muscles with a standard method using a solution of paraformaldehyde also induced the Z-band to A-band relocalization of Unc45b and Hsp90a. The Z-band protein CapZ remained localized in the Z band. This observation raises the cautionary note that paraformaldehyde fixation may cause redistribution of some cellular proteins. Including a permeabilizing agent like acetone with the fixative, as the authors did, could serve to confirm results obtained with sequential use of paraformaldehyde followed by permeabilizing reagent.

The association of these two GFP-chaperones with A bands was not observed in live wild-type embryos under normal conditions. To determine if a transient association of Unc45b-GFP and myosin could be detected during myofibrillar assembly, Etard et al. (11) expressed Unc45b-GFP in unc-45b mutant embryos in which Unc45b was truncated and myofibril assembly was defective. This partially rescued the myofibril assembly defect of the mutant, and Unc45b-GFP associated with the forming A bands. Over the next 48 hpf, Unc45b-GFP localized to Z bands. Hsp90a-GFP showed the same transition from A band to Z band in the mutant embryos. Future work will be needed to determine what proteins are responsible for the Z-band binding of the two chaperones, Unc45b and Hsp90a, in the zebrafish skeletal muscle cells (11).

In general, the extent and half-time of the exchange of fluorescent molecules analyzed with FRAP techniques are a function of the binding interaction of the proteins in the cellular complex where they are concentrated, with shorter half-times of fluorescence recovery suggesting lower affinity (16). FRAP measurements of the two chaperones in the Z bands revealed that the associations of these two molecules with Z-band elements are very dynamic, with half-times of fluorescence recovery of less than a second (11). These recovery times are about 100 to 1000 times as fast as those reported for seven structural Z-band proteins, suggesting that the two GFP-chaperones have low affinities for the Z bands (14). FRAP analysis was not performed when the chaperones were in the A bands. It might be expected that the two proteins will be less dynamic when acting as myosin chaperones than when localized at the Z band. The authors propose that "differential affinity" may explain the movement of the chaperones between the A band and the Z band. This might occur when the myosin-binding proteins like titin (Fig. 1) associate with the forming A bands and compete with the chaperones for binding the myosin filaments.

A second example of sarcomeric proteins that change localization is illustrated by two Z-band proteins that bind α-actinin and move between the sarcomere and the nucleus: muscle LIM protein (MLP) (17) and myopodin (10). Faul et al. (10) showed how phosphorylation and dephosphorylation regulate myopodin’s localization to the Z bands or nucleus. Phosphorylation of myopodin by either protein kinase A (PKA) or calcium-calmodulin–dependent protein kinase II (CaMKII) induces myopodin’s release from α-actinin, its binding partner in the Z band, and its entry into the nucleus. Dephosphorylation of myopodin by the calcium-activated phosphatase calcineurin permits myopodin to remain or relocalize to the Z band. If the phosphatase activity of calcineurin is inhibited, myopodin moves into the nuclei of cardiac myocytes. Moreover, pharmacological inhibition of the phosphorylation action of PKA reduced or abolished the import of myopodin into the nucleus. Activation of PKA by pharmacological agents leads to diffuse Z-band localization and nuclear localization of myopodin in adult cardiomyocytes. The mechanism of MLP movement from the Z band into the nucleus has not been determined (7, 17, 18).

The role of phosphorylation of other proteins in the Z bands needs further study. It is known that telethonin can be phosphorylated by the kinase region of titin (19). This domain of titin is, surprisingly, located in the M-band region of the A band, about as far apart as two partners might be in muscle (19). These disparate locations led to speculation that titin might be released from the middle of the thick filaments to phosphorylate the telethonin in the Z band. However, the demonstration by photobleaching experiments that telethonin can move in and out of the Z band (14) suggests that unbound telethonin could be phosphorylated by the kinase region of titin in the M band and then diffuse to the Z band. It is not known if telethonin needs to be phosphorylated to enter or leave the Z band.

In conclusion, Z bands are critically important for development and differentiation of the myofibril (15, 20), a nodal point for contractions and signaling, but a sensitive site easily disrupted in myopathies (4). Moreover, the Z band is an area of the sarcomere where new proteins with unexpected properties continue to be discovered (10, 11).

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