Jean M. Sanger

The Dynamic Z Bands of Striated Muscle Cells

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.

Cardiac myofibrillogenesis inside intact embryonic hearts

How proteins assemble into sarcomeric arrays to form myofibrils is controversial. Immunostaining and transfections of cultures of cardiomyocytes from 10-day avian embryos led us to propose that assembly proceeded in three stages beginning with the formation of premyofibrils followed by nascent myofibrils and culminating in mature myofibrils. However, premyofibril and nascent myofibril arrays have not been detected in early cardiomyocytes examined in situ in the forming avian heart suggesting that the mechanism for myofibrillogenesis differs in cultured and uncultured cells. To address this question of in situ myofibrillogenesis, we applied non-enzymatic procedures and deconvolution imaging techniques to examine early heart forming regions in situ at 2- to 13-somite stages (beating begins at the 9-somite stage), a time span of about 23 h. These approaches enabled us to detect the three myofibril stages in developing hearts supporting a three-step model of myofibrillogenesis in cardiomyocytes, whether they are present in situ, in organ cultures or in tissue culture. We have also discovered that before titin is organized the first muscle myosin filaments are about half the length of the 1.6 mum filaments present in mature A-bands. This supports the proposal that titin may play a role in length determination of myosin filaments.

Tracking Changes in Z-Band Organization During Myofibrillogenesis With FRET Imaging

There are a large number of proteins associated with Z-bands in myofibrils, but the precise arrangements of most of these proteins in Z-bands are largely unknown. Even less is known about how these arrangements change during myofibrillogenesis. We have begun to address this issue using Sensitized Emission Fluorescence Resonance Energy Transfer (SE-FRET) microscopy. Cultured skeletal muscle cells from quail embryos were transfected to express fusions of alpha-actinin, FATZ, myotilin, or telethonin with cyan and yellow fluorescent proteins in various pair wise combinations. FATZ and myotilin were selected because previous biochemical studies have suggested that they bind to alpha-actinin, the major protein of the Z-band. Telethonin was selected for its reported ability to bind FATZ. Statistical analysis of data from FRET imaging studies yield results that are in agreement with published biochemical data suggesting that FATZ and myotilin bind to alpha-actinin near its C-terminus as well as to each other and that a region near the amino-terminus of FATZ is responsible for its interaction with telethonin. In addition, our analysis has revealed changes in the arrangement of alpha-actinin and FATZ that take place during the transition as the z-bodies of premyofibrils fuse to form the Z-bands of mature myofibrils. There was no evidence for a change in the arrangement of myotilin as z-bodies transformed into Z-bands. Myotilin is one Z-band protein that does not exhibit decreased dynamics as z-bodies fuse to form Z-bands. These FRET results from living cells support a stepwise model for the assembly of myofibrils.

Tropomyosin expression and dynamics in developing avian embryonic muscles

The expression of striated muscle proteins occurs early in the developing embryo in the somites and forming heart. A major component of the assembling myofibrils is the actin-binding protein tropomyosin. In vertebrates, there are four genes for tropomyosin (TM), each of which can be alternatively spliced. TPM1 can generate at least 10 different isoforms including the striated muscle-specific TPM1alpha and TPM1kappa. We have undertaken a detailed study of the expression of various TM isoforms in 2-day-old (stage HH 10-12; 33 h) heart and somites, the progenitor of future skeletal muscles. Both TPM1alpha and TPM1kappa are expressed transiently in embryonic heart while TPM1alpha is expressed in somites. Both RT-PCR and in situ hybridization data suggest that TPM1kappa is expressed in embryonic heart whereas TPM1alpha is expressed in embryonic heart, and also in the branchial arch region of somites, and in the somites. Photobleaching studies of Yellow Fluorescent Protein-TPM1alpha and -TPM1kappa expressed in cultured avian cardiomyocytes revealed that the dynamics of the two probes was the same in both premyofibrils and in mature myofibrils. This was in sharp contrast to skeletal muscle cells in which the fluorescent proteins were more dynamic in premyofibrils. We speculate that the differences in the two muscles is due to the appearance of nebulin in the skeletal myocytes premyofibrils transform into mature myofibrils.

Localization of Sarcomeric Proteins During Myofibril Assembly in Cultured Mouse Primary Skeletal Myotubes

It is important to understand how muscle forms normally in order to understand muscle diseases that result in abnormal muscle formation. Although the structure of myofibrils is well understood, the process through which the myofibril components form organized contractile units is not clear. Based on the staining of muscle proteins in avian embryonic cardiomyocytes, we previously proposed that myofibrils formation occurred in steps that began with premyofibrils followed by nascent myofibrils and ending with mature myofibrils. The purpose of this study was to determine whether the premyofibril model of myofibrillogenesis developed from studies developed from studies in avian cardiomyocytes was supported by our current studies of myofibril assembly in mouse skeletal muscle. Emphasis was on establishing how the key sarcomeric proteins, F‐actin, nonmuscle myosin II, muscle myosin II, and α‐actinin were organized in the three stages of myofibril assembly. The results also test previous reports that nonmuscle myosins II A and B are components of the Z‐bands of mature myofibrils, data that are inconsistent with the premyofibril model. We have also determined that in mouse muscle cells, telethonin is a late assembling protein that is present only in the Z‐bands of mature myofibrils. This result of using specific telethonin antibodies supports the approach of using YFP‐tagged proteins to determine where and when these YFP‐sarcomeric fusion proteins are localized. The data presented in this study on cultures of primary mouse skeletal myocytes are consistent with the premyofibril model of myofibrillogenesis previously proposed for both avian cardiac and skeletal muscle cells. Anat Rec, 297:1571–1584, 2014.

Myotilin dynamics in cardiac and skeletal muscle cells

Myotilin cDNA has been cloned for the first time from chicken muscles and sequenced. Ectopically expressed chicken and human YFP‐myotilin fusion proteins localized in avian muscle cells in the Z‐bodies of premyofibrils and the Z‐bands of mature myofibrils. Fluorescence recovery after photobleaching experiments demonstrated that chicken and human myotilin were equally dynamic with 100% mobile fraction in premyofibrils and Z‐bands of mature myofibrils. Seven myotilin mutants cDNAs (S55F, S55I, T57I, S60C, S60F, S95I, R405K) with known muscular dystrophy association localized in mature myofibrils in the same way as normal myotilin without affecting the formation and maintenance of myofibrils. N‐ and C‐terminal halves of human myotilin were cloned and expressed as YFP fusions in myotubes and cardiomyocytes. N‐terminal myotilin (aa 1–250) localized weakly in Z‐bands with a high level of unincorporated protein and no adverse effect on myofibril structure. C‐terminal myotilin (aa 251–498) localized in Z‐bands and in aggregates. Formation of aggregated C‐terminal myotilin was accompanied by the loss of Z‐band localization of C‐terminal myotilin and partial or complete loss of alpha‐actinin from the Z‐bands. In regions of myotubes with high concentrations of myotilin aggregates there were no alpha‐actinin positive Z‐bands or organized F‐actin. The dynamics of the C‐terminal‐myotilin and N‐terminal myotilin fragments differed significantly from each other and from full‐length myotilin. In contrast, no significant changes in dynamics were detected after expression in myotubes of myotilin mutants with single amino acid changes known to be associated with myopathies.

Assembly and Maintenance of Myofibrils in Striated Muscle

In this chapter, we present the current knowledge on de novo assembly, growth, and dynamics of striated myofibrils, the functional architectural elements developed in skeletal and cardiac muscle. The data were obtained in studies of myofibrils formed in cultures of mouse skeletal and quail myotubes, in the somites of living zebrafish embryos, and in mouse neonatal and quail embryonic cardiac cells. The comparative view obtained revealed that the assembly of striated myofibrils is a three-step process progressing from premyofibrils to nascent myofibrils to mature myofibrils. This process is specified by the addition of new structural proteins, the arrangement of myofibrillar components like actin and myosin filaments with their companions into so-called sarcomeres, and in their precise alignment. Accompanying the formation of mature myofibrils is a decrease in the dynamic behavior of the assembling proteins. Proteins are most dynamic in the premyofibrils during the early phase and least dynamic in mature myofibrils in the final stage of myofibrillogenesis. This is probably due to increased interactions between proteins during the maturation process. The dynamic properties of myofibrillar proteins provide a mechanism for the exchange of older proteins or a change in isoforms to take place without disassembling the structural integrity needed for myofibril function. An important aspect of myofibril assembly is the role of actin-nucleating proteins in the formation, maintenance, and sarcomeric arrangement of the myofibrillar actin filaments. This is a very active field of research. We also report on several actin mutations that result in human muscle diseases.

Jasplakinolide reduces actin and tropomyosin dynamics during myofibrillogenesis

The premyofibril model proposes a three‐stage process for the de novo assembly of myofibrils in cardiac and skeletal muscles: premyofibrils to nascent myofibrils to mature myofibrils. FRAP experiments and jasplakinolide, a drug that stabilizes F‐actin, permitted us to determine how decreasing the dynamics of actin filaments affected the dynamics of tropomyosin, troponin‐T, troponin‐C, and two Z‐Band proteins (alpha‐actinin, FATZ) in premyofibrils versus mature myofibrils. Jasplakinolide reduced markedly the dynamics of actin in premyofibrils and in mature myofibrils in skeletal muscles. Two isoforms of tropomyosin‐1 (TPM1α, TPM1κ) are more dynamic in premyofibrils than in mature myofibrils in control skeletal muscles. Jasplakinolide reduced the exchange rates of tropomyosins in premyofibrils but not in mature myofibrils. The reduced tropomyosin recoveries did not match the YFP‐actin recoveries in premyofibrils in jasplakinolide. There were no significant differences in the effects of jasplakinolide on the dynamics of troponins in the thin filaments or of two Z‐band proteins in premyofibrils or skeletal mature myofibrils. Cardiac control mature myofibrils lack nebulin, and small decreases in actin (∼5%) and two tropomyosin isoforms (∼10–15%) dynamics are detected in premyofibril to mature myofibril transformations compared with skeletal muscle. In contrast to skeletal muscle, jasplakinolide lowered the dynamics of actin and tropomyosin isoforms in the cardiac mature myofibrils. These results suggest that the dynamics of tropomyosins in control muscle cells are related to actin exchange. These results also suggest a stabilizing role for nebulin, an actin and tropomyosin‐binding protein, present in mature myofibrils but not in premyofibrils of skeletal muscles.

Arg/Abl-binding protein, a Z-body and Z-band protein, binds sarcomeric, costameric, and signaling molecules.

ArgBP2 (Arg/Abl‐Binding Protein) is expressed at high levels in the heart and is localized in the Z‐bands of mature myofibrils. ArgBP2 is a member of a small family of proteins that also includes vinexin and CAP (c‐Cbl‐associated protein), all characterized by having one sorbin homology (SOHO) domain and three C‐terminal SH3 domains. Antibodies directed against ArgBP2 also react with the Z‐bodies of myofibril precursors: premyofibrils and nascent myofibrils. Expression in cardiomyocytes of plasmids encoding Yellow Fluorescent Protein (YFP) fused to either full length ArgBP2, the SOHO, mid‐ArgBP or the SH3 domains of ArgBP2 led to Z‐band targeting of the fusion proteins, whereas an N‐terminal fragment lacking these domains did not target to Z‐bands. Although ArgBP2 is not found in skeletal muscle cells, YFP‐ArgBP2 did target to Z‐bodies and Z‐bands in cultured myotubes. GST‐ArgBP2‐SH3 bound actin, α‐actinin and vinculin proteins in blot overlays, cosedimentation assays, and EM negative staining techniques. Over‐expression of ArgBP2 and ArgBP2‐SH3 domains, but not YFP alone, led to loss of myofibrils in cardiomyocytes. Fluorescence recovery after photobleaching was used to measure the rapid dynamics of both the full length and some truncated versions of ArgBP2. Our results indicate that ArgBP2 may play an important role in the assembly and maintenance of myofibrils in cardiomyocytes.

Clock is not a component of Z-bands.

The process of Z‐band assembly begins with the formation of small Z‐bodies composed of a complex of proteins rich in alpha‐actinin. As additional proteins are added to nascent myofibrils, Z‐bodies are transformed into continuous bands that form coherent discs of interacting proteins at the boundaries of sarcomeres. The steps controlling the transition of Z‐bodies to Z‐bands are not known. The report that a circadian protein, Clock, was localized in the Z‐bands of neonatal rat cardiomyocytes raised the question whether this transcription factor could be involved in Z‐band assembly. We found that the anti‐Clock antibody used in the reported study also stained the Z‐bands and Z‐bodies of mouse and avian cardiac and skeletal muscle cells. YFP constructs of Clock that were assembled, however, did not localize to the Z‐bands of muscle cells. Controls of Clocks activity showed that cotransfection of muscle cells with pYFP‐Clock and pCeFP‐BMAL1 led to the expected nuclear localization of YFP‐Clock with its binding partner CeFP‐BMAL1. Neither CeFP‐BMAL1 nor antibodies directed against BMAL1 localized to Z‐bands. A bimolecular fluorescence complementation assay (VC–BMAL1 and VN–Clock) confirmed the absence of Clock and BMAL1 from Z‐bands, and their nuclear colocalization. A second anti‐Clock antibody stained nuclei, but not Z‐bands, of cells cotransfected with Clock and BMAL1 plasmids. Western blots of reactions of muscle extracts and purified alpha‐actinins with the two anti‐Clock antibodies showed that the original antibody cross‐reacted with alpha‐actinin and the second did not. These results cannot confirm Clock as an active component of Z‐bands.