This review takes readers back to 1949, when two Australian scientists, Draper and Hodge, reported the first high-resolution electron microscopy images of striated muscle. In 1953, Jean Hanson and Hugh Huxley published phase-contrast microscopy and electron microscopy images that established the filamentous nature of the sarcomere, namely the myosin-containing thick filaments and actin-containing thin filaments. They discussed a putative third filament system, possibly a thinner actin-containing S filament, that appeared to connect one Z disc to the next. The next year, two back-to-back papers appeared in Nature, the first by Andrew Huxley and Rolf. Niedergerke, the second by Hugh Huxley with Jean Hanson. Independently, they proposed the sliding of actin filaments and myosin filaments. These two filaments quickly became firmly established in the literature and, even today, they remain the basis for the sliding filament hypothesis. The putative third filament concept was dropped, mainly through the lack of evidence but also because it was difficult to accommodate in the hypothesis where two sets of filaments maintained their lengths constant while sliding produced sarcomere shortening. The view that actin and myosin comprise more than 80% of the myofibril proteins also made it difficult to accommodate a major new protein. In the following years, using selective extraction of myosin and actin, dos Remedios (PhD thesis, University of Sydney, 1965) revealed a residual filament system in the sarcomere, and, once again, a third filament system re-entered the literature. Filaments were reported crossing the gap between the ends of thick and thin filaments in highly stretched muscle fibres. These and other early studies necessarily focussed on light and electron microscopy, and set the scene for investigations into the chemical nature and biophysical functions of the third filament system for striated muscles. Further progress had to wait for the improvement and/or development of a number of techniques. For example, in 1970, Laemmli (Nature 227:680-685, 1970) published an often cited method for improving SDS polyacrylamide gel electrophoresis. The Lowry et al. (J Biol Chem 193:265-275, 1951) protein assay method developed in 1950 was both unstable and insensitive in comparison, but we had to wait until 1976 for the development of the Bradford method (1976). Atomic force microscopy was not known before 1986, but it eventually enabled the direct measurement of single molecules of titin. This extraordinarily large (>106 Da) elastic protein became known as connectin (Maruyama in J Biochem 80:405-407, 1976) and was subsequently named titin (Wang et al. in Proc Natl Acad Sci U S A 76:3698-3702, 1979). Prior to the discovery of titin/connectin, biophysicists found it difficult to understand how a single polypeptide chain could could stretch from the Z disc to the M line, a distance of more than 1 μm. It was quite literally the 'elephant in the room'. In this review, we follow the trail of microscopy-based reports that led to the emergence of what is now known and accepted as titin, an elastic third filamentous protein that is the focus of this Special Issue.
Keywords: Connectin; Giant proteins; Historical review; Sarcomere filament structure; Titin.