1980; Perry, 1979). ... ing the method used previously (Bullard et al., 1988). About ... muscle Tn-H (MAC 143) has been described (Bullard et al.,. 1988).
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Journal of Cell Science 101, 503-508 (1992) Printed in Great Britain © The Company of Biologists Limited 1992
A method for determining the periodicity of a troponin component in isolated insect flight muscle thin filaments by gold/Fab labelling R. NEWMAN1-*, G. W. BUTCHER2, B. BULLARD1 and K. R. LEONARD 1 ! European Molecular Biology Laboratory, Meyerhofstrafie 1, D-6900 Heidelberg, Germany Department of Immunology, AFRC Institute of Animal Physiology and Genetics Research, Cambridge Research Station, Babraham, Cambridge CB2 4AT, UK
2
•Present address: Imperial Cancer Research Fund, Lincolns Inn Fields, London, UK tAuthor for correspondence
Summary Insect flight muscle has a large component (Tn-H) in the tropomyosin-troponin complex that is not present in vertebrate striated muscle thin filaments. Tn-H is shown by gold/Fab labelling to be present at regular intervals in insect flight muscle thin filaments. The Fab fragment of a monoclonal antibody to Tn-H was conjugated directly with colloidal gold and this probe used to label isolated thin filaments from the flight muscle of Lethocerus indicus (water bug). The distribution of gold particles seen in electron microscope images of negatively stained thin filaments was analysed to show that the probe bound to sites having a periodicity of approximately 40 nm, which is the expected value for the tropomyosintroponin repeat. Conjugates of Fab with colloidal gold particles of 3 nm diameter labelled almost all sites. Conjugates with gold particles of 5 nm and 10 nm diameter labelled less efficiently (70% and 30%, respectively) but analysis of the distribution of inter-particle
intervals among a number of filaments again gave the same fundamental spacing of 40 nm. The error in the measurements (standard deviation approximately ±4.2 for 5 nm gold/Fab) is less than earlier estimates for the size of the gold/Fab complex. Measurements on gold/Fab in negative stain suggest that the bound Fab contributes a shell about 2 nm in thickness around the gold particle. The radius of the probe (about 4.5 nm for 5 nm gold/Fab) would then be consistent with the value of error found. The size of the probe suggests that the gold particle binds to the side of the Fab molecule, rather close to the antibody combining site. The potential resolution of the technique may thus be better than originally expected.
Introduction
filaments and it is readily applicable to other specimens where periodicity is thought to exist. Isolated flight muscle thin filaments from the giant water bug, Lethocerus, have regular projections spaced at the tropomyosin periodicity of about 40 nm, which are thought to be the site of the large troponin complex (Bullard et al., 1988). We have labelled these thin filaments with gold-conjugated Fab fragment of antibody to Tn-H. The smallest gold particle that is electron-dense enough to be distinguished readily by conventional transmission electron microscopy is about 3 nm in diameter. For 2.5 nm gold particles conjugated directly to monovalent Fab fragments, the maximum distance between the centre of the gold particle and the antibody combining site is estimated to be about 8 nm (Baschong and Wrigley, 1990). This resolution would be sufficient to establish a 40 nm periodicity, but since the troponin sites are not always labelled fully by this method, we have also carried out an analysis of the
The regulatory proteins on the thin filaments of vertebrate skeletal muscle, tropomyosin and troponin, are arranged periodically along the actin helix (Ebashi, 1980; Perry, 1979). Insect flight muscle has a protein, heavy troponin (Tn-H) isolated with other troponin components, which is not found in vertebrate muscle (Bullard et al., 1988). If this protein is part of the troponin complex it would be expected to be arranged at regular intervals on the thin filament. We have investigated the distribution of Tn-H in two ways. The first was to label whole muscle fibres with antibody to Tn-H before embedding and sectioning for electron microscopy (Reedy et al., in preparation). A simpler approach, which is described here, is to label isolated thin filaments with antibody and to measure the distribution of label in electron microscope images. This method does not require full labelling of the
Key words: troponin, thin filaments, insect muscle, gold/Fab, analysis of periodicity.
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R. Newman and others
distribution of label spacings along partially labelled filaments. Materials and methods
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