Characterization of components of Z-bands in the fibrillar flight muscle of Drosophila melanogaster.

Abstract

Abstract. Twelve monoclonal antibodies have been raised against proteins in preparations of Z-disks isolated from Drosophila melanogaster flight muscle. The monoclonal antibodies that recognized Z-hand components were identified by immunofluorescence microscopy of flight muscle myofibrils. These antibodies have identified three Z-disk antigens on immunoblots of myofibrillar proteins. Monoclonal antibodies c~:1-4 recognize a 90-100-kD protein which we identify as o~-actinin on the basis of cross-reactivity with antibodies raised against honeybee and vertebrate ot-actinins. Monoclonal antibodies P:l-4 bind to the high molecular mass protein, projectin, a component of connecting filaments that link the ends of thick filaments to the Z-band in insect asynchronous flight muscles. The anti-projectin antibodies also stain synchronous muscle, but, surprisingly, the epitopes here are within the A-bands, not between the A-and Z-bands, as in flight muscle. Monoclonal antibodies Z(210):1-4 recognize a 210-kD protein that has not been previously shown to be a Z-band structural component. A fourth antigen, resolved as a doublet (,o400/600 kD) on immunoblots of Drosophila fibrillar proteins, is detected by a cross reacting antibody, Z(400):2, raised against a protein in isolated honeybee Z-disks. On Lowicryl sections of asynchronous flight muscle, indirect immunogold staining has localized ot-actinin and the 210-kD protein throughout the matrix of the Z-band, projectin between the Z: and A-bands, and the 400/600-kD components at the I-band/Z-band junction. Drosophila ol-actinin, projectin, and the 400/600-kD components share some antigenic determinants with corresponding honeybee proteins, but no honeybee protein interacts with any of the Z(210) antibodies.T HE Z-band is an electron-dense structural component of striated muscle. It serves as an attachment site for thin filaments and transmits tension between neighboring sarcomeres during contraction. Electron micrographs of both vertebrate muscle and insect fibrillar muscle show Z-bands with a highly ordered, almost crystalline, appearance in cross section (for reviews see 1, 4, 12, 40, 42). Several Z-band proteins have been identified from both vertebrate and insect species (2, 3, 6, 7, 18, 20-22, 25, 29, 30, 33-38); however, the manner in which these proteins are organized within the Z-band lattice is poorly understood. Moreover, the developmental programs that lead to the early organization of the Z-band are only beginning to be clarified.The study of insect Z-bands has been carried out primarily on the flight muscles of the honeybee (Apis) and the giant water bug (Lethocerus). These insects are particularly favorable for biochemical studies of muscle because their size and the predominance of the flight muscle permit isolation of reasonable amounts of homogeneous muscle tissue. The much smaller size of Drosophila presents obstacles for biochemical analyses but facilitates the genetic analyses that are proving to be another useful approach to the study of muscle structure ...

Flight muscle function in Drosophila requires colocalization of glycolytic enzymes.

“…We verified that the site of localization of GPDH fluorescence along the sarcomere coincides with Zdiscs and M-lines by using isolated myofibrillar preparations simultaneously reacted with anti-GPDH and anti-a-actinin. a-Actinin is a known Z-disc protein (Saide et al, 1989). Micrographs showing myofibrils obtained from wild-type and GPDH null flies reacted with anti-GPDH and anti-a-actinin and photographed first using Nomarski optics and then fluorescent microscopy are shown in Figure 3.…”

“…Thoraces were gently homogenized and allowed to stand on ice for 30 min. Myofibrils were then centrifuged and washed twice in relaxing buffer, incubated in 10% goat serum, washed again, and incubated for 1 h in a mixture of 1:2000 diluted rabbit anti-GPDH serum and 1:100 diluted monoclonal mouse anti a-actinin (Saide et al, 1989). The fibers were washed and then reacted for 1 h in a mixture of rhodamine-labeled goat anti-rabbit serum and fluorescein-labeled goat anti-mouse serum, each at a final dilution of 1:200.…”

Aberrant Splicing of an Alternative Exon in the Drosophila Troponin-T Gene Affects Flight Muscle Development

“…Protein techniques: One-dimensional gel electrophoresis and Western blotting of muscle proteins were conducted as outlined in Nongthomba et al (2001) with antibodies described in Saide et al (1989) or Nongthomba et al (2004). For 2-D gel electrophoresis, the IFM from 30 flies were dissected in 50% ethanol and transferred to 250 ml of rehydration buffer ½8 m urea, 2% CHAPS in dH 2 O, 0.018 m DTT, and 5 ml immobilized pH gradients (IPG) buffer, pH 4-7.…”

Tropomyosin and Troponin Regulation of Wild Type and E93K Mutant Actin Filaments from Drosophila Flight Muscle

“…A myofibrillar preparation was made from 50 g (50,000) of flies according to the method of Saide et al (23). After a high salt extraction (20 mM KPO 4 , 500 mM KCl, pH 7.0) on ice for 10 min, the myofibril pellet was dehydrated by resuspending first in 6 volumes of ice-cold 50% acetone, then in 6 volumes of ice-cold 100% acetone, and finally allowed to air dry overnight at room temperature.…”