Z-band proteins in the flight muscle and leg muscle of the honeybee

Abstract

Monoclonal antibodies (mAb's) have been raised against proteins in preparations of Z-discs isolated from honeybee fibrillar flight muscle. These antibodies have identified four Z-disc antigens on immunoblots of honeybee fibrillar proteins. Antibody alpha binds to the 90-100 kD protein, alpha-actinin; mAb P interacts with the protein, projectin, an extremely large polypeptide (greater than 600kD) found in the connecting filaments which link thick filaments to the Z-band in insect asynchronous flight muscle. Two other mAb's recognize previously uncharacterized insect Z-band proteins. Monoclonal antibody Z(400) binds to a pair of proteins with molecular masses near 400 kD and 600 kD. Antibody Z(175) recognizes two components, 158 kD and 175 kD, that are not only immunologically similar but have nearly identical peptide maps. Indirect immunofluorescence microscopy studies show that the proteins recognized by mAb's alpha, Z(175) and Z(400) are located at the Z-band, while the mAb P antigen is found on either side of it. Three of the four antibodies we have obtained recognize leg muscle proteins. Monoclonal antibodies alpha and P comigrate on SDS gels with analogous components from flight muscle. Only the smaller of the two proteins identified in flight muscle by mAb Z(400) is found in leg muscle, however. Furthermore, no Z(175) antigens have been detected in the non-fibrillar tissue by either monoclonal or polyclonal antibodies. Immunofluorescence microscopy studies localize the alpha and Z(400) antigens at the Z-line in leg muscle fibrils. Surprisingly, however, mAb P binds within the A-bands of synchronous fibres, not between the A- and Z-bands as in asynchronous fibrillar muscle.

The NH2-terminal Ig Domains Of Insect Projectin Could Serve As Elastic Elements

“…This distribution indicates that in dragonfly flight muscles, projectin has a similar topography to the one described in more derived insects such as D. melanogaster and A. mellifera [6][7][8] .…”

“…Several projectin antibodies for which the epitope localization is known were used in order to ascertain the myofibrillar position of the protein (See Materials and Methods for list of antibodies). The localization was established in comparison with actin detected by phalloidin staining, and several other proteins, such as α-actinin and kettin using specific antibodies 7,8,26 .…”

“…In insects, this filament, which is known as the connecting filament (C-filament) has been described in the Indirect Flight Muscles (IFMs) of several derived insect species, including Lethocerus indicus (waterbug), Apis mellifera (honeybee) and Drosophila melanogaster (reviewed in 2). The C-filaments provide a mechanical link between the Z-discs and the ends of the thick filaments and are composed of two large modular proteins, kettin/Sallimus [3][4][5] (abbreviated as Sls), and projectin [6][7][8] . The C-filaments have been proposed as one of the elements generating muscle stiffness in the IFMs of derived insect, a property necessary for the stretch activation mechanism and asynchronous flight physiology (reviewed in 2,9).…”

The Insect Z-Band

“…Larger variants are also found in honeybee and Lethocerus IFM. 35,3 Domain targeting studies suggest that sis sequences are variably spliced to produce the larger isoforms. The largest predicted transcript of the sis gene would encode a 1.9-2 MDa protein, but an antigen of this size has not been detected in either the IFM or other fiber types.…”

“…35 but since monoclonal antibodies directed against Lethocerus kettin and honeybee Z(400/600) recognize identical isoforms on immunoblots of Drosophila IFM myofibrils, (ref. 34 and unpublished observations) the antigens from these three insects are likely to be kettin family members.…”