Judith D. Saide

Structurally different Drosophila striated muscles utilize distinct variants of Z-band-associated proteins

SummaryMonoclonal antibodies raised against four proteins from insect asynchronous flight muscle have been used to characterize the cross-reacting proteins in synchronous muscle ofDrosophila melanogaster. Two proteins,α-actinin and Z(400/600), are found at the Z-band of every muscle examined. A larger variant ofα-actinin is specific for the perforated Z-bands of supercontractile muscle. A third Z-band protein, Z(210), has a very limited distribution. It is found only in the asynchronous muscle and in the large cells of the jump muscle (tergal depressor of the trochanter). The absence of Z(210) from the anterior four small cells of the jump muscle demonstrates that cells within the same muscle do not have identical Z-band composition. The fourth protein, projectin, > 600 kDa polypeptide component of the connecting filaments in asynchronous muscle, is also detected in all synchronous muscles studied. Surprisingly, projectin is detected in the region of the thick filaments in synchronous muscles, rather than between the thick filaments and the Z-band, as in asynchronous muscles. Despite their different locations, the projectins of synchronous and asynchronous muscles are very similar, but not identical, as judged by SDS-PAGE and by peptide mapping. Projectin shows immunological cross-reactivity with twitchin, a nematode giant protein that is a component of the body wall A-band and shares similarities with vertebrate titin.

Identification of a connecting filament protein in insect fibrillar flight muscle

Abstract A major component on sodium dodecyl sulfate-containing gels of solubilized isolated Z-discs, purified from honeybee flight muscle, migrates with an apparent molecular weight of 360,000. Antibodies to this high molecular weight polypeptide have been prepared by injecting rabbits with homogenized gel slices containing the protein band. With indirect immunofluorescence microscopy these antibodies are localized to a region extending from the edge of the Z-band to the A-band in shortened or stretched sarcomeres. Similarly, glycerinated flight muscle treated with antiserum and prepared for electron microscopy shows enhanced density from the ends of the thick filaments to the I-Z junction regardless of sarcomere length. Evidence indicates that antiserum is directed toward a structural protein of connecting filaments, which link thick filaments to the Z-band in insect fibrillar muscle, rather than to a thin filament component. In Ouchterlony double-diffusion experiments a single precipitin band is formed when antiserum is diffused against solubilized Z-discs; no reaction occurs between antiserum and proteins from native thin filaments prepared from honeybee flight muscle. Further, antibody stains the I-band in flight muscle fibrils from which thin filaments are removed. Finally, honeybee leg muscle myofibrils, in which connecting filaments have not been observed, are not labelled with antibody. Since antibody binds to the short projections which extend from the flat surfaces of isolated Z-discs, these projections are assumed to be remnants of connecting filaments and the source of the 360,000 Mr protein. The amino acid composition of this high molecular weight material, purified by Sepharose chromatography, is presented. The protein has been named “projectin”.

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

SummaryMonoclonal antibodies (mAbs) 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 α binds to the 90–100 kD protein,α-actinin; mAb P interacts with the protein, projectin, an extremely large polypeptide (>600kD) found in the connecting filaments which link thick filaments to the Z-band in insect asynchronous flight muscle. Two other mAbs 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 mAbsα, 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 antibodiesα 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 a 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.

Purification and properties of the isolated honeybee Z-disc

Abstract Z-discs have been isolated from honeybee indirect flight muscle fibers with 0.43% lactic acid, and have been purified with differential and sucrose density-gradient centrifugation. In the light microscope, isolated Z-discs are pale, round, homogeneous structures with diameters ranging from 2 μm to 9 μm, depending on the nature of the suspending medium. In the electron microscope, small Z-discs (2 μm in diameter) examined on Formvar-coated grids are thick, dense and lacking in detail; swollen Z-discs (6 μm in diameter) have a reticular pattern with 3-fold symmetry. Sectioned isolated Z-discs show fine projections extending about 1300 A from both surfaces. These projections may represent insoluble stubs of thin filaments or “C” filaments, which connect thick filaments to the Z-band. Although honeybee isolated Z-discs are very resistant structures that remain insoluble in a number of protein solvents and in solutions reported to extract Z-band material from vertebrate fibrils, it has been possible to solubilize them in 7 m -guanidine-HCl, 2.5 m m -dithiothreitol, 2.5 m m -EDTA (pH 7.5), and to resolve their components electrophoretically. Sodium dodecyl sulfate gel electrophoresis studies indicate that there are at least four polypeptides, with molecular weights of 87,000, 113,000, 158,000, and 175,000, localized in the isolated Z-disc. The Z-disc backbone contains no significant quantity of lipid as earlier reports have suggested. Total lipid extracted from Z-disc preparations with chloroform/ methanol comprises less than 1% of the Z-disc protein.

Fine structure of the vertebrate Z-disc

The fine structure of Z-discs from frog, chameleon, rabbit, rat and human muscles was studied. Our data lead us to conclude that the basket-weave (woven) lattice represents the fundamental en face pattern of the vertebrate Z-disc, regardless of the manner of fixation, and we suggest that the large and small-“square” lattices are fixation artifacts. We also find that the woven lattice pattern remains essentially unchanged throughout physiological ranges of resting sarcomere length, and is not detectably altered by active contraction. A model of the vertebrate Z-line, based on anatomical and possible functional considerations, is presented. It presumes that a thin filament, as it enters the Z-line, is continuous with three curved strands which unite with other I-filaments of the same sarcomere. The I-filaments and extending strands from the opposite sarcomere are proposed to be similarly arranged, with the main Z-line substance consisting of the two sets of strands from adjacent sarcomeres. The anatomical features of the Z-line and the phenomenon of “Z-line splitting” are explained by the proposed model. In addition, a potential hexagonal structural arrangement of the Z-line is retained so that a consistent geometrical organization persists throughout the entire sarcomere. Thus, the model also presents a means of understanding the recently suggested role of the Z-line in forming new sarcomeres.

Fine structure of the honeybee Z-disc☆

Abstract Z-discs from the dorsal longitudinal indirect flight muscles of the honeybee (Apis mellifera) are perforated with hundreds of triangular-shaped tubes ordered into an hexagonal array. Each tube is surrounded by 80 A thick rims which incorporate six thin filaments, three from each bordering sarcomere. Although the triangular rims of the tubes are oriented identically in any plane perpendicular to the fibril axis, this orientation changes as the tubes cross the Z-line. The tubes rotate approximately 60 ° about an axis parallel to that of the fibril in passing from one I-Z junction to another. On the basis of filament counting in the A (overlap zone) and I bands of stretched myofibrils, it is concluded that the primary filaments are physically continuous with the Z-lines by material which appears to participate both in the formation of Z-rim substance and the surrounding matrix. Finally, evidence is presented to support the view that filament lattices of adjacent sarcomeres are displaced from one another, so that each thick filament faces the trigonal position of three thick filaments on the other side of the Z-disc.

In indirect flight muscles Drosophila projectin has a short PEVK domain, and its NH2-terminus is embedded at the Z-band

Insect indirect flight muscles (IFM) contain a third filament system made up of elastic connecting or C-filaments. The giant protein projectin is the main, if not the only, component of these structures. In this study we found that projectin is oriented within the IFM sarcomere with its NH2−terminus embedded in the Z-bands. We demonstrate that this protein has an elastic region that can be detected by the movement of specific epitopes following stretch. One possible elastic region is the PEVK-like domain located close to the NH2−terminus. The amino acid length of this region is short, and 52% of its residues are P, E, V or K. We propose a model in which projectin extends from the Z-band to the lateral borders of the A-band. The PEVK-like domain and a series of Ig domains spanning the intervening I-band may provide the elastic properties of projectin.

The Insect Z-Band

The Z-band is an electron dense structure that borders sarcomeres in striated muscle. It is a complex assembly of proteins that organizes and stabilizes both thick and thin filament arrays in the contractile apparatus. By anchoring actin filaments and protein extensions of myosin filaments, the Z-band ensures that active as well as passive tensions are transmitted from one sarcomere to the next along the length of muscle.