Immunocytochemical electron microscopic study and western blot analysis of myosin, paramyosin and miniparamyosin in the striated muscle of the fruit fly Drosophila melanogaster and in obliquely striated and smooth muscles of the earthworm Eisenia foetida

Abstract

Miniparamyosin is a paramyosin isoform (55-60 kDa) that has been isolated in insects (Drosophila) and immunolocalized in several species of arthropods, molluscs, annelids and nematodes. In this study, the presence and distribution of this protein, in comparison with that of paramyosin and myosin, has been examined in the striated muscle (tergal depressor of trochanter) of Drosophila melanogaster, and the obliquely striated muscle (body wall) and the smooth muscle (outer layer of the pseudoheart) of the earthworm Eisenia foetida by means of immunocytochemical electron microscopic study and Western blot analysis miniparamyosin paramyosin and myosin antibodies from Drosophila. In the striated muscle of D. melanogaster, the three proteins were immunolocalized along the length of the thick filaments (A-bands). The distribution of immunogold particles along these filaments was uniform. The relative proportions miniparamyosin/paramyosin/myosin (calculated by counting the number of immunogold particles) were: 1/10/68. In the obliquely striated muscle of E. foetida, immunoreactions to the three proteins were also found in the thick filaments, and the relative proportions miniparamyosin/paramyosin/myosin were 1/2.4/6.9. However, whereas the distribution of both myosin and miniparamyosin along the thick filament length was uniform, paramyosin immunolabelling was more abundant in the extremes of thick filaments (the outer zones of A-bands in the obliquely striated muscle), where the thick filaments become thinner than in the centre (the central zone of A-bands), where these filaments are thicker. The relative proportions of paramyosin in the outer and of paramyosin in the central zones of A-bands were 4/1. This irregular distribution of paramyosin along the thick filament length might be actual but it may also be explained by the fusiform shape of thick filaments in the earthworm: assuming that paramyosin is covered by myosin, paramyosin antigens would be more exposed in the tips than in the centre of thick filaments. If miniparamyosin is, in turn, covered by paramyosin, the exposure of miniparamyosin antigens would be low even in the tips of thick filaments, and this might explain the scanty immunoreaction observed for this protein and the absence of a higher number of immunogold particles in the extremes of thick filaments. The distribution of the three proteins in the earthworm smooth muscle was as in the obliquely striated muscle, although the proportions miniparamyosin/paramyosin/myosin were 1/1.5/5.2; this is, immunoreactions to paramyosin and miniparamyosin were lower than in the obliquely striated muscle.