Faculty Bibliography

Fluorescently labelled contractile proteins (alpha-actinin and filamin) were used to study the dynamic nature of three types of microfilament bundles: myofibrils, stress fibres and polygonal networks. Cultured muscle and non-muscle cells that were microinjected with fluorescent alpha-actinin rapidly incorporated the labelled protein into Z-bands, stress fibre densities and the polygonal foci. Living, injected cells were then observed for varying periods of time, and changes in orientation and periodicity of the myofibrils, stress fibres and polygonal networks were recorded. Permeabilized cells were also reacted with fluorescently labelled proteins and with contractile protein antibodies in order to analyse further the changes taking place in the myofibrils and stress fibres. In both living cardiac myocytes and living skeletal muscle myotubes, contractile myofibrils were present in the same cell with non-contractile nascent myofibrils. The periodicities of small Z-bodies in the nascent non-contractile myofibrils were shorter than the Z-band spacings in the contractile myofibrils, yet both types of myofibrils contained muscle myosin. Over a period of 24 h, a nascent myofibril in a living, microinjected myotube was observed to grow from Z-body spacings of 0.9-1.3 micron to full sarcomere spacings (2.3 microns). During the same time, nascent myofibrils appeared de novo and Z-band alignment became more ordered in the fully formed myofibrils. Stress fibres were not observed to undergo the predictable type of growth seen in myofibrils, but stress fibre periodicities did change in some fibres; some shortened while others lengthened. The orientation of fibres shifted in cytoplasm of both mobile cells and stationary cells. Attachment plaques and foci also changed position and in some cases subdivided and/or disappeared. Models of stress fibres and polygonal networks are presented that suggest that the changes in the periodicities of the dense bodies in stress fibres and the distances between polygonal foci are related to the movement of the interdigitating actin and myosin filaments.

Fluorescently labeled alpha-actinin, isolated from chicken gizzards, breast muscle, or calf brains, was microinjected into cultured embryonic myotubes and cardiac myocytes where it was incorporated into the Z-bands of myofibrils. The localization in injected, living cells was confirmed by reacting permeabilized myotubes and cardiac myocytes with fluorescent alpha-actinin. Both living and permeabilized cells incorporated the alpha-actinin regardless of whether the alpha-actinin was isolated from nonmuscle, skeletal, or smooth muscle, or whether it was labeled with different fluorescent dyes. The living muscle cells could beat up to 5 d after injection. Rest-length sarcomeres in beating myotubes and cardiac myocytes were approximately 1.9-2.4 microns long, as measured by the separation of fluorescent bands of alpha-actinin. There were areas in nearly all beating cells, however, where narrow bands of alpha-actinin, spaced 0.3-1.5 micron apart, were arranged in linear arrays giving the appearance of minisarcomeres. In myotubes, alpha-actinin was found exclusively in these closely spaced arrays for the first 2-3 d in culture. When the myotubes became contraction-competent, at approximately day 4 to day 5 in culture, alpha-actinin was localized in Z-bands of fully formed sarcomeres, as well as in minisarcomeres. Video recordings of injected, spontaneously beating myotubes showed contracting myofibrils with 2.3 microns sarcomeres adjacent to noncontracting fibers with finely spaced periodicities of alpha-actinin. Time sequences of the same living myotube over a 24-h period revealed that the spacings between the minisarcomeres increased from 0.9-1.3 to 1.6-2.3 microns. Embryonic cardiac myocytes usually contained contractile networks of fully formed sarcomeres together with noncontractile minisarcomeres in peripheral areas of the cytoplasm. In some cells, individual myofibrils with 1.9-2.3 microns sarcomeres were connected in series with minisarcomeres. Double labeling of cardiac myocytes and myotubes with alpha-actinin and a monoclonal antibody directed against adult chicken skeletal myosin showed that all fibers that contained alpha-actinin also contained skeletal muscle myosin. This was true whether alpha-actinin was present in Z-bands of fully formed sarcomeres or present in the closely spaced beads of minisarcomeres. We propose that the closely spaced beads containing alpha-actinin are nascent Z-bands that grow apart and associate laterally with neighboring arrays containing alpha-actinin to form sarcomeres during myofibrillogenesis.

Cytokinesis consists of a contractile phase followed by sealing of the connecting midbody to form two separated cells. To determine how soon the midbody sealed after cleavage furrow contraction, the fluorescent dye Lucifer Yellow CH(457.3 M.W.) was microinjected into cells at various intervals after cleavage had begun. Mitotic PtK2 cells were recorded with video-microscopy so that daughter cells in the epithelial sheet could be identified for several hours after cell division. One daughter cell of each pair followed was microinjected to determine whether the dye diffused into the other daughter cell. For intervals up to four hours after the beginning of cytokinesis, diffusion took place between daughter cells. After this time the dye did not spread between daughter cells. In sea urchin blastomeres of the first, second and third divisions, Lucifer Yellow passed between daughter blastomeres only during the first 15 min after cytokinesis. If one cell of a two-cell, four-cell or eight-cell embryo was microinjected more than 15 min after the last cleavage, the dye remained in the injected cell and was distributed to all progeny of that cell, resulting in blastulae that were eigher one-half, one-quarter or one-eighth fluorescent, respectively. Thus, although cleavage furrow contraction takes approximately the same amount of time in sea urchin blastomeres and PtK2 cells, the time of midbody sealing differs dramatically in the two cell types. Our results also indicate the importance of knowing the mitotic history of cells when injecting dyes into interphase cells for the purpose of detecting gap junctions.

Eggs and embryos of Arbacia punctulata were microinjected with the fluorescent dye, Lucifer yellow CH, using a simple pressure injection system. When injected into eggs that were subsequently fertilized, the dye was distributed throughout all cells of the developing embryo. If one cell of a two-cell embryo was injected, dye did not diffuse into the uninjected blastomere. During subsequent development, all progeny of the injected cell contained dye resulting in an embryo that was half-fluorescent. Blue light irradiation of a two-cell embryo, one cell of which had been injected with Lucifer yellow, caused the injected blastomere to stop further divisions while the uninjected blastomere developed normally and was free of dye. These results indicate that the first two blastomeres of Arbacia embryos are not electrically coupled, nor up to the time of hatching, is there any coupling between cells in one half of the first cleavage plane and cells in the other half.