Microinjection of Smooth Muscle Myosin in Cultured Cells

Abstract

Our current knowledge on the ultrastructure of smooth muscle (SM) cells does not satisfactorily account for the particular length-tension behavior of SM, which seems to be able to adapt its contractile apparatus to generate great force over a wide range of lengths. A key question in the field remains: Are contractile filaments in SM cells a fixed array or dynamic structures? There is insufficient knowledge as to whether the myosin filaments can indeed reorganize themselves in vivo as has been shown through proposed models. We are proposing here to develop a microinjection technique to address this question.Methods: Smooth muscle myosin was purified from chicken gizzard using serial precipitation. As a control, monomeric myosin and self-assembled filaments of the purified myosin were tested for functionality using an in vitro motility assay. The myosin was then labeled with a rhodamine-derivative fluorophore, tetramethylrhodamine-5-iodoacetamide dihydroiodide (5-TMRIA). The labeled myosin was then self-assembled in vitro into filaments and images were taken using a spinning-disk confocal microscope. The labeled filaments, as well as monomeric myosin were then respectively microinjected into cultured A5R7 vascular SM cells. The volume of the cells was estimated by measuring them using a calibrated microscopic ruler. The volume of myosin solution that was microinjected represented 2 to 5% of the estimated volume of the cells. The cells' viability was estimated by observing their morphology after the microinjection.Results: The purified myosin was functional in the in vitro motility assay, both in a monomeric and filamentous form. The microinjection technique could be done with little modifications of the morphology of the injected cells in a reasonable number of cells.Conclusion: The microinjection technique of purified myosin can be used for further studies on the ultrastructure and dynamics of myosin filaments in smooth muscle cells.