Association of microinjected myosin and its subfragments with myofibrils in living muscle cells.

Abstract

Abstract. Purified skeletal muscle myosin was labeled with iodoacetamidofluorescein and microinjected into cultured chick myotubes. The fluorescent myosin analogue became incorporated within 10-15 min after injection, into either periodic (mean periodicity = 2.23 + 0.02 ~tm) bands or apparently continuous fibrillar structures. Comparison of rhodamine-labeled alphaactinin with coinjected fluorescein-labeled myosin suggested that myosin fluorescence was localized at the A-bands of myofibrils. In addition, close examination of the fluorescent myosin bands indicated that they were composed of two fluorescent bars separated by a nonfluorescent line that corresponded to the H-zone. Once incorporated, the myosin underwent a relatively slow exchange along myofibrils as indicated by fluorescence recovery after photobleaching. Glycerinated myofibrils were able to bind fluorescent myosin in a similar pattern in the presence or absence of MgATP, indicating that actin-myosin interactions had little effect on this process. Fluorescent heavy meromyosin did not incorporate into myofibrillar structures after injection. Light meromyosin, however, associated with A-bands as did whole myosin. These results suggest that microinjected myosin, even with its relatively low solubility under the cytoplasmic ionic condition, is capable of association with physiological structures in living muscle cells. Additionally, the light meromyosin portion of the molecule appears to be mainly responsible for the incorporation.

Fluorescent analogues of myosin II for tracking the behavior of different myosin isoforms in living cells

“…It will be interesting to see if either form is preferentially incorporated into the contractile ring during cytokinesis, or if transient differences in localization occur during the contraction of endothelia that occurs in response to thrombin and various other stimuli. It should be noted that the similarity between the distribution of platelet and smooth muscle myosin II is in stark contrast to what is observed when smooth and skeletal muscle myosins are injected into fibroblasts [Johnson et al, 1988]. Skeletal muscle myosin II fails to distribute into many structures and tends to form filamentous aggregates of a morphology not ordinarily observed in fibroblasts.…”

“…Because rhodamine-labeled analogues of smooth muscle myosin II are routinely used to study myosin dynamics in living cells, we first set out to make a complementary fluorescent analogue of smooth muscle myosin II. Fluorescein is frequently used with rhodamine in paired fluorescence, and fluorescein-labeled skeletal muscle myosin II has been prepared by Johnson et al [1988]. However, fluorescein is prone to photobleaching and its fluorescence is sensitive to local pH.…”

“…Fluorescent analogues of myosin II that have been previously described [Hahn et al, 1993;McKenna et al, 1989;Sanger, 1989;Johnson, 1988] were labeled with iodoacetamides of rhodamine or fluorescein, which react with sulfhydryl groups. Cy5-labeled analogues were prepared in the present study using an ester of cy5 that reacts with lysines, rather than sulfhydryls.…”

In vivo dynamics of myosin II inDictyostelium by fluorescent analogue cytochemistry

“…This technique has been used to study various cytoskeletal components, including actin [Wang and Taylor, 1979;Kreis et al, 1979;Cao and Wang, 1990 a,b;Theriot and Mitchison, 1991;Theriot et al, 19921, a-actinin [Feramisco, 19791, tubulin [Schulze and Kirschner, 1986;Sammak et al, 19871, myosin [Mittal et al, 1987;Johnson et al, 1988;McKenna et al, 19891, profilin [Cao et al, 19921, and vimentin [Vikstrom et al, 19891. To date, however, this technique has not been successfully applied to Dictyostelium amoeba because of such technical difficulties as its small size, rapid movement, unstable cell-substrate adhesion, and stickiness to the needle.…”

Disruption of microfilament organization in living nonmuscle cells by microinjection of plasma vitamin D-binding protein or DNase I.

“…Microinjection of trace amounts of fluorescently labeled actins (2,3) and myosins (4,5) into living nonmuscle and muscle cells have indicated how rapidly these labeled molecules are incorporated into stress fibers and myofibrils. The cycling of cells through the cell cycle also has revealed that the cells can disassemble and reassemble their stress fibers or myofibrils (6,7).…”