We propose to combine fluorescent analogue cytochemistry photobleaching, and image processing to study the behavior of actin and its accessory proteins in living cells during oncogenic transformation as well as after various treatments which result in the disruption of actin-containing structures. Actin, alpha-actinin, vinculin and tropomyosin will be fluorescently labeled in vitro and microinjected into living cells. The labeled molecules will incorporate into endogenous structures and allow us to see the dynamic properties of specific molecules in living cells. We will first record sequences of images during the disruption of actin-containing structures. The distribution of proteins in various parts of the structures and the relationship between various proteins will be analyzed to determine the mechanism of disruption. Similar observations will be performed on cells recovering from the treatments and from transformation, in order to determine the pathway of structural assembly and the proteins involved in he initiation process. Next, we will use laser microbeams to mark various parts of the structures during disruption and reassembly. The subsequent movement of the bleached spot will yield information on the polarity of elongation or shortening, and on the possible occurrence of stretching or contraction. In addition, the recovery of fluorescence after photobleaching will be measured to determine the mobility and exchangeability of proteins in various structures. Finally, intense laser beams will be used to sever stress fibers or disrupt adhesion plaques, in order to study the possible requirement of adhesion plaques for the stability of stress fibers. The information should help us to understand how actin-containing structures are assembled and disassembled under various conditions, and how transformation is related to other conditions which also induce changes in actin-containing structures.
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