Transient assembly-disassembly of actin structures is known to play a key role in many non-muscle cellular functions. In order to understand the underlying mechanism, it is important to characterize the organization of actin in intact cells, and to identify molecular interactions that are involved in actin assembly and regulation. The goal of this project is to address these issues using a combination of fluorescence imaging, biophysical, and molecular biological approaches. The study will cover a number of cellular activities, including locomotion, oncogenic transformation, cytokinesis, and neurite outgrowth. Regulatory mechanisms will be analyzed by directly examining the response of cellular actin organization to possible second messengers. The first part of the project is focused on nonfilamentous actin. First, a new ratio imaging approach will be used to map the extent of actin assembly in different regions of cultured cells. The distribution and dynamics of nonfilamentous actin will then be characterized in living cells microinjected with fluorescent actin analogs. Experiments will also be performed to determine the cellular localization of profilin molecules, using a combination of fluorescent analogs and energy transfer techniques. In addition, the physiological role of profilin in regulating actin assembly will be assessed with antisense technology and site-specific mutagenesis. The second part of the project will focus on the organization of filamentous actin in intact cells. The global orientation of actin filaments in relation to various cellular activities will be studied with polarized fluorescence optics. New optical techniques such as fluorescence anisotropy and differential circular dichroism, in combination with electron microscopy, will be used to probe the structural regularity and rotational mobility. Finally, possible interactions between actin filaments and microtubules will be examined by imaging fluorescence resonance energy transfer in both dividing and interphase cells. The project should shed light on important aspects of cancer cells, including reduced adhesion, active division, and unregulated migration.
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