Measuring orientation of actin filaments within a cell: orientation of actin in intestinal microvilli.

Orientational distribution of actin filaments within a cell is an important determinant of cellular shape and motility. To map this distribution we developed a method of measuring local orientation of actin filaments. In this method actin filaments within cells are labeled with fluorescent phalloidin and are viewed at high magnification in a fluorescent microscope. Emitted fluorescence is split by a birefringent crystal giving rise to two images created by light rays polarized orthogonally with respect to each other. The two images are recorded by a high-sensitivity video camera, and polarization of fluorescence at any point is calculated from the relative intensity of both images at this point. From the value of polarization, the orientation of the absorption dipole of the dye, and thus orientation of F-actin, can be calculated. To illustrate the utility of the method, we measured orientation of actin cores in microvilli of chicken intestinal epithelial cells. F-actin in microvillar cores was labeled with rhodamine-phalloidin; measurements showed that the orientation was the same when microvillus formed a part of a brush border and when it was separated from it suggesting that "shaving" of brush borders did not distort microvillar structure. In the absence of nucleotide, polarization of fluorescence of actin cores in isolated microvilli was best fitted by assuming that a majority of fluorophores were arranged with a perfect helical symmetry along the axis of microvillus and that the absorption dipoles of fluorophores were inclined at 52 degrees with respect to the axis. When ATP was added, the shape of isolated microvilli did not change but polarization of fluorescence decreased, indicating statistically significant increase in disorder and a change of average angle to 54 degrees. We argue that these changes were due to mechanochemical interactions between actin and myosin-I.