MEASURING ORIENTATION OF ACTIN-FILAMENTS WITHIN A CELL - ORIENTATION OF ACTIN IN INTESTINAL MICROVILLI

Orientational distribution of actin filaments within a cell is an impo rtant determinant of cellular shape and motility. To map this distribu tion we developed a method of measuring local orientation of actin fil aments. In this method actin filaments within cells are labeled with f luorescent phalloidin and are viewed at high magnification in a fluore scent microscope. Emitted fluorescence is split by a birefringent crys tal giving rise to two images created by light rays polarized orthogon ally with respect to each other. The two images are recorded by a high -sensitivity video camera, and polarization of fluorescence at any poi nt is calculated from the relative intensity of both images at this po int. 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-ac tin in microvillar cores was labeled with rhodamine-phalloidin; measur ements showed that the orientation was the same when microvillus forme d a part of a brush border and when it was separated from it suggestin g that ''shaving'' of brush borders did not distort microvillar struct ure. In the absence of nucleotide, polarization of fluorescence of act in cores in isolated microvilli was best fitted by assuming that a maj ority of fluorophores were arranged with a perfect helical symmetry al ong the axis of microvillus and that the absorption dipoles of fluorop hores were inclined at 52-degrees with respect to the axis. When ATP w as added, the shape of isolated microvilli did not change but polariza tion of fluorescence decreased, indicating statistically significant i ncrease in disorder and a change of average angle to 54-degrees. We ar gue that these changes were due to mechanochemical interactions betwee n actin and myosin-1.