This project concerns the transport of ion channels in nerve axoplasm by intracellular organelles. Previous work in this project has demonstrated distinct populations of organelles isolated from the axoplasm of squid giant axons; a putative anterograde organelle population having diameters in the 40-60 nm range, and a putative retrograde population having diameters in the 100-150 nm range. During the past year various microscopic techniques have been applied in an attempt to visualize the organelles in various different settings. For example, laser confocal microscopy has recently been used to observe Brownian motion of organelles which had been labeled with the fluorescent marker, Texas Red. These results are being used to determine the relative yield of organelles in the various fractions from the column, and also to provide some information on organelle size from the Brownian motion. The Texas Red treated preparations are also being used in an attempt to visualize motility of exogenous organelles along microtubules and perhaps also actin filaments in an in vitro motility assay with the differential interference contrast microscopy technique, and they are also being pressure injected into squid axons to determine the targeting of organelles to the axonal membrane with a fluorescence microscope.
