Axoplasm extruded from the squid giant axon contains at least seven members of the kinesin family of microtuble motors. An antibody that recognizes many family members of this family has been for immunolabeling of freeze substituted squid axoplasm. It was necessary to develop and apply cryogenic methods to prevent displacement of soluble kinesin during tissue processing. This method had the unanticipated advantage of reducing background label to very low levels, making the method both sensitive and semiquantitative. Using the broad spectrum antibody, we found that kinesins are widely distributed in the cytoplasm but several fold concentrated around cytoplasmic organelles. The greatest concentration is on vesicles (69.6-fold, but increases in gold particles over the cytoplasmic level was also seen around ER cisternae (29.5-fold), microtubles (15.2-fold), and mitochondria (6.2- fold). Thus, a third of the kinesin in the axon is on organelles while the rest is free in the axoplasm or association of microtubles. Axons incubated with a polyclonal antibody against squid neurofilament failed, as expected, to show a selective distribution around vesicles. A manuscript describing this new method and its application to synaptic and junctional proteins has been submitted. We are currently applying this method to show the distribution of particular kinesins and kinesin family proteins in axoplasm. This work is directed to understanding what different function are served by the different kinesins in axoplasm.
