Antibodies to defined domains of the light and heavy chains of the motor protein kinesin (from squid axon) have been used 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. We found that kinesins are widely distributed in the cytoplasm but several-fold concentrated around cytoplasmic organelles. The higher concentration is on vesicles (69.6-fold), but increase in gold particles over the cytoplasmic level was also seen around ER cisternae (29.5-fold), microtubules (15.2-fold), and mitochondria (6.2-fold). New experiments using affinity purified polyclonal antibodies against a defined protein fragment of the functional head of the kinesin heavy chain confirmed the previous kinesin location. Western blots performed with the same antibodies, using pure squid kinesin, whole axoplasm, and pellet and supernatant of the centrifuged axoplasm, confirmed the results showing the 116 Kd kinesin band on all the samples. Sections incubated with a polyclonal antibody against squid neurofilament, failed, as expected, to show a selective distribution around vesicles. This work is now complete and ready to publish. It shows that a third of the kinesin in the axon is on organelles while the rest is free in the axoplasm or associated with microtubules. Since the antibody was to the conserved head domain this overall distribution should include several kinesin isoforms.
