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 endoplasmic reticulum (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. Preliminary results using antibodies which distinguish different kinesin light chain isoforms detected similar gold distribution as shown for the heavy chain but comparatively higher on the cytosol and lower on organelles. Hindrance due to the larger volume of the heavy chain as the light chain attaches to organelles it may explain the lower label on organelles and higher label of free kinesin on the cytosol. Further work is needed for the interpretation of these results. Processing of this data is expected to suggest whether the differences are due to different exposition of the kinesin light chain on organelles or cytosol. This work is expected to elucidate where different members of the kinesin family are found in the axon, leading to a better understanding of how kinesins actually function in the nerve cell.
