Biophysical properties of stable microtubules in neurites revealed by optical techniques

We tested the stability of microtubules in the neurites of cultured dorsal root ganglion cells by dissolving the cytoplasmic membrane with detergent and exposing them to defined extracellular medium under the microscope. Smooth cytoplasmic filaments visualized after membrane removal were suggested to be microtubules by the preservation of all of the filaments in the presence but not in the absence of taxol. They were further confirmed to be microtubules by immunostaining with anti-tubulin antibody. Significant number of microtubules in the established neurites remained longer than 1 hour after membrane removal. To investigate their stabilization mechanism, we transected the exposed microtubules by laser microbeam irradiation and observed their length changes with video-enhanced microscopy. Microtubule fragments started to shorten on both sides of the transection site, more rapidly from the newly generated plus ends than from the minus ends. The maximal rate as well as the pattern of shortening correlated with the time of transection; microtubules transected later than 30 min after membrane removal shortened at rates less than 20 microm/min and typically with intermittent pauses, while the more labile microtubules included in the earlier transections shortened continuously at higher rates. Microtubules in neurites were thus stabilized by (1) stopping disassembly at local sites including the plus ends, and (2) slowing disassembly along the length. Observations of the course of disassembly also suggested the presence of specialized points along microtubules which is involved in anchoring microtubules to the substratum or transiently stopping disassembly.