Journal of Cell Science, Vol 104, Issue 2 339-352, Copyright © 1993 by Company of Biologists

JOURNAL ARTICLES

Composite microtubules of the axon: quantitative analysis of tyrosinated and acetylated tubulin along individual axonal microtubules

A Brown, Y Li, T Slaughter and MM Black
Department of Anatomy and Cell Biology, Temple University School of Medicine, Philadelphia, PA 19140.

We have shown previously, using immunoelectron microscopy, that axonal microtubules (MTs) are composite, consisting of distinct domains that differ in their content of tyrosinated alpha-tubulin (tyr-tubulin). Here, we extend these studies using a novel preparation that permits visualization of individual axonal MTs over distances of several tens of micrometers using conventional immunofluorescence procedures. Neurons are cultured on a substratum of poly-lysine and laminin and then extracted with a MT stabilizing solution containing Triton X-100 and NaCl. These extraction conditions cause a loosening of the axonal MT array so that individual MTs separate from each other for variable distances along their length. We call this phenomenon fraying. Within the axon shaft, individual MTs can often be traced for several tens of micrometers, but fraying is most extensive in the distal 100-200 microns of the axon, where individual MTs can frequently be traced for distances of 50 to 100 microns or more to their plus ends. In some cases MTs separate completely from the axon, permitting visualization of both of their ends. Double-staining of frayed preparations with various combinations of antibodies against tyr-tubulin, acetylated alpha-tubulin (Ac-tubulin) or beta-tubulin, clearly revealed the composite nature of axonal MTs. Composite MTs consisted of two distinct domains, one that was relatively rich in tyr-tubulin and poor in Ac-tubulin, and the other that was relatively poor in tyr-tubulin and rich in Ac-tubulin. The transition between these domains was relatively abrupt, with the tyr-tubulin-rich domain extending from the transition to the plus-end of the MT. Quantitative analyses of fluorescence intensity along individual MTs using digital image processing revealed that the relative amount of tyr-tubulin increased by approximately 800% across the transition, whereas the relative amount of Ac-tubulin decreased by approximately 60%. Within the tyr-tubulin-rich domains, the relative amount of tyr-tubulin was generally not constant, but increased from the transition to the plus-end of the MT in a nonlinear manner. We propose that the specific pattern of variation in the extent of post-translational modification along an individual MT represents a snapshot of that polymer's growth history.

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