Biology Reference
In-Depth Information
As noted earlier, our original interest in the neuronal centrosome was as a
''generator'' of new microtubules for axons and dendrites. However, we should
note that there are exceptions to the rule of microtubule nucleation being con-
strained to centrosomes, as gamma-tubulin can redistribute to new locations in
certain cell types (Bugnard et al.
2005
). In fact, we previously proposed that the
non-uniform orientation of dendritic microtubules might result from centrosomal
proteins being relocated from the centrosome into dendrites at early stages of their
differentiation (Baas et al.
1989
). There are also examples in the literature where
de novo nucleation of microtubules has been observed (Yvon and Wadsworth
1997
) but not commonly, as haphazard nucleation of new microtubules would
probably make more problems for cells than solutions. In studies directed at testing
for de novo nucleation of microtubules in axons, we found no evidence for it, as all
new assembly was observed to occur via elongation of existing polymers (see
below).
18.4 Older Data on Microtubules and the Neuronal
Centrosome
Our studies positing the neuronal centrosome as a generator of microtubules for
the axon were conducted in the 1990s, and utilized tools that had been previously
used in other cell types. The classic method for identifying sites of microtubule
nucleation in cells is to depolymerize existing microtubules with nocodazole, and
then remove the drug so that microtubules can reassemble from their sites of
origin. This method, first used to identify the centrosome as a site of microtubule
nucleation in other cell types, (De Brabander et al.
1977
,
1980
), was used by our
laboratory to identify potential sites of microtubule nucleation in the axons of
cultured rat sympathetic neurons (Baas and Ahmad
1992
). After drug removal, all
new microtubule assembly arose from the plus ends of the stable microtubules that
resisted depolymerization. No microtubules arose independently of existing
microtubules, suggesting that the plus ends of pre-existing microtubules are the
exclusive sites of microtubule assembly in the axon. These findings were con-
sistent with previous work on cultured sensory neurons demonstrating that when
all microtubule polymer is pharmacologically depolymerized from isolated axons,
no reassembly occurred after removal of the drug (Baas and Heidemann
1986
).
Based on these findings, we concluded that entirely new microtubules destined for
the axon must be nucleated within the cell body. To explore the issue further, we
investigated the distribution of gamma-tubulin in these neurons (Baas and Joshi
1992
). Using both biochemical and immunoelectron microscopic assays, we found
no evidence for gamma-tubulin in the axon. In addition, we found no appreciable
levels of gamma-tubulin anywhere in the cell body except at the centrosome,
suggesting that the centrosome is the sole site for the nucleation of new micro-
tubules for the entire neuron. On the basis of these findings, we proposed that
