Biology Reference
In-Depth Information
There are two potential solutions to overcome this limitation. One would
be to use a total protein stain to label isoelectrically focused samples of β-
and γ-actin followed by densitometry, thus enabling direct comparisons of
actin isoform levels. This experiment has several caveats, however, in that
separating β- and γ-actin, which differ only minutely in isoelectric points,
is inherently difficult not to mention the all too common and overlooked
potential for artifactual spots. Furthermore, the actin must be purified from
the total protein to enable visualization with protein stains, which is not
without the potential for artifacts as well. The second method would be to
generate standard curves of pure β- and γ-actin antibody staining to allow
for direct determination of actual molar quantities of protein present in
extracts. Until recently, there was no way to obtain biochemically relevant
amounts of pure β- and γ-actin in order to generate these curves. How-
ever, with the recent report of baculovirus-mediated expression of β- and
γ-actin in insect cells, this may now be possible, although contamination
from endogenous insect actin remains a problem (
Bergeron et al., 2010
).
In summary, data from a small number of studies suggest β- and γ-actin
levels likely increase during early neuronal differentiation and may be
accompanied by a relative change in the actin isoform composition (
Chang
et al., 1986
). With this shift, β-actin levels appear to decrease in total brain
and neuron-like cell lysates later in development and differentiation, while
γ-actin levels remain steady or increase subtly (
Micheva et al., 1998
;
Wein-
berger et al., 1996
). As for the actual molar composition of actin isoforms
during these phases of neuronal differentiation, the available data are frag-
mentary at best and based on isoelectric focusing. Lysates from chick sym-
pathetic and sensory neurons isolated at embryonic day (E) 10 and cultured
for 24-36 h to allow for neurite outgrowth showed an equivalent amount
of β- and γ-actin present (
Choo and Bray, 1978
). Actin isolated from whole
brain embryonic chicks of the same age showed a similar 1:1 ratio of β- to
γ-actin (
Flanagan and Lin, 1979
). In lysates from adult rat brains, however,
the ratio was much closer to 2:1 β- to γ-actin (
Otey et al., 1987
). Thus, the
evidence available indicates that actin isoform levels exhibit significant fluc-
tuations during development and potentially to different extents, eventually
resulting in β- to γ-actin ratios between 1:1 and 2:1.
5.1.2. Spatial Localization
As with fibroblasts, distinct localization of actin isoforms in neurons has
been difficult to pin down. In early work with cultured embryonic rat
cortical neurons and antibodies generated against β- and γ-actin-specific
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