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treatment ( Chang et al., 1986 ). Interestingly, we observed a larger increase in
γ-actin expression compared to β-actin during hippocampal neuron differ-
entiation in culture (unpublished results), which may also result in a similar
shift in the cytoplasmic actin composition toward a lower β- to γ-actin
ratio. Yet this is in contrast to similar experiments performed with whole
rodent brain extracts. In one study, β-actin protein levels increased rap-
idly during the second half of embryonic development followed by a sharp
decrease after birth. γ-actin levels increased to a much smaller percentage
but did not exhibit a similar drop off, and were maintained at similar levels
into adulthood ( Weinberger et al., 1996 ).
The conflicting nature of the reports described above emphasizes the
difficulty in executing informative studies examining individual actin iso-
form expression in rapidly changing cells. First, in some cases the reports
described above were performed decades before KO tissue was available
to validate the specificity of the antibodies. In addition, the cell culture
experiments present the significant problem of normalization of protein
levels. Neurons in culture exhibit dramatic and profound morphological
changes during differentiation, with cells progressing from small spheres to
extending long processes across vast distances. Thus, cytoskeletal proteins
used to normalize expression studies such as these become almost useless as
their levels most certainly change along with actin. In addition, commonly
used alternative loading controls such as GAPDH, ribosomal subunits, or
histone proteins likely also change due to the increased metabolic demands
of rapidly growing and differentiating neurons. The in vivo studies with
whole brain extracts can have similar normalization issues along with the
added complexity of the makeup of the tissue. For example, although glial
cells make up a relatively small proportion of the cells within the developing
brain, their proliferation continues through late development to the point
where they can substantially outnumber neurons in the mammalian adult
brain.Vasculature and microglia numbers also increase during development,
which may skew the actin isoform composition within the brain and com-
plicate interpretation of data. Microglia, in particular, may be especially
problematic as they have been reported to be the highest β-actin expressing
cells within the brain ( Plantier et al., 1998 ).
Direct molar comparisons between β- and γ-actin levels within neurons
would be even more informative than the relative comparisons described
above, but this type of experiment has only recently become even possible.
While antibodies can reveal relative changes in actin isoform expression, it
is not possible to directly compare levels across antibodies for β- and γ-actin.
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