Actin Isoforms in Neuronal Development and Function - International Review of Cell and Molecular Biology - page 171

by two independent groups using an additional set of actin isoform anti-
bodies ( Bunnell et al., 2011 ; Cheever et al., 2012 ; Dugina et al., 2009 ; Perrin
et al., 2010 ; Perycz et al., 2011 ). We hypothesize that brief treatment with
methanol may serve to “loosen” actin-based structures, thereby unmasking
binding sites for actin-isoform-specific antibodies.
Taking into account the factors just discussed, we have used primary
antibodies with specificity most definitively demonstrated on β- and γ-actin
KO tissue ( Bunnell et al., 2011 ; Cheever et al., 2012 ; Perrin et al., 2010 ) to
stain both dissociated neurons and brain sections to confirm the localization
patterns of β- and γ-actin. Importantly, we used primary antibodies directly
conjugated to fluorescent dyes and co-stained with both β- and γ-actin
antibodies simultaneously in order to limit any epitope accessibility issues
and allow for direct comparison of localization patterns within the same
neuron or tissue section. Surprisingly, in cultured hippocampal neurons, we
found β- and γ-actin to be colocalized with no evidence for preferential
enrichment of β-actin at the growth cone ( Cheever et al., 2012 ). Stain-
ing of E18 hippocampal sections also revealed no evidence for differential
localization of β- and γ-actin within tissue sections ( Cheever et al., 2012 ).
We further examined the localization patterns of β- and γ-actin in three
distinct regions of the adult brain ( Fig. 4.2 ). In the hippocampus, β- and
γ-actin staining was absent from nuclei but strongly localized to the stratum
radiatum and stratum lacunosum moleculare, both areas rich in synapses and
dendritic spines. We observed similar localization patterns for β- and γ-actin
in the cerebellum as well, with both isoforms colocalized in synapse-rich
regions like the molecular layer and notably absent from the cell body and
nuclei-rich granule cell layer.
There was, however, one specific case where we did observe distinct
localization patterns of the cytoplasmic actins. Strong staining for γ-actin
Figure 4.2 Localization of cytoplasmic actin isoforms in adult brains. Representative
images from control and CNS- Actb KO brain sections to demonstrate antibody speci-
ficity for β -actin. Sagittal sections through the hippocampus, cerebellum, and striatum
were costained with DAPI and cytoplasmic actin-specific primary antibodies directly
conjugated to fluorescent dyes. In the hippocampus, cytoplasmic actin staining colo-
calized in synapse-dense areas including the stratum radiatum and stratum lacunosum
moleculare, while staining was noticeably absent from cell bodies and nuclei. This was
also true in the cerebellum with the strongest staining localized to the molecular layer.
Interestingly, β -actin staining was virtually absent from the large white matter tracts in
the cerebellum, whereas γ -actin was still present (arrows). A similar apparent exclusion
of β -actin from large axonal tracts was also observed in the striatum (arrows). Scale bar
300 µm. For color version of this figure, the reader is referred to the online version of
this topic.