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A Boyden chamber‐based method for characterization of astrocyte protrusion localized RNA and protein
Author(s) -
Thomsen Rune,
Lade Nielsen Anders
Publication year - 2011
Publication title -
glia
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.954
H-Index - 164
eISSN - 1098-1136
pISSN - 0894-1491
DOI - 10.1002/glia.21223
Subject(s) - astrocyte , biology , microbiology and biotechnology , rna , pseudopodia , glial fibrillary acidic protein , cell migration , cell , neuroscience , actin , immunology , biochemistry , central nervous system , gene , immunohistochemistry
The Boyden chamber assay has been developed for various cell migration and invasion protocols. One variant of the Boyden chamber assay is the pseudopodium isolation assay, which has been developed to identify RNA and proteins localized in pseudopodia cell protrusions. Astrocytes are the most abundant cell type in the CNS and typically extend long cellular protrusions. Increasing interest emerges concerning for example the growth mechanisms and functions of astrocytes in respect to brain development, re‐uptake of neurotransmitters in the synaptic cleft and glial scar formation. Protein and RNA localization mechanisms have been extensively examined in neurons and shown to play pivotal roles for the functional presence of specific protein components in neuronal protrusions. Here we present a simple Boyden chamber based method to isolate astrocyte cell protrusions for biochemical analysis. We have succeeded in isolating protrusion localized RNA and protein from the mouse astrocyte cell line, C8‐S, and mouse primary astrocytes. This is exemplified in biochemical analyses showing specific localization of the mRNA for Ras‐related protein (Rab13), Plakophilin‐4 (Pkp4), Ankyrin Repeat Domain 25 (Ankrd25), and inositol polyphosphate‐1‐phosphatase (Inpp1) in the protrusions of both C8‐S cells and primary astrocytes. Concordant, the Pkp4 protein was also predominantly localized in the protrusions of C8‐S and primary astrocytes. The described methodology can be the basis for both genome wide and specific descriptive and functional studies of RNA and protein localization in the protrusions of astrocytes which could contribute considerably to the existing knowledge of astrocyte functions in the CNS. © 2011 Wiley‐Liss, Inc.