title: A Boyden chamber-based method for characterization of astrocyte protrusion localized RNA and protein.
abstract: 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.
authors.affiliation: Department of Biomedicine, Faculty of Health Sciences, Aarhus University, Aarhus, Denmark. rune@humgen.au.dk
authors.forename: Rune
authors.initials: R
authors.lastname: Thomsen
authors.forename: Anders
authors.initials: A
authors.lastname: Lade Nielsen
mesh_terms: D000818:Animals; D017089:Ankyrin Repeat / Q000235:genetics; D001253:Astrocytes / Q000378:metabolism*; D015153:Blotting, Western; D002465:Cell Movement; D022081:Cell Surface Extensions / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism / Q000648:ultrastructure; D003627:Data Interpretation, Statistical; D015194:Diffusion Chambers, Culture; D005455:Fluorescent Antibody Technique; D007091:Image Processing, Computer-Assisted; D051379:Mice; D041681:NIH 3T3 Cells; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000096:biosynthesis / Q000235:genetics; D051191:Plakophilins / Q000096:biosynthesis / Q000235:genetics; D011554:Pseudopodia / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction; D016622:Silver Staining; D020691:rab GTP-Binding Proteins / Q000096:biosynthesis / Q000235:genetics
chemical_list: D009419:Nerve Tissue Proteins; D051191:Plakophilins; D012333:RNA, Messenger; D010744:Phosphoric Monoester Hydrolases; C053087:inositol-1,4-bisphosphate 1-phosphatase; C484177:Rab13 protein, mouse; D020691:rab GTP-Binding Proteins