Subcellular localization of specific mRNAs and their protein products in Purkinje cells by combined fluorescence in situ hybridization and immunocytochemistry.
In this study we have investigated the subcellular distribution of two mRNAs coding for the Purkinje cell-specific proteins, calbindin D28K and L7 (L7/pcp-2). Whereas calbindin mRNA was found to be in the cell body only, L7 transcripts could be detected within the molecular layer, corresponding to Purkinje cell dendrites. We have now combined a highly sensitive fluorescence-based in situ hybridization protocol with immunofluorescence in conjunction with confocal optical sectioning to analyze the precise localization of these mRNAs in individual Purkinje neurons. We show that L7 mRNA is localized in clusters within the proximal and distal branches of dendrites, but also in the proximal part of Purkinje cell axons. In contrast, calbindin transcripts are restricted to the axonal pole of the perikaryon. Purkinje cells grown in primary cultures reveal similar mRNA distribution patterns for the two transcripts. Thus, the mechanism underlying localization of mRNA within Purkinje cells seems to function in a cell-intrinsic manner, guiding specific transcripts, such as L7 mRNA, to neuronal processes while restricting others, such as calbindin mRNA, to the perikaryon.
Wanner
I
I
Abteilung Anatomie und Zellbiologie, der Universität Ulm, Germany.
Baader
S L
SL
Brich
M
M
Oberdick
J
J
Schilling
K
K
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D064092:Calbindin 1; D064026:Calbindins; D002478:Cells, Cultured; D005456:Fluorescent Dyes / Q000737:chemistry; D007150:Immunohistochemistry; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D051379:Mice; D008810:Mice, Inbred C57BL; D009419:Nerve Tissue Proteins / Q000032:analysis* / Q000235:genetics; D011689:Purkinje Cells / Q000737:chemistry*; D012333:RNA, Messenger / Q000032:analysis*; D064030:S100 Calcium Binding Protein G / Q000032:analysis* / Q000235:genetics; D012680:Sensitivity and Specificity
C576499:Calb1 protein, mouse; D064092:Calbindin 1; D064026:Calbindins; D005456:Fluorescent Dyes; D009419:Nerve Tissue Proteins; C091104:Purkinje cell protein L7; D012333:RNA, Messenger; D064030:S100 Calcium Binding Protein G; D000469:Alkaline Phosphatase