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. 2007 Jul 30;178(3):489–502. doi: 10.1083/jcb.200702072

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Copyright © 2007, The Rockefeller University Press

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Figure 2. — Exogenously expressed Ca_vβ_1-4 subunits distribute in different patterns in hippocampal neurons and colocalize to various degrees with presynaptic marker proteins. (A) Fluorescence pattern of neurons from low density hippocampal cultures infected with the indicated GFP-tagged Ca_vβ subunit (i.e., Ca_vβ_1–4) reveal either a punctate (Ca_vβ_2a and Ca_vβ_4b) or a more diffuse, cytosolic staining (Ca_vβ_1b and Ca_vβ₃). (B) Increased magnification of hippocampal neurites reveal that Ca_vβ_2a and Ca_vβ_4b are clustered, whereas Ca_vβ_1b and Ca_vβ₃ are diffusely distributed. (C) The bar graph indicates that neurons overexpressing Ca_vβ_2a and Ca_vβ_4b mainly reveal punctate staining similar to the endogenous distribution of Ca_vβ subunits, whereas the majority of neurons infected with Ca_vβ_1b and Ca_vβ₃ or GFP alone do not reveal punctate staining (n = 110–153 for each subunit; *, P < 0.01 compared with GFP). Quantification of the percentage of transfected neurons showing that punctate staining was performed by generating 11–13 randomly chosen fields within each group of neurons analyzed. In each field, the number of total infected neurons was counted, and the percentage of those showing punctate patterns was calculated. The percentages in each group were then averaged. n is equal to the total number of infected neurons counted. (D and E) Ca_vβ_2a and Ca_vβ_4b reveal differences in their percentages to colocalize with presynaptic marker proteins. (D) Cultured hippocampal neurons were infected with GFP-tagged Ca_vβ_2a and Ca_vβ_4b and costained with synaptobrevin-II or synapsin-I (red). Representative confocal images of hippocampal neurites reveal that the fluorescent puncta in Ca_vβ_2a- and Ca_vβ_4b (green)-expressing neurons reveal a different colocalization percentage with the presynaptic marker (red). (E) Quantification of the GFP puncta containing either Ca_vβ_2a or Ca_vβ_4b that colocalize with synaptobrevin-II. The percentage of synaptic colocalization is given as the number of Ca_vβ GFP puncta, which colocalized with the number of synaptobrevin-II puncta relative to the amount of GFP puncta within the region of interest analyzed (n = 20–21; *, P < 0.01). Error bars represent SEM. Bars (A), 15 μm; (B and D) 2 μm.