Journal: PLoS ONE
Article Title: An SK3 Channel/nWASP/Abi-1 Complex Is Involved in Early Neurogenesis
doi: 10.1371/journal.pone.0018148
Figure Lengend Snippet: SK3 channels, Abi-1 and nWASP are found in neuronal complexes and colocalize in defined subcompartments of NSCs and hippocampal neurons (HNs). (A-B)Triple staining experiments show that Abi-1 as well as nWASP (red) colocalize with the SK3 channel (green) in distinct lamellipodia like structures of NSCs that are also rich of actin fibers stained by phalloidin (magenta), DAPI (blue) is used to label nuclei. (C–D) In hippocampal neurons SK3 channels are densely targeted to dendritic spines/PSDs and co-localize with nWASP as well as with Abi-1 (see arrows in insets). To visualize the cytoskeleton tubulin antibodies (Tubb, magenta) are used, DAPI staining (blue) show neuronal nuclei. (E) Co-immunoprecipitation experiments show the presence of all three molecules in one neuronal complex also in vivo . Magnetic beads loaded with SK3 channels antibodies were employed to precipitate the SK3 channel complex from rat brain. Within this precipitate Abi-1 as well as nWASP can be detected. (F) Schematic drawing of the fusions proteins and Abi-1 deletion constructs used for transfection experiments with their respective domain regions. The SK3 channel codes for a conserved N-terminal proline rich stretch that mediates the interaction with the Abi-1 SH3 domain. (G) Co-transfection of the nWASP with the SK3 channel results in a complete colocalization in NSCs. This is also observed when full length Abi-1-myc is co-transfected with the SK3 channel GFP fusion protein. The co-expression of SK3 channels with an Abi-1 SH3 domain deletion construct (Abi-1 ΔSH3-RFP) results in no co-localization of the proteins while the SK3 channel perfectly co-localizes with the Abi-1-SH3 domain fusion protein. Moreover, the full length SK3 channel protein can co-precipitate the GFP-SH3 domain from the co-transfected cell lysate but fails to bind to the Abi-1 protein that is not expressing the SH3 domain. Scale bars as indicated.
Article Snippet: The following primary antibodies were used: rabbit anti-SK3 diluted 1∶200, rabbit (Alomone Labs), mouse anti-Nestin monoclonal diluted 1∶500 (BD Biosciences), mouse anti-Abi-1 diluted 1∶250 (MBL), mouse anti-myc antibody diluted 1∶500 (Invitrogen), rabbit anti-PSD95 diluted 1∶1000, rabbit anti-SV2 diluted 1∶300 (both abcam, Cambridge, USA) and rabbit anti-nWASP diluted 1∶500 (Santa Cruz) or chicken anti-nWASP diluted 1∶500 (abcam); fluorescence labeled secondary antibodies were Alexa Fluor® 488 (green, used filter set: excitation BP 450 – 490, FT 510, emission BP 515 - 565), Alexa Fluor® 568 (red, used filter set: excitation BP 534 nm–558 nm, FT 560, emission BP 575 - 640) and Alexa Fluor® 647 (magenta used filter set: excitation BP 610 nm–670 nm, FT 660, emission BP 640–740 (all from Invitrogen) all diluted 1∶500.
Techniques: Staining, Immunoprecipitation, In Vivo, Magnetic Beads, Construct, Transfection, Cotransfection, Expressing