Journal: Development (Cambridge, England)
Article Title: WNT7A-positive dendritic cytonemes control synaptogenesis in cortical neurons
doi: 10.1242/dev.202868
Figure Lengend Snippet: Membrane-tethered WNT7A-GFP can cluster Wnt and synaptic components. (A) Schematic of the tethering experiment using double transfection of WNT7A-GFP and the morphotrap nanobody construct, variable heavy domain of the heavy chain (Vhh)-CD8-mCherry, which binds GFP-tagged proteins. (B,C) AGS cells transfected with soluble-GFP (secGFP) and mem-mCherry (B) and AGS cells transfected with secGFP and morphotrap (C). Orange arrow indicates membrane localisation of secGFP. (D) Quantification identifies a significant colocalisation between the normally secreted secGFP and the morphotrap at the plasma membrane. An unpaired, one-tailed Student's t -test (**** P <0.001). (E) The colocalisation of mem-mCherry and WNT7A-GFP on protrusions is shown in white. (F,G) Significantly higher WNT7A-GFP colocalisation on filopodia is observed when co-transfected with morphotrap (F), quantified by Pearson's correlation coefficient (PCC; G). Unpaired, one-tailed Student's t -test (* P <0.05). (H,H′) Super-resolution imaging of iPSC-derived cortical neurons transfected with mem-GFP and mem-mCherry, followed by post-staining for LRP6 and the actin cytoskeleton (phalloidin, Phn). (I,I′) Transfection with WNT7A-GFP and mem-mCherry, followed by post-staining for LRP6 and the actin cytoskeleton identified WNT7A-GFP-positive protrusions that cluster LRP6 in apposed cells (I′, yellow circle). (J,J′) Transfection with WNT7A-GFP and morphotrap, followed by post-staining for LRP6 and cytoskeleton, identified protrusions harbouring membrane-tethered WNT7A-GFP that cluster LRP6 in apposed cells (J′, yellow circles). (K) Quantification of co-clustering of LRP6 with WNT7A-GFP identified a significant increase in both the morphotrap and mem-mCherry conditions compared to mem-Ch alone. Unpaired, one-tailed Student's t -test (* P <0.05, ** P <0.01). (L,L′) Transfection with mem-GFP and mem-mCherry, followed by post-staining for PSD95 and the actin cytoskeleton (Phn). (M,M′) Transfection with WNT7A-GFP and mem-mCherry, followed by post-staining for PSD95 and actin, revealed colocalisation on dendritic protrusions (M′, yellow circle). (N,N′) WNT7A-GFP co-transfected with morphotrap and colocalised with PSD95 on dendritic protrusions (N′, yellow circle). (O) Quantification of the colocalisation of PSD95 with WNT7A-GFP also identified a significant increase in both the morphotrap and mem-mCherry conditions compared to mem-Ch alone. Experiments were performed on three independent biological replicates. A minimum of three transfected neurons were quantified per condition. Statistical significance was addressed using one-way ANOVA with Dunnett's post-hoc test for multiple comparisons, comparing groups to the mem-mCherry/mem-GFP control group. * P <0.05. In H-J,L-N, boxed areas indicate the area shown at higher magnification in H′-J′,L′-N′.
Article Snippet: The following plasmids were used in transfections: pCS2+-membrane-mCherry , pCAG-mGFP membrane-bound GFP (Addgene # 14757 ), pcDNA3.1-WNT7A-GFP, pcDNA3.2-WNT7A-V5 (Addgene # 43816 ), 7×TRE-SuperTOPFlash-NLS-mCherry , pCS2+-LifeAct-GFP, pCAG-PSD95.FingR-eGFP-CCR5TC (Addgene # 46295 ), Lck-mScarlet-I (Addgene # 98821 ), pCS2+-Vhh-CD8-mCherry [cloned from Gal4/LexA-Vhh-CD8-mCherry (M. Affolter, University of Basel, Switzerland) into pCS2+ vector using XhoI/XbaI], pCS2+-GAP43-jGCaMP7 s [cloned from pGP-CMV-jGCaMP7 s (Addgene # 104463 ) into pCS2+-GAP43-GFP using XbaI/SnaBI], pCS2+LRP6-eGFP (gift from G. Davidson, KIT, Karlsruhe, Germany), GFP-Bsn 95-3938 (gift from Eckart Gundelfinger, Leibniz Institute for Neurobiology, Magdeburg, Germany), pcDNA3.1-Notum-CD8-mCh [assembled by Gibson cloning with pcDNA3.1-hNotumFL (gift from J. P. Vincent, Francis Crick Institute, London, UK) and pCS2+-Vhh-CD8-mCherry], PSD-95-pTagRFP (Addgene # 52671 ).
Techniques: Membrane, Transfection, Construct, One-tailed Test, Imaging, Derivative Assay, Staining, Control
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