Journal: Nature Communications

Article Title: Cdk5 and GSK3β inhibit fast endophilin-mediated endocytosis

doi: 10.1038/s41467-021-22603-4

Figure Lengend Snippet: a Top, diagram showing the recruitment of CRMP2-5 adaptor complex (CRMP4 highlighted in blue) to Plexin A1 upon stimulation with Semaphorin 3A. Bottom, human CRMP4 protein sequence (aa 508–530). Amino acids phosphorylated by GSK3β (T509, T514, and S518) and by Cdk5 (S522) are shown in blue and red, respectively. The Endophilin binding motif established in this study is shown in orange (underlined). b Co-immunoprecipitation of CRMP4-EGFP and Endophilin A2-Myc from cells treated with 5 μM CHIR-99021 (GSK3i) or Dinaciclib (Cdk5i) as indicated. I, input (10% of the cell extracts), U, unbound (10% of total) and B, bound fractions (80% of total), respectively. c Co-immunoprecipitation of CRMP4-EGFP wild-type (WT), S22D or S522A and Endophilin A2-Myc. I, input (10% of the cell extracts), and B, bound fractions (90% of total), respectively. d Pull-down using GST-SH3 domains of Endophilin A1, A2, or A3 and cell extracts expressing the indicated EGFP-tagged CRMP proteins. GST was used as negative control. Binding proteins were detected by immunoblotting with an anti-EGFP antibody. input lanes correspond to 5% of the cell extracts. e Recruitment of EGFP-tagged CRMP4 WT, S522A, S522D, or R525E onto FEME carriers (cytoplasmic Endophilin-positive assemblies, EPAs) in HUVEC cells. Arrowheads point at FEME carriers. Scale bars, 5 μm. Histograms show the mean ± SEM from three independent biological experiments ( n = 30 cells per condition). Statistical analysis was performed by one-way ANOVA; ns non significant; * P < 0.05, ** P < 0.01, *** P < 0.001. f Recruitment of endogenous CRMP4 onto FEME carriers in HUVEC cells treated for 10 min with 5μM Dinaciclib (Cdk5i) and/or CHIR-99021 (GSK3i), or left untreated (resting). Arrowheads point at FEME carriers. Scale bars, 5 μm. Histograms show the mean ± SEM from three independent biological experiments ( n = 45 cells per condition). Statistical analysis was performed by one-way ANOVA; ns non significant; ***, P < 0.001. g Endogenous Plexin A1 uptake into FEME carriers in HUVEC cells depleted of Endophilin A1, A2, and A3 (Endophilin TKD), Cdk5 and GSK3α and β (CDK5 + GSK3α/β TKD), CRMP4 (CRMP4 KD) or AP2 (AP2 KD) or pre-treated with Cdk5i and/or GSK3i for 5 min. Cells were stimulated by 20 nM Semaphorin 3A (Sema3A) for 5 min in presence of 10 μg/mL anti-PlexinA1 antibodies (recognizing the ectodomain of PlexinA1) or left untreated (resting). Arrowheads point at FEME carriers. Scale bars, 5 μm. Histograms show the mean ± SEM from three independent biological experiments ( n = 30 cells per condition). Statistical analysis was performed by one-way ANOVA; ns non significant; *** P < 0.001. h Axon length of mouse hippocampal neurons expressing EGFP-CRMP4 wild type (WT), EGFP-CRMP4 S522A or EGFP-CRMP4 R525E mutants. Histograms show the mean ± SEM of 73, 93, and 38 axons from 4 independent biological experiments, respectively. Statistical analysis was performed by one-way ANOVA; *, P < 0.05, **, P < 0.01. i, Total (Left) or Interstitial (Right) axon branching of mouse hippocampal neurons expressing EGFP-CRMP4 wild type (WT), EGFP-CRMP4 S522A or EGFP-CRMP4 R525E mutants. Histograms show the mean ± SEM of 12, 12 and 24 captures from 3 independent biological experiments, respectively. Statistical analysis was performed by one-way (Right) two-way (Left) ANOVA; ns non significant; * P < 0.05, ** P < 0.01. j Axon growth cone collapse in resting or neurons treated with 5 nM of semaphorin 3A and expressing EGFP-CRMP4 wild type (WT), EGFP-CRMP4 S522A or EGFP-CRMP4 R525E mutants. Scale bar, 20 μm. Histograms show the mean ± SEM of 13, 10, and 12 captures from three independent biological experiments, respectively. Statistical analysis was performed by two-way ANOVA; ns non significant; *** P < 0.001.

Article Snippet: Full length and truncated genes (all human , unless specified) were amplified and cloned into pDONR201 (Invitrogen) and transferred into pEGFP, pTagRFP-T (called RFP in this study), pMyc, or pGEX-6P2 vectors converted into the Gateway system (pDEST vectors made from a pCI backbone), as appropriate: Endophilin-A2 ( SH3GL1 , IMAGE 3458016) full length and SH3 domain (aa 311-end); Endophilin-A1 ( SH3GL2 iso1 , FLJ 92732) full length and SH3 domain (aa 295-end); Endophilin-A3 ( SH3GL3 iso 1, IMAGE 5197246) full length and SH3 domain (aa 291-end); Bin1, also known as Amphiphysin-II ( BIN1 iso9 , cloned from human brain cDNA library) full length and SH3 domain (aa 366-end); full length CRMP2 ( DPYSL2 , DNASU HsCD00513405), full length CRMP3 ( DPYSL4 , NM_006426 Origene), full length mouse CRMP4 ( DPYSL3 , Origene 1197294), full length CRMP5 ( DPYSL5 , amplified from human brain cDNA library, Novagen), Ephrin receptor A1 cytoplasmic tail (aa 568-976) ( EPHA1 , DNASU HsCD00516390), Ephrin receptor A6 cytoplasmic tail (aa 572-1036) ( EPHA6 , DNASU HsCD00350501), Ephrin receptor B1 cytoplasmic tail (aa 259-346) ( EPHB1 , DNASU HsCD00038738), Ephrin receptor B4 cytoplasmic tail (aa 561-987) ( EPHB4 , DNASU HsCD00021508), Ephrin receptor B6 cytoplasmic tail (aa 616-1021) ( EPHB6 , DNASU HsCD00505529), Semaphorin 4D cytoplasmic tail (aa 756–862) ( SEMA4D , amplified from human brain cDNA library, Novagen), Semaphorin 4F cytoplasmic tail (aa 681–770) ( SEMA4F , DNASU HsCD00041427), Semaphorin 6A cytoplasmic tail (aa 671–1030) ( SEMA6A , Sino Biologica HG11189-M), Semaphorin 6B cytoplasmic tail (aa 616–1021) ( SEMA6B , amplified from human brain cDNA library, Novagen); Semaphorin 6D cytoplasmic tail (aa 684–1073) ( SEMA6D , DNASU HsCD00516397); Plexin B1 cytoplasmic tail (aa 1512–2135) ( PLXNB1 , Addgene 25352), mouse Roundabout homolog 1 cytoplasmic tail (aa 880–1612) ( ROBO1 , DNASU HsCD00295416); Roundabout homolog 3 cytoplasmic tail (aa 912–1386) ( ROBO3 , DNASU HsCD00302878) and Netrin receptor UNC5B cytoplasmic tail (aa 398–945) ( UNC5B , DNASU HsCD294959), EGFP-p27 (Addgene #15192); EGFP-p150Glued (Addgene #36154).

Techniques: Sequencing, Binding Assay, Immunoprecipitation, Expressing, Negative Control, Western Blot

Journal: Nature Communications

Article Title: Cdk5 and GSK3β inhibit fast endophilin-mediated endocytosis

doi: 10.1038/s41467-021-22603-4

Figure Lengend Snippet: a Colocalization of named EGFP-tagged BAR proteins on FEME carriers marked by endogenous Endophilin in BSC1 cells stimulated with additional 10% serum for 10 min prior to fixation. Histograms show the mean ± SEM from three independent biological experiments ( n > 100 puncta per condition). Statistical analysis was performed by one-way ANOVA; NS non significant; P values as indicated. b Colocalization of Bin1-EGFP on FEME carriers marked by endogenous Endophilin in BSC1 cells stimulated with additional 10% serum for 10 min prior to fixation. Arrowheads point at FEME carriers. Scale bar, 20 μm (main) and 10 μm (inset). c Colocalization of endogenous Bin1 and Endophilin upon stimulation with dobutamine, after treatment with 5 μM Dinaciclib (Cdk5i) and CHIR-99021 (GSK3i) for 10 min, or in cells depleted of Bin1 Amphiphysin (Amph + Bin1 DKD). Arrowheads point at FEME carriers. Scale bars, 5 μm. Histograms show the mean ± SEM from three independent biological experiments ( n > 150 puncta per condition). Statistical analysis was performed by one-way ANOVA; * P < 0.05, *** P < 0.001. d Pull-down experiments using beads with GST-SH3 domains of Endophilin A2 or Bin1, in resting cells or cells treated with extra 10% FBS for 10 min. GST beads were used as negative control. Inputs correspond to 4% of cell extracts. Bottom, histograms show the mean ± SEM of Dynein binding, normalized to resting GST levels from three independent biological experiments. Statistical analysis was performed by two-way ANOVA; ns non significant; *** P < 0.001. e Colocalisation between Bin1 and Dynein in cells stimulated with extra 10% serum (top), and between endophilin and dynein upon Amphiphysin/Bin1 double knock-down (DKD)(bottom). Arrowheads point at FEME carriers. Scale bars, 5 μm. Histograms show the mean ± SEM from three independent biological experiments ( n > 50 puncta per condition). Statistical analysis was performed by one-way ANOVA; ***, P < 0.001 . f Model: multi-layered regulation of FEME by Cdk5 and GSK3β: 1) obstruction of CRMP4 binding to Endophilin and thus PlexinA1 sorting into FEME carriers upon Semaphorin 3A stimulation, 2) inhibiton of Dynamin recruitment onto FEME carriers, thus inhibiting vesicle budding and 3) hinderance of Dynein recruitment by Bin1, thereby reducing FEME carriers movement. GSK3β binds to Endophilin and acts locally to hold off FEME. In cells exposed to growth factors, PI3K-mediated signaling activates AKT and other kinases that controls GSK3β activity, and thus license cells for FEME.

Article Snippet: Full length and truncated genes (all human , unless specified) were amplified and cloned into pDONR201 (Invitrogen) and transferred into pEGFP, pTagRFP-T (called RFP in this study), pMyc, or pGEX-6P2 vectors converted into the Gateway system (pDEST vectors made from a pCI backbone), as appropriate: Endophilin-A2 ( SH3GL1 , IMAGE 3458016) full length and SH3 domain (aa 311-end); Endophilin-A1 ( SH3GL2 iso1 , FLJ 92732) full length and SH3 domain (aa 295-end); Endophilin-A3 ( SH3GL3 iso 1, IMAGE 5197246) full length and SH3 domain (aa 291-end); Bin1, also known as Amphiphysin-II ( BIN1 iso9 , cloned from human brain cDNA library) full length and SH3 domain (aa 366-end); full length CRMP2 ( DPYSL2 , DNASU HsCD00513405), full length CRMP3 ( DPYSL4 , NM_006426 Origene), full length mouse CRMP4 ( DPYSL3 , Origene 1197294), full length CRMP5 ( DPYSL5 , amplified from human brain cDNA library, Novagen), Ephrin receptor A1 cytoplasmic tail (aa 568-976) ( EPHA1 , DNASU HsCD00516390), Ephrin receptor A6 cytoplasmic tail (aa 572-1036) ( EPHA6 , DNASU HsCD00350501), Ephrin receptor B1 cytoplasmic tail (aa 259-346) ( EPHB1 , DNASU HsCD00038738), Ephrin receptor B4 cytoplasmic tail (aa 561-987) ( EPHB4 , DNASU HsCD00021508), Ephrin receptor B6 cytoplasmic tail (aa 616-1021) ( EPHB6 , DNASU HsCD00505529), Semaphorin 4D cytoplasmic tail (aa 756–862) ( SEMA4D , amplified from human brain cDNA library, Novagen), Semaphorin 4F cytoplasmic tail (aa 681–770) ( SEMA4F , DNASU HsCD00041427), Semaphorin 6A cytoplasmic tail (aa 671–1030) ( SEMA6A , Sino Biologica HG11189-M), Semaphorin 6B cytoplasmic tail (aa 616–1021) ( SEMA6B , amplified from human brain cDNA library, Novagen); Semaphorin 6D cytoplasmic tail (aa 684–1073) ( SEMA6D , DNASU HsCD00516397); Plexin B1 cytoplasmic tail (aa 1512–2135) ( PLXNB1 , Addgene 25352), mouse Roundabout homolog 1 cytoplasmic tail (aa 880–1612) ( ROBO1 , DNASU HsCD00295416); Roundabout homolog 3 cytoplasmic tail (aa 912–1386) ( ROBO3 , DNASU HsCD00302878) and Netrin receptor UNC5B cytoplasmic tail (aa 398–945) ( UNC5B , DNASU HsCD294959), EGFP-p27 (Addgene #15192); EGFP-p150Glued (Addgene #36154).

Techniques: Negative Control, Binding Assay, Activity Assay