Journal: eLife

Article Title: CRMP4-mediated fornix development involves Semaphorin-3E signaling pathway

doi: 10.7554/eLife.70361

Figure Lengend Snippet: ( A ) Coronal sections of embryonic day 17.5 (E17.5) wild-type (WT) (columns a , c, d ) and CRMP4-KO (columns b , e ) brain were immunolabeled with anti-CRMP4 and anti-Nrp1 antibodies. White squares (columns a and b) indicate the positions of fields of view shown in columns c–e. Abbreviations: ac: anterior commissure, fi: fimbria; pf: post-commissural fornix. Scale bars, 100 µm. ( B ) Cultured subicular neurons at 2 days in vitro (DIV) were immunolabeled with anti-CRMP4 and anti-Nrp1 antibodies. The conventional confocal plane shows the entire neuron. Images from the confocal Airyscan detail the growth cone on two serial acquisition planes. Scale bars, 10 µm.

Article Snippet: The plasmid coding for human CRMP4-tGFP (aa 1–570, in pCMV6-AC-tGFP) was from Origene (Rockville, MD) (RG230865, # NM_001197294, NP_001184223).

Techniques: Immunolabeling, Cell Culture, In Vitro

Journal: eLife

Article Title: CRMP4-mediated fornix development involves Semaphorin-3E signaling pathway

doi: 10.7554/eLife.70361

Figure Lengend Snippet: ( A ) Schematic representation of the fornix tract (red region) on a sagittal diagram. Grey lines indicate the Bregma positions, in mm, of the coronal planes presented in B, C, and D. ( B–D ) Coronal brain sections from adult wild-type (WT) and CRMP4-knockout (KO) brains were stained with gold chloride. Diagrams of adult brain coronal slices illustrating the anatomical levels selected to measure surfaces covered by the fornix (pf) and mammillo-thalamic tract (mt). Square in the diagrams indicate the regions shown on the coronal sections stained with gold chloride. Histograms show quantifications of fiber areas and fasciculation index, mean ± s.e.m. WT = 5, KO = 4. Mann-Whitney test, *p < 0.05. Scale bar, 250 µm. Abbreviations: HF: hippocampal formation; MB: mammillary body; AH: anterior hypothalamus; SPT: septum; pre-f: pre-commissural fornix; pf: post-commissural fornix; mt: mammillo-thalamic tract; mcht, medial cortico-hypothalamic tract; ac: anterior commissure; S: subiculum. Figure 5—source data 1. Raw data of panels A-D.

Article Snippet: The plasmid coding for human CRMP4-tGFP (aa 1–570, in pCMV6-AC-tGFP) was from Origene (Rockville, MD) (RG230865, # NM_001197294, NP_001184223).

Techniques: Knock-Out, Staining, MANN-WHITNEY

Journal: eLife

Article Title: CRMP4-mediated fornix development involves Semaphorin-3E signaling pathway

doi: 10.7554/eLife.70361

Figure Lengend Snippet: Coronal sections of the subiculum from adult WT and CRMP4-knockout (KO) animals expressing the reporter eYFP in the subiculum. The dorsal subiculum shown in the diagram of Bregma –2.80 mm section ( A ) was delimited on the NeuN immunolabeling slice (B, dashed region of interest [ROI]). The density of eYFP-stained subicular neurons in WT and CRMP4-KO dorsal subicula was quantified, and the results are shown in ( C ). Difference between genotypes was not significant (n = 4 animals for each genotype).

Article Snippet: The plasmid coding for human CRMP4-tGFP (aa 1–570, in pCMV6-AC-tGFP) was from Origene (Rockville, MD) (RG230865, # NM_001197294, NP_001184223).

Techniques: Knock-Out, Expressing, Immunolabeling, Staining

Journal: eLife

Article Title: CRMP4-mediated fornix development involves Semaphorin-3E signaling pathway

doi: 10.7554/eLife.70361

Figure Lengend Snippet: ( A ) Adult half-brains from wild-type (WT)- and CRMP4-KO-Thy1-eYFP-H mice were cleared by a hybrid uDISCO method (see Material and methods section). Representative broad panoramic views of z projections of WT and CRMP4-KO are shown. White squares indicate regions shown in B. Scale bar, 1 mm. Voxel size, x = 2.07, y = 2.07, z = 5.77 µm. ( B ) Higher magnifications of z projections show post-commissural fornix fibers (pf) entering mammillary bodies (MB) and mcht fibers emerging from the fornix. Arrows and arrowheads indicate the mcht fibers emerging from the anterior and posterior sides, respectively, of the anterior commissure (ac). Scale bar, 200 µm. Abbreviations: HF: hippocampal formation, MB: mammillary body; S: subiculum; SPT: septum; ac: anterior commissure; mcht: medial cortico-hypothalamic tract; pf: post-commissural fornix; and pre-f: pre-commissural fornix.

Article Snippet: The plasmid coding for human CRMP4-tGFP (aa 1–570, in pCMV6-AC-tGFP) was from Origene (Rockville, MD) (RG230865, # NM_001197294, NP_001184223).

Techniques:

Journal: bioRxiv

Article Title: Cdk5 and GSK3β inhibit Fast Endophilin-Mediated Endocytosis

doi: 10.1101/2020.04.11.036863

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 5μM 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, S522D, S522A or R525E onto FEME carriers (cytoplasmic Endophilin-positive assemblies, EPAs) in HUVEC cells. Arrowheads point at FEME carriers. Right, histograms show the mean ± SEM from biological triplicates ( n =30 cells per condition). f , Recruitment of endogenous CRMP4 onto FEME carriers in HUVEC cells treated for 10min with 5μM Dinaciclib (Cdk5i) and/or CHIR-99021 (GSK3i), or not (resting). Arrowheads point at FEME carriers. Histograms show the mean ± SEM from biological triplicates ( n =45 cells per condition). g , Endogenous Plexin A1 uptake into FEME carriers in HUVEC cells depleted of Endophilin A1, A2 and A3 (‘Endophilin TKD), Cdk5 and GSK3α and b (‘CDK5+GSK3α/β TKD’), CRMP4 (‘CRMP4 KD’) or AP2 (‘AP2 KD’) or pre-treated with Cdk5i and/or GSK3i for 5min. Cells were stimulated by 20nM Semaphorin 3A (Sema3A) for 5min in presence of 10 μg/mL anti-PlexinA1 antibodies (recognizing the ectodomain of PlexinA1) or not (resting). Arrowheads point at FEME carriers. Histograms show the mean ± SEM from biological triplicates ( n =30 cells per condition). All experiments were repeated at least three times with similar results. Statistical analysis was performed by one-way ANOVA; NS , non significant; *, P <0.05, ***, P <0.001. Scale bars, 5μm.

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’ elsewhere), 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; 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) ( EFNB4 , DNASU HsCD00021508), Ephrin receptor B6 cytoplasmic tail (aa 616-1021) ( EPHB6 , DNASU HsCD00505529), 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 25252), 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: bioRxiv

Article Title: Cdk5 and GSK3β inhibit Fast Endophilin-Mediated Endocytosis

doi: 10.1101/2020.04.11.036863

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). 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. c , Colocalization of endogenous Bin1 and Endophilin upon stimulation with dobutamine, after treatment with 5μM Dinaciclib (Cdk5i) and CHIR-99021 (GSK3i) for 10min, or in cells depleted of Bin1 Amphiphysin (‘Amph+Bin1 DKD’). Arrowheads point at FEME carriers. Right, histograms show the mean ± SEM from three independent biological experiments ( n >150 puncta per condition). 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. 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). Right, histograms show the mean ± SEM from three independent biological experiments ( n >50 puncta per condition). 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’ elsewhere), 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; 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) ( EFNB4 , DNASU HsCD00021508), Ephrin receptor B6 cytoplasmic tail (aa 616-1021) ( EPHB6 , DNASU HsCD00505529), 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 25252), 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

Journal: The Journal of Neuroscience

Article Title: Mice Deficient in Collapsin Response Mediator Protein-1 Exhibit Impaired Long-Term Potentiation and Impaired Spatial Learning and Memory

doi: 10.1523/JNEUROSCI.4497-06.2007

Figure Lengend Snippet: Generation and characterization of CRMP-1−/− mice. A, The genomic structure of CRMP-1 and the targeted alleles. The CRMP-1 exons 2–6 (filled boxes) are depicted along with restriction enzyme sites (A, AflII; Ap, ApaI; B, BamHI; C, ClaI; H, HindIII; N, NcoI; X, XhoI; R, EcoRI). The wild-type allele (+) targeted by vector I generated the allele (“t”) harboring a loxP (filled triangle)-flanked HPRT (hatched box) in intron 5. Cre excision generated the one-loxP allele. Subsequent transfection of the one-loxP-containing ES cells with vector II generated the floxed allele (fl). Subsequent excision by Cre deleted the HPRT and exons 4 and 5 [the null allele (−)]. Open bars, Probes A and B for Southern blotting. Primers (2U, 3U, 3D, 4U, 4D, 5D, and 6D) for genotyping by PCR. C* in vector I, Deleted ClaI site. B, Southern blotting of recombinant ES cell clones digested by AflII and AflII plus BamHI and detected by probes A and B. DNA in each lane, 5–10 μg. Only probe A results are shown. Lane 1, Wild-type ES cells (showing 8.3 kb “+” allele). Wild-type allele is omitted in the following descriptions. Lane 2, Vector I targeted clone (10.8 kb; “t” allele). Lanes 3–5, DNA from ES cells of lane 2 after cre-excision and successful vector II recombination (lane 3, 9.8 kb floxed “fl” allele), unsuccessful vector II recombination in cis (lane 4, 6.8 kb “loxP” allele), and successful vector II recombination in trans (lane 5, a loxP allele plus a t* allele denoting the clone with the loxP-flanked HPRT fragment in intron 3 of the wild-type allele of lane 4). Lane 6, ES cell clone in lane 7 after Cre transfection to generate the CRMP-1−/− allele, the (−) allele. Lane 7, ES cell clone in lane 3 by AflII. C, Southern blotting of AflII-digested tail DNA from pups derived from ES cell clones of lane 6 of panel B. +/+, +/−, and −/−, Wild-type, heterozygous, and homozygous CRMP-1−/− mice. Both probes A and B gave the same pattern. D, Northern blotting of 20 μg of total RNA from P7 mouse brains and kidneys, using exons 1–11 CRMP-1 cDNA and a GAPDH probe. M, RNA size maker (MBI Fermentas, Hanover, MD). E, RT-PCR analysis. Total RNA (P7) of brains and PCR primers 2U and 6D were used. The 582 and 356 bp bands represent the wild-type and knock-out alleles, respectively. F, Western blot analysis of CRMPs in whole-brain extracts from CRMP-1+/+ and CRMP-1−/− mice at P1. Tissue extract samples (15 μg) were loaded in each lane. The membrane was first reacted with anti-CRMP-1 antibody (in-house), followed by anti-CRMP-2, anti-CRMP-4, and then anti-actin antibodies (internal control) with stripping and washing steps in between each antibody reaction. Rat P1 brain, Positive control. Lanes +/+, +/−, and −/−, Wild-type, heterozygous, and homozygous CRMP-1 knock-out mice, respectively. Multiple CRMP bands were attributable to phosphorylation events.

Article Snippet: The antibodies used were anti-CRMP-1 (prepared in-house or purchased from Millipore), anti-CRMP-2 (prepared in-house), anti-CRMP-4 (BD PharMingen, San Diego, CA), anti-growth-associated protein-43 (GAP-43) (Abcam, Cambridge, UK), and anti-microtubule-associated protein 2 (MAP2) and postsynaptic density-95 (PSD95) (Millipore) and anti-actin polyclonal antibodies (Sigma, St. Louis, MO).

Techniques: Plasmid Preparation, Generated, Transfection, Southern Blot, Recombinant, Clone Assay, Derivative Assay, Northern Blot, Reverse Transcription Polymerase Chain Reaction, Knock-Out, Western Blot, Stripping Membranes, Positive Control