Journal: eLife

Article Title: Proteolytically released Lasso/teneurin-2 induces axonal attraction by interacting with latrophilin-1 on axonal growth cones

doi: 10.7554/eLife.37935

Figure Lengend Snippet: ( A ) Recombinant Lasso constructs used in this work (FS, full size). The three proteolytic cleavage sites and the SS splice site are indicated. The antibody recognition sites/epitopes are shown by bars above the structure. Scale bar, 200 amino acids. ( B ) Intracellular processing and release of TENs. Left, TEN2 is constitutively cleaved in the trans-Golgi vesicles by furin at site 1. Middle, when delivered to the cell surface, the ECD remains tethered to the membrane and functions as a cell-surface receptor. Right, regulated cleavage at site 3 releases the ECD into the medium. ( C ) Expression of Lasso and release of its ECD fragment in hippocampal neurons in culture. Rat hippocampal neurons were cultured for 3, 7 and 14 days, and proportionate amounts of the conditioned media and cell lysates were separated by SDS-PAGE. A Western blot (representative of three independent experiments, which all gave similar results) was stained for Lasso, LPHN1, neurofilament-H (NF-H), and actinin. The doublet bands corresponding to splice variants of full-size Lasso (FS) and the fragment of ECD (Frag.) cleaved at site 1 are indicated by arrowheads. ( D ) Quantification of Western blots (as in C), using Lasso C-terminus staining data. ( E ) Axonal growth cones (white arrowheads) do not express Lasso/teneurin-2. Neurons in a 9 DIV hippocampal culture were permeabilized and stained for the axonal protein Tau (green) and Lasso (TN2C, red) (representative image from n = 5 experiments). ( F ) A detailed study of growth cones. Hippocampal neurons were transfected with a vector encoding GFP, then, after 14 DIV, stained for LPHN1 (PAL1 and Alexa 647-conjugated secondary antibody, magenta), and axonal growth cones were visualized by GFP fluorescence (green). ( G, H ) Correlation of LPHN1 polarization within a growth cone with its recent travel trajectory. G left, a fluorescent image of a growth cone stained for LPHN1 (magenta). G right, the same image in false color (contour based on GFP staining), demonstrating LPHN1 polarization on the right side. H left, the contours of 13 roughly symmetrical growth cones and their preceding axons were aligned to locate the stronger LPHN1 staining on the right. Note, that all axons approach growth cones from the right low quadrant. H right, the proportion of right- and left-turning growth cones plotted with Jeffreys 99.73% confidence intervals for a binomial parameter; ***, p<0.001; n = 13. ( I ). LPHN1 is found within filopodia and lamellipodia on the leading edge (left, arrowheads), but not on the trailing edge (right) of a growth cone. Green, GFP fluorescence; magenta, PAL1 staining for LPHN1. 10.7554/eLife.37935.004 Figure 1—source data 1. Source data for , Panels D and H.

Article Snippet: The following antibodies were used in this work: Rabbit anti-NF-H (Neuromics, RA22116); mouse anti-MAP-2 (Neuromics, MO22116); mouse monoclonal anti-V5 (clone SV5-Pk1, AbD Serotec/Bio-Rad, MCA1360); rabbit anti-V5 (Thermo Fisher Scientific, PA1-29324; RRID: AB_1961277 ); mouse monoclonal anti-myc (clone 9E10, Millipore, 05–419; RRID: AB_309725 ); chicken anti-myc (Millipore, AB3252; RRID: AB_2235702 ); mouse anti-FLAG M2 (Sigma-Aldrich, F3165; RRID: AB_259529 ); anti-FLAG M2 affinity gel (Sigma-Aldrich, A2220); mouse anti-actinin (Sigma-Aldrich, A7811); rabbit polyclonal anti-LPHN1-peptide (PAL1, ( ); rabbit polyclonal anti-LPHN1 NTF (RL1) ( ); mouse anti-Lasso/TEN2 C-terminus (TN2C, dmAb) ( ); sheep anti-TEN2 N-terminus (TN2N, R and D systems, AF4578; RRID: AB_10719438 ); mouse anti-synapsin (Santa-Cruz Biotechnology, sc-376623; RRID: AB_11150313 ); rabbit anti-PSD-95 (Millipore, AB9708; RRID: AB_11212529 ); rabbit anti-Tau (Synaptic Systems, 314 002; RRID: AB_993042 ); rabbit anti-GFP (Thermo Fisher Scientific, A-11122; RRID: AB_221569 ).

Techniques: Recombinant, Construct, Cell Surface Receptor Assay, Expressing, Cell Culture, SDS Page, Western Blot, Staining, Transfection, Plasmid Preparation, Fluorescence

Journal: eLife

Article Title: Proteolytically released Lasso/teneurin-2 induces axonal attraction by interacting with latrophilin-1 on axonal growth cones

doi: 10.7554/eLife.37935

Figure Lengend Snippet:

Article Snippet: The following antibodies were used in this work: Rabbit anti-NF-H (Neuromics, RA22116); mouse anti-MAP-2 (Neuromics, MO22116); mouse monoclonal anti-V5 (clone SV5-Pk1, AbD Serotec/Bio-Rad, MCA1360); rabbit anti-V5 (Thermo Fisher Scientific, PA1-29324; RRID: AB_1961277 ); mouse monoclonal anti-myc (clone 9E10, Millipore, 05–419; RRID: AB_309725 ); chicken anti-myc (Millipore, AB3252; RRID: AB_2235702 ); mouse anti-FLAG M2 (Sigma-Aldrich, F3165; RRID: AB_259529 ); anti-FLAG M2 affinity gel (Sigma-Aldrich, A2220); mouse anti-actinin (Sigma-Aldrich, A7811); rabbit polyclonal anti-LPHN1-peptide (PAL1, ( ); rabbit polyclonal anti-LPHN1 NTF (RL1) ( ); mouse anti-Lasso/TEN2 C-terminus (TN2C, dmAb) ( ); sheep anti-TEN2 N-terminus (TN2N, R and D systems, AF4578; RRID: AB_10719438 ); mouse anti-synapsin (Santa-Cruz Biotechnology, sc-376623; RRID: AB_11150313 ); rabbit anti-PSD-95 (Millipore, AB9708; RRID: AB_11212529 ); rabbit anti-Tau (Synaptic Systems, 314 002; RRID: AB_993042 ); rabbit anti-GFP (Thermo Fisher Scientific, A-11122; RRID: AB_221569 ).

Techniques: Immunocytochemistry, Western Blot, Purification, Isolation, Recombinant, Blocking Assay, Expressing, Sequencing, Negative Control, Software