gift fromthierrygalli  (Addgene inc)

Journal: Nature Communications

Article Title: Spatiotemporal proteomics reveals the biosynthetic lysosomal membrane protein interactome in neurons

doi: 10.1038/s41467-024-55052-w

Figure Lengend Snippet: a Heatmap in which the Log 2 Fold Change across time points of axonal synaptic proteins and members of the SNARE complex is plotted (two-sided t-test, no multiple comparison test was used). b Live neuron expressing RUSH-LAMP2A-mNG, RUSH-SYT1-Halo and scramble, imaged during 1 h of biotin addition. Still images show part of the Golgi and a budding event. Intensity profile graph in the bottom. c Neurons expressing RUSH-LAMP2A-mNG and RUSH-SYT1-Halo at 1 and 4 h post-release. Kymographs from live cell imaging along the axon every 1 s for 180 s are shown. Colocalized anterograde (blue), retrograde (orange) or stationary (grey) trajectories were traced on the right. d Quantification of the number of trajectories for 1 and 4 h. n = 15 and 19 neurons; each from 3 independent experiments ( N = 3; **** p < 0.0001, * p = 0.0433, ns p = 0.6092) e Confocal images of neurons expressing RUSH-LAMP1-V5 and EGFP-VAMP4, 1 h after release. Blue and orange boxes indicate magnified areas shown on the right, with corresponding intensity profile graph. f , g Confocal images of neurons expressing RUSH-LAMP1-V5 ( f ) or RUSH-SYT1-mNG ( g ) plus shRNA against VAMP4, or scramble. Quantifications of the number of RUSH-LAMP1 ( n = 19 and 27 neurons; each from 4 independent experiments N = 4, **** p < 0.0001) and SYT1 ( n = 27 and 22 cells; each from 3 independent experiments N = 3, **** p < 0.0001) positive compartments are shown on the right. h Still images from the soma of neuron expressing RUSH-LAMP2a-mNG, RUSH-SYT1-Halo and shRNA against VAMP4; control scramble in ( b ). Images show a Golgi budding event after 1 h release. Corresponding intensity profile graph on the right. i Neurons expressing RUSH-LAMP2A-mNG and shRNA against VAMP4 or scramble after 1 h release and immunostained for LAMTOR4 with respective intensity profile graphs are shown. j Neurons in ( h ) were labeled for SirLyso and imaged live after 1 h release. Still images from time points indicated in images and respective intensity profile graphs are shown. k Temporal intensity profile graph for RUSH-SYT1 and SirLyso from image in ( j ). Scale bars, 2 µm in ( b ), ( h – j ), 5 µm in ( f ), ( g ), and 10 µm in ( e ). Data are presented as mean values ± SD, plus individual points. Mann-Whitney test was used in ( d ), ( f ), and ( g ). See also Supplementary Figs. and . Representative images in b , e , and i were repeated in at least 3 independent experiments. Source data are provided as a Source Data file.

Article Snippet: The following vectors were used: FUGW was a gift from David Baltimore (Addgene plasmid # 14883) , pLKO.1 puro was a gift from Bob Weinberg (Addgene plasmid # 8453) , psPAX2 and pMD2.G were gifts from Didier Trono (Addgene plasmids # 12260 and # 12259) pmScarlet-i_C1 was a gift from Dorus Gadella (Addgene plasmid # 85044) , H2B-mNeonGreen-IRESpuro2 was a gift from Daniel Gerlich (Addgene plasmid # 183745) , LAMP1-RFP was a gift from Walther Mothes (Addgene plasmid # 1817) , pEGFP-VAMP4 was a gift from Thierry Galli (Addgene plasmid # 42313) , Str-KDEL_SBP-EGFP-E-cadherin was a gift from Franck Perez (Addgene plasmid # 65286) , mito-V5-APEX2 was a gift from Alice Ting (Addgene plasmid # 72480) , pAAV hSyn GFP-FXR1 was a gift from Martin Beaulieu (Addgene plasmid # 112732) , LAMP1-GFP was a gift from Dr. Juan Bonifacino, GFP-RAB6A, GFP-RAB7A and GFP-RAB11A were gifts from Casper Hoogenraad , pAAV ORANGE Gria1-HaloTag was a gift from Harold MacGillavry, PB-Ef1a-PCP-Halo (Addgene plasmid # 198337) and PB-Ef1a-β-actin-UTR-PP7 mRNA were gifts from Michael Ward.

Techniques: Comparison, Expressing, Live Cell Imaging, shRNA, Control, Labeling, MANN-WHITNEY