Journal: eLife
Article Title: Rho GTPase signaling and mDia facilitate endocytosis via presynaptic actin
doi: 10.7554/elife.92755
Figure Lengend Snippet: Figure 4. mDia facilitates synaptic vesicle (SV) endocytosis by regulating presynaptic F-actin. (A) Representative three-channel time-gated stimulated emission depletion (STED) images of synapses from hippocampal cultures transduced with shCTR or shmDia1 +3, targeting Diaph1/2 genes, fixed and immunostained for Bassoon (presynaptic marker, magenta), F-Actin (cyan) and Homer1 (postsynaptic marker, green). Scale bar, 250 nm. (B) Averaged normalized line profiles for synaptic distribution of F-Actin and Homer1 relative to Bassoon (Maximum set to 0 nm). Data are expressed as mean ± SEM. N=4 independent experiments from n=154 synapses. (C) Presynaptic F-Actin levels in synapses treated with 0.1% dimethyl sulfoxide (DMSO) (100±4.8) or 80 µM Dynasore (134.7±6.8; p=0.001, one sample Wilcoxon test) for 10 min before fixation (Representative images in Figure 4—figure supplement 1A). Cells were immunostained for Bassoon (magenta), F-Actin (cyan), and Homer1 (green). Absolute line profiles of F-Actin overlapping with Bassoon (presynapse) distribution were integrated. Data shown are normalized to DMSO (set to 100) and expressed as mean ± SEM. N=3 independent experiments from nDMSO = 207 synapses, nDynasore = 211 synapses. (D) Endocytic decay constants of vesicular glutamate transporter 1 (vGLUT1)-pHluorin traces from hippocampal neurons transduced with lentiviral particles encoding shCTR (τshCTR = 20.7 ± 0.9 s) or shmDia1 (τshmDia1 = 26.4±2.0 s) in response to 200 action potential (AP) (40 Hz, 5 s) stimulation. For rescue experiments, neurons were co-transduced with lentiviruses encoding mDia1-WT-SNAP (τshmDia1 + mDia1-WT=16.1±1.9 s) or mDia1-K994A-SNAP (τshmDia1 + mDia1-K994A=29.0±1.9 s) (pshmDia1 vs shmDia1 + mDia1-WT<0.01; pshmDia1 +
Article Snippet: resource Designation Source or reference Identifiers Additional information Recombinant DNA reagent vGlut1- mCherry Franck Polleux Kwon et al., 2016; https://doi.org/ 10.1371/journal.pbio.1002516 Expresses rat vGlut1- mCherry under a CAG promotor Recombinant DNA reagent Rac1- CA Addgene Cat# 12983; RRID:Addgene_12983 Expresses Q61L variant of human myc- Rac1 under a CMV promotor Recombinant DNA reagent Rac1- DN Addgene Cat# 12984; RRID:Addgene_12984 Expresses T17N variant of human myc- Rac1 under a CMV promotor Recombinant DNA reagent RhoA- WT Theofilos Papadopoulos Reddy- Alla et al., 2010; https://doi. org/10.1111/j.1460-9568.2010.07149.x Expresses human 3xHA- RhoA- WT under a CMV promotor Recombinant DNA reagent RhoA- DN Theofilos Papadopoulos Reddy- Alla et al., 2010; https://doi. org/10.1111/j.1460-9568.2010.07149.x Expresses T19N variant of human 3xHA- RhoA under a CMV promotor Recombinant DNA reagent RhoB- WT Harry Mellor Mellor, 1998; https://doi.org/10.
Techniques: Transduction, Marker
Addgene inc is a verified supplier 
