expression plasmids encoding memerald tagged gfap  (Addgene inc)

Journal: Nature Communications

Article Title: Multiplexed volumetric CLEM enabled by scFvs provides insights into the cytology of cerebellar cortex

doi: 10.1038/s41467-024-50411-z

Figure Lengend Snippet: Fluorescent scFv immuno-probes generated in this work

Article Snippet: After adherence, cells were transfected with mammalian expression plasmids encoding mEmerald-tagged GFAP (Addgene #54107 or Flag-tagged human calbindin (Origene # RC201358) using Lipofectamine 2000 (Thermo Fisher # 11668500) or Lipofectamine 3000 (Thermo Fisher # L3000001) transfection reagent following the manufacturer’s protocol.

Techniques: Generated, Expressing, Plasmid Preparation

Journal: Nature Communications

Article Title: Multiplexed volumetric CLEM enabled by scFvs provides insights into the cytology of cerebellar cortex

doi: 10.1038/s41467-024-50411-z

Figure Lengend Snippet: a Representative confocal images ( n = 3 experiments in each category) of different sections from the cerebellum labeled with: a calbindin-specific scFv probe conjugated with Alexa Fluor 488, a VGluT1-specific scFv probe conjugated with Alexa Fluor 532, a GFAP-specific scFv probe conjugated with 5-TAMRA, a K v 1.2-specific scFv probe conjugated with Alexa Fluor 594, and a parvalbumin-specific scFv probe conjugated with Alexa Fluor 647. The double dotted lines delineate the Purkinje cell layer. (see Supplementary Figs. and for larger fields of view). CB calbindin, VGluT1 vesicular glutamate transporter 1, GFAP glial fibrillary acidic protein, K v 1.2 potassium voltage-gated channel subfamily A member 2, PV parvalbumin, TAM 5-TAMRA. b Workflow of multicolor imaging enabled by scFv probes and linear unmixing (see the text). c Representative maximum intensity projection of the multicolor fluorescence image stack acquired by linear unmixing of confocal images ( n = 3 experiments). The signal of each fluorescent dye was pseudo-colored for better visualization. d Enlarged boxed inset from ( c ). The arrow indicates a Bergmann fiber (GFAP-positive) adjacent to the main dendrite of a Purkinje cell. Arrowhead indicates sites where axons form a pinceau structure labeled by the K v 1.2-specific scFv probe.

Article Snippet: After adherence, cells were transfected with mammalian expression plasmids encoding mEmerald-tagged GFAP (Addgene #54107 or Flag-tagged human calbindin (Origene # RC201358) using Lipofectamine 2000 (Thermo Fisher # 11668500) or Lipofectamine 3000 (Thermo Fisher # L3000001) transfection reagent following the manufacturer’s protocol.

Techniques: Labeling, Imaging, Fluorescence

Journal: Nature Communications

Article Title: Multiplexed volumetric CLEM enabled by scFvs provides insights into the cytology of cerebellar cortex

doi: 10.1038/s41467-024-50411-z

Figure Lengend Snippet: a The high-resolution EM volume acquired from the cerebellar lobule, Crus 1 with multicolor immunofluorescence from scFv probes separated by linear unmixing ( n = 1 experiment). The multicolor fluorescence data was co-registered with the high-resolution EM data. The Neuroglancer link to access the dataset is provided in the source data file. Numbers 1–4 indicate approximate regions where the ultrastructure was examined at high resolution ( n = 12 experiments). Owing to the absence of detergent in immunofluorescence labeling, fine ultrastructure was preserved throughout the EM volume, such as in the molecular layer (1), in the Purkinje cell layer (2), in the glomeruli in the granule cell layer (3), and in the granule cell bodies (4). b Demonstration of the overlay between fluorescence signals and EM ultrastructure. Left panel shows the multicolor six-channel fluorescent image of slice 250 ( n = 848 slices) of the spatially transformed fluorescence image volume. The middle panel shows three fluorescence channels corresponding to the labeling of CB, GFAP, and Hoechst overlaid onto the EM micrograph of slice 250. Right panel shows four fluorescent channels corresponding to the labeling of VGluT1, K v 1.2, PV, and Hoechst overlaid onto the EM micrograph of slice 250. Other examples of fluorescence overlay are shown in Supplementary Fig. .

Article Snippet: After adherence, cells were transfected with mammalian expression plasmids encoding mEmerald-tagged GFAP (Addgene #54107 or Flag-tagged human calbindin (Origene # RC201358) using Lipofectamine 2000 (Thermo Fisher # 11668500) or Lipofectamine 3000 (Thermo Fisher # L3000001) transfection reagent following the manufacturer’s protocol.

Techniques: Immunofluorescence, Fluorescence, Labeling, Transformation Assay

Journal: Nature Communications

Article Title: Multiplexed volumetric CLEM enabled by scFvs provides insights into the cytology of cerebellar cortex

doi: 10.1038/s41467-024-50411-z

Figure Lengend Snippet: a 2D CLEM image showing the fluorescence signal (green) of the calbindin-specific scFv probe overlapping with the cell body of a Purkinje cell. b EM image showing 2D segmentation (green) of the calbindin-positive Purkinje cell ( n = 1). c 3D reconstruction of the Purkinje cell labeled in a ( n = 1), with the cell body in dark green and a dendritic branch in light green; three parallel fibers (red) make synapses on three spine heads of the dendritic branch (arrow indicates a parallel fiber (PF); arrowhead indicates a synapse). d EM image showing 2D segmentation of the synapse (arrowhead) between a parallel fiber (red) and a spine head of the dendritic branch (green) ( n = 1). e 2D CLEM image showing fluorescence signals (red) of the GFAP-specific scFv probe overlapping with the cell body of a velate astrocyte in the granule cell layer ( n = 1). f EM image showing 2D segmentation (red) of the velate astrocyte in ( e ) ( n = 1). g 3D reconstruction of the velate astrocyte (red) labeled in ( e ) and two nearby granule cells (GC1 and GC2, light and dark blue) ( n = 2); the astrocyte extends a veil-like glial process (arrowhead) between the two granule cells. h EM image showing 2D segmentation of the glial process (arrowhead) between GC1 and GC2 ( n = 1). i 2D CLEM image showing fluorescence signals (red) of the GFAP-specific scFv probe overlapping with a Bergmann fiber ( n = 1). j EM image showing 2D segmentation (red) of the Bergmann fiber in ( i ) ( n = 1). k 3D reconstruction of two Bergmann glial cells (BG1 and BG2) ( n = 2) traced from their Bergmann fibers labeled by the GFAP-specific scFv probe. l EM image showing 2D segmentation of the cell body of BG2 ( n = 1) and a nearby basket cell ( n = 1), noting the lack of infoldings in BG2’s nuclear membrane compared to the basket cell. n indicates an example.

Article Snippet: After adherence, cells were transfected with mammalian expression plasmids encoding mEmerald-tagged GFAP (Addgene #54107 or Flag-tagged human calbindin (Origene # RC201358) using Lipofectamine 2000 (Thermo Fisher # 11668500) or Lipofectamine 3000 (Thermo Fisher # L3000001) transfection reagent following the manufacturer’s protocol.

Techniques: Fluorescence, Labeling, Membrane

Journal: eLife

Article Title: Rho GTPase signaling and mDia facilitate endocytosis via presynaptic actin

doi: 10.7554/eLife.92755

Figure Lengend Snippet: ( A ) Averaged normalized Synaptophysin-pHluorin (Syph-pH) fluorescence traces from transfected hippocampal neurons stimulated with 200 action potentials (APs) (40 Hz, 5 s) at physiological temperature (37.5 °C). Neurons were treated with 0.1% dimethyl sulfoxide (DMSO) or JLY cocktail (containing 8 µM Jasplakinolide, 5 µM Latrunculin A, and 10 µM Y-27632) as indicated. Data shown represent the mean ± SEM. N=4 independent experiments from n DMSO = 23 videos; n JLY = 36 videos. ( B ) Endocytic decay constants (τ) of Synaptophysin-pHluorin traces in A: τ DMSO = 29.1±3.4 s; τ JLY = 55.8±7.2 s; p<0.05, two-tailed student’s t-test. Data shown represent mean ± SEM. ( C ) Averaged normalized bleach-corrected vGAT-CypHer fluorescence traces from hippocampal neurons treated with DMSO or JLY cocktail in response to 200 AP (40 Hz, 5 s) stimulation. Data shown represent the mean ± SEM. N=4 independent experiments from n DMSO = 23 videos; n JLY = 29 videos. ( D ) Endocytic decay constants of vGAT-CypHer traces in C: τ DMSO = 10.9±0.7 s, τ JLY = 24.6±2.0 s; p<0.001, two-tailed unpaired student’s t-test. Data shown represent the mean ± SEM. ( E ) Averaged normalized Synaptophysin-pHluorin fluorescence traces from hippocampal neurons transfected with shRNA-encoding plasmids against no mammalian target ( shCTR ) or Diaph1 ( shmDia1 ) in response to 200 AP (40 Hz, 5 s) stimulation. Neurons were co-transfected with mDia1-mCherry (mDia1-WT) or mCherry alone ( shCTR & shmDia1 ) to exclude artifacts from overexpression. Data shown represent the mean ± SEM. N=3 independent experiments from n shCTR = 28 videos, n shmDia1 =21 videos, n shmDia1 + mDia1-WT =21 videos. ( F ) Endocytic decay constants of Synaptophysin-pHluorin traces in E: τ shCTR = 29.7±1.9 s; τ shmDia1 = 64.7 ± 3.9 s; τ shmDia1 + mDia1-WT = 30.6±3.7 s; p shCTR vs shmDia1 <0.001, p shmDia1 vs shmDia1 + mDia1-WT <0.001, one-way ANOVA with Tukey’s post-test. Data shown represent mean ± SEM. ( G ) Endocytic decay constants of averaged normalized vesicular glutamate transporter 1 (vGLUT1)-pHluorin fluorescence traces from hippocampal neurons transduced with shCTR (τ shCTR = 9.1±0.8 s), shmDia1 (τ shmDia1 = 14.3±1.5 s), or shmDia1 +3 (τ shmDia1+3 = 16.4±1.3 s) in response to 40 AP (20 Hz, 2 s) stimulation (p shCTR vs shmDia1 <0.05, p shCTR vs shmDia1+3 < 0.01, one-way ANOVA with Tukey’s post-test). Data shown represent mean ± SEM. N=4 independent experiments from n shCTR = 17 videos; n shmDia1 =19 videos; n shmDia1+3 = 18 videos. ( H ) Endocytic decay constants of averaged normalized vGLUT1-pHluorin fluorescence traces of neurons transduced with lentiviral vectors encoding shCTR (τ shCTR = 13.6±1.6 s), shmDia1 (τ shmDia1 = 22.0±3.2 s) or shmDia1 +3 (τ shmDia1+3 = 26.9±3.6 s) in response to 80 AP (40 Hz, 2 s) stimulation (p shCTR vs shmDia1+3 < 0.05, one-way ANOVA with Tukey’s post-test). Data shown represent mean ± SEM. N=4 independent experiments from n shCTR = 12 videos, n shmDia1 =15 videos, n shmDia1+3 = 18 videos. ( I ) Averaged normalized bleach-corrected vGAT-CypHer fluorescence traces from hippocampal neurons transduced with shCTR or shmDia1 +3 in response to 200 AP (40 Hz, 5 s) stimulation. Data shown represent the mean ± SEM. N=8 independent experiments from n shCTR = 37 videos, n shmDia1+3 = 35 videos. ( J ) Endocytic decay constants of vesicular γ aminobutyric acid transporter (vGAT)-CypHer traces in I: τ shCTR = 14.1±1.3 s; τ shmDia1+3 = 27.3±2.6 s; p<0.001, two-tailed unpaired student’s t-test. Data shown represent the mean ± SEM. ( K ) Averaged normalized vGLUT1-pHluorin fluorescence traces from transduced neurons in response to 80 AP (40 Hz, 2 s) stimulation. Cells were treated with 0.1% DMSO or 10 µM mDia activator (IMM) in the imaging buffer. Data shown represent mean ± SEM. N=3 independent experiments from n DMSO = 18 videos; n IMM = 16 videos. ( L ) Endocytic decay constants of vGLUT1-pHluorin traces in K: τ DMSO = 14.9±0.8 s; τ IMM = 9.8±0.5 s; p<0.05, two-tailed unpaired student’s t-test. Data shown represent mean ± SEM. Figure 1—source data 1. Numerical source data for .

Article Snippet: To generate mDia1-WT-mCherry, the sequence encoding mDia1 was cut from mDia1-mEmerald-N1 (Addgene, Cat#54157) and pasted into pmCherry-N1 by AgeI and XhoI digestion. mDia1-WT-SNAP was cloned by cutting out the coding sequence of mDia1 from mDia1-mEmerald-N1 and pasting it into pSNAP-N1 by AgeI and NheI digest of both vector and insert.

Techniques: Fluorescence, Transfection, Two Tailed Test, shRNA, Over Expression, Transduction, Imaging

Journal: eLife

Article Title: Rho GTPase signaling and mDia facilitate endocytosis via presynaptic actin

doi: 10.7554/eLife.92755

Figure Lengend Snippet: ( A ) Maxima of background-corrected Synaptophysin-pHluorin (Syph-pHluorin) fluorescence traces (surface normalized) for neurons treated with 0.1% dimethyl sulfoxide (DMSO) (1.7 ± 0.1) or JLY cocktail (1.8 ± 0.2) in response to 200 action potential (AP) stimulation (40 Hz, 5 s). Data represent mean ± SEM. N=4 independent experiments from n DMSO = 23 videos; n JLY = 36 videos. ( B ) Minima of background-corrected vesicular glutamate transporter 1 (vGAT)-CypHer fluorescence traces (surface normalization) for neurons treated with 0.1% DMSO or JLY cocktail (1.0± 0.1) in response to 200 AP stimulation (40 Hz, 5 s). Data represent mean ± SEM. Values for DMSO were set to 1. N=4 independent experiments from n DMSO = 23 videos; n JLY = 29 videos. ( C ) Averaged normalized Syph-pH fluorescence traces from transfected hippocampal neurons treated with 0.1% DMSO, JY or JL combinations (containing 8 µM Jasplakinolide, 5 µM Latrunculin A, and 10 µM Y-27632) stimulated with 200 APs (40 Hz, 5 s). Data shown represent the mean ± SEM. N=4 independent experiments from n DMSO = 24 videos; n JY = 19 videos; n JL = 21 videos. ( D ) Endocytic decay constants of fluorescence traces in C: τ DMSO = 19.0±1.9 s; τ JY = 28.5±1.6 s; τ JL = 18.2 ± 1.6 s; p DMSO vs JY <0.01, one-way ANOVA with Tukey’s post-test. Data shown represent mean ± SEM. ( E ) Averaged normalized Syph-pH fluorescence traces from transfected hippocampal neurons treated with 0.1% DMSO or 10 µM Y-27632 following 200 AP (40 Hz, 5 s) stimulation. Data shown represent the mean ± SEM. N=4 independent experiments from n DMSO = 25 videos; n Y = 18 videos. ( F ) Endocytic decay constants of fluorescence traces in E: τ DMSO = 17.0±2.2 s; τ Y = 20.2±4.3 s. Data shown represent mean ± SEM. ( G ) Maxima of background-corrected Syph-pHluorin fluorescence traces (surface normalized) for neurons transfected with shCTR (1.8±0.1) or shmDia1 (1.8 ± 0.1) in response to 200 AP stimulation (40 Hz, 5 s). Data represent mean ± SEM. N=9 independent experiments from n shCTR = 49 videos; n shmDia1 =42 videos. ( H ) Analysis of knockdown efficiency of lentiviral particles carrying shRNA against no mammalian target ( shCTR ) or Diaph1 and Diaph2 genes ( shmDia1 +3 ) in mouse hippocampal cultures harvested 12 days after transduction. Protein abundance of mDia1, mDia3, and Tubulin were immunoblotted with specific antibodies. ( I ) Averaged normalized vGLUT1-pHluorin fluorescence traces from stimulated (40 APs; 20 Hz, 2 s) hippocampal neurons transduced with lentiviruses encoding shCTR , shmDia1, or both shmDia1 and shmDia3 combined ( shmDia1 +3 ). Data represent mean ± SEM. N=4 independent experiments from n shCTR = 17 videos, n shmDia1 =19 videos, n shmDia1+3 = 18 videos. The corresponding endocytic decay constants are shown in . ( J ) Maxima of background-corrected vGLUT1-pHluorin fluorescence traces (surface normalized) for neurons transduced with shCTR (1.9±0.1) or shmDia1 +3 (1.9±0.1) in response to 40 AP stimulation (20 Hz, 2 s). Data represent mean ± SEM. N=22 independent experiments from n shCTR = 105 videos and n shmDia1+3 = 128 videos. ( K ) Averaged normalized vGLUT1-pHluorin fluorescence traces for neurons transduced with shCTR, shmDia1, or shmDia1 +3 in response to 80 AP stimulation (40 Hz, 2 s). Data represent mean ± SEM. N=4 independent experiments from n shCTR = 12 videos; n shmDia1 =15 videos; n shmDia1+3 = 18 videos. Corresponding endocytic decay constants are shown in . ( L ) Maxima of background-corrected vGLUT1-pHluorin fluorescence traces (surface normalized) for neurons transduced with shCTR (2.8±0.2), shmDia1 (2.6±0.2), or shmDia1 +3 (2.4±0.1) in response to 80 AP stimulation (40 Hz, 2 s). Data represent mean ± SEM. N=4 independent experiments from n shCTR = 12 videos; n shmDia1 =15 videos and n shmDia1+3 = 18 videos. ( M ) Minima of background-corrected vGAT-CypHer fluorescence traces (surface normalized) for neurons transduced with shCTR or shmDia1 +3 (1.0±0.2) in response to 200 AP stimulation (40 Hz, 5 s). Data represent mean ± SEM. Values for shCTR were set to 1. N=11 independent experiments from n shCTR = 45 videos and n shmDia1+3 = 42 videos. ( N ) Schematic representation of the regulation of mDia1. Binding of RhoA-GTP to the Rhotekin-Rho binding domain (RBD) (green) or application of mDia1 activator (IMM) competes with the intramolecular interaction of the N-terminal Diaphanous inhibitory domain (DID) (yellow) with the C-terminal Diaphanous autoinhibitory domain (DAD) (red) domain (see for domain structure). The release of autoinhibition leads to the dimerization of mDia formins in solution. ( O ) Maxima of background-corrected vGLUT1-pHluorin fluorescence traces (surface normalized) for neurons treated with 0.1% DMSO (1.7±0.1) or mDia activator (IMM; 1.6±0.1) in response to 80 AP stimulation (40 Hz, 2 s). Data represent mean ± SEM. N=3 independent experiments from n DMSO = 18 videos; n IMM = 16 videos. Figure 1—figure supplement 1—source data 1. Numerical source data of , B, C, D, E, F, G, I, J, K, L, M, O. Figure 1—figure supplement 1—source data 2. Original scan for the anti-mDia1 immunoblot from . Figure 1—figure supplement 1—source data 3. Original scan for the anti-mDia3 immunoblot from . Figure 1—figure supplement 1—source data 4. Original scan for the anti-tubulin immunoblot from . Figure 1—figure supplement 1—source data 5. Original scans for immunoblots from with highlighted bands and sample labels.

Article Snippet: To generate mDia1-WT-mCherry, the sequence encoding mDia1 was cut from mDia1-mEmerald-N1 (Addgene, Cat#54157) and pasted into pmCherry-N1 by AgeI and XhoI digestion. mDia1-WT-SNAP was cloned by cutting out the coding sequence of mDia1 from mDia1-mEmerald-N1 and pasting it into pSNAP-N1 by AgeI and NheI digest of both vector and insert.

Techniques: Fluorescence, Transfection, Knockdown, shRNA, Transduction, Quantitative Proteomics, Binding Assay, Western Blot

Journal: eLife

Article Title: Rho GTPase signaling and mDia facilitate endocytosis via presynaptic actin

doi: 10.7554/eLife.92755

Figure Lengend Snippet: ( A ) Representative synaptic electron micrographs from hippocampal neurons transduced with lentiviruses encoding shCTR or shmDia1 +3 , targeting Diaph1/2 genes. Postsynapse and postsynaptic cleft are colored in green and maroon, respectively. Scale bar, 250 nm. ( B ) Average number of SVs per μm 2 in boutons from shCTR (92.2±2.5) and shmDia1 +3 (81.4±2.9; p<0.0001, Mann-Whitney test) treated neurons. Data shown represent mean ± SEM. N=3 independent experiments from n shCTR = 326 synapses, n shmDia1+3 = 321 synapses. ( C ) Average number of SVs per μm 2 in synaptic boutons from hippocampal neurons transduced with lentiviruses encoding shmDia1 +3 and treated with 0.1% Vehicle (10 µM NaOAc; 83.2±2.9) or 1 µM Tetrodotoxin (TTX; 93.8±3.1; p<0.01, Mann-Whitney test) for 36 hr before chemical fixation. Data shown represent mean ± SEM from two independent experiments and n Vehicle = 225 synapses, n TTX = 221 synapses. Representative synaptic electron micrographs are shown in . The dotted line represents the average SV numbers/μm 2 in shCTR boutons treated with dimethyl sulfoxide (DMSO) from the same experiments as a reference , ( B ). ( D ) Representative electron micrographs of synapses in hippocampal neurons from wild-type (WT) or Diaph1 (encoding mDia1) knockout (KO) mice. Postsynapse and postsynaptic cleft are colored in green and maroon, respectively. Scale bar, 250 nm. ( E ) Average number of SVs per μm 2 in WT (117.6±5.3) and mDia1 KO (84.6±5.2; p<0.0001, Mann-Whitney test) boutons. Data shown represent the mean ± SEM from n WT = 103, n mDia1KO = 96 synapses (N=1). Figure 2—source data 1. Numerical source data for .

Article Snippet: To generate mDia1-WT-mCherry, the sequence encoding mDia1 was cut from mDia1-mEmerald-N1 (Addgene, Cat#54157) and pasted into pmCherry-N1 by AgeI and XhoI digestion. mDia1-WT-SNAP was cloned by cutting out the coding sequence of mDia1 from mDia1-mEmerald-N1 and pasting it into pSNAP-N1 by AgeI and NheI digest of both vector and insert.

Techniques: Transduction, MANN-WHITNEY, Knock-Out

Journal: eLife

Article Title: Rho GTPase signaling and mDia facilitate endocytosis via presynaptic actin

doi: 10.7554/eLife.92755

Figure Lengend Snippet: ( A ) Schematic representation of functional domains of mDia1. Rho-binding domain (RBD), Diaphanous inhibitory domain (DID), Dimerization domain (DD), Coiled coil domain (CC), Formin homology domain 1 (FH1), Formin homology domain 2 (FH2), Diaphanous autoinhibitory domain (DAD). The unstructured N-terminus (first 60 amino acids) contains three basic stretches and was truncated in the ΔN mutant. ( B ) Endocytic decay constants of Synaptophysin-pHluorin traces from hippocampal neurons transfected with shRNAmiR against no mammalian target ( shCTRmiR ) or Diaph1 ( shmDia1miR ) in response to 200 action potential (AP) (40 Hz, 5 s) stimulation. For rescue experiments, neurons were co-transfected with plasmids encoding mDia1-WT-mCherry (τ shmDia1miR + mDia1-WT =20.0±0.8 s), mDia1-ΔN-mCherry (τ shmDia1miR + mDia1-ΔN =34.5±2.9 s), or mCherry alone (τ shCTRmiR = 21.8±1.1 s, τ shmDia1miR = 30.4±1.9 s) to exclude artifacts from overexpression (p shCTRmiR vs shmDia1miR < 0.05; p shmDia1miR vs shmDia1miR + mDia1-WT <0.01; p shmDia1miR + mDia1-WT vs shmDia1miR + mDia1-ΔN <0.01, one-way ANOVA with Tukey’s post-test). Data shown represent mean ± SEM. N=5 independent experiments from n shCTRmiR = 41 videos, n shmDia1miR = 51 videos, n shmDia1miR + mDia1-WT =35 videos, n shmDia1miR + mDia1-ΔN =37 videos. ( C ) Representative three-channel time-gated stimulated emission depletion (STED) images of synapses from hippocampal cultures treated with 0.1% dimethyl sulfoxide (DMSO) or 80 μM Dynasore for 10 min before fixation and immunostained for Bassoon (presynaptic marker, magenta), mDia1 (cyan) and Homer1 (postsynaptic marker, green). Scale bar, 250 nm. ( D ) Averaged normalized line profiles for synaptic distribution of mDia1 and Homer1 relative to Bassoon (Maximum set to 0 nm). Data represent mean ± SEM. N=3 independent experiments from n=235 synapses. ( E ) Volcano plot of proteins associating with synaptic mDia1 analyzed by label-free proteomics of anti-mDia1 versus control (CTR) immunoprecipitates from detergent-extracted mouse synaptosomes (P2’ fraction). The logarithmic ratios of protein intensities are plotted against negative logarithmic p-values derived from a two-tailed student’s t-test. N=3 independent experiments. Each dot represents one protein. Selected cytoskeletal hits include: Actin, Myosin IIB (MyoIIB), and Rac1. Selected endocytic hits include Amphiphysin (p<0.05), Dynamin1, Endophilin-A1, PACSIN1, PACSIN2 (p<0.05), and Synaptojanin1. ( F ) Endogenous immunoprecipitation of mDia1 from detergent-extracted mouse synaptosomes (P2’ fraction) using mDia1-specific antibodies. Immunoprecipitates were analyzed by immunoblotting for mDia1, Dynamin1, and β-Actin. ( G ) Representative three-channel time-gated STED images of synapses from hippocampal cultures transduced with wild-type Dynamin1 (WT) or GTPase-deficient Dynamin1 (K44A) in response to 200 AP (40 Hz, 5 s) stimulation. Cells were immunostained for Bassoon (magenta), mDia1 (cyan), and Homer1 (green). Scale bar, 250 nm. ( H ) Presynaptic mDia1 levels in synapses treated with 0.1% DMSO (100±7.3) or 80 µM Dynasore (145.8±9.3; p=0.0001; one sample Wilcoxon test) for 10 min in response to 200 AP (40 Hz, 5 s) stimulation. Absolute line profiles of mDia1 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 n DMSO = 92 synapses, n Dynasore = 135 synapses. ( I ) Presynaptic mDia1 levels in synapses from hippocampal neurons transduced with wild-type Dynamin1 (WT; 100±6.2) or GTPase-deficient Dynamin1 (K44A; 142.9±8.3, p<0.0001, one sample Wilcoxon test) in response to 200 AP (40 Hz, 5 s) stimulation. Line profiles of mDia1 overlapping with Bassoon distribution were integrated. Data shown are normalized to Dynamin1-WT (set to 100) and expressed as mean ± SEM. N=2 independent experiments from n WT = 43 synapses, n K44A = 51 synapses. Figure 3—source data 1. Numerical source data for . Figure 3—source data 2. Original scan for the anti-mDia1 and anti-Dynamin1 immunoblots from . Figure 3—source data 3. Original scan for the anti-Actin immunoblot from . Figure 3—source data 4. Original scans for immunoblots from with highlighted bands and sample labels.

Article Snippet: To generate mDia1-WT-mCherry, the sequence encoding mDia1 was cut from mDia1-mEmerald-N1 (Addgene, Cat#54157) and pasted into pmCherry-N1 by AgeI and XhoI digestion. mDia1-WT-SNAP was cloned by cutting out the coding sequence of mDia1 from mDia1-mEmerald-N1 and pasting it into pSNAP-N1 by AgeI and NheI digest of both vector and insert.

Techniques: Functional Assay, Binding Assay, Mutagenesis, Transfection, Over Expression, Marker, Control, Derivative Assay, Two Tailed Test, Immunoprecipitation, Western Blot, Transduction

Journal: eLife

Article Title: Rho GTPase signaling and mDia facilitate endocytosis via presynaptic actin

doi: 10.7554/eLife.92755

Figure Lengend Snippet: ( A ) Membrane levels of mDia1-WT-mCherry versus mDia1-ΔN-mCherry proteins overexpressed in HEK293T cells. Membrane and cytosolic cellular fractions were isolated by ultracentrifugation and analyzed by immunoblotting with specific antibodies (LAMP1) and in-gel fluorescence of mCherry tags. ( B ) Densitometric quantification of mDia1-WT versus mDia1-ΔN (0.6±0.1; p<0.05, one sample t-test) membrane-associated protein levels. Data shown are normalized to mDia1-WT (set to 1) and expressed as mean ± SEM. Representative immunoblot is shown in A. N=5 independent experiments. ( C ) Averaged normalized Synaptophysin-pHluorin fluorescence from stimulated (200 action potentials (APs), 40 Hz, 5 s) hippocampal neurons transfected with shCTRmiR or shmDia1miR . For rescue experiments, neurons were co-transfected with plasmids encoding mDia1-WT-mCherry, mDia1-ΔN-mCherry or mCherry alone (shCTRmiR & shmDia1miR). Endocytic decay constants are shown in . ( D ) Full volcano plot of proteins from associating with synaptic mDia1 analyzed by label-free proteomics of anti-mDia1 versus control (CTR) immunoprecipitates from detergent-extracted mouse synaptosomes (P2’ fraction). The cyan dot shows the specific enrichment of mDia1 as the bait protein of the immunoprecipitation (p<0.001, two-tailed student’s t-test). N=3 independent experiments. ( E ) Endogenous co-immunoprecipitation of Myosin IIB by mDia1 from detergent-extracted mouse synaptosomes (P2’ fraction). Samples were analyzed by immunoblotting using specific antibodies against mDia1, Myosin IIB (MyoIIB), and β-Actin. ( F ) Representative three-channel time-gated STED image of a synapse from hippocampal cultures fixed and immunostained for Bassoon (presynaptic marker, magenta), Myosin IIB (cyan), and Homer1 (postsynaptic marker, green). Scale bar, 250 nm. ( G ) Averaged normalized line profiles for synaptic distribution of Myosin IIB and Homer1 relative to Bassoon (Maximum set to 0 nm). Data are expressed as mean ± SEM. N=3 independent experiments from n=267 synapses. ( H ) Rationale for quantification of presynaptic protein levels of interest. The presynapse was defined by the normalized Bassoon distribution (purple fraction, cut off at the cross-section with the Homer1 profile), and corresponding absolute individual synaptic line profiles were integrated. ( I ) Postsynaptic F-Actin levels in synapses treated with 0.1% dimethyl sulfoxide (DMSO) (42.6±3.4) or 80 µM Dynasore (56.5±3.3; p<0.001, Mann-Whitney test) for 10 min before fixation from . Data shown are normalized to presynaptic DMSO values from (set to 100) and expressed as mean ± SEM. N=3 independent experiments from n DMSO = 92 synapses, n Dynasore = 135 synapses. ( J ) Quantification of Bassoon and Homer1 levels in synapses treated with 0.1% DMSO (100.0±4.5 for Bassoon; 100.0±4.3 for Homer1) or 80 µM Dynasore (103.7±4.1 for Bassoon; 98.6±5.3) for 10 min before fixation. Data shown are normalized to DMSO values (set to 100) and expressed as mean ± SEM. N=3 independent experiments from n DMSO = 132 synapses, n Dynasore = 128 synapses. Figure 3—figure supplement 1—source data 1. Original scan for in-gel mCherry fluorescence from . Figure 3—figure supplement 1—source data 2. Original scan for the anti-LAMP1 immunoblot from . Figure 3—figure supplement 1—source data 3. Original scans from with highlighted bands and sample labels. Figure 3—figure supplement 1—source data 4. Numerical source data of , C, D, G, I, J. Figure 3—figure supplement 1—source data 5. Original scans for mCherry fluorescence in gels used for analysis are shown in . Figure 3—figure supplement 1—source data 6. Original scans for mCherry fluorescence in gels used for analysis are shown in with highlighted bands and sample labels. Figure 3—figure supplement 1—source data 7. Original scan for the anti-mDia1 immunoblot from . Figure 3—figure supplement 1—source data 8. Original scan for the anti-Myosin IIB immunoblot from . Figure 3—figure supplement 1—source data 9. Original scan for the anti-Actin immunoblot from . Figure 3—figure supplement 1—source data 10. Original scans for immunoblots in with highlighted bands and sample labels.

Article Snippet: To generate mDia1-WT-mCherry, the sequence encoding mDia1 was cut from mDia1-mEmerald-N1 (Addgene, Cat#54157) and pasted into pmCherry-N1 by AgeI and XhoI digestion. mDia1-WT-SNAP was cloned by cutting out the coding sequence of mDia1 from mDia1-mEmerald-N1 and pasting it into pSNAP-N1 by AgeI and NheI digest of both vector and insert.

Techniques: Membrane, Isolation, Western Blot, Fluorescence, Transfection, Control, Immunoprecipitation, Two Tailed Test, Marker, MANN-WHITNEY

Journal: eLife

Article Title: Rho GTPase signaling and mDia facilitate endocytosis via presynaptic actin

doi: 10.7554/eLife.92755

Figure Lengend Snippet: ( 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 ). 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 n DMSO = 207 synapses, n Dynasore = 211 synapses. ( D ) Endocytic decay constants of vesicular glutamate transporter 1 (vGLUT1)-pHluorin traces from hippocampal neurons transduced with lentiviral particles encoding shCTR (τ shCTR = 20.7 ± 0.9 s) or shmDia1 (τ shmDia1 = 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 (τ shmDia1 + mDia1-WT =16.1±1.9 s) or mDia1-K994A-SNAP (τ shmDia1 + mDia1-K994A =29.0±1.9 s) (p shmDia1 vs shmDia1 + mDia1-WT <0.01; p shmDia1 + mDia1-WT vs shmDia1 + mDia1-K994A <0.001, one-way ANOVA with Tukey’s post-test). Data are expressed as mean ± SEM. N=6 independent experiments from n shCTR = 21 videos; n shmDia1 =21 videos, n shmDia1 + mDia1-WT =16 videos, n shmDia1 + mDia1-K994A =19 videos. ( E ) Presynaptic F-Actin levels in synapses from hippocampal cultures transduced with shCTR (100±6.4) or shmDia1 +3 (58.1±2.9; p<0.001, one sample Wilcoxon test). Line profiles of F-Actin overlapping with Bassoon (presynapse) distribution were integrated. Data shown are normalized to shCTR (set to 100) and expressed as mean ± SEM. N=4 independent experiments from n shCTR = 155 synapses, n shmDia1+3 = 158 synapses. ( F ) Representative confocal and two-channel time-gated STED images of endogenous β-Actin (cyan) in vGLUT1 (magenta) positive boutons from hippocampal neurons transduced with lentiviruses encoding shCTR or shmDia1 +3 . Scale bar, 250 nm. ( G ) Analysis of presynaptic endogenous β-Actin levels in vGLUT1 positive boutons from shCTR (100±6.3) and shmDia1 +3 (47.7±4.3; p<0.0001 one-sample Wilcoxon test) transduced neurons. β-Actin STED mean intensity was measured using a confocal vGLUT1 signal as a mask. Data shown are normalized to shCTR (set to 100) and expressed as mean ± SEM from two independent experiments and n shCTR = 67 synapses, n shmDia1+3 = 53 synapses. ( H ) Endocytic decay constants of vGLUT1-pHluorin traces for neurons transduced with shCTR or shmDia1 +3 in response to 40 AP (20 Hz, 2 s) stimulation. Neurons were pre-incubated with 0.1% DMSO or 1 µM Jasplakinolide (Jasp) for 30 min in the media before imaging (τ shCTR + DMSO = 13.4±1.0 s, τ shCTR + Jasp = 15.0±2.2 s, τ shmDia1+3 + DMSO =25.0±2.7 s, τ shmDia1+3 + Jasp =15.6±2.4 s; p shCTR vs shmDia1+3 < 0.01; p shmDia1+3 + DMSO vs shmDia1+3 + Jasp <0.05, one-way ANOVA with Tukey’s post-test). Data are expressed as mean ± SEM. N=6 independent experiments from n shCTR + DMSO = 32 videos, n shmDia1+3 + DMSO =35 videos, n shCTR + Jasp = 33 videos; n shmDia1+3 + Jasp =34 videos. Figure 4—source data 1. Numerical source data for .

Article Snippet: To generate mDia1-WT-mCherry, the sequence encoding mDia1 was cut from mDia1-mEmerald-N1 (Addgene, Cat#54157) and pasted into pmCherry-N1 by AgeI and XhoI digestion. mDia1-WT-SNAP was cloned by cutting out the coding sequence of mDia1 from mDia1-mEmerald-N1 and pasting it into pSNAP-N1 by AgeI and NheI digest of both vector and insert.

Techniques: Transduction, Marker, Incubation, Imaging

Journal: eLife

Article Title: Rho GTPase signaling and mDia facilitate endocytosis via presynaptic actin

doi: 10.7554/eLife.92755

Figure Lengend Snippet: ( A ) Representative three-channel time-gated stimulated emission depletion (STED) images of synapses from hippocampal cultures treated with 0.1% dimethyl sulfoxide (DMSO) or 80 µM Dynasore for 10 min. Cells were fixed and stained for Bassoon (magenta), F-Actin (cyan), and Homer1 (green). Scale bar, 250 nm. Corresponding analysis of presynaptic F-Actin levels is shown in . ( B ) Representative three-channel time-gated STED images of synapses from hippocampal cultures transduced with Dynamin1-WT or Dynamin1-K44A. Cells were fixed and stained for Bassoon (magenta), F-Actin (cyan), and Homer1 (green). Scale bar, 250 nm. ( C ) Presynaptic F-Actin levels in synapses from neurons transduced with Dynamin1-WT (100±5.9) or Dynamin1-K44A (119.8±6.2, p<0.01, one sample t-test) in B. Absolute line profiles of F-Actin overlapping with Bassoon (presynapse) distribution were integrated. Data shown are normalized to WT (set to 100) and expressed as mean ± SEM. n WT = 54 synapses, n K44A = 49 synapses. ( D ) Averaged normalized vesicular glutamate transporter 1 (vGLUT1)-pHluorin fluorescence traces for neurons transduced with shCTR or shmDia1 in response to 200 action potential (AP) (40 Hz, 5 s) stimulation. For rescue purposes, cells were co-transduced with mDia1-WT-SNAP or mDia1-K994A-SNAP. Data are expressed as mean ± SEM. N=6 independent experiments from n shCTR = 21 videos; n shmDia1 =21 videos; n shmDia1 + mDia1-WT =16 videos; n shmDia1 + mDia1-K994A =19 videos. Corresponding endocytic decay constants are shown in . ( E ) Postsynaptic F-Actin levels in synapses transduced with shCTR (100.0±6.4) or shmDia1 +3 (89.3±6.4) from . Data shown are normalized to shCTR values (set to 100) and expressed as mean ± SEM. N=3 independent experiments from n shCTR = 206 synapses, n shmDia1+3 = 135 synapses. ( F ) Quantification of Bassoon and Homer1 levels in synapses transduced with shCTR (100.0±4.7 for Bassoon; 100.0±4.5 for Homer1) or shmDia1 +3 (101.4±4.8 for Bassoon; 92.4±4.0). Data shown are normalized to DMSO values (set to 100) and expressed as mean ± SEM. N=3 independent experiments from n shCTR = 158 synapses and n shmDia1+3 = 159 synapses. ( G ) Representative STED images of endogenous β-Actin in vGLUT1 positive synapses in hippocampal neurons transduced with shCTR or shmDia1 +3 and treated with 0.1% DMSO or 1 µM Jasplakinolide for 45 min. Neurons were co-transfected with pOrange-GFP-β-Actin knock-in and vGLUT1-mCherry plasmids before fixation and immunostaining. Scale bar, 2.5 µm. ( H ) Averaged normalized vGLUT1-pHluorin fluorescence traces for neurons transduced with shCTR or shmDia1 +3 in response to 40 AP (20 Hz, 2 s) stimulation. Neurons were pre-incubated with 0.1% DMSO or 1 µM Jasplakinolide (Jasp) for 30 min in the cell media before imaging. Data are expressed as mean ± SEM. N=6 independent experiments from n shCTR + DMSO = 32 videos, n shmDia1+3 + DMSO =35 videos, n shCTR + Jasp = 33 videos; n shmDia1+3 + Jasp =34 videos. The corresponding endocytic decay constants are shown in . Figure 4—figure supplement 1—source data 1. Numerical source data of , D, E, F, H.

Article Snippet: To generate mDia1-WT-mCherry, the sequence encoding mDia1 was cut from mDia1-mEmerald-N1 (Addgene, Cat#54157) and pasted into pmCherry-N1 by AgeI and XhoI digestion. mDia1-WT-SNAP was cloned by cutting out the coding sequence of mDia1 from mDia1-mEmerald-N1 and pasting it into pSNAP-N1 by AgeI and NheI digest of both vector and insert.

Techniques: Staining, Transduction, Fluorescence, Transfection, Knock-In, Immunostaining, Incubation, Imaging

Journal: eLife

Article Title: Rho GTPase signaling and mDia facilitate endocytosis via presynaptic actin

doi: 10.7554/eLife.92755

Figure Lengend Snippet: ( A ) Schematic representation of activation of mDia1 by RhoA-GTP and positive feedback loop of mDia1 on RhoA-GTP levels through GEF stimulation. ( B ) Representative three-channel time-gated STED image of synapses from hippocampal cultures, fixed and immunostained for Bassoon (magenta), RhoA (cyan), and Homer1 (green). Scale bar, 250 nm. ( C ) Averaged normalized line profiles for synaptic distribution of RhoA and Homer1 relative to Bassoon (Maximum set to 0 nm). Data are expressed as mean ± SEM. N=5 independent experiments from n=230 synapses. ( D ) Endocytic decay constants of averaged normalized Synaptophysin-pHluorin fluorescence traces in response to 200 action potential (AP) (40 Hz, 5 s) stimulation. Neurons were transfected with the annotated combinations of plasmids encoding wild-type (WT) or dominant-negative (DN, T19N mutation) RhoA and RhoB (τ RhoA-WT + RhoB-WT =18.4±0.7 s, τ RhoA-WT + RhoB-DN = 16.0±1.0 s, τ RhoA-DN + RhoB-WT =19.8±2.4 s, τ RhoA-DN + RhoB-DN =30.1±1.0 s; p RhoA-WT + RhoB-WT vs RhoA-DN + RhoB-DN <0.01, one-way ANOVA with Tukey’s post-test). Data shown represent mean ± SEM. N=3 independent experiments from n RhoA-WT + RhoB-WT =21 videos, n RhoA-DN + RhoB-WT =31 videos, n RhoA-WT + RhoB-DN =23 videos, n RhoA-DN + RhoB-DN =22 videos. ( E ) Analysis of RhoA activity by RhoA-GTP pulldown (PD) from whole-cell lysates (input) of mouse hippocampal neurons expressing shCTR or shmDia1 +3 using immobilized Rhotekin as a bait. Samples were analyzed by immunoblotting for mDia1, mDia3, RhoA, and Tubulin using specific antibodies. Input, 10% of material used for the pulldown. The contrast of pulldown and input blots was seperately adjusted for visualization purposes. ( F ) Densitometric quantification of RhoA-GTP normalized to total RhoA levels (input) in lysates from neurons transduced with shCTR or shmDia1 +3 (0.7±0.0, p<0.001, one sample t-test) from immunoblots exemplified in E. Values for shCTR were set to 1. Data are expressed as mean ± SEM from N=3 independent experiments. Figure 5—source data 1. Numerical source data for . Figure 5—source data 2. Original scans for the anti-mDia1, anti-Tubulin, anti-RhoA, and anti-mDia3 immunoblots from . Figure 5—source data 3. Original scans for immunoblots from with highlighted bands and sample labels. Figure 5—source data 4. Original scans for the anti-RhoA immunoblots used for analysis are shown in . Figure 5—source data 5. Original scans for immunoblots used for analysis are shown in with highlighted bands and sample labels.

Article Snippet: To generate mDia1-WT-mCherry, the sequence encoding mDia1 was cut from mDia1-mEmerald-N1 (Addgene, Cat#54157) and pasted into pmCherry-N1 by AgeI and XhoI digestion. mDia1-WT-SNAP was cloned by cutting out the coding sequence of mDia1 from mDia1-mEmerald-N1 and pasting it into pSNAP-N1 by AgeI and NheI digest of both vector and insert.

Techniques: Activation Assay, Fluorescence, Transfection, Dominant Negative Mutation, Mutagenesis, Activity Assay, Expressing, Western Blot, Transduction

Journal: eLife

Article Title: Rho GTPase signaling and mDia facilitate endocytosis via presynaptic actin

doi: 10.7554/eLife.92755

Figure Lengend Snippet: ( A ) Schematic of the interplay between RhoA and Rac1 signaling via GTPase regulatory proteins (e.g. GTPase activating proteins (GAPs) among others) common for RhoA and Rac1. ( B ) Analysis of Rac1 activity by Rac1-GTP pulldown (PD) from whole-cell lysates (input) of mouse hippocampal neurons expressing shCTR or shmDia1 +3 utilizing immobilized PAK as a bait. Samples were analyzed by immunoblotting for mDia1, mDia3, Rac1, and Tubulin using specific antibodies. Input, 10% of material used for the pulldown. The contrast of pulldown and input blots was seperately adjusted for visualization purposes. ( C ) Densitometric quantification of Rac1-GTP normalized to total Rac1 levels (input) in lysates from neurons transduced with shCTR or shmDia1 +3 (2.2±0.2; p<0.05, one sample t-test) from immunoblots exemplified in ( B ). Values for shCTR were set to 1. Data are expressed as mean ± SEM from N=3 independent experiments. ( D ) Representative three-channel time-gated stimulated emission depletion (STED) image of synapses from hippocampal cultures, fixed and immunostained for Bassoon (magenta), Rac1 (cyan), and Homer1 (green). Scale bar, 250 nm. ( E ) Averaged normalized line profiles for synaptic distribution of Rac1 and Homer1 relative to Bassoon (Maximum set to 0 nm). Data represent mean ± SEM. N=3 independent experiments from n=79 synapses. ( F ) Averaged normalized vGAT-CypHer fluorescence traces for neurons transduced with shCTR or shmDia1 +3 in response to 200 AP (40 Hz, 5 s) stimulation. Cells were acutely treated with 0.1% DMSO or 10 µM Rac1 Inhibitor (EHT 1864) in the imaging buffer. Data shown represent the mean ± SEM. N=8 independent experiments from n shCTR + DMSO = 46 videos, n shmDia1+3 + DMSO = 45 videos, n shCTR + EHT 1864 = 42 videos, n shmDia1+3 + EHT 1864 = 43 videos. ( G ) Endocytic decay constants of vGAT-CypHer traces in F: τ shCTR + DMSO = 14.7±0.9 s, τ shmDia1+3 + DMSO =27.5±2.3 s, τ shCTR + EHT 1864 = 30.3±6.7 s, τ shmDia1+3 + EHT 1864 = 41.0±4.3 s; p shCTR + DMSO vs shmDia1+3 + DMSO <0.05, p shCTR + DMSO vs shmDia1+3 + EHT 1864 < 0.0001, Kruskal-Wallis test with Dunn’s post-test. Data represent mean ± SEM. ( H ) Endocytic decay constants of Synaptophysin-pHluorin traces of neurons transduced with shCTR (τ shCTR = 12.0±0.7 s) or shmDia1 +3 (τ shmDia1+3 = 22.7±2.0 s) and transfected with constitutively active Rac1 (Rac1-CA; Q61L variant; τ shCTR + Rac1-CA =13.6±1.2 s, τ shmDia1+3 + Rac1-CA =13.3±1.4 s) or dominant negative Rac1 (Rac1-DN; T17N variant; τ shCTR + Rac1-DN = 27.8±1.3 s, τ shmDia1+3 + Rac1-DN = 33.4±1.6 s) in response to 200 AP (40 Hz, 5 s) stimulation (p shCTR vs shmDia1+3 < 0.01; p shCTR vs shCTR + Rac1-DN <0.0001, p shCTR vs shmDia1+3 + Rac1-DN <0.01, p shmDia1+3 vs shmDia1+3 + Rac1-DN <0.01, one-way ANOVA with Tukey’s post-test). Data are expressed as mean ± SEM. N=3 independent experiments from n shCTR = 12 videos, n shmDia1+3 = 23 videos; n shCTR + Rac1-CA =10 videos, n shmDia1+3 + Rac1-CA =14 videos, n shCTR + Rac1-DN = 9 videos; n shmDia1+3 + Rac1-DN = 13 videos. Figure 6—source data 1. Original scans for the anti-mDia3, anti-Tubulin, and anti-Rac1 immunoblots from . Figure 6—source data 2. Original scan for the anti-mDia1 immunoblot from . Figure 6—source data 3. Original scans for immunoblots from with highlighted bands and sample labels. Figure 6—source data 4. Numerical source data for . Figure 6—source data 5. Original scans for the anti-Rac1 immunoblots used for analysis are shown in . Figure 6—source data 6. Original scans for immunoblots used for analysis are shown in with highlighted bands and sample labels.

Article Snippet: To generate mDia1-WT-mCherry, the sequence encoding mDia1 was cut from mDia1-mEmerald-N1 (Addgene, Cat#54157) and pasted into pmCherry-N1 by AgeI and XhoI digestion. mDia1-WT-SNAP was cloned by cutting out the coding sequence of mDia1 from mDia1-mEmerald-N1 and pasting it into pSNAP-N1 by AgeI and NheI digest of both vector and insert.

Techniques: Activity Assay, Expressing, Western Blot, Transduction, Fluorescence, Imaging, Transfection, Variant Assay, Dominant Negative Mutation

Journal: eLife

Article Title: Rho GTPase signaling and mDia facilitate endocytosis via presynaptic actin

doi: 10.7554/eLife.92755

Figure Lengend Snippet: ( A ) Average number of invaginations per μm 2 in WT (0.1±0.1) and Diaph1 KO ( mDia1 KO; 0.4±0.1; p<0.01, Mann-Whitney test) boutons. Data shown represent the mean ± SEM from n WT = 103 synapses, n mDia1KO = 96 synapses. ( B ) Average number of endosome-like vacuoles (ELVs) per μm 2 in wild-type (WT) (1.3±0.2) and mDia 1KO (3.1±0.5; p<0.001, Mann-Whitney test) boutons. Data shown represent the mean ± SEM from n WT = 103 synapses, n mDia1KO = 96 synapses. ( C ) Average invagination length in shCTR and shmDia1 +3 boutons treated with 0.1% dimethyl sulfoxide (DMSO) (97.6±4.5 nm for shCTR ; 136.8±6.0 nm for shmDia1 +3 , p shCTR + DMSO vs shmDia1+3 + DMSO <0.0001) or 10 µM EHT 1864 (130.9±4.5 nm for shCTR, p shCTR + DMSO vs shCTR + EHT 1864 < 0.001; 143.1±4.9 nm for shmDia1 +3, p shCTR + DMSO vs shmDia1+3 + EHT 1864 < 0.0001, Kruskal-Wallis test with Dunn’s post-test) for 2 hr before chemical fixation. Data represent mean ± SEM from n shCTR + DMSO = 77 invaginations, n shmDia1+3 + DMSO =141 invaginations, n shCTR + EHT 1864 = 176 invaginations, n shmDia1+3 + EHT 1864 = 189 invaginations. ( D ) Average invagination width in shCTR and shmDia1 +3 boutons treated with 0.1% DMSO (124.5±5.6 nm for shCTR ; 184.1±6.6 nm for shmDia1 +3 , p shCTR + DMSO vs shmDia1+3 + DMSO <0.0001) or 10 µM EHT 1864 (179.0±5.8 nm for shCTR , p shCTR + DMSO vs shCTR + EHT 1864 < 0.0001; 191.0±5.4 nm for shmDia1 +3 , p shCTR + DMSO vs shmDia1+3 + EHT 1864 < 0.0001, Kruskal-Wallis test with Dunn’s post-test) for 2 hr before chemical fixation. Data represent mean ± SEM from n shCTR + DMSO = 77 invaginations, n shmDia1+3 + DMSO =141 invaginations, n shCTR + EHT 1864 = 176 invaginations, n shmDia1+3 + EHT 1864 = 189 invaginations. Figure 7—figure supplement 1—source data 1. Numerical source data of .

Article Snippet: To generate mDia1-WT-mCherry, the sequence encoding mDia1 was cut from mDia1-mEmerald-N1 (Addgene, Cat#54157) and pasted into pmCherry-N1 by AgeI and XhoI digestion. mDia1-WT-SNAP was cloned by cutting out the coding sequence of mDia1 from mDia1-mEmerald-N1 and pasting it into pSNAP-N1 by AgeI and NheI digest of both vector and insert.

Techniques: MANN-WHITNEY

Journal: eLife

Article Title: Rho GTPase signaling and mDia facilitate endocytosis via presynaptic actin

doi: 10.7554/eLife.92755

Figure Lengend Snippet:

Article Snippet: To generate mDia1-WT-mCherry, the sequence encoding mDia1 was cut from mDia1-mEmerald-N1 (Addgene, Cat#54157) and pasted into pmCherry-N1 by AgeI and XhoI digestion. mDia1-WT-SNAP was cloned by cutting out the coding sequence of mDia1 from mDia1-mEmerald-N1 and pasting it into pSNAP-N1 by AgeI and NheI digest of both vector and insert.

Techniques: Knock-Out, Control, Recombinant, Plasmid Preparation, Variant Assay, Transfection, shRNA, Binding Assay, Mutagenesis, Software, Sequencing