Journal: The Journal of Cell Biology

Article Title: EVL and MIM/MTSS1 regulate actin cytoskeletal remodeling to promote dendritic filopodia in neurons

doi: 10.1083/jcb.202106081

Figure Lengend Snippet: Characterization of synapse precursor dendritic filopodia in cultured mouse primary cortical neurons. (A) Schematic of in vitro cortical neuron development and synaptogenesis. (B) Live primary mouse cortical neurons at indicated days in vitro expressing mRuby2-LifeAct and synapse reporter PSD95-FingR-EGFP, demonstrating synapse formation through dendritic filopodia and synapse maturation (asterisks). Left: full cell image. Right: indicated segment of dendrite. (C) Immunofluorescence labeling of primary cortical neurons for the conventional filopodia marker fascin at D11 in vitro. Left: Intensity-coded LUT of fascin labeling in full cell image. Insets: Indicated regions showing fascin labeling at the axonal growth cone (A′), DF (B′), soma (C′), and dendritic growth cones (D′ and E′). White arrows indicate fascin-negative DF, red arrows indicate fascin-positive filopodia. (D) Immunofluorescence labeling of primary cortical neurons for the branched actin marker Arp2/3 and phalloidin at D11 in vitro. Top: Full cell image. Insets: Indicated segments of dendrite (left) and intensity-coded LUT of Arp2/3 labeling (right). White arrows indicate dendritic filopodia, red arrows indicate synapses. (E) Live D11 neurons expressing iRFP670-LifeAct and mRuby2-Arp3 or MRLC-mRuby2 showing localization of Arp2/3 complex and myosin-II in dendritic filopodia. Top: Full cell image. Inset: Indicated segment of dendrite (left) and intensity-coded LUT of localization (right). Scale bars = 10 µm.

Article Snippet: To reduce expression of EVL, the CMVie promoter was replaced with the Ef1a short promoter EFS in some constructs. cDNAs were tagged with mEmerald (a gift from Michael Davidson, Florida State University, Tallahassee, FL, USA; RRID:Addgene_53975), mRuby2 (a gift from Michael Davidson; ; RRID:Addgene_54768), or piRFP670 (a gift from Vladislav Verkhusha, Albert Einstein College of Medicine, The Bronx, NY, USA; ; RRID:Addgene_45457) on the N-terminus of EVL or Arp3, or C-terminus of MTSS1 or MRLC.

Techniques: Cell Culture, In Vitro, Expressing, Immunofluorescence, Labeling, Marker

Journal: The Journal of Cell Biology

Article Title: EVL and MIM/MTSS1 regulate actin cytoskeletal remodeling to promote dendritic filopodia in neurons

doi: 10.1083/jcb.202106081

Figure Lengend Snippet: MENA and EVL overexpression enhance DF motility. (A) Representative Western blot of protein lysates from D11 mouse cortical neuron cultures expressing EGFP-MENA or EGFP-EVL, and probed with antibodies targeting MENA or EVL for detection of endogenous and overexpressed species. (B) Examples of extreme phenotypes observed in live primary mouse cortical neurons at day in vitro 11 (D11) expressing mRuby2-LifeAct with high expression (SNR > 1.5) of EGFP-MENA or EGFP-EVL. Left: full cell image. Closed arrowheads indicate abnormal structures. Open arrowhead indicates magnified region (right). (C) Examples and quantification of DF morphological phenotypes observed in D11 neurons expressing mRuby2-LifeAct with low-expression (SNR < 1.5) of EGFP-MENA or EGFP-EVL. n = 83–133 DF per condition, N = 3 neurons. Scale bars = 10 µm. See also and . Source data are available for this figure: .

Article Snippet: To reduce expression of EVL, the CMVie promoter was replaced with the Ef1a short promoter EFS in some constructs. cDNAs were tagged with mEmerald (a gift from Michael Davidson, Florida State University, Tallahassee, FL, USA; RRID:Addgene_53975), mRuby2 (a gift from Michael Davidson; ; RRID:Addgene_54768), or piRFP670 (a gift from Vladislav Verkhusha, Albert Einstein College of Medicine, The Bronx, NY, USA; ; RRID:Addgene_45457) on the N-terminus of EVL or Arp3, or C-terminus of MTSS1 or MRLC.

Techniques: Over Expression, Western Blot, Expressing, In Vitro

Journal: The Journal of Cell Biology

Article Title: EVL and MIM/MTSS1 regulate actin cytoskeletal remodeling to promote dendritic filopodia in neurons

doi: 10.1083/jcb.202106081

Figure Lengend Snippet: MENA and EVL overexpression enhance DF motility. (A) Live primary mouse cortical neurons at day in vitro 11 (D11) expressing mRuby2-LifeAct and EGFP (left column), EGFP-MENA (middle column), or EGFP-EVL (right column). Row 1: Full cell image. Row 2: Indicated segment of dendrite. Rows 3 and 4: Individual fluorescent channels presented with intensity-coded LUTs. Row 5: Maximum intensity projection of temporally color-coded binary mask outline, illustrating DF dynamics during imaging (5 s interval, 5 min duration). (B) Scatterplot of average speed of DF tips, calculated as the average absolute tip displacement between successive timepoints. Gray shaded region indicates average speed less than 0.0128 µm/s (non-motile DFs). Median ± interquartile range (IQR). Mixed-effects model; n = 368–389 total DF from 4–6 neurons per biological replicate, N = 3 biological replicates. (C) Scatterplot of average length reached during the duration of imaging. Median ± IQR. Mixed-effects model; n = 368–389 total DF from 4–6 neurons per biological replicate, N = 3 biological replicates. (D) Bar graph of percent time motile (percent of time per DF in which instantaneous speed was greater than 0.0128 µm/s). Median ± IQR. Kruskal-Wallis test corrected for multiple comparisons; n = 368–389 total DF from 4–6 neurons per biological replicate, N = 3 biological replicates. (E) Scatterplot of median protrusion and retraction rates of DFs (the median of values when instantaneous change in length was greater than ±0.0128 µm/s [motile]). Median ± IQR. Mixed-effects model; n = 368–389 total DF from 4–6 neurons per biological replicate, N = 3 biological replicates. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, n.s. is not significant. Scale bars = 10 µm. See also and .

Article Snippet: To reduce expression of EVL, the CMVie promoter was replaced with the Ef1a short promoter EFS in some constructs. cDNAs were tagged with mEmerald (a gift from Michael Davidson, Florida State University, Tallahassee, FL, USA; RRID:Addgene_53975), mRuby2 (a gift from Michael Davidson; ; RRID:Addgene_54768), or piRFP670 (a gift from Vladislav Verkhusha, Albert Einstein College of Medicine, The Bronx, NY, USA; ; RRID:Addgene_45457) on the N-terminus of EVL or Arp3, or C-terminus of MTSS1 or MRLC.

Techniques: Over Expression, In Vitro, Expressing, Imaging

Journal: The Journal of Cell Biology

Article Title: EVL and MIM/MTSS1 regulate actin cytoskeletal remodeling to promote dendritic filopodia in neurons

doi: 10.1083/jcb.202106081

Figure Lengend Snippet: Live primary mouse cortical neurons at day in vitro 11 (D11) expressing mRuby2-LifeAct and EGFP (top row), EGFP-MENA (middle row), or EGFP-EVL (bottom row). Left: segment of dendrite with merge of EGFP (cyan) and LifeAct (magenta). Right: rainbow intensity-coded LUT of EGFP with outline of LifeAct-positive borders. Red = highest intensity, purple = lowest intensity. Scale bar = 10 µm. 5 s interval, 5 min duration, 10 fps. See also and .

Article Snippet: To reduce expression of EVL, the CMVie promoter was replaced with the Ef1a short promoter EFS in some constructs. cDNAs were tagged with mEmerald (a gift from Michael Davidson, Florida State University, Tallahassee, FL, USA; RRID:Addgene_53975), mRuby2 (a gift from Michael Davidson; ; RRID:Addgene_54768), or piRFP670 (a gift from Vladislav Verkhusha, Albert Einstein College of Medicine, The Bronx, NY, USA; ; RRID:Addgene_45457) on the N-terminus of EVL or Arp3, or C-terminus of MTSS1 or MRLC.

Techniques: In Vitro, Expressing

Journal: The Journal of Cell Biology

Article Title: EVL and MIM/MTSS1 regulate actin cytoskeletal remodeling to promote dendritic filopodia in neurons

doi: 10.1083/jcb.202106081

Figure Lengend Snippet: Tip enrichment of EVL precedes DF protrusion. (A) Filmstrip of live primary mouse cortical neurons at day in vitro 11 (D11) expressing EGFP (top row), EGFP-MENA (middle row), or EGFP-EVL (bottom row) in a representative DF showing localization dynamics presented with an intensity-coded LUT. Line scan of normalized fluorescence intensity over time at tracked tip (right; 5 s interval, 1 min duration). Scale bar = 1 µm. (B) Scatterplot of tip fluorescence variance at DF tips normalized to local background demonstrates range of tip enrichment per DF and hence magnitude of on-off dynamics. Median ± interquartile range (IQR). Mixed-effects model; n = 368–389 total DF from 4–6 neurons per biological replicate, N = 3 biological replicates. (C) Live D11 neurons expressing mRuby2-LifeAct and EGFP (top row), EGFP-MENA (middle row), or EGFP-EVL (bottom row). Segment of dendrite (left); scale bar = 10 µm. Numbers indicate DF position analyzed by kymograph (right), highlighting protein localization dynamics during DF motility (5 s interval, 5 min duration). Vertical scale bar = 1 µm, horizontal scale bar = 1 min, dashed line indicates dendrite. (D) Line plots of cross-correlation function (CCF) of normalized tip fluorescence intensity and tip motility as a function of time offset for top-correlating subcluster (TCS; color lines) versus non-top-correlating subcluster (non-TCS; gray lines) determined in , of DFs from D11 neurons expressing EGFP, EGFP-MENA, or EGFP-EVL. Peak cross-correlation at negative offset values indicates that fluorescence enrichment precedes motility, while peak cross-correlation at positive offset values indicates fluorescence enrichment follows motility. Mean ±95% CI. Peak cross-correlation GFP = 0.43 at offset 0.413 at offset −5s; MENA = 0.475 at offset 0, 0.464 at offset +5 s; EVL =, 0.482 at offset −10 s, 0.480 at offset −5 s. Statistical significance determined by peak cross-correlation >2/√(n-|offset|). (E) Percent of area under the curve at positive and negative offset values for indicated conditions. (F) Line plots (left) and scatterplots (right) for one representative DF from the top-correlating subcluster in D11 neurons from indicated conditions. Left: Line plot of tip motility (gray, left y-axis) and normalized tip fluorescence (color, right y-axis) during imaging (5 s interval, 5 min duration). Right: Scatterplot of normalized tip fluorescence and tip motility at individual timepoints demonstrating strength of relationship by Pearson’s correlation test. (G) Scatterplot of median protrusion rates for top-correlating subcluster versus non-top-correlating subcluster DFs from indicated conditions. Protrusion rate is the median of values when instantaneous change in length was greater than +0.0128 µm/s (motile, protruding). Median ± interquartile range (IQR). Kruskal-Wallis test corrected for multiple comparisons. TCS n = 42 (MENA) and 39 (EVL); non-TCS n = 320 (MENA) and 321 (EVL) total DF from 4–6 neurons per biological replicate, N = 3 biological replicates. (H) Bar graph of percent of time in protrusion during the duration of imaging for top-correlating subcluster versus non-top-correlating subcluster DFs from indicated conditions. Percent time in protrusion is calculated as the percent of time per DF in which positive change in length between successive timepoints was greater than 0.0128 µm/s. Median ± IQR. Kruskal-Wallis test corrected for multiple comparisons. TCS n = 42 (MENA) and 39 (EVL); non-TCS n = 320 (MENA) and 321 (EVL) total DF from 4–6 neurons per biological replicate, N = 3 biological replicates. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, n.s. is not significant. See also .

Article Snippet: To reduce expression of EVL, the CMVie promoter was replaced with the Ef1a short promoter EFS in some constructs. cDNAs were tagged with mEmerald (a gift from Michael Davidson, Florida State University, Tallahassee, FL, USA; RRID:Addgene_53975), mRuby2 (a gift from Michael Davidson; ; RRID:Addgene_54768), or piRFP670 (a gift from Vladislav Verkhusha, Albert Einstein College of Medicine, The Bronx, NY, USA; ; RRID:Addgene_45457) on the N-terminus of EVL or Arp3, or C-terminus of MTSS1 or MRLC.

Techniques: In Vitro, Expressing, Fluorescence, Imaging

Journal: The Journal of Cell Biology

Article Title: EVL and MIM/MTSS1 regulate actin cytoskeletal remodeling to promote dendritic filopodia in neurons

doi: 10.1083/jcb.202106081

Figure Lengend Snippet: EVL is required for DF morphogenesis and influences dendritic spine plasticity. (A) Live primary cortical neurons derived from wild-type (WT) or EVL knockout (KO) mice at indicated days in vitro expressing mRuby2-LifeAct. Top: Full cell image. Middle: Indicated segment of dendrite presented with an intensity-coded LUT. Bottom: Maximum intensity projection of temporally color-coded binary mask outline, illustrating DF dynamics during imaging (5 s interval, 5 min duration). Scale bars = 10 µm. (B) Scatterplot of average length reached during the duration of imaging for indicated conditions. Median ± interquartile range (IQR). Mixed-effects model; n = 280–641 total DF from 4–6 neurons per biological replicate, N = 3 biological replicates. (C) Scatterplot of median protrusion rates of DFs (the median of values when instantaneous change in length was greater than +0.0128 µm/s (motile), protruding). Median ± IQR. Mixed-effects model; n = 280–641 total DF from 4–6 neurons per biological replicate, N = 3 biological replicates. (D) High-density cultures of wild-type, EVL knockout, or wild-type overexpressing EVL neurons at day in vitro 14 (D14) expressing EGFP-LifeAct and synapse reporter PSD95-FingR-mRuby2. Left: Full cell image. Inset: EVL expression. Right: Indicated segment of dendrite. Scale bars = 10 µm. (E) Scatterplot of average dendritic spine density per neuron of indicated conditions. Median ± IQR. One-way ANOVA corrected with Holm-Sidak multiple comparisons; n = 38 total neurons for each condition, N = 3 biological replicates. (F) Filmstrip of segment of dendrite from live cortical neurons derived from wild-type or EVL knockout mice at D14 expressing EGFP-LifeAct and PSD95-FingR-mRuby2 showing synapse formation and dynamics. Inset: Illustration of region indicated by asterisks. Scale bars = 10 µm. (G) Scatterplot of average duration of synapse persistence per neuron as quantified by PSD95-positive foci tracked in time. Median ± IQR. Unpaired t test; n = 12 (wild-type) and 13 (knockout) total neurons; N = 3 biological replicates. (H) Scatterplot of average speed per neuron of PSD95-positive foci tracked in time. Median ± IQR. Unpaired t test; n = 12 (wild-type) and 13 (knockout) total neurons; N = 3 biological replicates. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, n.s. is not significant. See also , , and .

Article Snippet: To reduce expression of EVL, the CMVie promoter was replaced with the Ef1a short promoter EFS in some constructs. cDNAs were tagged with mEmerald (a gift from Michael Davidson, Florida State University, Tallahassee, FL, USA; RRID:Addgene_53975), mRuby2 (a gift from Michael Davidson; ; RRID:Addgene_54768), or piRFP670 (a gift from Vladislav Verkhusha, Albert Einstein College of Medicine, The Bronx, NY, USA; ; RRID:Addgene_45457) on the N-terminus of EVL or Arp3, or C-terminus of MTSS1 or MRLC.

Techniques: Derivative Assay, Knock-Out, In Vitro, Expressing, Imaging

Journal: The Journal of Cell Biology

Article Title: EVL and MIM/MTSS1 regulate actin cytoskeletal remodeling to promote dendritic filopodia in neurons

doi: 10.1083/jcb.202106081

Figure Lengend Snippet: Live primary cortical neurons derived from wild-type (WT) or EVL knockout (KO) mice at indicated days in vitro expressing mRuby2-LifeAct. Left: segment of dendrite with overlay of brightfield and intensity-coded LUT of mRuby2-LifeAct. Right: mRuby2-LifeAct alone. Scale bar = 10 µm. 5 s interval, 5 min duration, 10 fps. See also and .

Article Snippet: To reduce expression of EVL, the CMVie promoter was replaced with the Ef1a short promoter EFS in some constructs. cDNAs were tagged with mEmerald (a gift from Michael Davidson, Florida State University, Tallahassee, FL, USA; RRID:Addgene_53975), mRuby2 (a gift from Michael Davidson; ; RRID:Addgene_54768), or piRFP670 (a gift from Vladislav Verkhusha, Albert Einstein College of Medicine, The Bronx, NY, USA; ; RRID:Addgene_45457) on the N-terminus of EVL or Arp3, or C-terminus of MTSS1 or MRLC.

Techniques: Derivative Assay, Knock-Out, In Vitro, Expressing

Journal: The Journal of Cell Biology

Article Title: EVL and MIM/MTSS1 regulate actin cytoskeletal remodeling to promote dendritic filopodia in neurons

doi: 10.1083/jcb.202106081

Figure Lengend Snippet: Live wild-type and EVL knockout (KO) cortical neurons at day in vitro 12 (D12) expressing EGFP-LifeAct and PSD95-FingR-mRuby2. Row 1: Segment of dendrite with merge of PSD95 reporter (green) and LifeAct (magenta). Row 2: LifeAct fluorescence intensity presented with an inverted LUT to highlight actin dynamics. Row 3: PSD95 reporter fluorescence intensity presented with an inverted LUT. Scale bar = 10 µm. 30 s interval, 50 min duration, 10 fps. See also .

Article Snippet: To reduce expression of EVL, the CMVie promoter was replaced with the Ef1a short promoter EFS in some constructs. cDNAs were tagged with mEmerald (a gift from Michael Davidson, Florida State University, Tallahassee, FL, USA; RRID:Addgene_53975), mRuby2 (a gift from Michael Davidson; ; RRID:Addgene_54768), or piRFP670 (a gift from Vladislav Verkhusha, Albert Einstein College of Medicine, The Bronx, NY, USA; ; RRID:Addgene_45457) on the N-terminus of EVL or Arp3, or C-terminus of MTSS1 or MRLC.

Techniques: Knock-Out, In Vitro, Expressing, Fluorescence

Journal: The Journal of Cell Biology

Article Title: EVL and MIM/MTSS1 regulate actin cytoskeletal remodeling to promote dendritic filopodia in neurons

doi: 10.1083/jcb.202106081

Figure Lengend Snippet: EVL regulates DF morphogenesis and motility through membrane-targeted actin polymerization. (A) Schematic of protein domains of wild-type EVL and EVL mutants used in this study. EVH1 (blue) = Enabled/VASP homology-1, P (yellow) = profilin binding region, G (orange) = G-actin binding region, F (red) = F-actin binding region, CC (gray) = coiled-coil domain, PRD = proline-rich region, EVH2 = Enabled/VASP homology-2. (B) Western blot of protein lysates from D11 cortical neurons derived from EVL knockout (KO) mice expressing mEmerald-EVL or various mutants of EVL as indicated. (C) Segment of dendrite from live primary cortical neurons derived from EVL knockout mice at indicated day in vitro 11 (D11) expressing mRuby2-LifeAct and mEmerald alone, or mEmerald-tagged wild-type EVL or mutants of EVL as indicated. From left to right, Panel 1: Merge of LifeAct and mEmerald-tagged proteins. Scale bar = 10 µm. Panel 2: mEmerald fluorescence intensity presented with an inverted LUT. Panel 3: Kymograph of DF position indicated by arrowhead (5 s interval, 5 min duration). Vertical scale bar = 1 µm, horizontal scale bar = 1 min, dashed line indicates dendrite. Panel 4: Maximum intensity projection of temporally color-coded binary mask outline (5 s interval, 5 min duration). (D) Scatterplot of average speed of DF tips, calculated as the average absolute tip displacement between successive timepoints. Gray shaded region indicates average speed less than 0.0128 µm/s (non-motile DFs). Median ± IQR. Mixed-effects model; n = 248–397 total DF from 3–6 neurons per biological replicate, N = 3–5 biological replicates. (E) Scatterplot of average length reached during the duration of imaging for indicated conditions. Median ± IQR. Mixed-effects model; n = 248–397 total DF from 3–6 neurons per biological replicate, N = 3–5 biological replicates. (F) Scatterplot of median protrusion rates of DFs for indicated conditions. Median ± IQR. Mixed-effects model; n = 248–397 total DF from 3–6 neurons per biological replicate, N = 3–5 biological replicates. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, n.s. is not significant. See also . Source data are available for this figure: .

Article Snippet: To reduce expression of EVL, the CMVie promoter was replaced with the Ef1a short promoter EFS in some constructs. cDNAs were tagged with mEmerald (a gift from Michael Davidson, Florida State University, Tallahassee, FL, USA; RRID:Addgene_53975), mRuby2 (a gift from Michael Davidson; ; RRID:Addgene_54768), or piRFP670 (a gift from Vladislav Verkhusha, Albert Einstein College of Medicine, The Bronx, NY, USA; ; RRID:Addgene_45457) on the N-terminus of EVL or Arp3, or C-terminus of MTSS1 or MRLC.

Techniques: Membrane, Binding Assay, Western Blot, Derivative Assay, Knock-Out, Expressing, In Vitro, Fluorescence, Imaging

Journal: The Journal of Cell Biology

Article Title: EVL and MIM/MTSS1 regulate actin cytoskeletal remodeling to promote dendritic filopodia in neurons

doi: 10.1083/jcb.202106081

Figure Lengend Snippet: Live EVL knockout (KO) cortical neurons at day in vitro 11 (D11) expressing mRuby2-LifeAct and mEmerald alone, or mEmerald-tagged wild-type EVL or mutants of EVL as indicated. Phase 1: Segment of dendrite with merge of mEmerald (cyan) and LifeAct (magenta). Phase 2: Intensity-coded LUT of mRuby2-LifeAct. Phase 3: Rainbow intensity-coded LUT of mEmerald with outline of LifeAct-positive borders. Red = highest intensity, purple = lowest intensity. Scale bar = 10 µm. 5 s interval, 5 min duration, 10 fps. See also .

Article Snippet: To reduce expression of EVL, the CMVie promoter was replaced with the Ef1a short promoter EFS in some constructs. cDNAs were tagged with mEmerald (a gift from Michael Davidson, Florida State University, Tallahassee, FL, USA; RRID:Addgene_53975), mRuby2 (a gift from Michael Davidson; ; RRID:Addgene_54768), or piRFP670 (a gift from Vladislav Verkhusha, Albert Einstein College of Medicine, The Bronx, NY, USA; ; RRID:Addgene_45457) on the N-terminus of EVL or Arp3, or C-terminus of MTSS1 or MRLC.

Techniques: Knock-Out, In Vitro, Expressing

Journal: The Journal of Cell Biology

Article Title: EVL and MIM/MTSS1 regulate actin cytoskeletal remodeling to promote dendritic filopodia in neurons

doi: 10.1083/jcb.202106081

Figure Lengend Snippet: MIM/MTSS1 cooperates with EVL to promote DF initiation and motility. (A) Table of select actin-regulating proteins identified by affinity purification-mass spectrometry (AP-MS). (B) Representative western blot of co-immunoprecipitation experiments demonstrating requirement of MIM’s 634 LPSPP 638 motif for interaction with EVL’s EVH1 domain. HEK293T overexpressing indicated constructs were lysed and immunoprecipitated (IP) with anti-His antibody to pull down EVH1-mychis6, resolved by SDS-PAGE, and immunoblotted (IB) with indicated antibodies. (C) Representative western blot and quantification of protein lysates from cortical neurons derived from wild-type or EVL knockout mice at indicated days in vitro, and probed with an antibody targeting MIM. Mean ± SD. Unpaired t test; N = 3 biological replicates. (D) Representative western blot of protein lysates from primary neurons at D11, transduced on D7 with indicated pLKO-shRNA-TurboRFP lentiviral particles targeting Mtss1 or non-targeting (n.t.) control. Fold change quantification of knockdown compared to non-targeting shRNA control. Mean ± SD. Unpaired t test; N = 3 biological replicates. (E) Segment of dendrite from live EVL knockout cortical neurons at D11 expressing mRuby2-LifeAct, MIM-iRFP670, or MIM together with mEmerald-tagged wild-type EVL or ΔEVH1 as indicated (left). Right: kymograph of DF position indicated by arrowhead in left column. 5 s interval, 5 min duration, dendrite segment: scale bar = 10 µm. Kymograph: Horizontal scale bar = 1 min, vertical scale bar = 1 µm. (F) Distribution of MIM and EVL or ΔEVH1 localization along the distal length of DF. Mean ±95% confidence interval, n = 25 total DF. (G) Scatterplot of average speed of EVL knockout DF tips for indicated expression conditions, calculated as the average absolute tip displacement between successive timepoints. Gray shaded region indicates average speed less than 0.0128 µm/s (non-motile DFs). Median ± interquartile range (IQR). Mixed-effects model; n = 101–195 total DF from 2–4 neurons per biological replicate, N = 3 biological replicates. (H) Scatterplot of median protrusion rates of EVL knockout DFs for indicated expression conditions (the median of values when instantaneous change in length was greater than +0.0128 µm/s [motile, protruding]). Median ± IQR. Mixed-effects model; total DF from 2–4 neurons per biological replicate, N = 3 biological replicates. (I) Scatterplot of average length of EVL knockout DFs reached during the duration of imaging for indicated expression conditions. Median ± IQR. Mixed-effects model; total DF from 2–4 neurons per biological replicate, N = 3 biological replicates. (J and K) Glial cells (J) and cortical neurons (K) derived from wild-type mice expressing iRFP670-LifeAct, MIM-iLID, and MITO-iLID. Cells were photostimulated with 488 nm light for 5 min, and imaged for 5 min before, during, and after photostimulation. Row 1: Localization of LifeAct and MIM-iLID during indicated phases of photostimulation. Inset: mVenus-MITO-iLID localization (J only). Rows 2–3: Localization of MIM-iLID and LifeAct in magnified region of lamellipodia indicated in Row 1 (J) or the full dendrite segment (K). Individual channels displayed with black subtraction for ease of morphological comparison. Arrowheads indicate regions which exhibited altered dynamics following photostimulation (lamellipodia = filled arrowheads, filopodia = empty arrowheads). Row 4: Maximum intensity projection of temporally color-coded binary mask outline. Row 5: Kymograph of cell’s edge (J) or representative DF position (K). Scale bar = 10 µm. 5 s interval, 15 min duration. (L) Scatterplot of fold change of average tip speed, calculated as the average absolute tip displacement between successive timepoints relative to average tip speed pre-photostimulation, for 2.5 min bins during or post-photostimulation, for wild-type neurons expressing MIM-iLID with MITO-iLID. Median ± IQR. Mixed-effects model; n = 39 total DF from 2 neurons per biological replicate, N = 2 biological replicates. *P <0.05, **P <0.01, ***P <0.001, ****P <0.0001, n.s. is not significant. See also , , and . Source data are available for this figure: .

Article Snippet: To reduce expression of EVL, the CMVie promoter was replaced with the Ef1a short promoter EFS in some constructs. cDNAs were tagged with mEmerald (a gift from Michael Davidson, Florida State University, Tallahassee, FL, USA; RRID:Addgene_53975), mRuby2 (a gift from Michael Davidson; ; RRID:Addgene_54768), or piRFP670 (a gift from Vladislav Verkhusha, Albert Einstein College of Medicine, The Bronx, NY, USA; ; RRID:Addgene_45457) on the N-terminus of EVL or Arp3, or C-terminus of MTSS1 or MRLC.

Techniques: Affinity Purification, Mass Spectrometry, Protein-Protein interactions, Western Blot, Immunoprecipitation, Construct, SDS Page, Derivative Assay, Knock-Out, In Vitro, shRNA, Control, Knockdown, Expressing, Imaging, Comparison

Journal: The Journal of Cell Biology

Article Title: EVL and MIM/MTSS1 regulate actin cytoskeletal remodeling to promote dendritic filopodia in neurons

doi: 10.1083/jcb.202106081

Figure Lengend Snippet: MIM/MTSS1 cooperates with EVL to promote DF initiation and motility. (A and B) Representative western blots of co-immunoprecipitation experiments confirming reciprocal interaction of MIM with EVL (A) and dependence of interaction on EVL’s EVH1 domain (B). HEK293T overexpressing indicated constructs were lysed and immunoprecipitated (IP) with FLAG antibody to pull down MIM-3xFLAG, resolved by SDS-PAGE, and immunoblotted (IB) with indicated antibodies. (C) Filmstrip showing localization dynamics of MIM-mRuby2 during protrusive motility in a wild-type cortical neuron DF at day in vitro 11 (D11; left), and maximum intensity projection of temporally color-coded binary mask outline (right; 5 s interval, 1 m duration). Scale bar = 5 µm. (D) Segment of dendrite from a wild-type cortical neuron at D11 co-expressing iRFP670-EVL and MIM-mRuby2 (top), and kymograph of DF indicated by arrowhead (bottom; 5 s interval, 5 min duration). Dendrite segment: Scale bar = 5 µm. Kymographs: Horizontal scale bar = 1 min, vertical scale bar = 1 µm, dashed line indicates dendrite. (E) Scatterplot of background-normalized EVL and MIM tip fluorescence at individual timepoints. Strong correlation observed by Spearman’s correlation test. Individual timepoints presented from 5 total DF. (F) Scatterplot of average speed of wild-type DF tips for indicated conditions, calculated as the average absolute tip displacement between successive timepoints. Gray shaded region indicates average speed less than 0.0128 µm/s (non-motile DFs). Median ± interquartile range (IQR). Mixed-effects model; n = 273–345 total DF from 3–5 neurons per biological replicate, N = 3 biological replicates. (G) Scatterplot of median protrusion rates of wild-type DFs for indicated conditions (the median of values when instantaneous change in length was greater than +0.0128 µm/s [motile, protruding]). Median ± IQR. Mixed-effects model; n = 273–345 total DF from 3-5 neurons per biological replicate, N = 3 biological replicates. (H) Scatterplot of average length of wild-type DFs reached during the duration of imaging for indicated conditions. Median ± IQR. Mixed-effects model; n = 273–345 total DF from 3–5 neurons per biological replicate, N = 3 biological replicates. (I) Wild-type neurons expressing EGFP-LifeAct and pLKO-shRNA-TurboRFP targeting Mtss1 or non-targeting as indicated. Cells were transduced with shRNA lentiviral particles on D7. Top: Full cell image. Inset: TurboRFP expression identifies shRNA-positive neurons. Middle: Indicated segment of dendrite. Bottom: Maximum intensity projection of temporally color-coded binary mask outline (5 s interval, 5 min duration). Scale bars = 10 µm. (J) Scatterplot of average speed of DF tips for indicated conditions. Median ± IQR. Mixed-effects model; n = 362–363 total DF from 2–4 neurons per biological replicate, N = 3 biological replicates. (K) Scatterplot of median protrusion rates of DFs for indicated conditions. Median ± IQR. Mixed-effects model; n = 362–363 total DF from 2–4 neurons per biological replicate, N = 3 biological replicates. (L) Scatterplot of average length of DFs reached during the duration of imaging for indicated conditions. Median ± IQR. Mixed-effects model; n = 362–363 total DF from 2–4 neurons per biological replicate, N = 3 biological replicates. (M) EVL knockout neurons expressing EGFP-LifeAct and pLKO-shRNA-TurboRFP targeting Mtss1 or non-targeting as indicated. Top: Full cell image. Inset: TurboRFP expression identifies shRNA-positive neurons. Middle: Indicated segment of dendrite. Bottom: Maximum intensity projection of temporally color-coded binary mask outline (5 s interval, 5 min duration). Scale bars = 10 µm. (N) Scatterplot of protrusion density along dendrites from indicated conditions. Protrusions were defined as any actin-rich proturbences visibly extending beyond the dendrite. Median ± IQR. Mann-Whitney test; n = 19–35 dendrites from 3–5 neurons per biological replicate, N = 3 biological replicates. (O) Example of spontaneous DF initiation. Row 1: Filmstrip indicating localization of mEmerald-EVL and MIM-mRuby2 during DF initiation in a D11 wild-type neuron. Scale bar = 0.5 µm. Rows 2–4: Kymograph of mEmerald-EVL and MIM-mRuby2 during initiation. Arrowheads indicate foci of protein enrichment preceding initiation or motility events. 5 s interval, 5 min duration, horizontal scale bar = 1 min, vertical scale bar = 1 µm, dashed line indicates dendrite. (P) Box-and-whisker plot of newly initiated DFs as a percent of total DFs during the duration of imaging. Median ± IQR. One-way ANOVA corrected Holm-Sidak multiple comparisons; n = 9–14 total neurons, N = 3 biological replicates. (Q) Example of EVL-iLID-elicited DF initiation. Row 1: Filmstrip indicating localization of EVL-iLID and MIM-iRFP670 during photostimulation in a D11 knockout neuron. Scale bar = 0.5 µm. Rows 2–4: Kymograph of EVL-iLID and MIM-iRFP670 during initiation. Arrowheads indicate foci of protein enrichment preceding initiation or motility events. 5 s interval, 15 min duration, horizontal scale bar = 1 min, vertical scale bar = 1 µm, dashed line indicates dendrite. (R) Box-and-whisker plot of newly initiated DFs as a percent of total DFs during the duration of imaging before, during, and following iLID stimulation with indicated constructs. One-way ANOVA corrected Holm-Sidak multiple comparisons; n = 11–16 total neurons, N = 3 biological replicates *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, n.s. is not significant. See also , , and . Source data are available for this figure: .

Article Snippet: To reduce expression of EVL, the CMVie promoter was replaced with the Ef1a short promoter EFS in some constructs. cDNAs were tagged with mEmerald (a gift from Michael Davidson, Florida State University, Tallahassee, FL, USA; RRID:Addgene_53975), mRuby2 (a gift from Michael Davidson; ; RRID:Addgene_54768), or piRFP670 (a gift from Vladislav Verkhusha, Albert Einstein College of Medicine, The Bronx, NY, USA; ; RRID:Addgene_45457) on the N-terminus of EVL or Arp3, or C-terminus of MTSS1 or MRLC.

Techniques: Western Blot, Immunoprecipitation, Construct, SDS Page, In Vitro, Expressing, Fluorescence, Imaging, shRNA, Transduction, Knock-Out, MANN-WHITNEY, Protein Enrichment, Whisker Assay

Journal: The Journal of Cell Biology

Article Title: EVL and MIM/MTSS1 regulate actin cytoskeletal remodeling to promote dendritic filopodia in neurons

doi: 10.1083/jcb.202106081

Figure Lengend Snippet: Live primary wild-type mouse cortical neurons at day in vitro 11 (D11) expressing mRuby2-LifeAct, together with mEmerald-EVL and/or MIM-iRFP670 as indicated. Left: Segment of dendrite with overlay of brightfield and intensity-coded LUT of mRuby2-LifeAct. Right, bottom: Rainbow intensity-coded LUT of indicated constructs with outline of LifeAct-positive borders. Red = highest intensity, purple = lowest intensity. Right (MIM + EVL only): Merge of mEmerald-EVL (cyan) and MIM-iRFP670 (red). Scale bar = 10 µm. 5 s interval, 5 min duration, 10 fps. See also and .

Article Snippet: To reduce expression of EVL, the CMVie promoter was replaced with the Ef1a short promoter EFS in some constructs. cDNAs were tagged with mEmerald (a gift from Michael Davidson, Florida State University, Tallahassee, FL, USA; RRID:Addgene_53975), mRuby2 (a gift from Michael Davidson; ; RRID:Addgene_54768), or piRFP670 (a gift from Vladislav Verkhusha, Albert Einstein College of Medicine, The Bronx, NY, USA; ; RRID:Addgene_45457) on the N-terminus of EVL or Arp3, or C-terminus of MTSS1 or MRLC.

Techniques: In Vitro, Expressing, Construct

Journal: The Journal of Cell Biology

Article Title: EVL and MIM/MTSS1 regulate actin cytoskeletal remodeling to promote dendritic filopodia in neurons

doi: 10.1083/jcb.202106081

Figure Lengend Snippet: Arp2/3 and myosin-II differentially contribute to DF initiation and motility. (A and B) Segment of dendrite from wild-type (A) or EVL knockout (B) cortical neurons at D11 expressing mRuby2-LifeAct, and treated with DMSO (0.1%), Arp2/3 inhibitor CK-666 (100 µM), or myosin-II inhibitor blebbistatin (50 µM). Neurons were imaged 5 min prior to (left) and up to 40 min following (right) drug treatment. Maximum intensity projection of temporally color-coded binary mask outline of 10 min bins following treatment (middle; 15 s interval, 45 min duration). Scale bars = 10 µm. (C) Scatterplot of fold change of average tip speed following treatment with indicated pharmacological inhibitors, calculated as the average absolute tip displacement during successive 5 min bins, relative to average tip speed pre-treatment, for wild-type and EVL knockout DF. Mean ±95% confidence interval (CI). Mixed-effects model; DMSO n = 48–90 total DF, CK-666 n = 58–80 total DF, and Blebbistatin n = 88–96 total DF from 2–4 neurons per biological replicate, N = 3–5 biological replicates. (D) Scatterplot of fold change of average length reached between successive 5 min bins following treatment with indicated pharmacological inhibitors, relative to average length pre-treatment, for wild-type and EVL knockout DF. Mean ±95% CI. Mixed-effects model; DMSO n = 48–90 total DF, CK-666 n = 58–80 total DF, and Blebbistatin n = 88–96 total DF from 2–4 neurons per biological replicate, N = 3–5 biological replicates. (E) Segment of dendrite from an EVL knockout cortical neuron at D11 expressing mEmerald-Arp3, MIM-mRuby2, and iRFP670-EVL (top; scale bar = 5 µm), and kymographs of protein localization during DF initiation (middle) and motility (bottom). 5 s interval, 2 min duration, horizontal scale bar = 1 min, vertical scale bar = 1 µm, dashed line indicates dendrite. (F) Segment of dendrite from an EVL knockout cortical neuron at D11 expressing MRLC-mRuby2, mEmerald-EVL, and iRFP670-LifeAct (top; scale bar = 5 µm), and kymographs of protein localization during DF motility. 5 s interval, 2 min duration, horizontal scale bar = 1 min, vertical scale bar = 1 µm, dashed line indicates dendrite. (G) Model of spatiotemporal dynamics of MIM, Arp2/3, myosin-II, and EVL during DF initiation and protrusive motility. P-values indicated above each plot in C and D assess fold change over time within WT or KO. P values represented by asterisks on the plots assess difference between WT and KO fold change speed (C) or length (D); *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, only P < 0.05 values are shown on the plot (all P values are in ). See also .

Article Snippet: To reduce expression of EVL, the CMVie promoter was replaced with the Ef1a short promoter EFS in some constructs. cDNAs were tagged with mEmerald (a gift from Michael Davidson, Florida State University, Tallahassee, FL, USA; RRID:Addgene_53975), mRuby2 (a gift from Michael Davidson; ; RRID:Addgene_54768), or piRFP670 (a gift from Vladislav Verkhusha, Albert Einstein College of Medicine, The Bronx, NY, USA; ; RRID:Addgene_45457) on the N-terminus of EVL or Arp3, or C-terminus of MTSS1 or MRLC.

Techniques: Knock-Out, Expressing