Figure 8.
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 Table S2). See also Video 9.

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 Table S2). See also Video 9.

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