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 ₆₃₄LPSPP₆₃₈ 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 Fig. 6, Video 7, and Video 8. Source data are available for this figure: SourceData FS7.