(A–C) TEM of neurons fixed in the process of migrating reveals CCPs and CCVs (arrows). (A) CCVs near and ahead of the nucleus. Direction of migration is toward the lower righthand corner. (B, C) CCPs at points of ECM attachment (arrowheads). (D) Immunostaining for components of CME (green): CHC (left), CLC (middle), and AP-2 (right). (E) Individual fluorescence intensity line scans were binned to create 50 averaged regions along the length of each measured cell to compare cells of different lengths. Averaged line scan data for CHC (n = 9), normalized for cytoplasm content, shows a peak of intensity in the dilation, just ahead of the nucleus (DAPI, blue). (F) After 2 min exposure, Alexa 594-transferrin partially colocalizes with eGFP-tagged CLC. (G) After 10 min, transferrin partially colocalizes with YFP-2xFYVE+ early endosomes. (H) After 2 min exposure, transferrin is primarily localized to the dilation region. (I) Immunostaining for CLC (green arrows) and an active conformation of integrin β1 (red arrows) show overlap (yellow arrows) in the dilation. (J) Average Pearson's coefficients for each subcellular region (p<0.01, n = 10, mean±SEM, Student's t-test).

(A) Fewer cells migrate out of explants expressing K44A-dyn compared to explants expressing WT-dyn. (B) Example time-lapse series of cells in the same culture expressing either WT-dyn with tdTomato (red arrow) or K44A-dyn with GFP (green arrow). Kymographs showing position (x-axis) over time (y-axis) highlight movement of the cell body. Leading process movement can be seen in the K44A-dyn/GFP cell. See Movie S1. (C) K44A-dyn expressing cells that migrate out of explants have a significantly lower migration rate than WT-dyn expressing cells. (D) A greater percentage of K44A-dyn cells do not migrate, or have slower rates than those expressing WT-dyn. Data presented as mean±SEM, *p<0.05, **p<0.01, Student's t-test.

(A) Images of explants treated with varying concentrations of MiTMAB. Fewer DAPI-stained nuclei are present outside explants at increasingly higher concentrations of MiTMAB. (B) Effect of MiTMAB treatment on the percentage of cell nuclei present in evenly-spaced concentric rings surrounding the explant at increasing distances. (C) Time-lapse series of two neurons treated with MiTMAB. The yellow line marks the position of the dilation at the time of MiTMAB addition, which becomes that of the cell rear after washout. (Bottom) Kymographs plotting time vs. position highlight the positions of the cell rears. See Movies S2, S3. (D) Average velocity (µm/hr) of neurons migrating before the addition of MiTMAB is significantly faster than that in the presence of the drug or after washout (n = 19). The amount of time a cell spends in the same position between translocations is significantly longer before the first translocation after washout (Washout-1, n = 16) compared to either before (Pre, n = 15) or in the presence of MiTMAB (During, n = 13). Cells that made additional translocations during washout (Washout-2, n = 6) showed inter-translocation interval values similar to those before MiTMAB addition. Data presented as mean ± SEM, *p<0.05, **p<0.01, ***p<0.001, Student's t-test.

(A) Fewer DAPI-stained nuclei are present outside explants at increasingly higher concentrations of MDC. (B) Time-lapse series of two neurons treated with MDC. The yellow line marks the position of the dilation at the time of MDC addition, which becomes that of the cell rear after washout. (Bottom) Kymographs plotting time vs. position highlight the positions of the cell rears. See Movies S4, S5. (C) Average velocity (µm/hr) of neurons migrating before the addition of MDC is significantly faster than that in the presence of the drug or after washout (n = 63). The amount of time a cell spends in the same position between translocations is significantly longer before the first translocation after washout (Washout-1, n = 66) compared to either before (Pre, n = 48) or in the presence of MDC (During, n = 40). Cells that made additional translocations during washout (Washout-2, n = 46) showed inter-translocation interval values similar to those before MDC addition. Data presented as mean ± SEM, *p<0.05, **p<0.01, ***p<0.001, Student's t-test.

(A,B) Cytoplasmic GFP and DAPI reveal the morphology of neurons treated with (A) 1% DMSO vehicle or (B) MiTMAB. (C–H) Average line scans of fluorescence intensity normalized by cytoplasmic GFP were aligned by nuclear position, as determined by maximum scaled DAPI value. Integrin β1 (C, control, n = 9; D, MiTMAB, n = 8), active integrin β1 (E, control, n = 7; F, MiTMAB, n = 6), and FAK (G, control, n = 9; H, MiTMAB, n = 10). (I) Average fluorescence intensity of adhesion markers for: (top) all bins across cell position; (middle) all bins posterior to maximum DAPI value; (bottom) all bins anterior to maximum DAPI value. Data presented as mean ± SEM, *p<0.05, **p< 0.01, ***p<0.001, Student's t-test.

(A–C) After 2 days migrating in the developing cortex, neurons expressing DN-Hub (B) did not populate the CP as did control cells expressing GFP only (A). DN-Hub-expressing cells in the IZ/SVZ (B′) show similar morphology to GFP-only-expressing cells (A′). (C) The distribution of cells expressing DN-Hub vs GFP differs significantly at E15 (p<0.01, Chi-square test). DN-Hub-expressing neurons in their final position in the CP (E, E′) resemble GFP-only expressing neurons (D, D′). (F) The distributions of cells expressing DN-Hub vs GFP do not differ significantly at E17 (Chi-square test). Mean ± SEM. Scale bars, 10 µm.

(A–C) Neurons expressing K44A-dyn (B,E) did not reach the CP after 2 days migrating in the developing cortex, unlike cells expressing WT-dyn (A,D). Higher power view of WT-dyn (A′) and K44A-dyn (B′) cells in the IZ at E15, and of WT-dyn cells in the CP (D′) and K44A-dyn cells in the IZ (E′) at E17. (C, F) The distributions of cells expressing K44A-dyn at E15 and E17 differ significantly from those of WT-dyn-expressing cells (p<0.01, Chi-square test). Mean ± SEM. Scale bars, 10 µm.

A proposed model of neuronal migration consistent with our findings, though future work should address adhesion dynamics directly. Neurons first extend an exploratory leading process that forms transient and dynamic adhesions (perforated blue lines) in the growth cone. The leading process pauses, strengthening adhesions (solid blue lines) to provide traction. A cytoplasmic dilation forms, where endocytosis (gray circles) weakens adhesion. Myosin II (yellow) mediated contractions at the rear squeeze the nucleus forward and disrupt remaining attachments. Nucleokinesis into the former dilation establishes a new cell rear.