Three CCs identified by TFM. (a) Steps in determining microbead displacements around a GC. a1, image of a GC. a2, superimposed image of fluorescent beads before (red) and after (green) osmotic lysis of the cell. Insets show the boxed regions at a higher resolution (bars, 1 μm). Solid traces, cell outline. a3, discrete displacement vectors, with arrow length and arrowheads representing displacement magnitude and direction, respectively. a4, continuous displacement field map (coded in grayscale) obtained by interpolation of discrete displacement vectors in a3, marked by regularly spaced vectors. (b) Displacement of the bead at different distances from the GC. (b, left) Displacement map. (b, middle) Beads close to (cyan and purple asterisks, <30 µm) and distant from (red asterisks, ≥30 µm) the neuron. Green arrow, direction of soma translocation. Broken line, points ≥30 µm away. (b, right) Changes of displacements with time for beads at different distances. No displacement was observed for distant beads (red). (c) Stress maps of substrates. c1–c3, stress maps computed from the continuous displacement field maps at different times for the same GC shown in a, and three distinct CCs (white circles) were observed at three different times. c1, stress map at t = 1 min (the same time as in a). c2, t = 21 min. c3, t = 38 min. c4, stress map caused by another GC showing two coexisting CCs. c5, each bifurcating process generated its own CC. (d) CC locations identified by a stress map generated by the same typical GC shown in a. (d, left) Outline (blue) and axis (red) of the GC. (d, middle) Dynamics of the stress along the axis of the migratory GC at different distances from the tip of the TP over a 60-min period, obtained from the stress map similar to that shown in c1. Line bins, 21 s. The location of the CCs (marked by green dots), determined as the site of the maximal absolute value of the angular difference between stress vectors of adjacent pixels (see Materials and methods). Cyan line, soma center. (d, right) Frequency distribution of CCs observed at different distances away from the soma during the observation period for the cell shown on the left. Red curve, best fit with a multi-peak Gaussian function (R = 0.61). Broken black line, soma center. Additionally, 18 more cases are shown in Fig. S2 and a total number of 72 GCs are summarized in e4. (e) CC locations identified by a stress differential map. e1–e3, changes in the stress (“stress differential,” ΔF over Δt = 21 s) at three different time points (t = 0, 26, and 38 min) for the same cell as in a, with ΔF color-coded. Note that three different CCs were identified (broken lines) as regions with convergent stress differential vectors. e4, percentage of cells showing CCs at three different locations. dLP, distal end of LP; pLP, proximal end of LP; TP, trailing process. Histograms, data from all 72 GCs, each for an ∼40-min observation period, with the CC location determined by stress map (orange) and stress differential map (blue).
