B cells within germinal centers preferentially move from DZ to LZ. A plane was fitted to the interface region between DZ and LZ, and various measures of cell migration were plotted as a function of the shortest distance to the estimated plane. All values represent running averages of movement steps. Plotted are the mean 3D speed (A), the mean turning angle (B), the mean angle to the normal vector that defines the DZ–LZ border plane and that points toward the LZ (D), the mean angle to the estimated DZ–LZ border plane (E), and the mean displacement rate toward the LZ (F). The relationship between the angles measured in D and in E is further explained by the drawing in C.

Modeling shows that directed B-cell migration from DZ to LZ strongly affects the time to reach the LZ. (A) Example of 20 in silico cells migrating in a simulated GC. The plane represents the border separating LZ and DZ. (B) Comparison of the experimentally measured and simulated distributions of speed, turning angle, and angle to the vector normal to the DZ–LZ border pointing toward the LZ. Note that the latter distribution deviates only slightly from the distribution expected for random migration (denoted by the solid line). (C) The spatial distribution of simulated cells in 10 simulations at the beginning and at the end of a 5-h simulation. Each dot represents the relative frequency of cells within distance bins of 10 μm wide in a single simulation of 1,000 cells. (D) The mean time to reach the LZ when starting at various depths within the DZ of the simulated GC. Simulations are either based on all DZ migration data (green and black line) or on the DZ migration data of B cells with straight tracks (maximum confinement ratio 1.75 min^1/2, red line). Simulations either incorporate the directed migration as observed experimentally (black and red line) or are based on random migration with similar speeds and turning angles as in the experimental data (green line). Error bars denote SD of the mean.

Estimating a plane through the interface region between DZ and LZ for each experiment separately. (A) Voxels forming the DZ–LZ interface are plotted as individual dots along with the plane fitted through these data. Dots are color coded by their depth in 3D space (z coordinate). (B) Comparison of the DZ–LZ border planes estimated at the first (in brown), middle (in green), and final (in blue) time point of imaging for each experiment. Planes are oriented such that all are visible.

Preferential migration from DZ to LZ is overrepresented within a large subset of straight moving cells. (A) The corrected confinement ratio (CR) as a function of track duration. (Left) The red lines represent the different tracks in an experiment, and the green line gives the average behavior of all cells. (Right) Demonstration of how the maximum CR is calculated for the example track shown in the Inset. Each black line represents the CR for one particular part of the track, and the maximum of all these lines is the maximum CR (denoted by arrow). The red line represents the average CR for this particular cell (i.e., this is one of the red lines on the Left. (B) The frequency distribution of maximum CR values for all cells in one experiment and the corresponding tracks for two subselections of the cells. (C and D) Mean displacement rate toward the LZ in the straightest part of the track as a function of the maximum CR value of the cell. In C, each dot represents one cell (red line represents a linear regression), whereas in D, running averages of 200 cells are presented. (E) The mean displacement rate toward the LZ as a function of the distance to the DZ–LZ border plane for a subpopulation with relatively straight tracks (red line, cells with max CR values above 1.75 min^1/2) and for one with relatively condensed tracks (blue line, cells with max CR values below 1.75 min^1/2). For comparison, the behavior of the entire B-cell population is plotted in black (same as Fig. 2F). Lines are running averages of 3,000 movement steps. The horizontal dotted line in D and in E gives the expected mean value for the displacement rate toward LZ in a randomly migrating population.