Fig. 4. CMT-causing YARS1 mutations disrupt cell migration, protrusion dynamics, and neurite outgrowth in SH-SY5Y cells.

a Phase-contrast time course imaging of a scratch closure after a mechanical wound induction in a confluent culture of undifferentiated SH-SY5Y cells stably expressing either YARS1^WT, YARS1^E196K, or YARS1^G41R. Yellow lines indicate the migration front at the specific time point while blue lines show the initial position of the wound. Scale bar – 400 µm. b Quantifications of the cell density at the scratch over the time course of 80 h and at specific time points (24 h and 48 h); n = 5 independent measurements, averaged from 10-16 individual scratch wounds, taken every 2 h. Data in the bar graph in b presented as mean values ± SEM; at 24 h – ns – non-significant (p > 0.9999), *p = 0.0317, **p = 0.0079; at 48 h – ns – non-significant (p = 0.2222), *p = 0.0159. c Phase-contrast image sequences (10 fps for 10 min) of individual lamellipodia used to generate kymographs from 15 µm line segments (yellow lines) perpendicular to the lamellipodia movement direction. d Representative kymographs from YARS1^WT and YARS1^CMT individual lamellipodia, used to determine the protrusion displacement (micrometers on the x axis), protrusion persistence (time in seconds on the y axis), and the protrusion rate (displacement to persistence ratio). e Graph representing the protrusion metrics calculated from the kymographs. Violin plots represent at least 185 individual protrusions per genotype. After a two-sided Mann-Whitney U test for wound distance ***p = 0.0004 and p = 0.0002 for YARS1^E196K and YARS1^G41R compared to YARS1^WT, respectively; for wound persistence ****p < 0.0001; for wound rate – ns – non-significant (p = 0.7920), **p = 0.0077 and p = 0.0028 for YARS1^E196K and YARS1^G41R compared to YARS1^WT, respectively. f Representative phase-contrast images of differentiated SH-SY5Y cells stably expressing YARS1^WT, YARS1^E196K or YARS1^G41R, on which neurite outgrowth and branching were analyzed; blue traces delineate soma, green traces mark projections (primary neurites and their secondary branches). g Quantifications of projections’ metrics in YARS1^WT, YARS1^E196K and YARS1^G41R cells. For neurite/soma area ratio, n = 144 random image fields for each genotype. Primary neurite length determined on n = 2342 (YARS1^WT), 1944 (YARS1^E196K), and 1723 (YARS1^G41R) individual cells from at least three independent differentiations. Secondary neurite length, n = 65 individual cells for each genotype. Percentage of secondary neurite presence (presented as mean values ± SEM), n = 18 random image fields for each genotype. Median and quartiles on the violin plots are indicated by thick and thin black lines, respectively. ns – non-significant, ****p < 0.0001; after a two-sided Mann-Whitney U test. Scale bar – 100 µm. Source data are provided as a Source data file.