Extended Data Fig. 7: Force clamp protocol for microsphere insertion into zebrafish embryonic cell nuclei.
a, Schematics of the nuclear insertion process using the force clamp modality of the optical micromanipulation platform. A bead is brought into contact with the nucleus and a force clamp is set at F0 = 100 ~ 150 pN. During some tens of seconds, the bead creeps against the nuclear envelope, eventually breaking it and entering the nucleus. b, Example of an unsuccessful bead insertion assay. The nuclear envelope is too stiff for the optical trap to enter the nucleus. At t∿5 s, the force is set to 120 pN (top) and the trap pushes the bead against the nucleus. After t∿40 s, the force clamp is turned off. c, Example of a successful insertion. Similar to (b), a force clamp of F0 = 120 pN is initiated at t∿5 s. Around t∿25 s, the nuclear envelope breaks and the trap accelerates into the nucleus. After this, the force clamp is turned off. d, Summary for control cells (i), cells incubated with latrunculin A, (ii) and cells with lamin A overexpression (iii). Trajectories have been normalized with respect to the force setpoint as x(t)/F0. Colored traces (red: CTR; black: LatA; blue: LMNA + ) indicate successful events in which the bead gets inserted into the nucleus. Gray lines are force compliance curves that didn’t succeed in inserting the bead into the nucleus. e, Normalized creep compliance curves over the first 5 s of creep test. Solid lines are medians and shaded areas correspond to the ±25% quantiles. Note, all curves show a fast initial compliance, followed by an inflection indicating an elastic plateau. f, Representative data showing the fitting procedure. Yellow line indicates the fit, dotted lines indicate the relevance of the extracted parameters. g, Quantification of the force applied to the nucleus during the constant force experiment. Error bars represent the 95% confidence interval of the median. h-j Leading parameters extracted from the fit. (h) Restoring stiffness of the nuclear envelope; (i) resistance to deformation on short time scales and (j) resistance to deformation on long time scales. Number of measurements for control N = 15 from n = 4 independent experiments. For LatA N = 13, n = 3 and for LMNA+ N = 20, n = 4. Numbers above the brackets in panel g-j indicate the p-value derived from two-tailed pairwise comparison using a Mann-Whitney U-test on the experimental data points (grey). Violin plots represent the distribution of the bootstrapped mean value (±95% confidence interval) calculated from 5000 virtual experiments, using the experimental data as a sample to describe the population.
