November 15, 2017;
Nuclear pore complex plasticity during developmental process as revealed by super-resolution microscopy.
Nuclear Pore Complex (NPC) is of paramount importance for cellular processes since it is the unique gateway for molecular exchange through the nucleus. Unraveling the modifications of the NPC structure in response to physiological cues, also called nuclear pore plasticity, is key to the understanding of the selectivity of this molecular machinery. As a step towards this goal, we use the optical super-resolution microscopy method called direct Stochastic Optical Reconstruction Microscopy (dSTORM), to analyze oocyte development impact on the internal structure and large-scale organization of the NPC. Staining of the FG-Nups proteins and the gp210 proteins allowed us to pinpoint a decrease of the global diameter by measuring the mean diameter of the central channel and the luminal ring of the NPC via autocorrelation image processing. Moreover, by using an angular and radial density function we show that development of the Xenopus laevis oocyte is correlated with a progressive decrease of the density of NPC and an ordering on a square lattice.
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dSTORM imaging of the nuclear envelopes from X. laevis oocytes. (A) dSTORM image of a spread nuclear envelope labelled with anti-gp210 primary antibody and Alexa647 secondary antibody. (B) dSTORM image of a spread nuclear envelope labelled with WGA-Alexa488. Scale bar 500 nm. (C) dSTORM image of a spread nuclear envelope labelled with WGA-Alexa488 (green) and gp210-Alexa647 (magenta). (D) Zoom on the image (A). (E) Zoom on the image (B).
Figure 2. Effect of oocyte development on the density, the number and the diameter of the nuclear pore complexes. (A–C) dSTORM images of nuclear envelopes from oocytes respectively at stage II, IV and VI. The central channel is labelled with fluorescent WGA-Alexa647. Scale bar 5 µm. Insets: Stereomicroscope images of the oocyte respectively at stage II, IV and VI. Scale bar 50 µm. (D) Effect of oocyte development on nuclear pore complex density. (E) Effect of oocyte development on nuclear pore number per nucleus. (F) Effect of oocyte development on central channel diameter. (G) Effect of oocyte development on gp210 diameter. For each condition, the number of investigated NPC is superior to 300 000. Errors are experimental standard errors. The precision of the measurements was assessed by bootstrapping and by comparing different rounds of experiments.
Figure 3. Effect of oocytes development on the organization of the nuclear pore complexes. (A–C) 2D histogram of the probability P(d,α) to observe a NPC on a given envelope with two neighbors at a distance d and forming an angle α respectively for oocytes at stage II, IV and VI. For stage VI oocytes the most probable coordinates are (135 ± 5 nm, 90 ± 6°). The frequency event is normalized by the maximum for each histogram. (D–F) First neighbor angle distribution evolution for respectively stage II, stage IV and stage VI oocytes. The red dashed line at 60° corresponds to the minimal angle possible between 3 NPCs. (G) Experimental most probable angle α* between three nuclear pore complexes as a function of the distance d between the central pore and its two neighbors (dashed line) and theoretical angle α between three nuclear pore complexes as a function of the distance d between the central pore and its two neighbors in the packing model (solid line). In this model all the nuclear pore complexes are in contact with their first neighbors but without any large-scale order.