In particular, the SR radius was taken as 0
In particular, the SR radius was taken as 0.01 (since the DR is assumed not to be influenced by mechanical deformations of the NE), and a basket length of 0.075 = (Baum et al., ZCL-278 2014), and the nuclear pore density (Bizzarri et al., 2012), with which we obtain a total of 2908 NPCs/nucleus. Results Nuclear envelope 3D reconstruction Table ?Table11 shows the pore diameters and areas obtained from the 3D reconstruction. the NPC deformation. We then compared the prediction of the model for two different cell configurations with roundish and spread nuclear topologies with those measured on cells cultured in both configurations. To measure the geometrical features of the NPC, using electron tomography we reconstructed three-dimensional portions of the envelope of cells cultured in both configurations. We found nonsignificant differences in both the shape and size of the transmembrane ring of single pores with envelope deformation. In the numerical model, we thus assumed that the changes in pore complex permeability, caused by the envelope strains, are due to variations in the opening configuration of the nuclear basket, which in turn modifies the porosity of the pore complex mainly on its nuclear side. To validate the model, we cultured cells on a substrate shaped as a spatial micro-grid, called the nichoid, which is nanoengineered by two-photon laser polymerization, and induces a roundish nuclear configuration in cells adhering to the nichoid grid, and a spread configuration in cells adhering to the flat substrate surrounding the grid. We then measured the diffusion through the nuclear envelope of an inert green-fluorescent protein, by fluorescence recovery after photobleaching (FRAP). Finally, we compared the diffusion times predicted by the numerical model for roundish vs. spread cells, with the measured times. Our data show that cell stretching modulates the characteristic time needed for the nuclear import of a small inert molecule, GFP, and the model predicts a faster import of diffusive molecules in the spread compared to roundish cells. (Rompolas et al., 2013) and (Nava et al., 2012). = 3) on glass coverslips (13 mm diameter) or 35 mm-Petri dishes. One day after plating, the culture medium was removed and cells were washed with phosphate buffered saline. To model the deformed (spread) configuration, MSCs were fixed for 2 h at room temperature with 1.5% glutaraldehyde in 0.1 M sodium cacodylate (pH 7.2), detached by scraping, centrifuged to recover the pellet, kept overnight at 4C in 1.5% glutaraldehyde in 0.1 M sodium cacodylate and finally rinsed in 0.1 M sodium cacodylate (pH 7.2). To model the undeformed (roundish) configuration, MSCs were detached with trypsin, ZCL-278 centrifuged to recover the pellet, fixed overnight with 1.5% glutaraldehyde in 0.1 M sodium cacodylate, and rinsed in 0.1 M sodium cacodylate. STEM analysis After chemical fixation, MSCs cells in the spread and roundish configurations were washed several times in 0.1 M sodium cacodylate (pH 7.2), post-fixed in 1% osmium tetroxide in distilled water for 2 h and stained overnight at 4C in an aqueous 0.5% uranyl acetate solution. After several washes in distilled water, the samples were dehydrated in a graded ethanol series, and embedded in EPON resin. Sections of about 70 nm were cut with a diamond knife (DIATOME) on a Leica EM UC6 ultramicrotome. Transmission electron microscopy (TEM) images were collected with an FEI Tecnai G2 F20 (FEI Company, The Netherlands). EM tomography was performed in scanning TEM (STEM) mode, using a high angular annular dark field (HAADF) detector on 400 nm thick sections of MSCs cells in both spread and roundish configurations. The tilt series were acquired from a 60 tilt range. The ZCL-278 resulting images had a pixel size of 1 1.85 nm as shown in Figure ?Figure2.2. The tomograms were computed with IMOD (version 4.8.40) (Kremer et al., 1996). Isosurface based segmentation and three-dimensional visualization on unbinned and unfiltered tomograms were performed using Amira (FEI Visualization Science Group, Bordeaux, France). Open in a separate window Figure 2 TEM image of the NE with NPCs (in circles). Nuclear envelope 3D reconstruction Open source image processing software, IMOD (Kremer et al., 1996), specialized in tomographic reconstruction developed by the University of Colorado was used to segment STEM images. Segmentation was performed manually on each slice. This process was guided by first locating the heterochromatin which is located very close to the membrane on the nuclear side (Figure ?(Figure2).2). Figure ?Figure3A3A shows a typical slice segmentation detailing the location of several nuclear pores in the membrane. This process was followed for each slice as shown in Figure ?Figure3B.3B. The nuclear envelope was then reconstructed by linear interpolation of the segmentation between consecutive slices (Figure ?(Figure3C3C). Open in a separate window Figure 3 STEM Cell segmentation of Bmp10 the Nuclear Envelope and Pores. (A) Cell electron.