Journal: Research Square

Article Title: Exploring Cellular Gateways: Unraveling the Secrets of Disordered Proteins within Live Nuclear Pores

doi: 10.21203/rs.3.rs-3504130/v1

Figure Lengend Snippet: A) Simplified optical setup for SPEED microscopy. Single-point illumination employs 488-nm, 561-nm, and 633-nm lasers, targeting individual NPCs at the nuclear envelope (NE) equator in live cells. DM: dichroic mirror, RM: reflection mirror, N: Nucleus, C: Cytoplasm. B) A single NPC is illuminated at the focal plane of SPEED microscopy, depicted in both axial and lateral dimensions. (N: Nucleus, C: Cytoplasm). C-E) Example single-molecule fluorescence are shown for Nup214C located on the cytoplasmic side, POM121C in the central scaffold, and Nup153C in the nuclear basket. The cartoons illustrate the labeling of these FG Nups. The time-series images and their corresponding photobleaching curves indicate the initiation and termination of fluorescence from JF dyes used to label these FG Nups, with the Y-axis representing the photon counts and the X-axis representing time. F) Single-molecule tracking and the localization of fluorescent spots in the time-series images, as presented in C-E, generated the 2D single-molecule trajectories for Nup214C (red), POM121C (green), and Nup153C (yellow). These trajectories were then superimposed and overlaid onto the NPC scaffold structure (light grey). The light grey structure represents the NPC scaffold structure obtained through Cryo-EM, sourced from the RCSB Protein Data Bank. It is noteworthy that the structure presented here is the original version publicly available. This structure contains three small fragments of Nup98 and a coiled-coil structure from each of the three central scaffold FG Nups: Nup62, Nup58, and Nup54, in addition to the structural information of scaffold Nups. G-I) Mean Squared Displacement (MSD) analyses were conducted on 2D single-molecule trajectories, which were compiled from over fifty single-molecule traces for the FG-rich and non-FG-rich domains of Nup214 (red), POM121C (green), and Nup153C (yellow), respectively. These analyses (ii) of the trajectories (i) provided essential insights, including the extension length (iv), the diffusion coefficient (iii), and the exponent α (iii), as determined by the relationship M S D ( t ) = 4 D t ∧ α for these FG Nups within live cell NPCs. The histogram of extension lengths was fitted with Gaussian functions, facilitating the quantification of the maximum extension length presented as mean ± SD. The blue and pink regions specifically delineate the range from −200 nm to 0 nm (cytoplasmic side) and from 0 nm to 200 nm (nuclear side) along the nucleocytoplasmic transport axis (the X dimension), respectively. In the perpendicular direction (the Y dimension) for both regions, the range extends from −100 nm to 100 nm. J) The distribution of localizations for the FG-rich domains of each FG-Nup, evaluated with respect to their 2D spatial locations on the cytoplasmic (blue) or nucleoplasmic (pink) side, is illustrated. The blue and pink areas correspond to the range from −200 nm to 0 nm and from 0 nm to 200 nm along the nucleocytoplasmic transport axis, respectively. K) Displays the average maximum extensions (in nm) for each FG-Nup, distinguishing between FG-rich (orange) and non-FG-rich (dark blue) regions. Nups denoted with * contain FG-rich regions (orange) at both ends. L) A scatter plot depicts the diffusion of FG Nups positioned on the cytoplasmic side (red), within the central scaffold (green), and on the nuclear side (yellow). The Y-axis represents α , while the X-axis displays the diffusion coefficient (μm 2 /s), presented on a logarithmic scale. N: Nucleus, C: Cytoplasm.

Article Snippet: Mean square displacement calculations were made using the MSD analyzer package for MATLAB .

Techniques: Microscopy, Fluorescence, Labeling, Generated, Cryo-EM Sample Prep, Diffusion-based Assay

Journal: Developmental cell

Article Title: Actin dynamics drive microvillar motility and clustering during brush border assembly

doi: 10.1016/j.devcel.2019.07.008

Figure Lengend Snippet: (A) LLSM of a CL4 cell stably expressing mCherry-Espin, reconstructed then viewed as a maximum intensity z-projection. Scale bar 10 μm, blue dashed boxes corresponds to B. (B) Enlarged images from A (left) with time projections (right) showing microvillar movement over 3 minutes. (C) SDCM of the apical surface of a CL4 cell stably expressing mCherry-Espin, viewed as a maximum intensity z-projection. Scale bar 10 μm, white dashed box corresponds to D, red dashed box corresponds to E. (D) 3-dimensional (3D) depth color coded z-stack viewed en face (xy plane, upper panel) or laterally (xz plane, lower panel). Scale bars are 1 μm, z-axis depth color code (lower panel) to scale with tick marks at 1 μm intervals. Microvilli exhibit a range of orientations from parallel (represented by a single color) to perpendicular to the cell surface (spanning multiple color bands, circled—top panel, arrowheads—bottom panel). (E) Time series of microvilli translocating across the cell surface; red and orange arrows highlight the paths of two distinct protrusions. Scale bar 2 μm. (F) Rose plot of trajectories measured from the tips of microvilli (n = 101) for the cell in C. (G) Microvillar trajectories from F were subject to MSD analysis; red open circles represent the mean MSD, error bars indicate standard error of the mean (SEM), grey area marks the weighted standard deviation (SD) over all MSD curves, and the solid line indicates a fit of the data to an active movement model (diffusion coefficient, D = 0.000283 μm2/s and velocity, V = 0.21 μm/min). (H) Trajectories from F were analyzed for normalized velocity autocorrelation, solid line. Dotted line at 0 indicates the velocity autocorrelation of random diffusive movement. (I-K) Average microvillar velocity, maximum microvillar length, and persistence, respectively. (L) Microvillar lifetime frequency histogram. Error bars indicate mean ± SD, (I-L) n = 171 microvilli from 7 cells for mCherry-Espin, n = 183 microvilli from 6 cells for EGFP-Lifeact, * p < 0.05, n.s. = not significant.

Article Snippet: Data was exported and analyzed with MATLAB using a package specifically developed for MSD analysis ( Tarantino et al., 2014 ), which is publicly available at http://www.mathworks.com/matlabcentral/fileexchange/40692-mean-square-displacement-analysis-of-particles-trajectories .

Techniques: Stable Transfection, Expressing, Standard Deviation, Diffusion-based Assay

Journal: Developmental cell

Article Title: Actin dynamics drive microvillar motility and clustering during brush border assembly

doi: 10.1016/j.devcel.2019.07.008

Figure Lengend Snippet: (A) SDCM of the apical surface of CL4 cells stably expressing mCherry-Espin showing the response to 20 μM Blebbistatin. Right, time series montage of a single protrusion highlighted with a 10% pseudo-colored overlay. Scale bars are 2 μm. Drug was added following the 5-minute time interval, yellow arrowhead. Black arrows indicate the baseline rate of microvillar movement. Blue arrow indicates the rate of microvillar movement after the addition of drug. (B) Rose plot shows the microvillar trajectories (n = 100) from a single cell treated with 20 μM Blebbistatin. (C) 25 representative microvillar trajectories are isolated for display. Scale bar is 5 μm. (D) MSD analysis of microvillar trajectories from B. (E) Normalized velocity autocorrelation analysis of microvillar trajectories from B; data were fit to an active movement model with D = 0.000093 μm2/s, V = 0.12 μm/min. (F) SDCM of the apical surface of CL4 cells stably expressing mCherry-Espin showing the response to 30 μM Cytochalasin D. Right, time series montage of a single protrusion highlighted with a 10% pseudo-colored overlay. Scale bars are 2 μm. Drug was added following the 5-minute time interval, yellow arrowhead. Black arrows indicate the baseline rate of microvillar movement. Orange arrow indicates the rate of microvillar movement after the addition of drug. (G) Rose plot shows the microvillar trajectories (n = 100) from a single cell treated with 500 nM Cytochalasin B. (H) 25 representative microvillar trajectories are isolated for display. Scale bar is 5 μm. (I) MSD analysis of microvillar trajectories from G; data could not be fit with an active movement model. The curve for mCherry-Espin with no drug treatment is plotted for comparison (grey dotted line). (J) Normalized velocity autocorrelation analysis of microvillar trajectories from G. (K-M) Average microvillar velocity, maximum microvillar length, and persistence, respectively, measured from untreated cells (from Figure 1), or cells exposed to Blebbistatin or Cytochalasin B. For Blebbistatin and Cytochalasin datasets, n = 178 and 175 microvilli, respectively, from 6-7 cells. Bars represent mean ± SD. *** p < 0.0001, n.s. = not significant.

Article Snippet: Data was exported and analyzed with MATLAB using a package specifically developed for MSD analysis ( Tarantino et al., 2014 ), which is publicly available at http://www.mathworks.com/matlabcentral/fileexchange/40692-mean-square-displacement-analysis-of-particles-trajectories .

Techniques: Stable Transfection, Expressing, Isolation, Comparison