n 21 max  (R&D Systems)

Journal: Nature Communications

Article Title: Compartmentalized cytoplasmic tradewinds direct soluble proteins

doi: 10.1038/s41467-026-70688-6

Figure Lengend Snippet: a Photoactivation of a diffraction-limited spot in a CAD cell expressing PaGFPactin shows asymmetric dispersion toward the front of the cell. b Graphic illustrating how the asymmetry is analyzed from fluorescence intensity perpendicular and parallel to the cell leading edge of control and blebbistatin cells. A transport metric is defined as the ratio of the area under each half of the intensity curve, indicated by different shading levels on either side of the center line. c Photoactivation of a diffraction-limited spot in a blebbistatin-treated CAD cell expressing PaGFPactin shows symmetric movement in all directions away from the activation spot. d Stacked line-intensity plots of data summed for t = 1.5, 3, 7.5 s after the initiation of spot activation indicate that the asymmetry in the control cell in ( a ) only occurs perpendicular to the direction of the cell's leading edge. e Box and whisker plots of the transport ratio, including all individual data points, indicate fluorescence dispersion toward the leading edge (perpendicular) in control cells. ( n = 28 control, 32 blebbistatin (0.2 μM)). f Stacked line-intensity data plots summed for t = 1.5, 3, 7.5 s after the initiation of spot activation indicate the symmetric fluorescence distribution in the blebbistatin-treated cell in ( c ). g Transport ratio analysis as described in ( b ) reveals asymmetry for the spread of actin mutants that cannot be incorporated into the network ( n = 13, G13R, 14, R62D). h Asymmetric spread was also observed for free dye, mEos3.2 ( n = 13), parallel to the edge and perpendicular to the edge. i Sum of 3891 single-molecule frames acquired at 8 ms/frame from spot activation of mEos-R62D, yielding 1451 tracks > 5 steps. p values are two-tailed t -tests in ( e , h ), and one-way ANOVA with post-hoc Tukey HSD test in ( g ). Box plots show median (center line), interquartile range (25th–75th percentiles; box), whiskers to the most extreme non-outlier values, and notches as 95% CIs. Scale bars, 5 μm. All panels represent at least three independently performed experiments, n= number of cells analyzed. Source data are provided as a Source Data file.

Article Snippet: PaGFP-Actin was constructed by exchanging the color from PaGFP-C1 (gift from George Patterson, Addgene no. 11910) and egfp-Actin using BsPE1 and BamH1 , . mNeon LC-Myosin-N7 was constructed by exchanging color with mEmerald-LC-Myosin-N7 (gift from Michael Davidson, Addgene no. 54146) and mNeon Lifeact (Addgene no. 9887) using BsrGI and AgeI. mEos3.2 ARC3 was constructed by exchanging color with egfp-ARC3, a gift from Matt Welch (Addgene no. 8462) and mEos3.2-N1 (Addgene no. 54525) using SmaI and Not I. mEos2 Paxillin-22 (Addgene no. 57409) and mEos3.2 Lifeact-7 (Addgene no. 54696) were gifts from Michael Davidson. mEos3.2 Vinculin-N-21 was constructed by exchanging color with mEos2-vinculin-14 (Addgene 57438) and mEos3.2-N1 (Addgene no. 54525) and is available as (Addgene no. 6692). pENTR-NLS-actin-R62D (Addgene no. 11831) was used to construct the mEos3.2 actin-R62 plasmids using SalI-HF and BmbG1 to combine the mutated region of actin with existing mEos3.2 actin plasmids.

Techniques: Expressing, Dispersion, Fluorescence, Control, Activation Assay, Whisker Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Compartmentalized cytoplasmic tradewinds direct soluble proteins

doi: 10.1038/s41467-026-70688-6

Figure Lengend Snippet: a , b Spot activation of mEos3.2 Lifeact in either the lamella or the cell body in CAD cells. Randomly colored trajectories 10 frames or longer were detected during a 50-frame window. Circles indicate a 2 μm boundary around the center of the activation spot. c , d Rose plots illustrate the angle at which trajectories moving away from the activation spot cross the bounding circle in the lamella or the cell body. Rayleigh Tests indicate that ( c ) is not uniformly distributed ( p = 0.0002, while ( d ) may be uniformly distributed (p = 0.36). Both the Mardia–Watson–Wheeler and Kuiper two-sample tests detect a significant difference between ( c ) and ( d ) p = 0.001. e Representative FCS autocorrelation curves (black lines) of Halo Actin labeled with JF549 fit to a model of two diffusion coefficients and flow. The model fits of the data (black trace) in the lamella ( D = 11.1 μm 2 s −1 , v = 1.8 μms −1 ) and cell body ( D = 9.64 μm 2 s −1 , v = 1.1 μms −1 ), and the residual panel indicates the accuracy of fit . f , g Primary diffusion is not significantly different in the cell lamella or body. The flow velocity is much larger in the lamella and statistically different from the cell body ( n = 40 cell body measurements in 24 cells, and 20 lamella measurements in 20 cells), over five independent replicates. p value values two-tailed t -tests. Box plots show median (center line), interquartile range (25th–75th percentiles; box), whiskers to the most extreme non-outlier values, and notches as 95% CIs. Source data are provided as a Source Data file.

Article Snippet: PaGFP-Actin was constructed by exchanging the color from PaGFP-C1 (gift from George Patterson, Addgene no. 11910) and egfp-Actin using BsPE1 and BamH1 , . mNeon LC-Myosin-N7 was constructed by exchanging color with mEmerald-LC-Myosin-N7 (gift from Michael Davidson, Addgene no. 54146) and mNeon Lifeact (Addgene no. 9887) using BsrGI and AgeI. mEos3.2 ARC3 was constructed by exchanging color with egfp-ARC3, a gift from Matt Welch (Addgene no. 8462) and mEos3.2-N1 (Addgene no. 54525) using SmaI and Not I. mEos2 Paxillin-22 (Addgene no. 57409) and mEos3.2 Lifeact-7 (Addgene no. 54696) were gifts from Michael Davidson. mEos3.2 Vinculin-N-21 was constructed by exchanging color with mEos2-vinculin-14 (Addgene 57438) and mEos3.2-N1 (Addgene no. 54525) and is available as (Addgene no. 6692). pENTR-NLS-actin-R62D (Addgene no. 11831) was used to construct the mEos3.2 actin-R62 plasmids using SalI-HF and BmbG1 to combine the mutated region of actin with existing mEos3.2 actin plasmids.

Techniques: Activation Assay, Labeling, Diffusion-based Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Compartmentalized cytoplasmic tradewinds direct soluble proteins

doi: 10.1038/s41467-026-70688-6

Figure Lengend Snippet: a Asymmetry analysis of spot activation of mEos3.2 Arp3, vinculin, and paxillin in CAD cells illustrates that they are targeted toward the leading edge, where polymerization and adhesions initiate ( n = 14, Arp3, 9 vinculin, 10 paxillin cells). b Temporal color-coded spot activation of mEos3.2 actin in an NG108 cell advancing preferentially on the right illustrates that this is the region where recently activated actin is targeted (arrows). c Maximum projection of 3D SIM images of Alexa 647-phalloidin and mEmerald MLC in control cells and cells treated with 0.2 μM nitro-blebbistatin before fixation illustrates the broadening and flattening of the actin-myosin arcs at the border between the lamella and cell body when myosin is inhibited. d These observations were quantified by fitting the arcs to ellipses and calculating ellipticity, which reveals flatter arcs in the blebbistatin-treated cells ( n = 19 control cells, 87 measurements, 17 blebbistatin cells, 100 measurements). Ellipticity =√(1-(minor) 2 major −2 )) e An overlay of two different mNeon MLC image time points ( t = 0, and t = 45 s) with arrows indicating where the edge retracts and extends during the 45 s. f Contour of the same two-time points in ( e ) with arrows indicating changes in the position and curvature of the arcs corresponding to the retraction and advancement of the leading edge. g CAD cell expressing mEmerald MLC were treated with a high-power localized beam of 405 light positioned over one arc, causing the arc to be disrupted and only the leading edge in front of that single arc to collapse. The yellow line indicates the position of the edge prior to MLC disruption. p values two-tailed t -tests. Box plots show median (center line), interquartile range (25th–75th percentiles; box), whiskers to the most extreme non-outlier values, and notches as 95% CIs. Scale bars, 5 μm. Source data are provided as a Source Data file.

Article Snippet: PaGFP-Actin was constructed by exchanging the color from PaGFP-C1 (gift from George Patterson, Addgene no. 11910) and egfp-Actin using BsPE1 and BamH1 , . mNeon LC-Myosin-N7 was constructed by exchanging color with mEmerald-LC-Myosin-N7 (gift from Michael Davidson, Addgene no. 54146) and mNeon Lifeact (Addgene no. 9887) using BsrGI and AgeI. mEos3.2 ARC3 was constructed by exchanging color with egfp-ARC3, a gift from Matt Welch (Addgene no. 8462) and mEos3.2-N1 (Addgene no. 54525) using SmaI and Not I. mEos2 Paxillin-22 (Addgene no. 57409) and mEos3.2 Lifeact-7 (Addgene no. 54696) were gifts from Michael Davidson. mEos3.2 Vinculin-N-21 was constructed by exchanging color with mEos2-vinculin-14 (Addgene 57438) and mEos3.2-N1 (Addgene no. 54525) and is available as (Addgene no. 6692). pENTR-NLS-actin-R62D (Addgene no. 11831) was used to construct the mEos3.2 actin-R62 plasmids using SalI-HF and BmbG1 to combine the mutated region of actin with existing mEos3.2 actin plasmids.

Techniques: Activation Assay, Control, Expressing, Disruption, Two Tailed Test