Myosin-X Acts Upstream of L-Plastin to Drive Stress-Induced Tunneling Nanotubes

Figures

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  Figure 1. L-plastin localizes within TNTs and proto-TNTs in CAD cells treated with hydrogen peroxide. CAD cells were treated with 100 μM H2O2 for 5 min to increase the number of TNTs between cells, fixed and labeled with anti-L-plastin antibody (red) and the membrane dye WGA-green to identify cellular protrusions (green). (a) A confocal microscopy 3D representative image of CAD cells connected with TNTs (white arrows). (b) 3D reconstruction image using Imaris software, employing “spot rendering” to identify L-plastin localization (red) within TNTs and along proto-TNTs. (c) 3D reconstruction image using Imaris software, employing “spot rendering” plus “filament tracer” to enhance the visualization of L-plastin puncta (in red) within TNTs (gray). (d) Zoom in of the 3D reconstruction using filament tracer and spot rendering in (c). All scale bars are 2 μm in length.

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  Figure 2. L-plastin upregulation increases the number of cells connected by TNTs. CAD cells were transiently transfected with either GFP-vector as a control or GFP-L-plastin for 24 h and lysed. (a) Representative stain free gel is shown as a loading control. Stain free gels were used to quantify the total protein loaded in each lane. (b) Representative fluorescent Western blot shows upregulation of L-plastin protein levels in GFP-L-plastin transfected cells compared to cells transfected with GFP-vector control. Parallel experiments were performed along with the Western blots. CAD cells transfected with GFP-vector or GFP-L-plastin were imaged and Z-stacks of random fields were acquired. (c) Representative 3D image of GFP transfected cells is shown for the green channel (GFP) and red channel (WGA-Rhod). (d) Zoom in of ROIs (dashed rectangles) outlined in (c), where TNTs (white arrowhead) are present. (e) Representative 3D image of GFP-L-plastin transfected cells is shown for the green channel (GFP) and red channel (WGA-Rhod). (f) Zoom in of ROIs (dashed rectangles) outlined in (e), where TNTs are present (white arrowhead). All the experiments were acquired and analyzed under blinded…

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  Figure 3. L-plastin downregulation decreases the number of cells connected by TNTs. (a) Representative stain free gel is shown as a loading control. (b) Representative fluorescent Western blot demonstrating successful downregulation of L-plastin protein levels in shRNA-L-plastin cells compared to the shRNA-Control. (c) Quantification of L-plastin downregulation from Western blots shows a 77.2% downregulation in L-plastin expression (22.8 ± 6.48) compared to control cells. Data are the average of 5 independent experiments. Graph show means (±s.e.m; p = 0.0121). (d) Top Z-stack representative 3D image of control shRNA transduced cells and (e) Zoom in of ROI outlined in (dashed rectangles) (d). (f) Representative 3D image of shRNA L-plastin transduced cells and (g) Zoom in of ROI outlined in (dashed rectangles) (f). TNTs are highlighted with white arrowheads and cells connected by TNTs are identified with a white star (*). Scale bars for (d,f) = 10 μm and 5 μm for (e,g). Scale bars = 5 μm. (h) Down regulation of L-plastin resulted in a significantly lower percentage of cells connected by TNT (32.7 ± 4.7%) compared to the shRNA-control cells (51.2 ± 3.8%). The quantification of cells connected…

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  Figure 4. L-plastin expression levels dos not affect Myo10 expression. (a) Representative stain free gel is shown as a loading control. (b) Representative fluorescent Western blot showing up-regulation of L-plastin expression and its effect on Myo10 protein levels. (c) Quantitative analysis showing no statistical significant changes (ns) in Myo10 expression (92.8% ± 2.3%; p = 0.9203) upon L-plastin upregulation. (d,e) Stain free gel and Western blot from Figure 3a,b showing successful down-regulation of L-plastin (~70 KDa) in ShRNA L-plastin cells. To see its effect on Myo10 expression, the top part of the blot was probed with Myo10 antibody to determine the effect of L-plastin down regulation on Myo10 protein levels (~250 KDa). (f) Quantification of relative levels of Myo 10 expression shows no significant changes (96.4%; (ns) with p = 0.1443) compared to control cells. Data are the average of 5 independent experiments, and the graph shows means (±s.e.m).

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  Figure 5. Myo10 expression levels affect L-plastin expression. (a) Representative stain free gel is shown as a loading control. (b) Representative fluorescent Western blot showing that down-regulation of Myo10 expression (61.0% ± 2.4% with p = 0.0121) using shRNA lentiviral particles (c) results in the reduction of L-plastin expression levels to 68% (±2.8%; p-value: 0.0121). Data are the average of 5 independent experiments. (d) Conversely, we looked at the effects of up-regulation of Myo10 using transient GFP-Myo10 transfection. (e) Representative stain free gel is shown as a loading control. To avoid that the very bright GFP signal affects the endogenous Myo10 signal, the GFP-vector experiments were loaded first, followed by the GFP-Myo10 transfected cells on the same gels. Here a representative experiment is shown, where the control lane (GFP-vector) and its matching experimental lane (GFP-Myo10) from the same gel were juxtaposed as shown by the black separation lane. The entire gel is shown in Figure S3; lanes 1 (e) and 4 (f) were used as a representative image here. (f) Representative fluorescent Western blot of the same samples as (e) are shown and it demonstrates that up-regulation…

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  Figure 6. Myo10 and L-plastin co-localize in TNTs and proto-TNTs. CAD cells were treated with H2O2 in order to increase the number of cells connected by TNTs, then labeled with anti-L-plastin (yellow) and anti-Myo10 antibodies (green), WGA-350 (Blue). (a) A confocal microscopy representative image of CAD cells connected with TNTs is shown. 3D reconstruction using Imaris software, employing “spot rendering” was used to highlight endogenous L-plastin (yellow punctae) and endogenous Myo10 (green punctae), and WGA-350 was used to delineate TNTs. Both proteins are found throughout TNTs and proto-TNTs. Scale bar = 5 μm. (b) Zoom in of (a) shows that endogenous L-plastin punctates (yellow) colocolize with Myo10 punctates (green) within these structures (white arrows). Scale bar = 2 μm.