mcherry arp2 n 14  (Addgene inc)

Journal: Journal of cell science

Article Title: A role for class I p21-activated kinases in the regulation of the excitability of the actin cytoskeleton.

doi: 10.1242/jcs.263763

Figure Lengend Snippet: Fig. 4. Formation of cortactin/actin rings is PI3K dependent. (A) Still from Movie 6 of an MEF transiently overexpressing mCherry–LifeAct and PI(3,4,5)P3 marker (GFP BTK PH) following treatment with 10 μM G5555 for at least an hour. Accompanying linescan demonstrates localisation PI(3,4,5)P3 marker relative to actin cytoskeleton. (B) Still from Movie 7 of an MEF transiently overexpressing mEGFP–LifeAct and PI(4,5)P2 marker (mCherry PH domain of PLCdelta1) following treatment with 10 μM G5555 for at least an hour. Accompanying linescan demonstrates localisation of PI(4,5)P2 marker relative to actin cytoskeleton. (C) Percentage of cells producing cortactin rings in the presence and absence of 10 μM PI3K inhibitor LY294002 alongside either cotreatment with 10 μM G5555 or overexpression of GFP–PAK1Δkin. At least 100 cells analysed per condition for each of at least three biological repeats. Inhibitors were added 1 h before fixation. (D) Schematic summarising the localisation of various proteins upon addition of G5555. Scale bars: 10 μm. All error bars indicate s.d. ***P≤0.001 (ordinary one-way ANOVA followed by a post hoc Tukey’s multiple comparison test). A.U., arbitrary units.

Article Snippet: All other plasmids were acquired from Addgene: mApple LifeAct (Addgene plasmid #54747), mEGFP LifeAct (Addgene plasmid #54610), mCherry N-WASp (Addgene plasmid #55164), Myo1e mCherry (Addgene plasmid #27698), EmeraldVinculin (Addgene plasmid #54303), mCherry Arp2 (Addgene plasmid #54980), GFP BTK PH (Addgene plasmid #51463), mCherry PH domain of PLCdelta1 (Addgene plasmid #36075), GFP-PH-Tapp1 (Addgene plasmid #161985), p40PX-EYFP (Addgene plasmid #19011), Cyfip GFP (Addgene plasmid #109139).

Techniques: Marker, Over Expression, Comparison

Journal: iScience

Article Title: Cryo-ET of actin cytoskeleton and membrane structure in lamellipodia formation using optogenetics

doi: 10.1016/j.isci.2025.112529

Figure Lengend Snippet: PA-Rac1-induced lamellipodia formation (A) Schematic representation of subdomains in lamellipodia. (B) COS-7 cells expressing PA-Rac1 and Lifeact-mCherry on glass-bottom dishes. Images were captured every 10 s, with PA-Rac1 activated by a 458 nm laser from time 0 at 10-s intervals. The fluorescence images of Lifeact-mCherry are shown. (C) Lifeact-mCherry image of COS-7 cells, highlighting the crescent-shaped cell edges in red. (D–F) COS-7 cells expressing PA-Rac1 and Lifeact-mCherry on EM grids. Images were captured every 10 s from −5 min, with PA-Rac1 activated by scanning with a single 458 nm laser within 0–10 min at 10-s intervals. (D and E) Representative time-lapse images. A merged image of DIC and fluorescence of Lifeact-mCherry (yellow) (D), and fluorescence images of Lifeact-mCherry (E) are shown. A yellow arrowhead indicates dotted actin structures within the interior of cells. (F) Quantification of cell area changes. Data are presented as the means ± SD from 6 cells. Blue background indicates the activation period; red dotted line marks the timing of freezing. Scale bar = 20 μm. See also and and .

Article Snippet: mCherry-ARP2-N-14 , Davidson lab – mCherry constructs, unpublished , RRID:Addgene 54980.

Techniques: Expressing, Fluorescence, Activation Assay

Journal: iScience

Article Title: Cryo-ET of actin cytoskeleton and membrane structure in lamellipodia formation using optogenetics

doi: 10.1016/j.isci.2025.112529

Figure Lengend Snippet: Experimental workflow from sample preparation to tomogram acquisition Laminin-coated EM grids were placed on the 3D-printed grid holders in 4-well dishes, and cells were seeded on the EM grids. At the same time, plasmids encoded mVenus-PA-Rac1 and Lifeact-mCherry were transfected into the cells. The next day, the cells were rapidly frozen using a plunge freezer after inducing lamellipodia formation by activating PA-Rac1 with blue light irradiation, controlling the timing of the freezing. The frozen cells were observed using cryo-fluorescence microscopy to identify the cells that had formed lamellipodia. By correlating the fluorescence images with low-magnification EM images, the regions for tomography were determined. Subsequently, continuous tilt series images were acquired by cryo-EM from −70° to +70°, and tomograms were reconstructed.

Article Snippet: mCherry-ARP2-N-14 , Davidson lab – mCherry constructs, unpublished , RRID:Addgene 54980.

Techniques: Sample Prep, Transfection, Irradiation, Fluorescence, Microscopy, Tomography, Cryo-EM Sample Prep

Journal: iScience

Article Title: Cryo-ET of actin cytoskeleton and membrane structure in lamellipodia formation using optogenetics

doi: 10.1016/j.isci.2025.112529

Figure Lengend Snippet: Cryo-CLEM images Cryo-CLEM images of three cells from which tomograms were captured in this study. Green: mVenus-PA-Rac1, purple: Lifeact-mCherry. See also .

Article Snippet: mCherry-ARP2-N-14 , Davidson lab – mCherry constructs, unpublished , RRID:Addgene 54980.

Techniques: