Journal: Molecular Biology of the Cell

Article Title: The LINC complex is required for endothelial cell adhesion and adaptation to shear stress and cyclic stretch

doi: 10.1091/mbc.E20-11-0698

Figure Lengend Snippet: Disruption of the LINC complex affects focal adhesion dynamics. (A) DN-KASH affects focal adhesion morphology. Cells expressing AV-mCherry or AV-DN-KASH-mCherry were immunostained for DAPI, vinculin, or vimentin (green) and actin (gray). (B) DN-KASH expression affects the vimentin cytoskeleton. Cells expressing AV-mCherry or DN-KASH-mCherry were immunostained for DAPI, vimentin (green), and actin (gray). (C) Cells were cotransfected with vinculin-TSmod and AV-mCherry or DN-KASH-mCherry. Force exerted across vinculin is reduced upon LINC disruption in HUVEC. Coinfected cells with adenovirus-mCherry or DN-KASH-mCherry with vinculin tension-sensor (TSmod). Mann–Whitney test (* p < 0.05), N = 3. (D) Altered phosphorylation states of focal adhesion proteins with DN-KASH expression. HUVEC-expressing AV-DN-KASH or AV-mCherry control were lysed for 36–48 h and blotted for pY1065 vinculin, pS425 Talin, and pY397 FAK ( N = 3). Mann–Whitney test (** p < 0.01).

Article Snippet: The design of VE-cadherin TSmod ( Conway et al. , 2013 ) (Addgene plasmid 45848) and vinculin TSmod ( Grashoff et al. , 2010 ) (Addgene plasmid 26019) were previously described.

Techniques: Disruption, Expressing, MANN-WHITNEY, Phospho-proteomics, Control

Journal: bioRxiv

Article Title: An enhanced molecular tension sensor based on bioluminescence resonance energy transfer (BRET)

doi: 10.1101/617696

Figure Lengend Snippet: Schematic of the genetically-encodable BRET molecular tension sensor (BRET-TS). (A) NanoLuc, the energy donor, is excited via addition of furimazine (Fz) in the presence of oxygen. In the absence of force (f), resonance energy transfer occurs to the fluorescent acceptor, mNeonGreen. With applied force, the donor-acceptor pair is separated, reducing resonance energy transfer. Unloaded spectral and resonance energy transfer properties of (B) BRET-TS in comparison to (C) FRET-based TSMod. Spectral components and resulting best-fit additive spectrum depicted. Axes in units of luminescence normalized at 460nm and relative fluorescence units (RFU). Energy transfer efficiencies E were calculated as described in Methods.

Article Snippet: The TSMod recombinant protein was made in the same context, except via PCR amplification of the TSMod DNA (Addgene #26019, courtesy of Martin Schwartz).

Techniques: Förster Resonance Energy Transfer, Comparison, Fluorescence