Feeling for Filaments: Quantification of the Cortical Actin Web in Live Vascular Endothelium

Abstract

Contact-mode atomic force microscopy (AFM) has been shown to reveal cortical actin structures. Using live endothelial cells, we visualized cortical actin dynamics simultaneously by AFM and confocal fluorescence microscopy. We present a method that quantifies dynamic changes in the mechanical ultrastructure of the cortical actin web. We argue that the commonly used, so-called error signal imaging in AFM allows a qualitative, but not quantitative, analysis of cortical actin dynamics. The approach we used comprises fast force-curve-based topography imaging and subsequent image processing that enhances local height differences. Dynamic changes in the organization of the cytoskeleton network can be observed and quantified by surface roughness calculations and automated morphometrics. Upon treatment with low concentrations of the actin-destabilizing agent cytochalasin D, the cortical cytoskeleton network is thinned out and the average mesh size increases. In contrast, jasplakinolide, a drug that enhances actin polymerization, consolidates the cytoskeleton network and reduces the average mesh area. In conclusion, cortical actin dynamics can be quantified in live cells. To our knowledge, this opens a new pathway for conducting quantitative structure-function analyses of the endothelial actin web just beneath the apical plasma membrane.

Intracellular nanoparticle dynamics affected by cytoskeletal integrity

“…In fibroblasts, escaping a primary mesh and traveling to a neighbouring mesh is a rare event, resulting in a drastically different environment experienced by the QD. In comparison to published research on the mesh size of the actin network, values of 100–200 nm 12,14 are more prevalent than values of 300–600 nm 13 for fibroblast cell lines. The actin mesh size is cell type dependent as well as locally heterogeneous as the network undergoes densification toward the cell periphery.…”

“…These relationships persist at shorter and longer delay times, namely t = 0.5 s and 3 s. For fibroblasts, the breadth and number of tracks with MSDs greater than 104 nm 2 significantly increased upon exposure to cytochalasin D suggesting an increase in mobility and less confinement of QDs during diffusion. Interestingly, MSD values for fibroblasts and cytochalasin D treatment correspond to the open spaces within the actin network measured by Kronalge 14 within endothelial cells.…”

“…The actin network has been reported to have a heterogeneous mesh size ranging from 30–100 nm (Luby-Phelps 12 ) to 300–600 nm (Kusumi et al 13 ) across mammalian cells. More recently, Kronlage et al 14 measured the open pore size within the actin cytoskeleton of endothelial cells under control conditions of approximately 10,000 nm 2 (about 100 nm square) and under cytochalasin D treatment, the open area increases to above 30,000 nm 2 (173 nm square). When particle size is similar to or greater than the cytoskeletal mesh, the resulting confinement allows for the measuring of the microrheological properties of the cell interior.…”

Morphology and force probing of primary murine liver sinusoidal endothelial cells

“…The best resolution was obtained for stiffer cantilever MLCT‐E (k = 100 mN/m), but dragging of the cell body often occurs. In sieve plates area in high‐magnification image, we noticed that fiber actin net structure, which indicates that not fenestrations but underlying fenestrae associated cytoskeleton ring structure, is observed …”

Calcium-mediated actin reset (CaAR) mediates acute cell adaptations

“…Apart from the experiment in Figure 2B labeled ‘serum’, all cells were starved in imaging buffer 1 hr prior to experiments. The following cell lines were used in this study: MDCK II (ECACC 0062107); MDCK II cells stably expressing Lifeact-mCherry (Klingner et al, 2014); MCF-7 (ECACC 86012803); MCF-7 cells stably expressing Lifeact-GFP or Lifeact-mCherry (this study); HeLa (ECACC 93021013); HeLa cells stably expressing Lifeact-GFP or Lifeact-mCherry (this study); HeLa INF2 KO cells, and HeLa INF2 KO cells stably expressing Lifeact-mCherry (this study); NIH 3T3 (ECACC 93061524); CCL-39 (ATCC-CCL-39); PANC-1 (ECACC 87092802); U-2 OS (ECACC 92022711); COS-7 (ECACC 87021302); GM7373 (DSMZ ACC109); GM7373 stably expressing Lifeact-mKate2 (Kronlage et al, 2015); AB8 podocytes (Saleem et al, 2002), AB8 cells stably expressing Lifeact-GFP (this study); HoxB8-immortalized mouse monocytes and neutrophils ([Wang et al, 2006] and this study); HEK293 cells (ECACC 85120602). All cell lines were checked for identity by visual inspection of morphologies and tested negative in Mycoplasma tests using the following primers: RWS2534: 5’-CGCCTGAGTAGTACGTTCGC-3’; RWS2535: 5’-CGCCTGAGTAGTACGTACGC-3’; RWS2536: 5’-TGCCTGAGTAGTACATTCGC-3’; RWS2537: 5’-CGCCTGGGTAGTACATTCGC-3’; RWS2538: 5’-CGCCTGAGTAGTAGTCTCGC-3’; RWS2539: 5’-TGCCTGGGTAGTACATTCGC-3’; RWS2540: 5’-GCGGTGTGTACAAGACCCGA-3’; RWS2541: 5’-GCGGTGTGTACAAAACCCGA-3’; RWS2542: 5’-GCGGTGTGTACAAACCCCGA-3’.…”