A multi-structural single cell model of force-induced interactions of cytoskeletal components

Several computational models based on experimental techniques and theories have been proposed to describe cytoskeleton (CSK) mechanics. Tensegrity is a prominent model for force generation, but it cannot predict mechanics of individual CSK components, nor explain the discrepancies from the different single cell stimulating techniques studies combined with cytoskeleton-disruptors. A new numerical concept that defines a multi-structural 3D finite element (FE) model of a single-adherent cell is proposed to investigate the biophysical and biochemical differences of the mechanical role of each cytoskeleton component under loading. The model includes prestressed actin bundles and microtubule within cytoplasm and nucleus surrounded by the actin cortex. We performed numerical simulations of atomic force microscopy (AFM) experiments by subjecting the cell model to compressive loads. The numerical role of the CSK components was corroborated with AFM force measurements on U2OS-osteosarcoma cells and NIH-3T3 fibroblasts exposed to different cytoskeleton-disrupting drugs. Computational simulation showed that actin cortex and microtubules are the major components targeted in resisting compression. This is a new numerical tool that explains the specific role of the cortex and overcomes the difficulty of isolating this component from other networks in vitro. This illustrates that a combination of cytoskeletal structures with their own properties is necessary for a complete description of cellular mechanics

Dynamic mechanical responses of Arabidopsis thylakoid membranes during PSII-specific illumination.

Remodeling of thylakoid membranes in response to illumination is an important process for the regulation of photosynthesis. We investigated the thylakoid network from Arabidopsis thaliana using atomic force microscopy to capture dynamic changes in height, elasticity, and viscosity of isolated thylakoid membranes caused by changes in illumination. We also correlated the mechanical response of the thylakoid network with membrane ultrastructure using electron microscopy. We find that the elasticity of the thylakoid membranes increases immediately upon PSII-specific illumination, followed by a delayed height change. Direct visualization by electron microscopy confirms that there is a significant change in the packing repeat distance of the membrane stacks in response to illumination. Although experiments with Gramicidin show that the change in elasticity depends primarily on the transmembrane pH gradient, the height change requires both the pH gradient and STN7-kinase-dependent phosphorylation of LHCII. Our studies indicate that lumen expansion in response to illumination is not simply a result of the influx of water, and we propose a dynamic model in which protein interactions within the lumen drive these changes

Dynamic mechanical responses of Arabidopsis thylakoid membranes during PSII-specific illumination.

Remodeling of thylakoid membranes in response to illumination is an important process for the regulation of photosynthesis. We investigated the thylakoid network from Arabidopsis thaliana using atomic force microscopy to capture dynamic changes in height, elasticity, and viscosity of isolated thylakoid membranes caused by changes in illumination. We also correlated the mechanical response of the thylakoid network with membrane ultrastructure using electron microscopy. We find that the elasticity of the thylakoid membranes increases immediately upon PSII-specific illumination, followed by a delayed height change. Direct visualization by electron microscopy confirms that there is a significant change in the packing repeat distance of the membrane stacks in response to illumination. Although experiments with Gramicidin show that the change in elasticity depends primarily on the transmembrane pH gradient, the height change requires both the pH gradient and STN7-kinase-dependent phosphorylation of LHCII. Our studies indicate that lumen expansion in response to illumination is not simply a result of the influx of water, and we propose a dynamic model in which protein interactions within the lumen drive these changes

Actin cytoskeleton of murine syncytiotrophoblast (SYN) contributes to its elastic strength.

<p>A. Microrheology with an atomic force microscope was used to measure elastic strength of mouse trophoblast stem cells (TSC) and SYN. Photos depict microscopic cantilever positioned above cultured live cells prior to measurement. <b>B.</b> The elastic modulus (Young's modulus) of SYN is significantly higher than that of TSC (p = 4.7×10⁻⁵ by Student's T-test). Elastic modulus of SYN was measured in the exact same spot prior to and after treatment with Cyto-D for 40–60 min. Disruption of the actin cytoskeleton with Cyto-D significantly decreased the elastic modulus of SYN (p = 0.001 by Student's T-test). Bars represent median values. Graph is based on three independent experiments performed in triplicate. <b>C.</b> Immunofluorescence images of the actin (red) in mSYN show that the characteristic actin meshwork (i–iii) is disrupted by 1 hr treatment with Cyto-D (iv–vi). Nuclei are shown in white. Bars in panels i and iv are 50 um. Panels ii–iii and v–vi are representative close-up images of untreated and Cyto-D treated mSYN, respectively. Panels iii and vi show just the actin channel of ii and v respectively. Aggregation of microfilaments in distinct puncta are observed upon treatment. Bars = 10 um.</p

Cell-to-cell spread of <i>L. monocytogenes</i> (LM) into human syncytiotrophoblast (SYN) is enhanced by syncytial actin network disruption.

<p>A. Immunofluorescence image of sectioned primary human placenta showing diffuse actin structure in the syncytium (outlined and marked by blue star.) Bar = 10 um. <b>B.</b> Untreated and 1 hr Cyto-D treated human placental organ cultures were incubated with LM-infected macrophages and foci of spread observed and quantified. Panels i and ii show representative images showing cell-to-cell spread occurs almost exclusively at the extravillous trophoblasts (EVT) in untreated placenta; Cyto-D treatment increases incidence of spread into SYN. Bar = 100 um. Panel iii shows representative image of bacterial presence in the syncytium in Cyto-D treated placenta. Bar = 10 um. LM is shown in green, nuclei in white, SYN (b-hCG staining) is shown in red. <b>C.</b> Quantification of cell-to-cell spread into SYN. Each data point represents average co-localization of LM with b-hCG from ten microscopic fields (10×); bar represents median, graph is based on seven independent experiments. Cyto-D treatment significantly increases bacterial cell-to-cell spread into placental syncytium (p = 0.04 by Student's T-test).</p

<i>L. monocytogenes</i> (LM) invades and replicates in murine trophoblast stem cells.

<p>A. Colony forming units (CFU) per coverslip at 2, 5, 8, and 24 hours post-inoculation (p.i.) for the following <i>L. monocytogenes</i> (LM) strains were determined: wild type (wt), InlA-deficient (del-InlA), and murinized InlA (InlA^m). Average CFU per coverslip at 2 hours p.i. for each strain is based on nine independent experiments. Average CFU per coverslip at time points 5, 8, and 24 hours p.i. for each strain are based on three independent experiments. Bars represent standard error. Average CFU at 2 hours for InlA^m is 30-fold higher than for wild type and the p-value denotes statistical significance (p = 0.008 by Student's T-test). There is no difference in invasion for wild type versus del-InlA (p = 0.3 by Student's T-test). <b>B.</b> Immunofluorescence image of mTSC at 2 hours p.i.: LM is shown in green, nuclei in white, actin in red. Arrowheads point to foci of infection, Bar = 50 um.</p

Cell-to-cell spread of <i>L. monocytogenes</i> (LM) into mouse syncytiotrophoblast (SYN) is enhanced by syncytial actin network disruption.

<p>Untreated and Cyto-D treated SYN was incubated with LM-infected murine macrophages and foci of bacterial spread were quantified. Bacterial foci were included in the analysis when multiple bacteria were observed unbounded by the outline of a macrophage membrane; many such foci were surrounded by actin clouds. <b>A.</b> Panel i: Representative immunofluorescence image of untreated differentiated TSCs shows bacterial infection of mononuclear trophoblasts (MNT) versus SYN. SYN is outlined and marked by blue star. Panel ii: One hour of Cyto-D treatment increases the number of bacterial foci in SYN. Arrowheads indicate adherence of infected macrophages to trophoblasts; arrows indicate spread events. Actin is shown in red, nuclei in blue, LM in green. Bars = 50 um. <b>B.</b> Quantification of bacterial foci per unit area in untreated versus Cyto-D treated SYN at 5 hours p.i. with macrophages. Each data point represents the average of bacterial foci in ten random fields (20×) with at least 50% syncytium. Treatment with Cyto-D significantly increases infection of SYN via cell-to-cell spread (p = 0.03 by Student's T-test); treatment does not significantly change infection of MNT (p = 0.33). Bars represent median.</p