Measuring Local Viscosities near Plasma Membranes of Living Cells with Photonic Force Microscopy

The molecular processes of particle binding and endocytosis are influenced by the locally changing mobility of the particle nearby the plasma membrane of a living cell. However, it is unclear how the particle’s hydrodynamic drag and momentum vary locally and how they are mechanically transferred to the cell. We have measured the thermal fluctuations of a 1 μm-sized polystyrene sphere, which was placed in defined distances to plasma membranes of various cell types by using an optical trap and fast three-dimensional (3D) interferometric particle tracking. From the particle position fluctuations on a 30 μs timescale, we determined the distance-dependent change of the viscous drag in directions perpendicular and parallel to the cell membrane. Measurements on macrophages, adenocarcinoma cells, and epithelial cells revealed a significantly longer hydrodynamic coupling length of the particle to the membrane than those measured at giant unilamellar vesicles (GUVs) or a plane glass interface. In contrast to GUVs, there is also a strong increase in friction and in mean first passage time normal to the cell membrane. This hydrodynamic coupling transfers a different amount of momentum to the interior of living cells and might serve as an ultra-soft stimulus triggering further reactions.     

Structural,Mechanical, and Dynamical Variability of the Actin Cortex in Living Cells

In eukaryotic cells, an actin-based cortex lines the inner leaflet of the plasma membrane, endowing the cells with crucial mechanical and functional properties. Unfortunately, it has not been possible to study the structural dynamics of the actin cortex at high lateral resolution in living cells. Here, we performed atomic force microscopy time-lapse imaging and mechanical mapping of actin in the cortex of living cells at high lateral and temporal resolution. Cortical actin filaments adopted discernible arrangements, ranging from large parallel bundles with low connectivity to a tight meshwork of short filaments. Mixing of these architectures resulted in attuned cortex networks with specific connectivity, mechanical responses, and marked differences in their dynamic behavior.     

Mechanical Impedance and Nutrient Acquisition in Rice

The aim of this work was to examine the effect of mechanical impedance on nutrient acquisition and gene expression in rice (Oryza sativa L.). Roots were mechanically impeded in a sand-core apparatus to vary impedance independently of aeration and water status. The effect of impedance on plant growth, anion concentration and expression of genes for anion transporters was compared for six varieties with differences in root penetration ability. Impedance decreased shoot growth more than root growth in all varieties, resulting in increased root/shoot ratios. Impedance substantially increased shoot tissue nitrate concentration in all varieties but only caused a small increase in shoot sulphate and phosphate concentrations. High impedance increased expression of the sulphate transporter OsST1 in five varieties, which was associated with decreased sulphate concentration in root tissues. In contrast, impedance decreased expression of the phosphate transporter OsPT2 expression in all varieties, which was associated with decreased phosphate concentration in root tissues. Localisation of expression of the sulphate transporter by in situ hybridisation indicated high levels of expression in lateral bud primordia. It was suggested that the decreased root phosphate concentrations of impeded roots were caused by low phosphate transporter gene expression, while the increase in sulphate transporter gene expression was due to a derepression mechanism of control.     

Impedance of Nitellopsis obtusa Cells at Low Frequencies

Measurements of impedance were made on the internodal cellsof Nitellopsis obtusa with the aid of external electrodes andcurrent frequencies of from 20 to 1000 Hz. The analysis of theresults points to the fact that the resistance (r) and capacity(c) of the membrane and the specific resistance (p) of the interiorof the cell are constant in the range of the frequencies used.The values of r, c, and p are: r = 26.2 k cm²; c = 0.55 F cm^–2;p = 78 em. The observed dispersion of the resistance and capacityof the system investigated at low frequencies is a result ofits complex structure, which consists of several phases withdifferent electrical parameters. These phases are: the membranes,the cytoplasm, the vacuolar sap, the external solution, andthe electrodes.     

Chromatin Texture Analysis in Living Cells

In recent years, there has been an increasing interest in applications of fluorescence measurements to studies on many physiological mechanisms in living cells. However, few studies have taken advantage of DNA quantification by fluorometry for dynamic assessment of chromatin organization. This type of approach involves both optimal conditions for DNA staining and the use of image cytometry. In this context, this report describes the application of an internal grey-level segmentation method for the assessment of real time modifications of chromatin organization in living cells. These developments are based on a specific, stoichiometric method for nuclear DNA content measurement. Preliminary data obtained from Hela cells suggests the possibility of following variations of nuclear texture (heterogeneity, granularity, condensation, radial distribution) related to the cell cycle progression of cells that are maintained alive.     

Fluorescence Fluctuation Spectroscopy of mCherry in Living Cells

The red fluorescent protein mCherry is of considerable interest for fluorescence fluctuation spectroscopy (FFS), because the wide separation in color between mCherry and green fluorescent protein provides excellent conditions for identifying protein interactions inside cells. This two-photon study reveals that mCherry exists in more than a single brightness state. Unbiased analysis of the data needs to account for the presence of multiple states. We introduce a two-state model that successfully describes the brightness and fluctuation amplitude of mCherry. The properties of the two states are characterized by FFS and fluorescence lifetime experiments. No interconversion between the two states was observed over the experimentally probed timescales. The effect of fluorescence resonance energy transfer between enhanced green fluorescent protein (EGFP) and mCherry is incorporated into the two-state model to describe protein hetero-oligomerization. The model is verified by comparing the predicted and measured brightness and fluctuation amplitude of several fusion proteins that contain mCherry and EGFP. In addition, hetero-fluorescence resonance energy transfer between mCherry molecules in different states is detected, but its influence on FFS parameters is small enough to be negligible. Finally, the two-state model is applied to study protein oligomerization in living cells. We demonstrate that the model successfully describes the homodimerization of nuclear receptors. In addition, we resolved a mixture of interacting and noninteracting proteins labeled with EGFP and mCherry. These results provide the foundation for quantitative applications of mCherry in FFS studies.     

Mechanics of Individual Keratin Bundles in Living Cells

Along with microtubules and microfilaments, intermediate filaments are a major component of the eukaryotic cytoskeleton and play a key role in cell mechanics. In cells, keratin intermediate filaments form networks of bundles that are sparser in structure and have lower connectivity than, for example, actin networks. Because of this, bending and buckling play an important role in these networks. Buckling events, which occur due to compressive intracellular forces and cross-talk between the keratin network and other cytoskeletal components, are measured here in situ. By applying a mechanical model for the bundled filaments, we can access the mechanical properties of both the keratin bundles themselves and the surrounding cytosol. Bundling is characterized by a coupling parameter that describes the strength of the linkage between the individual filaments within a bundle. Our findings suggest that coupling between the filaments is mostly complete, although it becomes weaker for thicker bundles, with some relative movement allowed.     

Phycomyces: Mechanical Analysis of the Living Cell Wall

Stress relaxation measurements were conducted on stage IVb Phycomycessporangiophores in order to correlate the effect of imposedstress on cell wall growth. It was found that the cell wallshowed maximum growth when subjected to maximum stress. Growthunder stress decreased as the stress decreased. This techniquewas used to measure the response of the sporangiophore to alight stimulus; the response is measured directly from the stressrelaxation curve. Stress/strain measurements were also conducted on the stageIVb Phycomyces sporangiophores in order to further characterizethe mechanical properties of the growing zone. It was foundthat the stress/strain ratio was invariant to the strain ratewithin the ranges tested but the stress/strain ratio did increasewith larger loads, i.e., the stress/strain ratio shows non-linearbehaviour.     

Local Loperamide Inhibits Thermal Hyperalgesia But Not Mechanical Allodynia Induced by Intratibial Inoculation of Melanoma Cells in Mice

The stimulation of peripheral opioid receptors counteracts thermal hyperalgesia produced by the intratibial inoculation of NCTC 2472 cells in mice, through the activation of the nitric oxide/cGMP/ATP-sensitive K⁺-channels (NO/cGMP/K⁺ _ATP) cascade (Menéndez et al. 2007, Neuropharmacology 53:71–80). We aimed to elucidate whether this peripheral opioid antihyperalgesic effect is exclusive to this model or might also occur in other types of bone neoplastic processes. In C57BL/6 mice intratibially inoculated with B16-F10 melanoma cells, the progressive tumoral damage was accompanied by the establishment of thermal hyperalgesia (unilateral hot plate test) and mechanical allodynia (von Frey test). Intraplantar administration of loperamide (15 μg, 30 min before) inhibited thermal hyperalgesia, but did not modify the intense mechanical allodynia. The fact that the coadministration of naloxone-methiodide (5 μg) completely suppressed the thermal antihyperalgesic effect induced by loperamide indicates its production through the stimulation of peripheral opioid receptors. Furthermore, its prevention by the coadministration of the non-selective inhibitor of the NO synthase, N^G-monomethyl-L-arginine (L-NMMA, 10 μg), the selective inhibitor of neural NOS, N-ω-propyl-L-arginine (1–10 μg), or the K⁺ _ATP channel blocker, glibenclamide (10 μg) demonstrated the involvement of the NO/cGMP/K⁺ _ATP pathway in the antihyperalgesic effect induced by loperamide. Overall, the present results show that the intratibial inoculation of B16-F10 cells to C57BL/6 mice evokes thermal hyperalgesia and mechanical allodynia and that, as occurred in the osteosarcoma model, the stimulation of peripheral opioid receptors is not effective in modifying neoplastic allodynia but completely inhibits thermal hyperalgesia through the activation of the NO/cGMP/K⁺ _ATP cascade.