Desmin Mutation in the C-Terminal Domain Impairs Traction Force Generation in Myoblasts

The cytoskeleton plays a key role in the ability of cells to both resist mechanical stress and generate force, but the precise involvement of intermediate filaments in these processes remains unclear. We focus here on desmin, a type III intermediate filament, which is specifically expressed in muscle cells and serves as a skeletal muscle differentiation marker. By using several complementary experimental techniques, we have investigated the impact of overexpressing desmin and expressing a mutant desmin on the passive and active mechanical properties of C2C12 myoblasts. We first show that the overexpression of wild-type-desmin increases the overall rigidity of the cells, whereas the expression of a mutated E413K desmin does not. This mutation in the desmin gene is one of those leading to desminopathies, a subgroup of myopathies associated with progressive muscular weakness that are characterized by the presence of desmin aggregates and a disorganization of sarcomeres. We show that the expression of this mutant desmin in C2C12 myoblasts induces desmin network disorganization, desmin aggregate formation, and a small decrease in the number and total length of stress fibers. We finally demonstrate that expression of the E413K mutant desmin also alters the traction forces generation of single myoblasts lacking organized sarcomeres.     

P-cadherin promotes collective cell migration via a Cdc42-mediated increase in mechanical forces

Collective cell migration (CCM) is essential for organism development, wound healing, and metastatic transition, the primary cause of cancer-related death, and it involves cell–cell adhesion molecules of the cadherin family. Increased P-cadherin expression levels are correlated with tumor aggressiveness in carcinoma and aggressive sarcoma; however, how P-cadherin promotes tumor malignancy remains unknown. Here, using integrated cell biology and biophysical approaches, we determined that P-cadherin specifically induces polarization and CCM through an increase in the strength and anisotropy of mechanical forces. We show that this mechanical regulation is mediated by the P-cadherin/β-PIX/Cdc42 axis; P-cadherin specifically activates Cdc42 through β-PIX, which is specifically recruited at cell–cell contacts upon CCM. This mechanism of cell polarization and migration is absent in cells expressing E- or R-cadherin. Thus, we identify a specific role of P-cadherin through β-PIX–mediated Cdc42 activation in the regulation of cell polarity and force anisotropy that drives CCM.     

TMA-DPH A fluorescent probe of membrane dynamics in living cells

Trimethylammonium-diphenylhexatriene (TMA-DPH), a hydrophobic fluorescent probe, has been shown in earlier studies to possess a variety of particular properties in interaction with intact living cells —specific and rapid incorporation into the plasma membrane and partition equilibrium between the membranes and the buffer. These properties offer promising applications in membrane fluidity studies and in monitoring exocytosis kinetics. Furthermore, these properties offer a method described here for quantitative monitoring of phago-cytosis kinetics, by means of simple fluorescence intensity measurements. This method is original in that it evaluates only the particles which have actually been internalized by phagocytosis, and not those adsorbed on the cell surface, and that it gives quantitative information on the amount of plasma membrane involved in the process. It has been tested on mouse bone marrow macrophages.     

Nonreductive Biomineralization of Uranium(VI) Phosphate Via Microbial Phosphatase Activity in Anaerobic Conditions

The remediation of uranium from soils and groundwater at Department of Energy (DOE) sites across the United States represents a major environmental issue, and bioremediation has exhibited great potential as a strategy to immobilize U in the subsurface. The bioreduction of U(VI) to insoluble U(IV) uraninite has been proposed to be an effective bioremediation process in anaerobic conditions. However, high concentrations of nitrate and low pH found in some contaminated areas have been shown to limit the efficiency of microbial reduction of uranium. In the present study, nonreductive uranium biomineralization promoted by microbial phosphatase activity was investigated in anaerobic conditions in the presence of high nitrate and low pH as an alternative approach to the bioreduction of U(VI). A facultative anaerobe, Rahnella sp. Y9602, isolated from soils at DOE's Oak Ridge Field Research Center (ORFRC), was able to respire anaerobically on nitrate as a terminal electron acceptor in the presence of glycerol-3-phosphate (G3P) as the sole carbon and phosphorus source and hydrolyzed sufficient phosphate to precipitate 95% total uranium after 120 hours in synthetic groundwater at pH 5.5. Synchrotron X-ray diffraction and X-ray absorption spectroscopy identified the mineral formed as chernikovite, a U(VI) autunite-type mineral. The results of this study suggest that in contaminated subsurfaces, such as at the ORFRC, where high concentrations of nitrate and low pH may limit uranium bioreduction, the biomineralization of U(VI) phosphate minerals may be a more attractive approach for in situ remediation providing that a source of organophosphate is supplied for bioremediation.     

Stabilization of T7-promoter-based pARHS expression vectors using the parB locus.

We describe a modification of the pAR3040 vector which results in its efficient stabilization during cell division. The parB locus of the plasmid R1 was introduced into the plasmid, pAR3040, to construct the pARHS vectors. These vectors are stable for at least 60 cell generations, even in the absence of selection by an antibiotic present in the culture media, both with or without IPTG induction.     

“Escherichia coli‐milk” biofilm removal from stainless steel surfaces: Synergism between ultrasonic waves and enzymes

Three different methods to standardize biofilm removal for in situ sanitary control of closed surfaces in the food industry have been developed and compared, i.e. sonication, enzymatic treatment and a combined treatment which involved the application of ultrasound to enzyme preparations. The biofilm studied was an Escherichia coli model biofilm, made with milk on stainless steel sheets. Plate counting and epifluorescence microscopy were used to assess the efficiency of each treatment. The results are expressed in percentages, 100% denoting total removal, obtained with a flat ultrasonic transducer (T1) developed and presented in a previous study. The application of ultrasound by a patented curved transducer, T2 (10 s, 40 kHz), specifically devised for closed surfaces, was not sufficient to completely remove the biofilm (30 ± 7%). This biofilm was dislodged by two proteolytic enzyme preparations tested by immersion, viz. a 15‐min application of protease (84±1%) and a 30‐min trypsin application (95±8%). Using a combined treatment, the results showed a synergism between ultrasonic waves and proteolytic or glycolytic enzyme preparations, with removal of a significant amount of biofilm, i.e. 61–96% depending on the conditions tested, i.e. two to three times greater compared to sonication alone (30%). This application was in agreement with an industrial control, i.e. a good reproducible recovery of the biofilm in 10 s compared with 30 or 15 min with the enzyme alone.