Eukaryotic CRFK Cells Motion Characterized with Atomic Force Microscopy.

We performed a time-lapse imaging with atomic force microscopy (AFM) of the motion of eukaryotic CRFK (Crandell-Rees Feline Kidney) cells adhered onto a glass surface and anchored to other cells in culture medium at 37 °C. The main finding is a gradient in the spring constant of the actomyosin cortex along the cells axis. The rigidity increases at the rear of the cells during motion. This observation as well as a dramatic decrease of the volume suggests that cells may organize a dissymmetry in the skeleton network to expulse water and drive actively the rear edge.

Nanomechanical detection of Escherichia coli infection by bacteriophage T7 using cantilever sensors.

Viruses that infect bacteria (bacteriophages) are a promising alternative treatment for bacterial diseases, especially in the case of antibiotic resistance. Due to a renewed interest in phage therapies, development of rapid and specific detection methods for bacteria/bacteriophage interaction are gaining attention for proper diagnosis and treatment. This paper describes a new method to detect the interaction between Escherichia coli and bacteriophage T7 in a sensitive and quantitative way, using the nanomechanical motion of bacteria adhered to a cantilever surface. Our approach combines both deflection and dynamic frequency-domain characterization. The device was able to determine the viability of a low amount of living bacteria attached to the cantilever, and was used to monitor T7 interaction with E. coli over a wide range of virus concentrations up to 109 PFU ml-1. The nanomechanical assay described here requires no protein labeling and can be performed in a single reaction without additional reagents. The system was able to detect the interaction between a few thousand particles through the fluctuation of mechanical energy over a broad range of frequencies. The presented data provides the basis for more detailed studies of the sequence of molecular events that contribute to the motion of the device.

Mechanics of Virus-like Particles Labeled with Green Fluorescent Protein.

Nanoindentation with an atomic force microscope was used to investigate the mechanical properties of virus-like particles (VLPs) derived from the avian pathogen infectious bursal disease virus, in which the major capsid protein was modified by fusion with enhanced green fluorescent protein (EGFP). These VLPs assemble as ∼70-nm-diameter T = 13 icosahedral capsids with large cargo space. The effect of the insertion of heterologous proteins in the capsid was characterized in the elastic regime, revealing that EGFP-labeled chimeric VLPs are more rigid than unmodified VLPs. In addition, nanoindentation measurements beyond the elastic regime allowed the determination of brittleness and rupture force limit. EGFP incorporation results in a complex shape of the indentation curve and lower critical indentation depth of the capsid, rendering more brittle particles as compared to unlabeled VLPs. These observations suggest the presence of a complex and more constrained network of interactions between EGFP and the capsid inner shell. These results highlight the effect of fluorescent protein insertion on the mechanical properties of these capsids. Because the physical properties of the viral capsid are connected to viral infectivity and VLP transport and disassembly, our results are relevant to design improved labeling strategies for fluorescence tracking in living cells.

The RNA-Binding Protein of a Double-Stranded RNA Virus Acts like a Scaffold Protein.

Infectious bursal disease virus (IBDV), a nonenveloped, double-stranded RNA (dsRNA) virus with a T=13 icosahedral capsid, has a virion assembly strategy that initiates with a precursor particle based on an internal scaffold shell similar to that of tailed double-stranded DNA (dsDNA) viruses. In IBDV-infected cells, the assembly pathway results mainly in mature virions that package four dsRNA segments, although minor viral populations ranging from zero to three dsRNA segments also form. We used cryo-electron microscopy (cryo-EM), cryo-electron tomography, and atomic force microscopy to characterize these IBDV populations. The VP3 protein was found to act as a scaffold protein by building an irregular, ∼40-Å-thick internal shell without icosahedral symmetry, which facilitates formation of a precursor particle, the procapsid. Analysis of IBDV procapsid mechanical properties indicated a VP3 layer beneath the icosahedral shell, which increased the effective capsid thickness. Whereas scaffolding proteins are discharged in tailed dsDNA viruses, VP3 is a multifunctional protein. In mature virions, VP3 is bound to the dsRNA genome, which is organized as ribonucleoprotein complexes. IBDV is an amalgam of dsRNA viral ancestors and traits from dsDNA and single-stranded RNA (ssRNA) viruses.IMPORTANCE Structural analyses highlight the constraint of virus evolution to a limited number of capsid protein folds and assembly strategies that result in a functional virion. We report the cryo-EM and cryo-electron tomography structures and the results of atomic force microscopy studies of the infectious bursal disease virus (IBDV), a double-stranded RNA virus with an icosahedral capsid. We found evidence of a new inner shell that might act as an internal scaffold during IBDV assembly. The use of an internal scaffold is reminiscent of tailed dsDNA viruses, which constitute the most successful self-replicating system on Earth. The IBDV scaffold protein is multifunctional and, after capsid maturation, is genome bound to form ribonucleoprotein complexes. IBDV encompasses numerous functional and structural characteristics of RNA and DNA viruses; we suggest that IBDV is a modern descendant of ancestral viruses and comprises different features of current viral lineages.

A protein with simultaneous capsid scaffolding and dsRNA-binding activities enhances the birnavirus capsid mechanical stability.

Viral capsids are metastable structures that perform many essential processes; they also act as robust cages during the extracellular phase. Viruses can use multifunctional proteins to optimize resources (e.g., VP3 in avian infectious bursal disease virus, IBDV). The IBDV genome is organized as ribonucleoproteins (RNP) of dsRNA with VP3, which also acts as a scaffold during capsid assembly. We characterized mechanical properties of IBDV populations with different RNP content (ranging from none to four RNP). The IBDV population with the greatest RNP number (and best fitness) showed greatest capsid rigidity. When bound to dsRNA, VP3 reinforces virus stiffness. These contacts involve interactions with capsid structural subunits that differ from the initial interactions during capsid assembly. Our results suggest that RNP dimers are the basic stabilization units of the virion, provide better understanding of multifunctional proteins, and highlight the duality of RNP as capsid-stabilizing and genetic information platforms.

Stepwise motion of a microcantilever driven by the hydrolysis of viral ATPases.

The biomolecular machines involved in DNA packaging by viruses generate one of the highest mechanical powers observed in nature. One component of the DNA packaging machinery, called the terminase, has been proposed as the molecular motor that converts chemical energy from ATP hydrolysis into mechanical movement of DNA during bacteriophage morphogenesis. However, the conformational changes involved in this energy conversion have never been observed. Here we report a real-time measurement of ATP-induced conformational changes in the terminase of bacteriophage T7 (gp19). The recording of the cantilever bending during its functionalization shows the existence of a gp19 monolayer arrangement confirmed by atomic force microscopy of the immobilized proteins. The ATP hydrolysis of the gp19 terminase generates a stepped motion of the cantilever and points to a mechanical cooperative effect among gp19 oligomers. Furthermore, the effect of ATP can be counteracted by non-hydrolyzable nucleotide analogs.

Label-free detection of DNA hybridization based on hydration-induced tension in nucleic acid films.

The properties of water at the nanoscale are crucial in many areas of biology, but the confinement of water molecules in sub-nanometre channels in biological systems has received relatively little attention. Advances in nanotechnology make it possible to explore the role played by water molecules in living systems, potentially leading to the development of ultrasensitive biosensors. Here we show that the adsorption of water by a self-assembled monolayer of single-stranded DNA on a silicon microcantilever can be detected by measuring how the tension in the monolayer changes as a result of hydration. Our approach relies on the microcantilever bending by an amount that depends on the tension in the monolayer. In particular, we find that the tension changes dramatically when the monolayer interacts with either complementary or single mismatched single-stranded DNA targets. Our results suggest that the tension is mainly governed by hydration forces in the channels between the DNA molecules and could lead to the development of a label-free DNA biosensor that can detect single mutations. The technique provides sensitivity in the femtomolar range that is at least two orders of magnitude better than that obtained previously with label-free nanomechanical biosensors and with label-dependent microarrays.

Study of the origin of bending induced by bimetallic effect on microcantilever.

An analytical model for predicting the deflection and force of a bimaterialcantilever is presented. We introduce the clamping effect characterised by an axial loadupon temperature changes. This new approach predicts a non linear thermal dependence ofcantilever strain. A profilometry technique was used to measure the thermal strain.Comparison with experimental results is used to verify the model. The concordance of theanalytical model presented with experimental measurements is better than 10.

Measurement of the Mass and Rigidity of Adsorbates on a Microcantilever Sensor.

When microcantilevers are used in the dynamic mode, the resonance shift uponmaterial adsorption depends on the position of the adsorbate along the microcantilever. Wehave previously described that the adsorbate stiffness needs to be considered in addition toits mass in order to correctly interpret the resonance shift. Here we describe a method thatallows obtaining the Young's modulus of the adsorbed bacteria derived from themeasurement of the frequency shift when adsorbates are placed close to the clampingregion. As a model system we have used E. Coli bacteria deposited on the cantileversurface by the ink-jet technique. We demonstrate that the correct information aboutadsorbed mass can be extracted by recording the cantilever profile and its resonanceresponse. Also, the position and extent of adsorbates is determined by recording themicrocantilever profile. We use a theoretical model based on the Euler - Bernouilliequation for a beam with both mass and flexural rigidity local increase due to the depositedmaterial.

Effects of temperature and pressure on microcantilever resonance response.

The variation in resonance response of microcantilevers was investigated as a function of pressure (10(-2)-10(6)Pa) and temperature (290-390K) in atmospheres of helium (He) and dry nitrogen (N(2)). Our results for a silicon cantilever under vacuum show that the frequency varies in direct proportion to the temperature. The linear response is explained by the decrease in Young's modulus with increasing the temperature. However, when the cantilever is bimaterial, the response is nonlinear due to differential thermal expansion. Resonance response as a function of pressure shows three different regions, which correspond to molecular flow regime, transition regime, and viscous regime. The deflection in flow transition regime resulting from thermal variation has minimal effect on frequency. The frequency variation of the cantilever is caused mainly by changes in the mean free path of gas molecules.