Andrea Alessandrini

AFM: a versatile tool in biophysics

Here we review the applications of atomic force microscopy to the study of samples of biological origin. Emphasis is given to provide the reader with information on the broad range of different biophysical applications that, to date, such a technique can deal with. After recalling briefly the operating principles of an atomic force microscope, the broad field of bio-imaging applications is faced (DNA, DNA–protein interaction, proteins, lipid membranes, cells); thereafter, the use of the atomic force microscope to measure forces is introduced and force mapping on living cells is discussed. This section is followed by the description of the use of force curves in assessing single-molecule inter- and intramolecular interactions. A paragraph on the perspectives of the technique in biophysical applications concludes the paper. We hope that this review can help the reader in appreciating how atomic force microscopy contributes to the current explosive growth of nanobiosciences, where biology, chemistry and physics merge.

A CMOS, fully integrated sensor for electronic detection of DNA hybridization

An integrated field-effect device for fully electronic deoxyribonucleic acid (DNA) detection was realized in a standard CMOS process. The device is composed of a floating-gate MOS transistor, a control-capacitor acting as integrated counterelectrode, and an exposed active area for DNA immobilization. The drain-current of the transistor is modulated by the electric charge carried by the DNA molecules. After DNA hybridization, this charge increases and a change in the output current is measured. Experimental results are provided. Full compatibility with a standard CMOS process opens the way to the realization of low-cost large-scale integration of fast electronic DNA detectors.

Single-metalloprotein wet biotransistor

Metalloproteins are redox molecules naturally shuttling electrons with high efficiency between molecular partners. As such, they are candidates of choice for bioelectronics. In this work, we have used bacterial metalloprotein azurin, hosted in a nanometer gap between two electrically biased gold electrodes, to demonstrate an electrochemically gated single-molecule transistor operating in an aqueous environment. Gold-chemisorbed azurin shows peaks in tunneling current upon changing electrode potential and a related variation in tunneling barrier transparency which can be exploited to switch an electron current through it. These results suggest the wet approach to molecular electronics as a viable method for exploiting electron transfer of highly specialized biomolecules.

Unravelling single metalloprotein electron transfer by scanning probe techniques

This review is intended to account for the experimental and theoretical achievements obtained in a period of about 15 years on the investigation of the electron transport through single redox metalloproteins by scanning probe techniques. A highly focussed research effort has been deployed by the scientists active in this particular field towards measuring and interpreting electronic current signals flowing via blue copper, redox metalloproteins (e.g. azurin). The field has taken a remarkable advantage of the use of electrochemically assisted scanning tunnelling microscope (EC-STM) which has allowed to probe single molecule signals under full control of all the potential values involved in the experiments. This experimental activity has both triggered more comprehensive theoretical interpretations and has been, in its turn, stimulated by theoreticians to test always new predictions. The authors hope to have succeeded in providing the reader with a valuable appraisal of this fascinating field.

Supported Lipid Bilayers on Mica and Silicon Oxide: Comparison of the Main Phase Transition Behavior

The usual biophysical approach to the study of biological membranes is that of turning to model systems. From these models, general physical principles ruling the lateral membrane structure can be obtained. A promising model system is the supported lipid bilayer (SLB) which could foresee the simultaneous investigation of the structure and physical properties of lipid bilayers reconstituted with membrane proteins. A complete exploitation of the model system to retrieve biologically relevant information requires an in-depth knowledge of the possible effect that experimental parameters could have on the behavior of the SLB. Here we used atomic force microscopy (AFM) to study the effect of different types of substrates on the behavior of SLBs as far as their main phase transition is concerned. We found that different substrates (mica and silicon oxide) can affect in dissimilar ways the interleaflet coupling of the bilayer, which might represent a sort of lipid signaling allowing communication between receptors on the extracellular leaflet and cytoplasmic components. By decreasing the interaction between the SLB and the substrate the interleaflet coupling is preserved independently of the bilayer preparation strategy. Moreover, we investigated by time-lapse AFM an isothermal phase transition induced by a pH change on a SLB. We established that the presence of a pH gradient across the bilayer can weaken the strength of the interleaflet coupling which is present in symmetrical pH conditions.

Effect of physical parameters on the main phase transition of supported lipid bilayers.

Supported lipid bilayers composed of 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE) and 1-palmitoyl-2-oleoyl-phosphatidylglycerol (POPG) were assembled by the vesicle fusion technique on mica and studied by temperature-controlled atomic force microscopy. The role of different physical parameters on the main phase transition was elucidated. Both mixed (POPE/POPG 3:1) and pure POPE bilayers were studied. By increasing the ionic strength of the solution and the incubation temperature, a shift from a decoupled phase transition of the two leaflets, to a coupled transition, with domains in register, was obtained. The observed behavior points to a modulation of the substrate/bilayer and interleaflet coupling induced by the environment and preparation conditions of supported lipid bilayers. The results are discussed in view of the role of different interactions in the system. The influence of the substrate on the lipid bilayers, in terms of interleaflet coupling, can also help us in understanding the possible effect that submembrane elements like the cytoskeleton might have on the structure and dynamics of biomembranes.

Early effects of AZT on mitochondrial functions in the absence of mitochondrial DNA depletion in rat myotubes

Zidovudine (AZT) is a potent inhibitor of human immunodeficiency virus (HIV) replication. In humans, as well as in animal models, long-term treatment with AZT induces a severe myopathy characterised by structural and functional alterations of mitochondria associated with depletion of mitochondrial DNA (mtDNA). In the present work, we compared the effects induced by AZT on mitochondria upon short- or long-term treatments of cultured rat myotubes. Morphological alterations were investigated by electron microscopy, and mtDNA depletion and deletions were analysed by Southern blot. Mitochondrial membrane potential was determined after JC-1 staining by laser-scanning confocal microscopy in whole cells, and by flow cytometry in isolated muscle mitochondria. We found that the early effects of AZT on mitochondrial functions were a marked, yet reversible reduction in mitochondrial membrane potential, in the absence of any effect on mtDNA. The long-term treatment, in addition to mitochondrial membrane potential alterations, induced morphological changes in mitochondria, and a remarkable reduction in the amount of mtDNA, without any significant evidence of mtDNA deletions. In both treatments, a block of the spontaneous contraction of myotubes was observed. To study in more detail the early effects induced by AZT, the ability of the drug to interact with cardiolipin, an important component of internal mitochondrial membrane, was investigated by atomic force microscopy (AFM) in an artificial membrane model system. The results suggest that the primary effects of AZT may be related to a physical interference with the membrane structure leading to a consequent modification of its physical characteristics.

Unraveling lipid/protein interaction in model lipid bilayers by Atomic Force Microscopy

The current view of the biological membrane is that in which lipids and proteins mutually interact to accomplish membrane functions. The lateral heterogeneity of the lipid bilayer can induce partitioning of membrane‐associated proteins, favoring protein–protein interaction and influence signaling and trafficking. The Atomic Force Microscope allows to study the localization of membrane‐associated proteins with respect to the lipid organization at the single molecule level and without the need for fluorescence staining. These features make AFM a technique of choice to study lipid/protein interactions in model systems or native membranes. Here we will review the technical aspects inherent to and the main results obtained by AFM in the study of protein partitioning in lipid domains concentrating in particular on GPI‐anchored proteins, lipidated proteins, and transmembrane proteins. Whenever possible, we will also discuss the functional consequences of what has been imaged by Atomic Force Microscopy. Copyright

Monitoring cell-cycle-related viscoelasticity by a quartz crystal microbalance

We have monitored viscoelasticity variation of a cell population during the cell cycle by a Quartz Crystal Microbalance (QCM). Balb 3T3 fibroblasts were synchronized in the G0/G1 phase and seeded in a QCM chamber placed in a cell incubator. After cell sedimentation, the frequency signal was characterized by an amplitude modulation attributed to the viscoelasticity variation of the cells proliferating in phase. A control experiment with nonsynchronized cells showed a similar signal trend, but without significant modulation. Interestingly, the system resulted also to perform as a device sensitive to the effect of drugs affecting the cell cycle, such as colchicine.

Study of elastic fiber organization by scanning force microscopy.

Elastic fibers of beef ligamentum nuchae were observed by atomic force microscopy and data compared with those obtained by conventional and freeze-fracture electron microscopy. Fresh isolated elastin fibers as well as thin sections of ligament fragments, which were fixed and embedded either in relaxed or in stretched conditions, were analysed. The results confirm that, at least in beef ligamentum nuchae, elastic fibers consist of beaded filaments which can be oriented by stretching in the direction of the force applied. Moreover, atomic force microscopy revealed that these beaded filaments are laterally connected by periodical bridges which become more pronounced upon stretching. The data clearly show that elastin molecules are organized in a rather ordered array, at least at the super-molecular level, and a depiction of the elastin organization in beef ligamentum nuchae is attempted.