Single-molecule experiments in biological physics: methods and applications

I review single-molecule experiments (SME) in biological physics. Recent technological developments have provided the tools to design and build scientific instruments of high enough sensitivity and precision to manipulate and visualize individual molecules and measure microscopic forces. Using SME it is possible to: manipulate molecules one at a time and measure distributions describing molecular properties; characterize the kinetics of biomolecular reactions and; detect molecular intermediates. SME provide the additional information about thermodynamics and kinetics of biomolecular processes. This complements information obtained in traditional bulk assays. In SME it is also possible to measure small energies and detect large Brownian deviations in biomolecular reactions, thereby offering new methods and systems to scrutinize the basic foundations of statistical mechanics. This review is written at a very introductory level emphasizing the importance of SME to scientists interested in knowing the common playground of ideas and the interdisciplinary topics accessible by these techniques. The review discusses SME from an experimental perspective, first exposing the most common experimental methodologies and later presenting various molecular systems where such techniques have been applied. I briefly discuss experimental techniques such as atomic-force microscopy (AFM), laser optical tweezers (LOT), magnetic tweezers (MT), biomembrane force probe (BFP) and single-molecule fluorescence (SMF). I then present several applications of SME to the study of nucleic acids (DNA, RNA and DNA condensation), proteins (protein-protein interactions, protein folding and molecular motors). Finally, I discuss applications of SME to the study of the nonequilibrium thermodynamics of small systems and the experimental verification of fluctuation theorems. I conclude with a discussion of open questions and future perspectives.Comment: Latex, 60 pages, 12 figures, Topical Review for J. Phys. C (Cond. Matt

The Use of PRV-Bartha to Define Premotor Inputs to Lumbar Motoneurons in the Neonatal Spinal Cord of the Mouse

The neonatal mouse has become a model system for studying the locomotor function of the lumbar spinal cord. However, information about the synaptic connectivity within the governing neural network remains scarce. A neurotropic pseudorabies virus (PRV) Bartha has been used to map neuronal connectivity in other parts of the nervous system, due to its ability to travel trans-neuronally. Its use in spinal circuits regulating locomotion has been limited and no study has defined the time course of labelling for neurons known to project monosynaptically to motoneurons.Here we investigated the ability of PRV Bartha, expressing green and/or red fluorescence, to label spinal neurons projecting monosynaptically to motoneurons of two principal hindlimb muscles, the tibialis anterior (TA) and gastrocnemius (GC). As revealed by combined immunocytochemistry and confocal microscopy, 24-32 h after the viral muscle injection the label was restricted to the motoneuron pool while at 32-40 h the fluorescence was seen in interneurons throughout the medial and lateral ventral grey matter. Two classes of ipsilateral interneurons known to project monosynaptically to motoneurons (Renshaw cells and cells of origin of C-terminals) were consistently labeled at 40 h post-injection but also a group in the ventral grey matter contralaterally. Our results suggest that the labeling of last order interneurons occurred 8-12 h after motoneuron labeling and we presume this is the time taken by the virus to cross one synapse, to travel retrogradely and to replicate in the labeled cells.The study establishes the time window for virally-labelling monosynaptic projections to lumbar motoneurons following viral injection into hindlimb muscles. Moreover, it provides a good foundation for intracellular targeting of the labeled neurons in future physiological studies and better understanding the functional organization of the lumbar neural networks

Solute-Vacancy Clustering In Al-Mg-Si Alloys Studied By Muon Spin Relaxation Technique

Zero-field muon spin relaxation experiments were carried out with Al-1.6%Mg2Si, Al-0.5%Mg, and Al-0.5%Si alloys. Observed relaxation spectra were compared with the calculated relaxation functions based on the Monte Carlo simulation to extract the dipolar width (Δ), trapping (νt), and detrapping rates (νd), with the initially trapped muon fraction (P0). The fitting analysis has elucidated that the muon trapping rates depended on the heat treatment and solute concentrations. The dissolved Mg in Al dominated the νt at lower temperatures below 120 K, therefore the similar temperature variations of νt were observed with the samples mixed with Mg. The νt around 200 K remarkably reflected the heat treatment effect on the samples, and the largest νt value was found with the sample annealed at 100°C among Al-1.6%Mg2Si alloys. The as-quenched Al-0.5%Si sample showed significant νt values between 80 and 280 K relating with Si-vacancy clusters, but such clusters disappeared with the natural aged Al-0.5%Si sample.Stopy Al-1,6%Mg2Si, Al-0,5%Mg i Al-0,5%S poddano badaniu relaksacji spinowej mionów w zerowym polu magnetycznym. Obserwowane widma relaksacyjne porównano z funkcjami relaksacji obliczonymi przy wykorzystaniu symulacji Monte Carlo w celu otrzymania informacji o szerokości dipolarnej Δ (ang. dipolar width) oraz szybkości uwięzienia νt (ang. trapping) i uwolnienia νd (ang. detrapping) z pierwotnie związaną frakcją mionów (P0). Z przeprowadzonej analizy wynika, że szybkość uwięzienia mionów zależy od zastosowanej uprzednio obróbki cieplnej oraz stężenia pierwiastków domieszki. W przypadku Mg rozpuszczonego w osnowie Al dominowała szybkość νt w temperaturach poniżej 120 K, dlatego też w próbkach domieszkowanych Mg obserwowano podobne odchylenia temperaturowe szybkości νt. Szybkość νt w temperaturze około 200 K doskonale ukazuje wpływ zastosowanej obróbki cieplnej na omawiane próbki, z których najwyższą wartość νt wykazały te wyżarzane w 100°C spośród stopów Al-1,6%Mg2Si. Próbka Al-0,5%Si wykazała znaczące wartości νt pomiędzy temperaturami 80 a 280 K wskazujące na aglomeracje wakancji Si, które jednak zanikają po naturalnym starzeniu stopu Al-0.5%Si