Su appuntamento via email, ufficio 125 Dipartimento di Fisica e Astronomia, Via Sansone 1, Sesto Fiorentino
Responsabilità e interessi scientifici attuali
Attualmente sono Ricercatore Senior a tempo determinato (RTDb) presso il dipartimento di Fisica e Astronomia dell’Università degli Studi di Firenze e Ricercatore Associato e Group Leader (Single Molecule Manipulation and Imaging Group) presso il Laboratorio Europeo di Spettroscopia Non-lineare (LENS). I miei interessi scientifici sono a cavallo tra la fisica e la biologia. Da un lato la mia ricerca è incentrata sullo sviluppo di tecniche per lo studio della biologia a livello molecolare. In particolare, mi occupo dello sviluppo di tecniche di microscopia e intrappolamento ottico per la rivelazione, localizzazione e manipolazione di singole molecole biologiche. Dall’altro lato sono particolarmente interessato alla biofisica e ai meccanismi di regolazione meccanica dei sistemi biologici. Ho in particolare studiato i motori molecolari e come in questi avvengano i processi di conversione e regolazione chemo-meccanica. Studio inoltre come i segnali meccanici vengono convertiti in segnali biochimici e in cambiamenti di espressione genica. La mia ricerca ha gettato luce su importanti meccanismi alla base del funzionamento dei motori molecolari e portato allo sviluppo di tecniche all’avanguardia per lo studio di singole molecole biologiche e dell’influenza che le forze hanno su di esse (Capitanio et al., PNAS, 2006; Capitanio et al.,Nature Methods, 2012; Gardini et al. Scientific Reports, 2015).
All’interno dell’area di ricerca di biofisica del LENS coordino il gruppo di ricerca di singola molecola. Al LENS ho sviluppato apparati sperimentali dedicati alla manipolazione e microscopia di singole molecole biologiche. Grazie a un finanziamento Futuro in Ricerca ho potuto iniziare una nuova linea di ricerca volta a comprendere come i segnali meccanici influenzino la differenziazione e lo sviluppo delle cellule staminali, un tema di particolare attualità e con importanti ricadute mediche. Grazie a questo ed altri fondi di ricerca ottenuti recentemente, ho potuto costruire un nuovo apparato sperimentale presso il Dipartimento di Fisica e Astronomia e far crescere il gruppo di singola molecola. Le collaborazioni formatesi negli anni all’interno del Dipartimento di Fisica e del LENS fungono da fondamentale supporto allo svolgimento della mia ricerca. All’esterno ho stabilito diverse importanti collaborazioni nazionali e internazionali.
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Single Molecule Biophysics Lab
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Selected Publications
Se siete studenti interessati ad una tesi o un dottorato sperimentale in biofisica molecolare contattatemi pure per email o telefono.
Legenda
My current position is Researcher and Adjunct Professor at the University of Florence and Group Leader (Single Molecule Biophysics Group) at LENS (European Laboratory for Non-linear Spectroscopy, Florence, Italy, http://www.lens.unifi.it). The main research focus of the single molecule group is the study of molecular motors, the role of force in regulating biological systems, and the molecular mechanisms of gene expression regulation. The group develops optical setups and single molecule techniques beyond state-of-the-art. Several experimental systems in the lab allow visualization and manipulation of single biological molecules, using fluorescence and scattering microscopy together with optical and magnetic tweezers. Application of these techniques gave new insight into important mechanisms underlying the functioning of molecular motors (Capitanio et al., Proc. Natl. Acad. Sci. USA 103, 87-92, 2006). I recently developed a technique (the ultrafast force-clamp spectroscopy) that allowed clarifying the molecular mechanism of load-dependence of muscle contraction. These results were worth a publication in Nature Methods (Capitanio et al., Nature Methods 9, 1013-1019, 2012), together with a profile of the author (M.Vivien, "THE AUTHOR FILE Marco Capitanio," Nature Methods 9, 933-933, 2012) and the cover page (http://www.nature.com/nmeth/journal/v9/n10/covers/index.html). I recently obtained a FIR (“Futuro in Ricerca”) funding, an extremely selective grant awarded by the Italian Ministry of Education, University and Research (MIUR) to foster high quality research and support the independence of young researchers. I am also involved in research projects funded by the EU (Joint Research Action Optobio within LaserLab), MIUR (Flagship Project Nanomax) and private foundations (Ente Cassa di Risparmio di Firenze). I am an expert reviewer of AERES (“Agence d'évaluation de la recherche et de l'enseignement supérieur”, France), reviewer of grant proposals for the National Science Foundation (NSF), USA and Netherlands Organisation for Scientific Research (NWO), as well as reviewer of several scientific journals. I gave several invited talks in international conferences and I am involved in the organization of conferences. I have lectured in several international schools of higher education and I have a well-proven experience in training PhD students and experienced researchers.
Optical manipulation and imaging of single molecules
The Single Molecule Biophysics group at LENS develops novel single molecule manipulation and imaging tools for molecular biology. Manipulation of single molecules is realized using high-resolution optical tweezers, which allow probing sub-nanometer conformational changes of proteins or nuclei acids with sub-millisecond time resolution. Imaging and 3D localization of single molecules with nanometer accuracy is performed through fluorescent probes and advanced microscopy approaches in vitro as well as in living cells. Such adavanced tools are applied to the study of molecular motors and gene expression regulation.
Ultra-fast optical tweezers
Force has a fundamental role in a wide array of biological processes. For example, it modulates enzymatic activity, induces structural changes in proteins and nucleic acids, alters kinetics of molecular bonds, regulates motion of molecular motors, and has a role in mechanical transduction and sensory functions. At the molecular level, these processes are ultimately related to the capacity of force to modulate lifetimes of molecular interactions and transition rates in biochemical reaction cycles that involve motion.Single-molecule force spectroscopy techniques such as atomic force microscopy, optical tweezers and magnetic tweezers have opened up the possibility of studying such fundamental processes at the molecular level. Protocols for single-molecule force spectroscopy have been devised for the study of stable and long-lived bonds between two molecules. When a molecular bond is weak, however, the unbinding kinetics becomes rapid and application of such protocols during the short lifetime of the molecular complex becomes challenging. Such molecular interactions include receptors with low-binding-affinity ligands, non-processive motors interacting with their tracks and nucleic acid–binding proteins interacting with nonspecific sequences during the target search. Molecular interactions on the millisecond time scale are very common, and single-molecule force spectroscopy of such short-lived molecular complexes requires sub-millisecond resolution and control of the applied load. We developed an ultra-fast force-clamp spectroscopy technique that uses a dual trap configuration to apply constant loads between a single intermittently interacting biological polymer and a binding protein [1]. Our system has a delay of only ~10 μs between formation of the molecular bond and application of the force, and can detect interactions as short as 100 μs. The force-clamp configuration in which our assay operates allows direct measurements of the load dependence of the lifetimes of single molecular bonds. Moreover, conformational changes of single proteins and molecular motors can be recorded with sub-nanometer accuracy and a temporal resolution in the tens of microseconds. We applied our technique to the study of molecular motors, using myosin II from fast skeletal muscle, and to protein-DNA interaction, for the lactose repressor (LacI) interaction with DNA.
[1] Capitanio M., Canepari M., Maffei M., Beneventi D., Monico C., Vanzi F., Bottinelli R., and F. S. Pavone, Ultrafast force-clamp spectroscopy of single molecules reveals load dependence of myosin working stroke, Nature Methods 9, 1013–1019 (2012) (full text, cover page, author's file)
[2] Capitanio M. and F. S. Pavone, Interrogating biology with force: single molecule high resolution, Biophysical Journal, Vol. 105, Issue 6, pp. 1293-1303, 2013, September 17. (full text)
Combined manipulation and imaging
Single molecule (SM) techniques have greatly developed over the past thirty years to respond to the need of overcoming some of the limitations of traditional, bulk solution measurements. The manipulation of single biological molecules has created the opportunity to measure mechanical properties of biopolymers and control the mechanical parameters of protein-protein and protein-DNA interactions. SM fluorescence detection, on the other hand, represents an incredibly versatile tool for studying protein activity in vitro and in vivo, leading to the possibility of localizing and tracking single molecules with nanometer precision. Through fitting of the instrument point-spread-function to the SM image, in fact, one can accomplish localization with a precision depending mainly on signal-to-noise ratio (SNR) and reaching a limit of about one nanometer. These methodologies find powerful applications in the study of the dynamics of motor proteins, as well as of the diffusion processes underlying target search in DNA-binding proteins. The capability of determining diffusion constants as a function of the DNA sequence, residence time on the target and accurately measuring the DNA length explored during one-dimensional diffusion events, represent a powerful tool for the study of protein-DNA interaction dynamics and for the investigation of the mechanisms of specific target search. Recently, the combination of these two techniques has produced a new generation of experimental setups enabling the simultaneous manipulation of a biological substrate (for example an actin filament or a DNA molecule) and detection/localization of an interacting partner enzyme (for example myosin or a DNA-binding protein). The advantages of these techniques mainly rest on the possibility of exerting mechanical control over the trapped polymer, thus enabling the study of interaction dynamics versus forces or torques. Also, the methodology allows measuring biochemical reactions far from the surface, avoiding one of the main limitations of classic SM methods, i.e., the need for immobilization of the molecules under study on a surface (glass slide or microspheres). The combination of two single molecules techniques requires overcoming several technical difficulties, mainly arising from the requirements of mechanical stability and adequate SNR (especially when requiring localization with nm precision). Particularly, when coupling SM fluorescence detection with optical tweezers, the reduction of noise and photobleaching from the trapping infrared lasers and the control of biochemical buffers for assembly of the biological complexes and performance of the experimental measurements are of paramount importance. We developed methods for performing successful measurements in a dual trapping/SM Fluorescence localization setup. The methodology has been applied to the study of lactose repressor protein (LacI), fluorescently labeled with Atto532, and detected as it binds to a DNA molecule (trapped between two optical tweezers) containing specific LacI binding sequences (i.e., operators). The method is effective in detecting binding of LacI to DNA and diffusion along its contour in the target search process. The method is applicable to any combination of DNA sequence and DNA-binding protein, as well as to other systems (microtubules or actin filaments and the motor proteins interacting with them).
[3] Monico C., Belcastro G., Vanzi F., Pavone F. S., and M. Capitanio. Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions. J. Vis. Exp. (90), e51446, doi:10.3791/51446 (2014). (full text)
[4] Monico C., Capitanio M., Belcastro G., Vanzi F., and F. S. Pavone, Optical methods to study protein-DNA interactions in vitro and in living cells at the single-molecule level, International Journal of Molecular Sciences, Vol. 14, n. 2, pp. 3961-3992, 2013, February 18. (full text)
[5] Capitanio M., Maggi D., Vanzi F., and F. S. Pavone, FIONA in the trap: the advantages of combining optical tweezers and fluorescence, J. Opt. A: Pure Appl. Opt., vol. 9, pp. S157-S163, DOI:10.1088/1464-4258/9/8/S07, Cit. 7, 2007 August 1. (full text)
