Su appuntamento via email, ufficio 125 Dipartimento di Fisica e Astronomia, Via Sansone 1, Sesto Fiorentino

Se siete studenti interessati ad una tesi o un dottorato sperimentale in biofisica molecolare o cellulare contattatemi pure per email o telefono. 

My current position is Associate Professor at the Department of Physics of the University of Florence and Group Leader (Group of Molecular and Cellular Mechanobiology) at the European Laboratory for Non-linear Spectroscopy (LENS), a renowned international and interdisciplinary center of excellence of the University of Florence.

My research interests lie across physics and biology. On one hand, my research is focused on the development of techniques for the study of biology at the molecular scale, with a particular interest on optical methods. On the other hand, I am particularly interested in the molecular mechanisms underlying mechanical regulation of biological systems. My biological interests encompass the study of the chemomechanical properties of molecular motors; the molecular mechanisms of mechanotransduction; and the conversion of mechanical signals into changes in gene expression and cell fate. I developed novel techniques for the imaging and manipulation of single biological molecules, using optical and magnetic tweezers combined with fluorescence and scattering microscopy. Application of these techniques gave new insight into important mechanisms underlying the functioning of molecular motors (Capitanio et al., PNAS 2006) and the load-dependence of muscle contraction (Capitanio et al., Nature Methods 2012). More recently, my techniques have been applied to investigate the earliest events in force generation by cardiac myosin (Woody et al., eLife 2019), mechanosensitivity of processive molecular motors (Gardini et al., Nature Communications 2018), target search by DNA-binding proteins (Tempestini et al., Nucleic Acids Research 2018), and the mechanosensitivity of α-catenin inside and outside adherens junctions (Arbore et al., Nature Communications, 2022).

I 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. My group is also involved in a PRIN grant awarded by MIUR, in research projects funded by the Human Frontier Science Program (HFSP), the Italian association for cancer research (AIRC), and the LaserLab-Europe H2020 Integrated Infrastructure Initiative.

Personal webpages

Single Molecule Biophysics Lab

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Selected Publications

1. Gardini, L., Tempestini, A., Pavone F.S., and M. Capitanio “High-speed optical tweezers for the study of single molecular motors”. In: Lavelle, C. (eds) Molecular Motors 2nd ed. Methods in Molecular Biology 1805. Humana Press, New York, NY (2018)
2. Gardini, L., Calamai, M., Hatakeyama, H., Kanzaki, M.,Capitanio, M., and F.S.Pavone, “three-dimensional tracking of quantum dot-conjugated molecules in living cells”. In: Lyubchenko Y. (eds) Nanoscale Imaging. Methods in Molecular Biology 1814. Humana Press, New York, NY (2018)
3. Sosa-Costa, A., Piechocka, I., Gardini, L., Pavone, F. S., Capitanio, M., Garcia-Parajo, M. F., Manzo, C. “PLANT, a method for detecting changes of slope in noisy trajectories”, Biophysical Journal 114, Issue 9, 2044–2051 (2018).
   
4. Woody, M.S., Capitanio,M., Ostap, M.E., and Y.E. Goldman, “Electro-optic deflectors deliver advantages over acousto-optical deflectors in a high resolution, ultra-fast force-clamp optical trap”, Optics Express 26, 11181-11193 (2018).
   
5. Tempestini, A., Monico, C., Gardini, L., Vanzi, F., Pavone F. S., and M. Capitanio, “Sliding of a single lacrepressor protein along DNA is tuned by DNA sequence and molecular switching”, Nucleic Acids Research, doi:10.1093/nar/gky208, (2018) 
   
6. Capitanio M.. Optical Tweezers. In “An Introduction to Single Molecule Biophysics”, Edited by Yuri L. Lyubchenko,Publisher: CRC Press,Taylor & Francis group; 1 edition (November 29, 2017), ISBN-10: 1439806942,ISBN-13: 978-1439806944
7. Gardini, L., Capitanio, M., Pavone F.S., “3D tracking of single nanoparticles and quantum dots in living cells by out-of-focus imaging with diffraction pattern recognition”, Sci. Rep. 5, 16088; doi: 10.1038/srep16088(2015).
8. 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).
   
9. Capitanio, M. and Pavone, F.S. “Interrogating biology with force: single-molecule high-resolution measurements with optical tweezers”,Biophys. J. 105, 1293-1303 (2013).
   
10. Monico, C., Capitanio, M., Belcastro, G., Vanzi, F., Pavone, F.S., “Optical Methods to Study Protein-DNA Interactions in Vitro and in Living Cells at the Single-Molecule Level”, Int. J. Mol. Sci. 14, 3961-3992 (2013).
    
11. Capitanio, M., Canepari, M., Maffei, M., Beneventi, D., Monico, C., Vanzi, F., Bottinelli, R. and Pavone F.S. “Ultrafast force-clamp spectroscopy of single molecules reveals load dependence of myosin working stroke”, Nature Methods, 9, 1013–1019 (2012).
    
12. Elangovan, R., Capitanio, M., Melli, L., Pavone, F.S., Lombardi, V., Piazzesi, G. An integrated in vitroand in situstudy of kinetics of myosin II from frog skeletal muscle. J Physiol., 590, 1227-1242  (2012).
    
13. Capitanio,M.,Maggi, D.,Vanzi, F. and Pavone, F. S. Fiona in the trap: the advantages of combining optical tweezers and fluorescence.J. Opt. A - Pure Appl. Opt.,9, S157-S163 (2007).
    
14. Capitanio,M.,Cicchi, R. and Pavone, F. S. Continuous and time-shared multiple optical tweezers for the study of single motor proteins.Opt. Las. Eng. 45,450-457 (2007).
    
15. F. Vanzi,Capitanio, M., Sacconi, L., Stringari, C., Cicchi, R., Canepari, M., Maffei, M., Bottinelli, R., Piroddi, N., Tesi, C., Poggesi, C., Nucciotti, V., Linari, M., Piazzesi, G., Lombardi, V., and Pavone, F.S. New Techniques in linear- and non-linear Laser Optics in Muscle Research. J. Muscle Res. Cell Mot. 27, 469-479 (2006).
    
16. Capitanio, M., Canepari, M., Cacciafesta, P., Lombardi, V., Cicchi, R., Maffei, M., Pavone, F.S. and Bottinelli, R. Two independent mechanical events in the interaction cycle of skeletal muscle myosin with actin.Proc. Natl. Acad. Sci. USA103, 87-92 (2006).
    
17. Capitanio,M.,Cicchi, R. and Pavone, F. S. Position control and optical manipulation for nanotechnology applications.Eur. Phys. J. B46, 1-8 (2005).
    
18. Capitanio, M.,Vanzi, F., Broggio, C., Cicchi, R., Normanno, D., Romano, G., Sacconi, L., and Pavone, F. S. Exploring Molecular Motors and Switches at the Single-Molecule Level.Micr. Res. Tech. 65, 194-204 (2004).
    
19. Normanno, D., Capitanio, M. and Pavone, F. S. Spin absorption, windmill and magneto-optics effects in optical angular momentum transfer.Phys. Rev. A70, 053829 (2004).
    
20. Capitanio, M., Normanno, D. and Pavone, F. S. High precision measurement of light-induced torque on absorbing micro-spheres. Opt. Lett. 29, 2231-2233 (2004).
    
21. Romano, G., Sacconi, L., Capitanio,M. and Pavone, F.S. Force and torque measurements using magnetic micro beads for single molecule biophysics. Opt. Comm. 215, 323-331 (2003).
    
22. Capitanio, M., Romano, G., Ballerini, R., Dunlap, D., Finzi, L., Giuntini, M. and Pavone, F. S. Calibration of optical tweezers with differential interference contrast signals. Rev. Sci. Instrum.73, 1687-1696 (2002).
    
23. Sacconi, L., Romano, G., Ballerini, R., Capitanio,M.,De Pas, M., Dunlap, D., Giuntini, M., Finzi, L. and Pavone,   F. S. Three-dimensional  magneto-optical trap for micro-object manipulation. Opt. Lett.26, 1359-1361 (2001).

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)