Molecular Imaging (Joint IBF-CNR)

Application in living cells of the combined pH and chloride sensor, ClopHensor. Simultaneous map for pH and chloride concentration at the plasma membrane (inner domain) and in secretory vesicles. In the schematic representation, chloride ions (yellow) binds to the molecular sensor via a static quenching mechanismOverview
Molecular imaging is a new integrative discipline that enables noninvasive in vivo investigation of cellular functions and molecular processes involved in physiological and pathological conditions. The laboratory is focused on the development of new biosensor capable to image particular functions, pathways and targets playing relevant roles in translational research, and in various microscopy and nanoscopy techniques including live imaging, total internal reflection microscopy fluorescence (TIRF)-microscopy, and single particle tracking.

 

Research directions
Two are our current aims:

  • Chloride imaging. By mutagenesis of the green fluorescent protein (GFP), we recently identified a mutation (T203Y) sufficient to create a highly specific anion-binding site that, when occupied leads to the complete loss of fluorescence via static quenching. We have exploited this GFP property within a ratiometric-design concept for the development of a combined pH and chloride concentration sensor. Specific goals are now to extend this imaging in living animals though 2-photon excitation microscopy and establish a high-throughput chloride flux assay.
  • Visualization of HIV-1 and single particle tracking of intranuclear dynamics in collaboration with the Molecular Virology laboratory. Nuclear import and subsequent integration of viral genome are emerging as tightly regulated aspects of the HIV-1 lifecycle. We are interested in defining the dynamics, energy-dependent motion and molecular interactions exploited by the virus to target specific integration sites. Specifically, we investigate the nuclear translocation and the nuclear trafficking of the HIV genome.

Group members

  • Daniele Arosio, PI
  • Sonja Lukowski, Postdoctoral Fellow
  • José Manuel Paredes, Postdoctoral Fellow
  • Ashwanth Francis, PhD student
  • Faraz Kurshid, PhD Student
  • Francesco Rocca, PhD Student

Collaborations

  • Gian Michele Ratto, Istituto NanoScienze, CNR, Pisa, Italy
  • Giorgio Carmignoto, Istituto di Neuroscienze, CNR, Padova, Italy
  • Tommaso Fellin, Italian Institute of Technology, IIT, Genova, Italy
  • Matteo Caleo, Istituto di Neuroscienze, CNR, Pisa, Italy
  • Laura Cancedda, Italian Institute of Technology, IIT, Genova, Italy
  • Marco Cecchini, Istituto NanoScienze, CNR, Pisa, Italy
  • Thomas Hope, Northwestern University, Chicago IL, USA

Selected publications

Bregestovski, P. & Arosio, D. Green Fluorescent Protein-Based Chloride Ion Sensors for In Vivo Imaging. Fluorescent Proteins II 12, 99–124 (2012).

Allouch, A. et al. The TRIM Family Protein KAP1 Inhibits HIV-1 Integration. Cell Host & Microbe 9, 484–495 (2011).

Arosio, D. et al. Simultaneous intracellular chloride and pH measurements using a GFP-based sensor. Nat Meth 7, 516-518 (2010).

Ricci, F., Arosio, D. & Beltram, F. A Fluorescent Indicator Monitors in Vivo Acetyl-Transferase Activity. Biophysical Journal 98, 582a-583a (2010).

Marchetti, L. et al. Homeotic proteins participate in the function of human-DNA replication origins. Nucleic Acids Research (2010).

Comelli, L. et al. The homeotic protein HOXC13 is a member of human DNA replication complexes. Cell Cycle 8, 454-459 (2009).

Albertazzi, L., Arosio, D., Marchetti, L., Ricci, F. & Beltram, F. Quantitative FRET analysis with the EGFP-mCherry fluorescent protein pair. Photochem. Photobiol 85, 287-297 (2009).

Arosio, D. et al. Nuclear trafficking of hiv-1 pre-integration complexes in living cells. Retrovirology 6, (2009).

Allouch, A. et al. HIV-1 acetylated integrase is targeted by KAP1 (TRIM28) to inhibit viral integration. Retrovirology 6, (2009).

Christ, F. et al. Transportin-SR2 imports HIV into the nucleus. Curr. Biol 18, 1192-1202 (2008).

Albanese, A., Arosio, D., Terreni, M. & Cereseto, A. HIV-1 Pre-Integration Complexes Selectively Target Decondensed Chromatin in the Nuclear Periphery. PLoS ONE 3, e2413 (2008).

Arosio, D. et al. Spectroscopic and structural study of proton and halide ion cooperative binding to gfp. Biophys. J 93, 232-244 (2007).

Bizzarri, R. et al. Green Fluorescent Protein Ground States: The Influence of a Second Protonation Site near the Chromophore. Biochemistry 46, 5494-5504 (2007).

Di Benedetto, F. et al. Two Dimensional Patterning of Fluorescent Proteins in Hydrogels. Langmuir 22, 29-31 (2006).

Bizzarri, R. et al. Development of a novel GFP-based ratiometric excitation and emission pH indicator for intracellular studies. Biophysical journal 90, 3300–3314 (2006).