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Spatiotemporal Fluorescence Correlation Spectroscopy of Inert Tracers: A Journey Within Cells, One Molecule at a Time

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Perspectives on Fluorescence

Part of the book series: Springer Series on Fluorescence ((SS FLUOR,volume 17))

Abstract

The fundamental unit of biology is unarguably the cell. Thus, as we move forward in our understanding of the processes occurring in the cell, it is crucial to reflect on how much of the cell biophysics remains unexplained or unknown. A ubiquitous observation in cell biology is that the translational motion of molecules within the intracellular environment is strongly suppressed as compared to that in dilute solutions. By contrast, molecular rotation is not affected by the same environment, indicating that the close proximity of the molecule must be aqueous. Theoretical models provide explanations for this apparent discrepancy pointing to the presence of macromolecular intracellular crowding, but with expectations that depend on the nanoscale organization assigned to crowding agents. A satisfactory experimental discrimination between possible scenarios has remained elusive due to the lack of techniques to explore molecular diffusion at the appropriate spatiotemporal scale in the 3D-intracellular environment. Here we discuss our recent experimental evidences for molecular diffusion in crowded biological media. By using monomeric GFP as a fluorescent tracer, and spatiotemporal fluorescence correlation spectroscopy (FCS) as main analytical tool, we reconstruct an imaginary journey, one molecule at a time, across intracellular compartments, such as cytoplasm and nucleoplasm, as well as within subcellular dynamic nanostructures, such as the nuclear pore complex. Results in cells are complemented by in vitro experiments where a variety of model systems mimic physiological crowding conditions. During this journey, Gregorio Weber intuitions on the nature of the cell protoplasm (see below) and on the intrinsic link between the spatial and temporal scales of diffusion processes both inspired our measurements and guided data interpretation. We do believe that the experimental observations on molecular diffusion collected in the interior of cells might influence the way biochemical reactions take place, with possible significant contributions to our understanding of crucial, still obscure phenomena, e.g., the biological benefit of anomalous transport, the regulation of protein folding/unfolding, intracellular signaling, target-search processes, and bimolecular reactions kinetics.

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Acknowledgments

This work ideally encompasses the research conducted across several years and, as such, reflects the invaluable contribution by other people. In particular, the authors are grateful to Dr. Elizabeth Hinde and Dr. Michelle Digman who conducted the research on chromatin, Dr. Luca Lanzano who contributed to the research on the nuclear pore, and Dr. Carmine Di Rienzo who conducted the research on protein diffusion in the cell. Also, the authors acknowledge the precious contribution to research of other colleagues, including Prof. Fabio Beltram, Dr. Vincenzo Piazza, Dr. Milka Stakic, Dr. Aaron Kershner, and Prof. Judith Kimble.

The research described in this work was supported by several grants, including: the Cell Migration Consortium Grant U54 GM064346, the National Institutes of Health Grants P41-RRO3155, P41-GM103540, RO1 DK066029, P50-GM076516, MIUR under FIRB-RBAP11X42L, and Fondazione Monte dei Paschi di Siena.

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Authors and Affiliations

  1. Center for Nanotechnology Innovation @NEST, Istituto Italiano di Tecnologia, Piazza San Silvestro 12, 56127, Pisa, Italy

    Francesco Cardarelli

  2. Laboratory for Fluorescence Dynamics, Department of Biomedical Engineering, University of California, Irvine, CA, 92697, USA

    Enrico Gratton

Authors
  1. Francesco Cardarelli
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  2. Enrico Gratton
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Correspondence to Francesco Cardarelli .

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Editors and Affiliations

  1. John A. Burns School of Medicine, University of Hawaii at Manoa, Honolulu, Hawaii, USA

    David M. Jameson

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Cardarelli, F., Gratton, E. (2016). Spatiotemporal Fluorescence Correlation Spectroscopy of Inert Tracers: A Journey Within Cells, One Molecule at a Time. In: Jameson, D. (eds) Perspectives on Fluorescence. Springer Series on Fluorescence, vol 17. Springer, Cham. https://doi.org/10.1007/4243_2016_6

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