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INTRACELLULAR NANOBIOPHYSICS

 

PI: Ranieri Bizzarri - ranieri.bizzarri@nano.cnr.it

 

People

Barbara Storti

Giovanni Signore (CNI-IIT)


 

Our understanding of cell machinery and of biological processes at subcellular level resides on the ability to detect biomolecular interactions as well as how the latters are modulated by the physicochemical features of the local environment. Positron-emission tomography, magnetic resonance imaging, and optical coherence tomography provide real-time read-out from animal or human subjects, but they cannot discern details smaller than ~1 mm, ~100 mm and ~10 mm, respectively. At the opposite end of the spectrum, electron microscopy provides near molecular-level spatial resolution, but cells must be fixed, which is invasive and prevents dynamic imaging. Between these two resolution extremes, fluorescence microscopy imaging with visible light is minimally invasive and 3D images of living cells can be effectively recorded at submicron spatial scale for times ranging from hundreds of μs to hours. Additionally, “super-resolution” techniques allow for discriminating emitters only a few nanometers apart. Fluorescence imaging relies on the spatially resolved contrast that can be achieved through the observation of four fluorescence parameters (intensity, wavelength, lifetime, and polarization) generated by intrinsic or added (extrinsic) labels to a given sample. On account of the energy separation between excitation and emission radiation, all fluorescence techniques are inherently ultrasensitive, in several cases allowing for single molecule detection. In addition, the usually “long” (>1ps) lifetime of the excited states makes fluorescence emission exquisitely sensitive to a plethora of factors such as the physicochemical properties of the environments (polarity, viscosity, electric field), structural rearrangements, molecular motion, and molecular interactions through energy, proton, or electron transfer. Thus, a fluorescence probe can in principle act as an ultrasensitive active reporter of biomolecules/bioprocesses at nanoscale. In this perspective, the goal of the intracellular nanobiophysics research line is:

         • the rational design of novel fluorescent probes and imaging methods capable to report,
           with high spatiotemporal resolution, on relevant biochemical targets in living cells


By following, a description of the three application areas covered by the research line is provided together with a short summary of the results of the last five-years.

 

1. Membrane organization
For several decades, membrane research was dominated by the idea that membrane proteins were the key factors for membrane functionality, whereas lipids were regarded as a passive, fluid solvent. Nowadays, membrane segregation into sub-μm domains is fully recognized as modulator of trafficking and signaling mediated by membrane proteins, thus conferring an active biological function to physicochemical properties and organization of the lipid bilayer.
In our activity, we seek to develop new fluorescent reporters of membrane organization at nanoscale. Our goals are:

         • Clarify how external mechanical forces or amyloid molecular aggregation modify plasma
           membrane (PM) organization thereby modulating receptor signaling and cell dysfunction.

         • Clarify how repairing processes of misfolded proteins occurring in the endoplasmic
           reticulum (ER) are influenced by ER membrane organization

 

To achieve these goals, we design and synthesize organic fluorescent reporters sensitive to the polarity and viscosity properties of their local environment, the latters being strictly connected to membrane organization. Typically, an environmentally sensitive reporter consists of three subunits, an electron donor unit, an electron acceptor unit, and an electron-rich spacer unit that is composed of a network of alternative single and double bonds (push-pull conjugated configuration). In this configuration, the molecule responds to photoexcitation with an intramolecular charge transfer (ICT) from the donor to the acceptor unit. The polar nature of ICT state induces solvent relaxation (on the ps scale in most solvents) and concomitantly a significant decrease of its energy (Fig. 1); this effect leads to an emission red-shift that is strictly dependent on the polarity of the nanoenvironment surrounding the molecule (solvatochromism). A second environmental effect is brought about by the possible stereochemical twist of the donor subunit with respect to the acceptor subunit once in ICT state (Fig. 1). Twisting motion is critically dependent on the environment viscosity and these reporters (molecular rotors) can act as local nanorehometers.

 

 

    

Fig 1. Photophysical processes conferring environmental sensitivity. Upon excitation, a push-pull hyperconjugated fluorophore goes to a high-energy state (Locally Excited, LE) with a stronger charge separation as compared to fundamental state. Owing to the very fast photoexcitation process (10-16 s) the surrounding polar solvent molecules have no time to rearrange to minimize their dipolar interaction energy with the fluorophore; however, after a few ps a solvent relaxed intramolecular charged state (ICT) is generated. ICT can decay back to the fundamental state by emitting a photon whose energy is dependent on solvent polarity. In some cases ICT can evolve to a twisted state (TICT) that decays fast to fundamental state non-radiatively. In such a case the temporal ICT->TICT evolution, and therefore the global quantum yield and lifetime of emission, is modulated by solvent viscosity.

 

 

1.1 Results 2009-2013

Coumarin-based polarity reporters By a rational approach starting from a computational screening, we developed fluorescent coumarins endowed with strong fluorescence emission in the blue-green region of the VIS spectrum (460-500 nm), excellent molar extinction coefficients (up to 40000) and fluorescence quantum yields (up to 0.95), and impressive solvatochromic properties (Fig. 2, left), such as 400-fold enhancement of their fluorescence emission going from polar to apolar environments (1,2). We demonstrated that our probes could effectively report on protein binding in vitro and report on polarity variations in living cells, particularly PM and ER membranes (1,3).

 

      

Fig 2.: Left: fluorescence emission of a coumarin derivative going from polar (water, right) to apolar (acetonitrile, left) environment (?exc: 405nm). Dye structure is reported in white in the upper-left side; center: absorption and fluorescence emission spectra of a butenolide derivative. The two channels selected for ratiometric analysis are evidenced in yellow and orange dashed boxes; right: dielectric constant map of butenolide dye in living cell. The dye is mostly localized in nuclear envelope and endoplasmic reticulum.

 

 

Butenolide-based polarity reporters Butenolide derivatives were easily assembled from commercial precursors (4), according to a simple synthetic procedure that allows for straightforward engineering of bio-derivatized structures for membrane and intracellular targeting. Some butenolide derivatives were demonstrated to possess strong solvatochromism (Fig. 2, center) characterized by a linear relationship between the fluorescence emission and the logarithm of the local dielectric constant (5). Accordingly, we measured the dielectric constant in different subcellular domains including PM and ER (Fig. 2, right), probing the existence of domains with different polarity properties and associating them to different organizations of the bilayer. Owing to the scarce cytotoxicity of these dyes, it was also possible to follow polarity changes occurring to the cell after external stimulation (5). Molecular rotors We developed new molecular rotors, based on styryl and coumarine scaffolds, which bear push-pull functional groups, as well as twisting side chains conferring viscosity dependent fluorescence to the dyes. For one of these molecules we demonstrated strong absorption in the green region of the spectrum, very little solvatochromism, and strong emission and lifetime sensitivity to local viscosity (6). By this rotor, intracellular viscosity measurements in intracellular compartments such as PM and mitochondria can be efficiently carried out by lifetime imaging following the “phasor approach”, an efficient method for measuring lifetime-related properties in biological contexts (Fig. 3).

 

Fig 3. Cultured HUVEC cells treated with our styryl molecular rotor. (a-b) Fluorescence intensity images. (c,d) “phasor” (lifetime) images corresponding to panels a,b; the color code is: red for membrane, green for lysosomes, blue for mitochondria. (e) Phasor plot for images in panels c,d; the three different regions corresponding to membrane (red), lysosomes (green) and mitochondria (blue) are enclosed in colored squares and the related viscosities are added.

 

 

2. Nucleocytoplasmic transport
The spatial separation of transcription and translation functions provides eukaryotes with powerful mechanisms to control gene expression, but demands finely-tuned transport mechanisms between nucleus and cytoplasm in order to maintain the distinctive composition of each compartment. This exchange occurs through nuclear pore complexes (NPCs), large protein complexes present on the nuclear envelope. Small molecules (<40-60 kDa) can freely diffuse through the NPC (passive translocation). Larger molecules such as proteins, RNAs, and their complexes are ushered selectively (active translocation) by dedicated transport receptors (importins for nuclear import and exportins for nuclear export) that can recognize specific nuclear-localization (NLS) or nuclear-export-signal (NES) peptides displayed by the cargo itself. Active translocation is an energy dependent process. Remarkably, several pathologies (e.g. tumors) are characterized by dysfunctional nucleocytoplasmic translocation and/ore NPC.
In our activity, we seek to develop new fluorescence imaging strategies to obtain a quantitative molecular picture of nucleocytoplasmic transport. Our goals are:

         • Clarify the linkage between selected cell dysfunctions (e.g. laminophaties) and the kinetics
           and thermodynamic properties of nucleocytoplasmic translocation.

         • Clarify the physicochemical properties of the molecular "sieve/gate" of the NPC

 

Concerning the first goal, we apply time-resolved microscopy imaging techniques such as Fluorescence Recovery After Photobleaching (FRAP) to measure molecular fluxes at variable cargo concentrations. By our method we are able to quantify: a) the import and export intrinsic rates of energy-dependent and/or passive translocations of proteins through the NPC, b) the binding affinity between the main transporter receptors and NLS or NES-bearing protein cargoes. These parameters describe fully the efficiency of the transport machinery, and can be quantitatively correlated to alterations of the cell physiological state. Concerning the second goal, we are studying the dynamic viscosity inside the NPC by molecular rotors and fluorescence depolarization (anisotropy) in both steady-state and time resolved imaging modes. Restricted motion inside the NPC is thought to account for the dependence of passive translocation on the molecular size of the cargo.

 

2.1 Results 2009-2013
We developed an original method relying on combination of time-resolved microscopy imaging techniques probing molecular fluxes (FRAP) and steady-state microscopy imaging techniques probing both protein binding and intracellular concentrations (7). In its simplest form, this method evaluates nucleocytoplasmic fluxes of target proteins fused to a genetically encoded fluorescent reporter (an autofluorescent protein) through FRAP, and correlates these fluxes with the concentration of the target proteins (Fig. 4). By Forster Resonance Energy Transfer (FRET) imaging it is also possible to identify the actual mediator of nucleocytoplasmic transport and to determine again the binding affinity between the two partners in an independent way (8). This approach is extremely general and can be applied to deletion variants of the target proteins to evaluate the role of sequence motifs on the nucleocytoplasmic translocation (9). By our method we quantified the active import and export rates, as well as the binding affinity to transporter receptors, for prototypical NLS (8) and NES sequences (2). Currently, we are comparing these parameters in cells whose nuclear lamina is normal or defective (Hutchinson-Gilford progeria syndrome), in view of correlating structural factors of nuclear envelope and transport through the NPCs.

 

Fig 4. FRAP experiments on NES-EGFP, a model of protein cargo actively exported from nucleus. Upon nuclear photobleaching, fluorescence recovery curves in both nucleoplasm and cytoplasm are fitted to simple monoexponential equations (left top). By our mathematical model of nucleocytoplasmic translocation (7), the fitting parameters are converted to molecular fluxes. Analysis of fluxes at different cargo concentrations (different expression levels found in several cells) afford the active and passive permeation parameters (right bottom) (2).

 

 

3. New fluorescence imaging approaches
In recent years many novel techniques have been proposed to improve fluorescence imaging of living specimens in terms of sensitivity/selectivity, and spatiotemporal resolution. Some of these advancements rely on specific photophysical or molecular properties of the fluorescent probes. In other cases, improvements over conventional imaging are brought about by a different fluorescence acquisition strategy.
In our activity, we seek to develop new fluorescence imaging approaches and related fluorescent reporters to conjugate effectively functional imaging/sensing with high sensitivity and spatiotemporal resolution. We focus on:

         • Photochromic fluorescent probes for super-resolution or optical lock-in detection
           and thermodynamic properties of nucleocytoplasmic translocation.

         • Phasor approach to lifetime imaging applied to intracellular reporters

         • Super-resolved spatiotemporal fluorescence correlation spectroscopy

 

3.1 Results 2009-2013
Photochromic probes. Photochromism is the reversible transformation of a chemical species between two forms by the absorption of electromagnetic radiation, where the two forms have different absorption spectra. Photochromic fluorescent probes are at basis of two relevant non-standard imaging techniques: photochromic FRET and optical lock-in detection (OLID). Additionally, photochromic fluorophores are widely employed in super-resolution techniques based on single molecule localization, such as PALM and STORM. In this field, we found out that the E222Q aminoacid replacement mutation in the primary sequence confers good photochromic properties (Fig. 5a) to otherwise photostable Aequorea Victoria fluorescent proteins (10,11), the latter being the most popular genetically-encodable probes for intracellular use. Actually we demonstrated that the photoswitching of the anionic cis protein chromophore yields a neutral non-fluorescent trans state, providing the optical basis for on<->off cycling of fluorescence (12). Photoswitchable fluorescence of any E222Q mutant is easily decoupled from that of a non-switchable probe emitting in the same wavelength region (Fig. 5b). Besides, E222Q mutants can be efficiently applied to super-resolution PALM imaging, as demonstrated for the fusion protein between the Transient Receptor Potential channel TRPV1 and the E222Q mutant EYQ1 (Fig. 5c).

 

Fig 5. (a) E222Q GFP mutants can be repeatedly photoswitched between an emissive and dark state, both in vitro and in living cells. (b) Cells expressing a membrane receptor labeled by the photochromic E222Q mutant EQ1 (green), a non-photochromic label of mitochondria (green), and a non photochromic label of chromatin (blue): in the green channel, the average intensity can not decouple membrane emission from mitochondrion emission, as they have similar spectrum; OLID decouples the switchable (membrane) from the non switchable (mitochondria) signal; the composite images shows membrane in green, mitochondria in red, and chromatin in blue. (c) PALM applied to TRPV1-EYQ1 (EYQ1 is a yellow E222Q photochromic mutant): localization of switching single molecules is applied to the cell region enclosed in the white square of epifluorescence image (left image) yielding the super-resolved PALM image (central image, scale bar 1 ?m); the rightmost panel shows the histogram of localization precision of single molecules, peaked at about 30 nm.

 

 

Phasor approach to lifetime imaging. Fluorescence Lifetime Imaging (FLIM) constitutes an elegant way to circumvent the most compelling problem of many reporters for fluorescence imaging, i.e. their failure to generate an intensity-based signal independent from their concentration, the latter being a quantity that cannot be easily controlled at nanoscale and particularly in the cellular context. The major drawback of conventional FLIM, however, is the low number of photons usually collected in each pixel of the image (~500-1000) to avoid long acquisition times that would hinder most time-dependent biological processes. Additionally, fitting of the emission decay at each pixel in a high-resolution image may constitute a demanding computational problem. These limitations are mostly addressed by the “phasor approach to FLIM”, a fit-free graphical method that requires no a priori knowledge on the photophysical system and provides unbiased representation of the FLIM raw data. We successfully combined the phasor approach to FLIM with a versatile intracellular pH reporter constituted by an engineered fluorescent protein (13,14); we showed that this method affords easily intracellular pH maps under resting or altered physiological conditions by using both single-photon confocal or two-photon microscopy (15). The phasor approach to FLIM was also employed to monitor the optical response of a molecular rotor, as reported at 1.1. Super-resolved spatiotemporal correlation spectroscopy Fluorescence Correlation Spectroscopy (FCS) represents an established technique to recover single-molecule diffusion and binding properties in cells. Scanning microscopy imaging adds a spatial dimension to the classic, purely temporal, FCS modality: spatiotemporal FCS (stFCS) provides details about the routes that are followed by the diffusing particles or molecules in the specimen. We combine spatiotemporal fluorescence correlation spectroscopy (stFCS) and stimulated emission depletion (STED) to monitor intracellular protein diffusion at spatial resolution below the optical diffraction limit (super-resolution). Our method was validated both in vitro and at intracellular level by following the diffusion of fluorescent nanocapsids and of GFP bound to SV40 Nuclear Localization Signal (NLS), respectively. We are currently applying this method to investigate nucleocytoplasmic translocation.

 

References

 

1. Polarity-sensitive coumarins tailored to live cell imaging

G Signore, R Nifosi, L Albertazzi, B Storti, and R Bizzarri

J. Am. Chem. Soc. 132, 1276-1288 (2010).

 

2. Fluorescent Recovery after Photobleaching (FRAP) Analysis of Nuclear Export Rates Identifies Intrinsic Features of Nucleocytoplasmic Transport

F Cardarelli, L Tosti, M Serresi, F Beltram, and R Bizzarri

J. Biol. Chem. 287, 5554-5561 (2012).

 

3. A novel coumarin fluorescent sensor to probe polarity around biomolecules

G Signore, R Nifosi, L Albertazzi, and R Bizzarri

J. Biomed. Nanotechnol. 5, 722-729 (2009).

 

4. Cis-trans photoisomerization properties of GFP chromophore analogs

G Abbandonato, G Signore, R Nifosi, V Voliani, R Bizzarri, and F Beltram

Eur. Biophys. J. 40, 1205-1214 (2011).

 

5. Imaging the static dielectric constant in vitro and in living cells by a bioconjugable GFP chromophore analog

G Signore, G Abbandonato, B Storti, M Stockl, V Subramaniam, and R Bizzarri

Chem. Comm. 49, 1723-1725(2013).

 

6. Imaging intracellular viscosity by a new molecular rotor suitable for phasor analysis of fluorescence lifetime

A Battisti, S Panettieri, G Abbandonato, E Jacchetti, F Cardarelli, G Signore, F Beltram, and R Bizzarri

Anal. Bioanal. Chem. 405, 6223-6233 (2013).

 

7. Fluorescence recovery after photobleaching reveals the biochemistry of nucleocytoplasmic exchange

R Bizzarri, F Cardarelli, M Serresi, and F Beltram

Anal. Bioanal. Chem. 403, 2339-2351 (2012).

 

8. Probing nuclear localization signal-importin alpha binding equilibria in living cells

F Cardarelli, R Bizzarri, M Serresi, L Albertazzi, and F Beltram

J. Biol. Chem. 284, 36638-36646 (2009).

 

9. Quantitative analysis of tat Peptide binding to import carriers reveals unconventional nuclear transport properties

F Cardarelli, M Serresi, A Albanese, R Bizzarri, and F Beltram

J. Biol. Chem. 286, 12292-12299 (2011).

 

10. Single amino acid replacement makes Aequorea victoria fluorescent proteins reversibly photoswitchable

R Bizzarri, M Serresi, F Cardarelli, S Abbruzzetti, B Campanini, C Viappiani, and F Beltram

J. Am. Chem. Soc. 132, 85-95 (2010).

 

11. Raman study of chromophore states in photochromic fluorescent proteins

S Luin, V Voliani, G Lanza, R Bizzarri, P Amat, V Tozzini, M Serresi, and F Beltram

J. Am. Chem. Soc. 131, 96-103 (2009).

 

12. Photoswitching of E222Q GFP mutants: "concerted" mechanism of chromophore isomerization and protonation

S Abbruzzetti, R Bizzarri, S Luin, R Nifosi, B Storti, C Viappiani, and F Beltram

Photochem. Photobiol. Sci. 9, 1307-1319 (2010).

 

13. Real-time measurement of endosomal acidification by a novel genetically encoded biosensor

M Serresi, R Bizzarri, F Cardarelli, and F Beltram

Anal. Bioanal. Chem. 393, 1123-1133 (2009).

 

14. Green fluorescent protein based pH indicators for in vivo use: a review

R Bizzarri, M Serresi, S Luin, and F Beltram

Anal. Bioanal. Chem. 393, 1107-1122 (2009).

 

15. Intracellular pH measurements made simple by fluorescent protein probes and the phasor approach to fluorescence lifetime imaging

A Battisti, M A Digman, E Gratton, B Storti, F Beltram, and R Bizzarri

Chem. Comm. 48, 5127-5129 (2012).


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