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Probing short-range protein Brownian motion in the cytoplasm of living cells.

Di Rienzo C, Piazza V, Gratton E, Beltram F, Cardarelli F - Nat Commun (2014)

Bottom Line: Here we show that fluorescence-fluctuation analysis of raster scans at variable timescales can provide this information.By using green fluorescent proteins in cells, we measure protein motion at the unprecedented timescale of 1 μs, unveiling unobstructed Brownian motion from 25 to 100 nm, and partially suppressed diffusion above 100 nm.We discuss the implications of these results for intracellular processes.

View Article: PubMed Central - PubMed

Affiliation: 1] Center for Nanotechnology Innovation @NEST, Istituto Italiano di Tecnologia, Piazza San Silvestro 12, 56127 Pisa, Italy [2] NEST, Scuola Normale Superiore and Istituto Nanoscienze-CNR, Piazza San Silvestro 12, 56127 Pisa, Italy.

ABSTRACT
The translational motion of molecules in cells deviates from what is observed in dilute solutions. Theoretical models provide explanations for this effect but with predictions that drastically depend on the nanoscale organization assumed for macromolecular crowding agents. A conclusive test of the nature of the translational motion in cells is missing owing to the lack of techniques capable of probing crowding with the required temporal and spatial resolution. Here we show that fluorescence-fluctuation analysis of raster scans at variable timescales can provide this information. By using green fluorescent proteins in cells, we measure protein motion at the unprecedented timescale of 1 μs, unveiling unobstructed Brownian motion from 25 to 100 nm, and partially suppressed diffusion above 100 nm. Furthermore, experiments on model systems attribute this effect to the presence of relatively immobile structures rather than to diffusing crowding agents. We discuss the implications of these results for intracellular processes.

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iMSD in dilute solution.Experimental iMSD values at the different timescales for differently sized molecules in dilute solution at 37 °C. Monomeric GFP (N=7 measurements, green dots) shows a linear behaviour in time, as expected for Brownian motion: fit by a free diffusion model (equation 13 in Supplementary Information) yields Dw=134±4 μm2 s−1 (, Hr=2.5 nm); Alexa488 (Dw=428±15 μm2 s−1, Hr=0.75 nm, , N=7 measurements; blue dots) and 30-nm-diameter fluorescent beads (Dw=22±0.5 μm2 s−1, , Hr=15 nm, N=7 measurements; black dots) are acquired under the same conditions. Data are mean values±s.d.
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f2: iMSD in dilute solution.Experimental iMSD values at the different timescales for differently sized molecules in dilute solution at 37 °C. Monomeric GFP (N=7 measurements, green dots) shows a linear behaviour in time, as expected for Brownian motion: fit by a free diffusion model (equation 13 in Supplementary Information) yields Dw=134±4 μm2 s−1 (, Hr=2.5 nm); Alexa488 (Dw=428±15 μm2 s−1, Hr=0.75 nm, , N=7 measurements; blue dots) and 30-nm-diameter fluorescent beads (Dw=22±0.5 μm2 s−1, , Hr=15 nm, N=7 measurements; black dots) are acquired under the same conditions. Data are mean values±s.d.

Mentions: Technically, we perform consecutive raster image correlation spectroscopy (RICS)21 measurements, in which the pixel dwell time is progressively increased (that is, from 0.5 to 100 μs as described in Methods). Thus, we segment the temporal scale in such a way that the observed portion of particle trajectory along the scan direction varies according to scan speed (Fig. 1a). In particular, at high scan speeds only short-range particle displacements can be detected (Fig. 1a, left panel). Then, by decreasing the scan speed, progressively larger displacements are observed (Fig. 1a, middle-right panels). As a consequence, at high scan speeds, the particle image is only slightly deformed by the scan process, that is, it almost coincides with the autocorrelation of the instrumental point spread function (PSF; Fig. 1b, left). On the contrary, for decreasing scanning speeds, the apparent particle image is deformed because of molecular movement (Fig. 1b, middle-right), that is, the spatial correlation function becomes much larger than the PSF. We used a Gaussian interpolation of experimental correlation functions as an algorithmic way to obtain an estimate of the width of the particle-displacement distribution for each scan speed. This is quantitatively described by equation 15 in Supplementary Information and allows the recovery of the particle MSD directly from the raster scan images (iMSD, Fig. 1c). Notably, this approach covers a hitherto unexplored dynamic range, from 0.5 μs to several milliseconds (Supplementary Fig. 1). We used untagged monomeric GFP as an example of fluorescent tracer with no specific interactions with the environment, and suitable for both in cell22 and in cuvette10 experiments. As a preliminary experiment, we performed measurement on GFP recombinant proteins in dilute solutions (Fig. 2, green line). The measured average GFP displacements are reported against the corresponding timescale of observation on a double-logarithmic representation, as a means to easily identify the linear dependence typical of Brownian motion and possible deviations from it. As reported in the plot in Fig. 2, the iMSD values show the expected linear behaviour in time, clear indication of GFP Brownian motion in the dilute solution over the entire spatiotemporal scale observed. This outcome allowed us to obtain a diffusion coefficient (Dw) of 134±4 μm2 s−1 at 37 °C (equation 13 in Supplementary Information), corresponding to the expected hydrodynamic radius (Hr) of the tracer (2.5 nm), in keeping with previous experimental estimates23242526. As expected, the experimental distributions of molecular displacements in dilute solution are well described by the Gaussian algorithm used for data interpolation (Supplementary Fig. 2). This accordance has been also verified by simulating the 3D diffusion of a point-like particle in solution (Supplementary Fig. 3). The capability to reveal the Brownian behaviour in dilute solution was further assessed with very differently sized molecules. In particular, the approach presented in this paper is suitable for tracking molecules with a wide range of diffusion coefficients (for example, from 500 μm2 s−1 typical of a small organic dye to 20 μm2 s−1 of a 30-nm particle; Fig. 2, black solid and dashed lines, respectively). It is also worth noting that average displacements down to 20–30 nm can be measured, thus demonstrating the ability of this approach to resolve molecular displacements well below the diffraction limit (dashed grey line in Fig. 2).


Probing short-range protein Brownian motion in the cytoplasm of living cells.

Di Rienzo C, Piazza V, Gratton E, Beltram F, Cardarelli F - Nat Commun (2014)

iMSD in dilute solution.Experimental iMSD values at the different timescales for differently sized molecules in dilute solution at 37 °C. Monomeric GFP (N=7 measurements, green dots) shows a linear behaviour in time, as expected for Brownian motion: fit by a free diffusion model (equation 13 in Supplementary Information) yields Dw=134±4 μm2 s−1 (, Hr=2.5 nm); Alexa488 (Dw=428±15 μm2 s−1, Hr=0.75 nm, , N=7 measurements; blue dots) and 30-nm-diameter fluorescent beads (Dw=22±0.5 μm2 s−1, , Hr=15 nm, N=7 measurements; black dots) are acquired under the same conditions. Data are mean values±s.d.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4281647&req=5

f2: iMSD in dilute solution.Experimental iMSD values at the different timescales for differently sized molecules in dilute solution at 37 °C. Monomeric GFP (N=7 measurements, green dots) shows a linear behaviour in time, as expected for Brownian motion: fit by a free diffusion model (equation 13 in Supplementary Information) yields Dw=134±4 μm2 s−1 (, Hr=2.5 nm); Alexa488 (Dw=428±15 μm2 s−1, Hr=0.75 nm, , N=7 measurements; blue dots) and 30-nm-diameter fluorescent beads (Dw=22±0.5 μm2 s−1, , Hr=15 nm, N=7 measurements; black dots) are acquired under the same conditions. Data are mean values±s.d.
Mentions: Technically, we perform consecutive raster image correlation spectroscopy (RICS)21 measurements, in which the pixel dwell time is progressively increased (that is, from 0.5 to 100 μs as described in Methods). Thus, we segment the temporal scale in such a way that the observed portion of particle trajectory along the scan direction varies according to scan speed (Fig. 1a). In particular, at high scan speeds only short-range particle displacements can be detected (Fig. 1a, left panel). Then, by decreasing the scan speed, progressively larger displacements are observed (Fig. 1a, middle-right panels). As a consequence, at high scan speeds, the particle image is only slightly deformed by the scan process, that is, it almost coincides with the autocorrelation of the instrumental point spread function (PSF; Fig. 1b, left). On the contrary, for decreasing scanning speeds, the apparent particle image is deformed because of molecular movement (Fig. 1b, middle-right), that is, the spatial correlation function becomes much larger than the PSF. We used a Gaussian interpolation of experimental correlation functions as an algorithmic way to obtain an estimate of the width of the particle-displacement distribution for each scan speed. This is quantitatively described by equation 15 in Supplementary Information and allows the recovery of the particle MSD directly from the raster scan images (iMSD, Fig. 1c). Notably, this approach covers a hitherto unexplored dynamic range, from 0.5 μs to several milliseconds (Supplementary Fig. 1). We used untagged monomeric GFP as an example of fluorescent tracer with no specific interactions with the environment, and suitable for both in cell22 and in cuvette10 experiments. As a preliminary experiment, we performed measurement on GFP recombinant proteins in dilute solutions (Fig. 2, green line). The measured average GFP displacements are reported against the corresponding timescale of observation on a double-logarithmic representation, as a means to easily identify the linear dependence typical of Brownian motion and possible deviations from it. As reported in the plot in Fig. 2, the iMSD values show the expected linear behaviour in time, clear indication of GFP Brownian motion in the dilute solution over the entire spatiotemporal scale observed. This outcome allowed us to obtain a diffusion coefficient (Dw) of 134±4 μm2 s−1 at 37 °C (equation 13 in Supplementary Information), corresponding to the expected hydrodynamic radius (Hr) of the tracer (2.5 nm), in keeping with previous experimental estimates23242526. As expected, the experimental distributions of molecular displacements in dilute solution are well described by the Gaussian algorithm used for data interpolation (Supplementary Fig. 2). This accordance has been also verified by simulating the 3D diffusion of a point-like particle in solution (Supplementary Fig. 3). The capability to reveal the Brownian behaviour in dilute solution was further assessed with very differently sized molecules. In particular, the approach presented in this paper is suitable for tracking molecules with a wide range of diffusion coefficients (for example, from 500 μm2 s−1 typical of a small organic dye to 20 μm2 s−1 of a 30-nm particle; Fig. 2, black solid and dashed lines, respectively). It is also worth noting that average displacements down to 20–30 nm can be measured, thus demonstrating the ability of this approach to resolve molecular displacements well below the diffraction limit (dashed grey line in Fig. 2).

Bottom Line: Here we show that fluorescence-fluctuation analysis of raster scans at variable timescales can provide this information.By using green fluorescent proteins in cells, we measure protein motion at the unprecedented timescale of 1 μs, unveiling unobstructed Brownian motion from 25 to 100 nm, and partially suppressed diffusion above 100 nm.We discuss the implications of these results for intracellular processes.

View Article: PubMed Central - PubMed

Affiliation: 1] Center for Nanotechnology Innovation @NEST, Istituto Italiano di Tecnologia, Piazza San Silvestro 12, 56127 Pisa, Italy [2] NEST, Scuola Normale Superiore and Istituto Nanoscienze-CNR, Piazza San Silvestro 12, 56127 Pisa, Italy.

ABSTRACT
The translational motion of molecules in cells deviates from what is observed in dilute solutions. Theoretical models provide explanations for this effect but with predictions that drastically depend on the nanoscale organization assumed for macromolecular crowding agents. A conclusive test of the nature of the translational motion in cells is missing owing to the lack of techniques capable of probing crowding with the required temporal and spatial resolution. Here we show that fluorescence-fluctuation analysis of raster scans at variable timescales can provide this information. By using green fluorescent proteins in cells, we measure protein motion at the unprecedented timescale of 1 μs, unveiling unobstructed Brownian motion from 25 to 100 nm, and partially suppressed diffusion above 100 nm. Furthermore, experiments on model systems attribute this effect to the presence of relatively immobile structures rather than to diffusing crowding agents. We discuss the implications of these results for intracellular processes.

Show MeSH
Related in: MedlinePlus