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Rapid measurement of molecular transport and interaction inside living cells using single plane illumination.

Hedde PN, Stakic M, Gratton E - Sci Rep (2014)

Bottom Line: To demonstrate the advantages of our approach, we quantified the dynamics of several different proteins in the cyto- and nucleoplasm of living cells.For example, from a single measurement, we were able to determine the diffusion coefficient of free clathrin molecules as well as the transport velocity of clathrin-coated vesicles involved in endocytosis.Used in conjunction with dual view detection, we further show how protein-protein interactions can be quantified.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Fluorescence Dynamics, Department of Biomedical Engineering, University of California, Irvine, CA, USA.

ABSTRACT
The ability to measure biomolecular dynamics within cells and tissues is very important to understand fundamental physiological processes including cell adhesion, signalling, movement, division or metabolism. Usually, such information is obtained using particle tracking methods or single point fluctuation spectroscopy. We show that image mean square displacement analysis, applied to single plane illumination microscopy data, is a faster and more efficient way of unravelling rapid, three-dimensional molecular transport and interaction within living cells. From a stack of camera images recorded in seconds, the type of dynamics such as free diffusion, flow or binding can be identified and quantified without being limited by current camera frame rates. Also, light exposure levels are very low and the image mean square displacement method does not require calibration of the microscope point spread function. To demonstrate the advantages of our approach, we quantified the dynamics of several different proteins in the cyto- and nucleoplasm of living cells. For example, from a single measurement, we were able to determine the diffusion coefficient of free clathrin molecules as well as the transport velocity of clathrin-coated vesicles involved in endocytosis. Used in conjunction with dual view detection, we further show how protein-protein interactions can be quantified.

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Related in: MedlinePlus

cciMSD in live cells.(A) Fluorescence images of an EGFP-paxillin-mCherry expressing CHO-K1 cell (top, green channel; middle, red channel; bottom, overlay). A series of SPIM images was acquired alternatingly in both channels. (B) The resulting spatiotemporal autocorrelations in each channel (at 16 ms lag time) as well as the cross-correlation (at 8 ms lag time) are shown for the region marked by the box in panel (A). (C) Amplitudes returned from Gaussian fitting of each STICS series plotted over the lag time (squares, green channel; dots, red channel; triangles, cross-correlation). (D) Fluorescence images of an EGFP-paxillin and mCherry-paxillin expressing CHO-K1 cell. (E,F) The absence of any cross-correlation indicates no binding between both fusion proteins. Scale bars, 10 μm.
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f4: cciMSD in live cells.(A) Fluorescence images of an EGFP-paxillin-mCherry expressing CHO-K1 cell (top, green channel; middle, red channel; bottom, overlay). A series of SPIM images was acquired alternatingly in both channels. (B) The resulting spatiotemporal autocorrelations in each channel (at 16 ms lag time) as well as the cross-correlation (at 8 ms lag time) are shown for the region marked by the box in panel (A). (C) Amplitudes returned from Gaussian fitting of each STICS series plotted over the lag time (squares, green channel; dots, red channel; triangles, cross-correlation). (D) Fluorescence images of an EGFP-paxillin and mCherry-paxillin expressing CHO-K1 cell. (E,F) The absence of any cross-correlation indicates no binding between both fusion proteins. Scale bars, 10 μm.

Mentions: To demonstrate the capability of SPIM-cciMSD to measure molecular interactions in living cells, we transfected CHO-K1 cells with a plasmid encoding for an EGFP-paxillin-mCherry fusion protein (Fig. 4A). As expected, the cross-correlation shows concurrent movement of green and red marker proteins (Fig. 4B,C). As a control, CHO-K1 cells expressing both EGFP and mCherry were subjected to SPIM imaging. While the fluorescence images show perfect colocalization (Fig. 4D), the absence of any cross-correlation proves that there is no interaction between both proteins (Fig. 4E,F).


Rapid measurement of molecular transport and interaction inside living cells using single plane illumination.

Hedde PN, Stakic M, Gratton E - Sci Rep (2014)

cciMSD in live cells.(A) Fluorescence images of an EGFP-paxillin-mCherry expressing CHO-K1 cell (top, green channel; middle, red channel; bottom, overlay). A series of SPIM images was acquired alternatingly in both channels. (B) The resulting spatiotemporal autocorrelations in each channel (at 16 ms lag time) as well as the cross-correlation (at 8 ms lag time) are shown for the region marked by the box in panel (A). (C) Amplitudes returned from Gaussian fitting of each STICS series plotted over the lag time (squares, green channel; dots, red channel; triangles, cross-correlation). (D) Fluorescence images of an EGFP-paxillin and mCherry-paxillin expressing CHO-K1 cell. (E,F) The absence of any cross-correlation indicates no binding between both fusion proteins. Scale bars, 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4231332&req=5

f4: cciMSD in live cells.(A) Fluorescence images of an EGFP-paxillin-mCherry expressing CHO-K1 cell (top, green channel; middle, red channel; bottom, overlay). A series of SPIM images was acquired alternatingly in both channels. (B) The resulting spatiotemporal autocorrelations in each channel (at 16 ms lag time) as well as the cross-correlation (at 8 ms lag time) are shown for the region marked by the box in panel (A). (C) Amplitudes returned from Gaussian fitting of each STICS series plotted over the lag time (squares, green channel; dots, red channel; triangles, cross-correlation). (D) Fluorescence images of an EGFP-paxillin and mCherry-paxillin expressing CHO-K1 cell. (E,F) The absence of any cross-correlation indicates no binding between both fusion proteins. Scale bars, 10 μm.
Mentions: To demonstrate the capability of SPIM-cciMSD to measure molecular interactions in living cells, we transfected CHO-K1 cells with a plasmid encoding for an EGFP-paxillin-mCherry fusion protein (Fig. 4A). As expected, the cross-correlation shows concurrent movement of green and red marker proteins (Fig. 4B,C). As a control, CHO-K1 cells expressing both EGFP and mCherry were subjected to SPIM imaging. While the fluorescence images show perfect colocalization (Fig. 4D), the absence of any cross-correlation proves that there is no interaction between both proteins (Fig. 4E,F).

Bottom Line: To demonstrate the advantages of our approach, we quantified the dynamics of several different proteins in the cyto- and nucleoplasm of living cells.For example, from a single measurement, we were able to determine the diffusion coefficient of free clathrin molecules as well as the transport velocity of clathrin-coated vesicles involved in endocytosis.Used in conjunction with dual view detection, we further show how protein-protein interactions can be quantified.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Fluorescence Dynamics, Department of Biomedical Engineering, University of California, Irvine, CA, USA.

ABSTRACT
The ability to measure biomolecular dynamics within cells and tissues is very important to understand fundamental physiological processes including cell adhesion, signalling, movement, division or metabolism. Usually, such information is obtained using particle tracking methods or single point fluctuation spectroscopy. We show that image mean square displacement analysis, applied to single plane illumination microscopy data, is a faster and more efficient way of unravelling rapid, three-dimensional molecular transport and interaction within living cells. From a stack of camera images recorded in seconds, the type of dynamics such as free diffusion, flow or binding can be identified and quantified without being limited by current camera frame rates. Also, light exposure levels are very low and the image mean square displacement method does not require calibration of the microscope point spread function. To demonstrate the advantages of our approach, we quantified the dynamics of several different proteins in the cyto- and nucleoplasm of living cells. For example, from a single measurement, we were able to determine the diffusion coefficient of free clathrin molecules as well as the transport velocity of clathrin-coated vesicles involved in endocytosis. Used in conjunction with dual view detection, we further show how protein-protein interactions can be quantified.

Show MeSH
Related in: MedlinePlus