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Localized topological changes of the plasma membrane upon exocytosis visualized by polarized TIRFM.

Anantharam A, Onoa B, Edwards RH, Holz RW, Axelrod D - J. Cell Biol. (2010)

Bottom Line: In this study, we report the implementation of a TIRFM-based polarization technique to detect rapid submicrometer changes in plasma membrane topology as a result of exocytosis.Experiments on diI-stained bovine adrenal chromaffin cells using polarized TIRFM demonstrate rapid and varied submicrometer changes in plasma membrane topology at sites of exocytosis that occur immediately upon fusion.We provide direct evidence for a persistent curvature in the exocytotic region that is altered by inhibition of dynamin guanosine triphosphatase activity and is temporally distinct from endocytosis measured by VMAT2-pHluorin.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmacology, University of Michigan, Ann Arbor, MI 48109, USA. arunanan@umich.edu

ABSTRACT
Total internal reflection fluorescence microscopy (TIRFM) images the plasma membrane-cytosol interface and has allowed insights into the behavior of individual secretory granules before and during exocytosis. Much less is known about the dynamics of the other partner in exocytosis, the plasma membrane. In this study, we report the implementation of a TIRFM-based polarization technique to detect rapid submicrometer changes in plasma membrane topology as a result of exocytosis. A theoretical analysis of the technique is presented together with image simulations of predicted topologies of the postfusion granule membrane-plasma membrane complex. Experiments on diI-stained bovine adrenal chromaffin cells using polarized TIRFM demonstrate rapid and varied submicrometer changes in plasma membrane topology at sites of exocytosis that occur immediately upon fusion. We provide direct evidence for a persistent curvature in the exocytotic region that is altered by inhibition of dynamin guanosine triphosphatase activity and is temporally distinct from endocytosis measured by VMAT2-pHluorin.

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Simulated Images. Based on Eq. 6 and a custom IDL program, the expected intensity patterns P’ and S’ for p-pol and s-pol excitation, respectively, are shown for a diI-labeled spherical granule fusing with (and truncated by) a diI-labeled planar plasma membrane. In this particular simulation, most of the sphere is still intact; only the lower one fourth of its radius is truncated off. The schematic line drawing (white) shows a side view of this configuration at the same scale as the simulated images. The effects of finite evanescent depth, optical resolution limit, and nonparallelism of the diI dipole with the membrane are all included in generating these P’ and S’ patterns, thereby simulating what appears at the CCD array image plane. The pixelation of the CCD array is superimposed along with an outline showing which pixels are actually used to integrate the total intensities P and S. The ratio P’/S’ and the sum P’+2S’ are also shown. The corresponding pixel by pixel ratios and sums on experimental data (as pixelated by the camera) are used to determine lateral positions of diI/membrane morphology features at the time of exocytosis. However, extended temporal tracking of the p-pol and s-pol ratios and sums uses the spatially integrated values P and S (without the primes) before forming the P/S and P+2S combinations. The predictions of the simulations are sensitive to the assumed parameters, which are set close to the actual or expected experimental values: granule radius = 150 nm; Airy disk half-width (out to first minimum) = 211 nm; evanescent field depth = 110 nm; side length of CCD array pixel (as projected onto the image) = 73 nm; angle β between membrane normal and diI dipole = 69°. The P’ and S’ images are shown with the same grayscale; the P’/S’ and P’+2S’ each have their own gray scales.
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fig3: Simulated Images. Based on Eq. 6 and a custom IDL program, the expected intensity patterns P’ and S’ for p-pol and s-pol excitation, respectively, are shown for a diI-labeled spherical granule fusing with (and truncated by) a diI-labeled planar plasma membrane. In this particular simulation, most of the sphere is still intact; only the lower one fourth of its radius is truncated off. The schematic line drawing (white) shows a side view of this configuration at the same scale as the simulated images. The effects of finite evanescent depth, optical resolution limit, and nonparallelism of the diI dipole with the membrane are all included in generating these P’ and S’ patterns, thereby simulating what appears at the CCD array image plane. The pixelation of the CCD array is superimposed along with an outline showing which pixels are actually used to integrate the total intensities P and S. The ratio P’/S’ and the sum P’+2S’ are also shown. The corresponding pixel by pixel ratios and sums on experimental data (as pixelated by the camera) are used to determine lateral positions of diI/membrane morphology features at the time of exocytosis. However, extended temporal tracking of the p-pol and s-pol ratios and sums uses the spatially integrated values P and S (without the primes) before forming the P/S and P+2S combinations. The predictions of the simulations are sensitive to the assumed parameters, which are set close to the actual or expected experimental values: granule radius = 150 nm; Airy disk half-width (out to first minimum) = 211 nm; evanescent field depth = 110 nm; side length of CCD array pixel (as projected onto the image) = 73 nm; angle β between membrane normal and diI dipole = 69°. The P’ and S’ images are shown with the same grayscale; the P’/S’ and P’+2S’ each have their own gray scales.

Mentions: This idealized calculation must be altered to include numerous real effects: finite evanescent field depth, exocytotic structures containing both truncated spherical and planar regions, granule size on the edge of optical resolvability, and pixelation. These effects, and a general approach to handling them, are considered in detail in Materials and methods. Based on those considerations, computer-simulated images (Fig. 3) and expected integrated intensities for P and S (Fig. 4) can be produced.


Localized topological changes of the plasma membrane upon exocytosis visualized by polarized TIRFM.

Anantharam A, Onoa B, Edwards RH, Holz RW, Axelrod D - J. Cell Biol. (2010)

Simulated Images. Based on Eq. 6 and a custom IDL program, the expected intensity patterns P’ and S’ for p-pol and s-pol excitation, respectively, are shown for a diI-labeled spherical granule fusing with (and truncated by) a diI-labeled planar plasma membrane. In this particular simulation, most of the sphere is still intact; only the lower one fourth of its radius is truncated off. The schematic line drawing (white) shows a side view of this configuration at the same scale as the simulated images. The effects of finite evanescent depth, optical resolution limit, and nonparallelism of the diI dipole with the membrane are all included in generating these P’ and S’ patterns, thereby simulating what appears at the CCD array image plane. The pixelation of the CCD array is superimposed along with an outline showing which pixels are actually used to integrate the total intensities P and S. The ratio P’/S’ and the sum P’+2S’ are also shown. The corresponding pixel by pixel ratios and sums on experimental data (as pixelated by the camera) are used to determine lateral positions of diI/membrane morphology features at the time of exocytosis. However, extended temporal tracking of the p-pol and s-pol ratios and sums uses the spatially integrated values P and S (without the primes) before forming the P/S and P+2S combinations. The predictions of the simulations are sensitive to the assumed parameters, which are set close to the actual or expected experimental values: granule radius = 150 nm; Airy disk half-width (out to first minimum) = 211 nm; evanescent field depth = 110 nm; side length of CCD array pixel (as projected onto the image) = 73 nm; angle β between membrane normal and diI dipole = 69°. The P’ and S’ images are shown with the same grayscale; the P’/S’ and P’+2S’ each have their own gray scales.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2819686&req=5

fig3: Simulated Images. Based on Eq. 6 and a custom IDL program, the expected intensity patterns P’ and S’ for p-pol and s-pol excitation, respectively, are shown for a diI-labeled spherical granule fusing with (and truncated by) a diI-labeled planar plasma membrane. In this particular simulation, most of the sphere is still intact; only the lower one fourth of its radius is truncated off. The schematic line drawing (white) shows a side view of this configuration at the same scale as the simulated images. The effects of finite evanescent depth, optical resolution limit, and nonparallelism of the diI dipole with the membrane are all included in generating these P’ and S’ patterns, thereby simulating what appears at the CCD array image plane. The pixelation of the CCD array is superimposed along with an outline showing which pixels are actually used to integrate the total intensities P and S. The ratio P’/S’ and the sum P’+2S’ are also shown. The corresponding pixel by pixel ratios and sums on experimental data (as pixelated by the camera) are used to determine lateral positions of diI/membrane morphology features at the time of exocytosis. However, extended temporal tracking of the p-pol and s-pol ratios and sums uses the spatially integrated values P and S (without the primes) before forming the P/S and P+2S combinations. The predictions of the simulations are sensitive to the assumed parameters, which are set close to the actual or expected experimental values: granule radius = 150 nm; Airy disk half-width (out to first minimum) = 211 nm; evanescent field depth = 110 nm; side length of CCD array pixel (as projected onto the image) = 73 nm; angle β between membrane normal and diI dipole = 69°. The P’ and S’ images are shown with the same grayscale; the P’/S’ and P’+2S’ each have their own gray scales.
Mentions: This idealized calculation must be altered to include numerous real effects: finite evanescent field depth, exocytotic structures containing both truncated spherical and planar regions, granule size on the edge of optical resolvability, and pixelation. These effects, and a general approach to handling them, are considered in detail in Materials and methods. Based on those considerations, computer-simulated images (Fig. 3) and expected integrated intensities for P and S (Fig. 4) can be produced.

Bottom Line: In this study, we report the implementation of a TIRFM-based polarization technique to detect rapid submicrometer changes in plasma membrane topology as a result of exocytosis.Experiments on diI-stained bovine adrenal chromaffin cells using polarized TIRFM demonstrate rapid and varied submicrometer changes in plasma membrane topology at sites of exocytosis that occur immediately upon fusion.We provide direct evidence for a persistent curvature in the exocytotic region that is altered by inhibition of dynamin guanosine triphosphatase activity and is temporally distinct from endocytosis measured by VMAT2-pHluorin.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmacology, University of Michigan, Ann Arbor, MI 48109, USA. arunanan@umich.edu

ABSTRACT
Total internal reflection fluorescence microscopy (TIRFM) images the plasma membrane-cytosol interface and has allowed insights into the behavior of individual secretory granules before and during exocytosis. Much less is known about the dynamics of the other partner in exocytosis, the plasma membrane. In this study, we report the implementation of a TIRFM-based polarization technique to detect rapid submicrometer changes in plasma membrane topology as a result of exocytosis. A theoretical analysis of the technique is presented together with image simulations of predicted topologies of the postfusion granule membrane-plasma membrane complex. Experiments on diI-stained bovine adrenal chromaffin cells using polarized TIRFM demonstrate rapid and varied submicrometer changes in plasma membrane topology at sites of exocytosis that occur immediately upon fusion. We provide direct evidence for a persistent curvature in the exocytotic region that is altered by inhibition of dynamin guanosine triphosphatase activity and is temporally distinct from endocytosis measured by VMAT2-pHluorin.

Show MeSH