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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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Lateral displacement of localized topological change in plasma membrane and the last granule position before fusion. (A, left) A dashed circle is centered on the last observed position of a fusing granule. (right) A solid circle is centered on the first observed location of a P/S spot. Bar, 800 nm. (B) The lateral displacement from the center of mass of the fusing granule to the center of mass of the P/S puncta for 23 fusion events is plotted in a frequency histogram. The mean displacement observed was 122 ± 15 nm.
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fig9: Lateral displacement of localized topological change in plasma membrane and the last granule position before fusion. (A, left) A dashed circle is centered on the last observed position of a fusing granule. (right) A solid circle is centered on the first observed location of a P/S spot. Bar, 800 nm. (B) The lateral displacement from the center of mass of the fusing granule to the center of mass of the P/S puncta for 23 fusion events is plotted in a frequency histogram. The mean displacement observed was 122 ± 15 nm.

Mentions: The granule membrane becomes continuous with the plasma membrane upon exocytosis, allowing diffusion of the diI from the plasma into the granule membrane. A high K+ (56 mM) depolarizing solution was perfused locally on the cell of interest to stimulate exocytosis. A short-lived local change in orientation of the diI-labeled membrane occurs around the time of exocytosis (Fig. 5). Fig. 5 A (top) shows a series of NPY-Cer images acquired at the indicated times. Time 0 represents the frame sequence just before exocytosis (i.e., the last frame before disappearance of the NPY-Cer–labeled granule). The corresponding P/S and P+2S images of diI-labeled membrane are also shown (Fig. 5 B, bottom). The P/S ratio increased locally within 260 nm of the last location of the granule immediately after release of NPY-Cer (see Fig. 9 A). The mean P/S ratio in a 292-nm radius region of interest (ROI) centered on the P/S spot increased 20% in the frame after fusion (Fig. 5 B, dotted lines) and declined back to baseline within 40 s.


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)

Lateral displacement of localized topological change in plasma membrane and the last granule position before fusion. (A, left) A dashed circle is centered on the last observed position of a fusing granule. (right) A solid circle is centered on the first observed location of a P/S spot. Bar, 800 nm. (B) The lateral displacement from the center of mass of the fusing granule to the center of mass of the P/S puncta for 23 fusion events is plotted in a frequency histogram. The mean displacement observed was 122 ± 15 nm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig9: Lateral displacement of localized topological change in plasma membrane and the last granule position before fusion. (A, left) A dashed circle is centered on the last observed position of a fusing granule. (right) A solid circle is centered on the first observed location of a P/S spot. Bar, 800 nm. (B) The lateral displacement from the center of mass of the fusing granule to the center of mass of the P/S puncta for 23 fusion events is plotted in a frequency histogram. The mean displacement observed was 122 ± 15 nm.
Mentions: The granule membrane becomes continuous with the plasma membrane upon exocytosis, allowing diffusion of the diI from the plasma into the granule membrane. A high K+ (56 mM) depolarizing solution was perfused locally on the cell of interest to stimulate exocytosis. A short-lived local change in orientation of the diI-labeled membrane occurs around the time of exocytosis (Fig. 5). Fig. 5 A (top) shows a series of NPY-Cer images acquired at the indicated times. Time 0 represents the frame sequence just before exocytosis (i.e., the last frame before disappearance of the NPY-Cer–labeled granule). The corresponding P/S and P+2S images of diI-labeled membrane are also shown (Fig. 5 B, bottom). The P/S ratio increased locally within 260 nm of the last location of the granule immediately after release of NPY-Cer (see Fig. 9 A). The mean P/S ratio in a 292-nm radius region of interest (ROI) centered on the P/S spot increased 20% in the frame after fusion (Fig. 5 B, dotted lines) and declined back to baseline within 40 s.

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
Related in: MedlinePlus