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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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Plasma membrane topological changes become long lived in the presence of dynasore. Dynasore was added to the bathing solution at a final concentration of 80 µM at least 10 min before the imaging. (A) Long-lived increases in P/S and P+2S are observed after fusion of a granule (release of NPY-Cer). Circles are centered over the last observed location of the granule. Bar, 1 µm. (B) Data from A is presented with the vertical dotted line indicating the frame before fusion. (C) Schematic interpretation of the event in A. In the presence of dynasore, a fused structure is formed with significant indentation and curvature that is connected to the plasma membrane.
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fig8: Plasma membrane topological changes become long lived in the presence of dynasore. Dynasore was added to the bathing solution at a final concentration of 80 µM at least 10 min before the imaging. (A) Long-lived increases in P/S and P+2S are observed after fusion of a granule (release of NPY-Cer). Circles are centered over the last observed location of the granule. Bar, 1 µm. (B) Data from A is presented with the vertical dotted line indicating the frame before fusion. (C) Schematic interpretation of the event in A. In the presence of dynasore, a fused structure is formed with significant indentation and curvature that is connected to the plasma membrane.

Mentions: The role of dynamin in the local topological changes after fusion was investigated by inhibiting the GTPase activities of dynamin with dynasore (Macia et al., 2006). Dynasore is a membrane-permeant inhibitor of dynamin 1 and dynamin 2, both of which are expressed in chromaffin cells. A brief preincubation with dynasore caused significant changes in the topological responses that follow exocytosis. An example of a typical response is shown in Fig. 8. P/S immediately increased after fusion then decreased somewhat but remained elevated for tens of seconds. Most remarkably, P+2S increased, a response that is rarely observed in control cells (Fig. S4).


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)

Plasma membrane topological changes become long lived in the presence of dynasore. Dynasore was added to the bathing solution at a final concentration of 80 µM at least 10 min before the imaging. (A) Long-lived increases in P/S and P+2S are observed after fusion of a granule (release of NPY-Cer). Circles are centered over the last observed location of the granule. Bar, 1 µm. (B) Data from A is presented with the vertical dotted line indicating the frame before fusion. (C) Schematic interpretation of the event in A. In the presence of dynasore, a fused structure is formed with significant indentation and curvature that is connected to the plasma membrane.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig8: Plasma membrane topological changes become long lived in the presence of dynasore. Dynasore was added to the bathing solution at a final concentration of 80 µM at least 10 min before the imaging. (A) Long-lived increases in P/S and P+2S are observed after fusion of a granule (release of NPY-Cer). Circles are centered over the last observed location of the granule. Bar, 1 µm. (B) Data from A is presented with the vertical dotted line indicating the frame before fusion. (C) Schematic interpretation of the event in A. In the presence of dynasore, a fused structure is formed with significant indentation and curvature that is connected to the plasma membrane.
Mentions: The role of dynamin in the local topological changes after fusion was investigated by inhibiting the GTPase activities of dynamin with dynasore (Macia et al., 2006). Dynasore is a membrane-permeant inhibitor of dynamin 1 and dynamin 2, both of which are expressed in chromaffin cells. A brief preincubation with dynasore caused significant changes in the topological responses that follow exocytosis. An example of a typical response is shown in Fig. 8. P/S immediately increased after fusion then decreased somewhat but remained elevated for tens of seconds. Most remarkably, P+2S increased, a response that is rarely observed in control cells (Fig. S4).

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