Real-Time Investigation of Plasma Membrane Deformation and Fusion Pore Expansion Using Polarized Total Internal Reflection Fluorescence Microscopy

The cytomatrix at the active zone (CAZ) has been implicated in defining the site of Ca2+-dependent exocytosis of neurotransmitters. Here, we demonstrate the expression and function of ELKS, a protein structurally related to the CAZ protein CAST, in insulin exocytosis. The results of confocal and immunoelectron microscopic analysis showed that ELKS is present in pancreatic β cells and is localized close to insulin granules docked on the plasma membrane-facing blood vessels. Total internal reflection fluorescence microscopy imaging in insulin-producing clonal cells revealed that the ELKS clusters are less dense and unevenly distributed than syntaxin 1 clusters, which are enriched in the plasma membrane. Most of the ELKS clusters were on the docking sites of insulin granules that were colocalized with syntaxin 1 clusters. Total internal reflection fluorescence images of single-granule motion showed that the fusion events of insulin granules mostly occurred on the ELKS cluster, where repeated fusion was sometimes observed. When the Bassoon-binding region of ELKS was introduced into the cells, the docking and fusion of insulin granules were markedly reduced. Moreover, attenuation of ELKS expression by small interfering RNA reduced the glucose-evoked insulin release. These data suggest that the CAZ-related protein ELKS functions in insulin exocytosis from pancreatic β cells.

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Imaging Constitutive Exocytosis with Total Internal Reflection Fluorescence Microscopy

The Journal of Cell Biology ◽

10.1083/jcb.149.1.23 ◽

2000 ◽

Vol 149 (1) ◽

pp. 23-32 ◽

Cited By ~ 126

Author(s):

Jan Schmoranzer ◽

Mark Goulian ◽

Dan Axelrod ◽

Sanford M. Simon

Keyword(s):

Plasma Membrane ◽

Membrane Protein ◽

Fluorescence Microscopy ◽

Total Internal Reflection ◽

Fluorescent Protein ◽

Internal Reflection ◽

Total Internal Reflection Fluorescence ◽

Green Fluorescent ◽

Kinetics Of ◽

Flattening Process

Total internal reflection fluorescence microscopy has been applied to image the final stage of constitutive exocytosis, which is the fusion of single post-Golgi carriers with the plasma membrane. The use of a membrane protein tagged with green fluorescent protein allowed the kinetics of fusion to be followed with a time resolution of 30 frames/s. Quantitative analysis allowed carriers undergoing fusion to be easily distinguished from carriers moving perpendicularly to the plasma membrane. The flattening of the carriers into the plasma membrane is seen as a simultaneous rise in the total, peak, and width of the fluorescence intensity. The duration of this flattening process depends on the size of the carriers, distinguishing small spherical from large tubular carriers. The spread of the membrane protein into the plasma membrane upon fusion is diffusive. Mapping many fusion sites of a single cell reveals that there are no preferred sites for constitutive exocytosis in this system.

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Role of Microtubules in Fusion of Post-Golgi Vesicles to the Plasma Membrane

Molecular Biology of the Cell ◽

10.1091/mbc.e02-08-0500 ◽

2003 ◽

Vol 14 (4) ◽

pp. 1558-1569 ◽

Cited By ~ 47

Author(s):

Jan Schmoranzer ◽

Sanford M. Simon

Keyword(s):

Plasma Membrane ◽

Fluorescence Microscopy ◽

Actin Filaments ◽

Total Internal Reflection ◽

Internal Reflection ◽

Total Internal Reflection Fluorescence ◽

Cell Periphery ◽

Golgi Vesicles ◽

Cytoskeletal Elements

Biosynthetic cargo is transported away from the Golgi in vesicles via microtubules. In the cell periphery the vesicles are believed to engage actin and then dock to fusion sites at the plasma membrane. Using dual-color total internal reflection fluorescence microscopy, we observed that microtubules extended within 100 nm of the plasma membrane and post-Golgi vesicles remained on microtubules up to the plasma membrane, even as fusion to the plasma membrane initiated. Disruption of microtubules eliminated the tubular shapes of the vesicles and altered the fusion events: vesicles required multiple fusions to deliver all of their membrane cargo to the plasma membrane. In contrast, the effects of disrupting actin on fusion behavior were subtle. We conclude that microtubules, rather than actin filaments, are the cytoskeletal elements on which post-Golgi vesicles are transported until they fuse to the plasma membrane.

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