Visualization of regulated exocytosis with a granule-membrane probe using total internal reflection microscopy

Area of Science:

• Cell Biology
• Biophysics

Background:

• Exocytosis is a fundamental cellular process for secretion.
• Understanding the dynamics of secretory granule fusion with the plasma membrane is crucial.

Purpose of the Study:

• To characterize the distinct visual signatures of secretory granule exocytosis.
• To quantify the dynamics of granule fusion and VAMP-GFP diffusion during exocytosis.

Main Methods:

• Total internal reflection fluorescence microscopy (TIR-FM) was used to visualize secretory granules labeled with Vamp-green fluorescent protein (GFP).
• Analysis of fluorescence intensity, shape changes (puncta, spreading cloud, ring-like structures), and diffusion of Vamp-GFP.

Main Results:

• Approximately 90% of fusion events showed increased fluorescence, spreading, and Vamp-GFP diffusion into the plasma membrane, indicating granule flattening.
• About 10% of events displayed ring-like structures, suggesting fusion with retained concavity.
• Exocytosis can occur rapidly, with some granules interacting with the plasma membrane for as little as 0.3 seconds.
• Evidence for sequential (piggy-back) exocytosis was observed at some sites.

Conclusions:

• Secretory granule exocytosis exhibits diverse morphological outcomes.
• Vamp-GFP diffusion dynamics provide insights into the mechanics of granule fusion and flattening.
• The rapid kinetics of granule-plasma membrane interaction highlight the efficiency of the exocytosis machinery.
• Observed exocytosis patterns are consistent with those in the constitutive secretory pathway.