Exocytosis, dependent on Ca2+ release from Ca2+ stores, is regulated by Ca2+ microdomains

Jiun T Low,Alka Shukla,Natasha Behrendorff,Peter Thorn

doi:10.1242/jcs.071225

Abstract

The relationship between the cellular Ca2+ signal and secretory vesicle fusion (exocytosis) is a key determinant of the regulation of the kinetics and magnitude of the secretory response. Here, we have investigated secretion in cells where the exocytic response is controlled by Ca2+ release from intracellular Ca2+ stores. Using live-cell two-photon microscopy that simultaneously records Ca2+signals and exocytic responses, we provide evidence that secretion is controlled by changes in Ca2+ concentration [Ca2+] in relatively large-volume microdomains. Our evidence includes: (1) long latencies (>2 seconds) between the rise in [Ca2+] and exocytosis, (2) observation of exocytosis all along the lumen and not clustered around Ca2+ release hot-spots, (3) high affinity (Kd=1.75 microM) Ca2+dependence of exocytosis, (4) significant reduction in exocytosis in the presence of cytosolic EGTA, (5) spatial exclusion of secretory granules from the cell membrane by the endoplasmic reticulum, and (6) inability of local Ca2+ responses to trigger exocytosis. These results strongly indicate that the control of exocytosis, triggered by Ca2+ release from stores, is through the regulation of cytosolic[Ca2+] within a microdomain.

Highlights

Ca2+-dependent exocytosis is an essential and widespread process (Sudhof, 2004)
To observe exocytic responses the tissue was bathed in sulforhodamine B (SRB), a fluorescent probe that surrounds the cells and diffuses into the lumens between the cells (Fig. 1A, lower images, colored red) (Nemoto et al, 2001; Thul and Falcke, 2004)
The average Fura-2 response in each cell is plotted in Fig. 1B and the time-course of the exocytic responses measured as normalized SRB changes – within regions of interest (ROIs) centered on each exocytic granule are shown in Fig. 1B

Summary

Introduction

Ca2+-dependent exocytosis is an essential and widespread process (Sudhof, 2004). An increase in cytosolic Ca2+ concentration ([Ca2+])triggers secretory vesicle fusion with the plasma membrane leading to the release of vesicle cargoes, such as neurotransmitters and proteins, for example, hormones and enzymes. Clusters of Ca2+ channels provide a localized ‘cloud’ of Ca2+, triggering exocytosis across microdomains (Beaumont et al, 2005; Borst and Sakmann, 1996; Chow et al, 1994). In the latter case, the secretory response is slower, but precise control of [Ca2+] within the microdomain is used to fine-tune the secretory output (Chow et al, 1994).

Methods

Results

Conclusion