Related Experiment Videos
Roles of microfilaments in exocytosis: a new hypothesis
1Department of Anatomy and Cell Biology, Faculty of Medicine, Kitasato University, Kanagawa, Japan.
Abstract:
We observed the dynamic changes in the localization of microfilaments during the exocytic secretion of rat parotid and submandibular gland acinar cells, and obtained results which led us to propose a new concept of microfilament function in exocytosis. With the electron microscopy, NBD-Phallacidin (NBD-PL) fluorescence technique and immunohistochemistry for myosin, microfilaments consisting of F-actin and myosin were localized mainly underneath the luminal plasma membrane. Microfilaments were not detectable around the secretory granules which were stored in the cytoplasm, but were clearly observed around them whose membranes were continuous with the luminal plasma membrane. When viewed with NBD-PL and myosin fluorescence, the area of fused granule membranes revealed bright fluorescence in association with the luminal border, so that the luminal membrane undergoing exocytosis appeared like a 'bunch of grapes'. When excess exocytosis was stimulated by isoproterenol (IPR), the number of individual 'grapes' increased dramatically, indicating that the secretory granules are surrounded by microfilaments after the fusion with the luminal membrane. Microfilaments thus continuously undercoat the luminal membrane during exocytosis although the exocytic process involves the dilation and subsequent reduction of the luminal membrane due to the addition and removal of secretory granule membranes. This reduction of the dilated luminal membrane following exocytosis was, however, inhibited when the microfilaments were disrupted by cytochalasin D. Following this treatment, the lumina was expanded extraordinarily and the secretory products remained in the enlarged lumina, showing that the release of secretory products is inhibited when the microfilament function is disturbed. These results indicate that 1) microfilaments are localized mainly underneath the luminal plasma membrane and act as an obstacle to exocytosis when cells are at the resting phase and 2) at the secretory phase microfilaments allow exocytosis by disorganizing their barrier system and then, by encircling the discharged secretory granule membranes, provide forces for the extrusion of secretory products through the action of the acto-myosin contractile system.
Insights
Microfilaments beneath the luminal membrane initially block exocytosis but later facilitate secretion by reorganizing and contracting, aiding product release from rat glands.
Area of Science:
- Cell Biology
- Exocytosis Mechanisms
- Cytoskeletal Dynamics
Background:
- Exocytic secretion is crucial for gland function.
- The role of microfilaments in exocytosis is not fully understood.
Purpose of the Study:
- To investigate the dynamic localization and function of microfilaments during exocytosis in rat parotid and submandibular gland acinar cells.
- To propose a new model for microfilament involvement in the exocytic process.
Main Methods:
- Electron microscopy
- NBD-Phallacidin (NBD-PL) fluorescence staining for F-actin
- Immunohistochemistry for myosin
- Stimulation of exocytosis with isoproterenol (IPR)
- Inhibition of microfilaments with cytochalasin D
Main Results:
- Microfilaments (F-actin and myosin) are primarily located beneath the luminal plasma membrane.
- Microfilaments surround secretory granules only after their fusion with the luminal membrane.
- Isoproterenol stimulation increases microfilament association with fused granule membranes, resembling a 'bunch of grapes'.
- Cytochalasin D treatment inhibits the reduction of the luminal membrane post-exocytosis, expanding the lumen and blocking product release.
Conclusions:
- Microfilaments act as a barrier to exocytosis in resting cells.
- During secretion, microfilaments reorganize, facilitating exocytosis and providing force for product extrusion via acto-myosin contraction.