Research Interests
The mechanisms of exocytosis and endocytosis currently represent one of the
most exciting topics in cell biology. The process of regulated exocytosis is
responsible for release of neurotransmitters and neuropeptides by nerve
terminals and endocrine cells, release of enzymes or cytotoxic proteins by
granulocytes, release of histamine and other mediators by mast cells, as
well as several other secretory processes. During exocytosis the membrane of
secretory granules fuses with the plasma membrane of the cell, allowing the
secretory granules to release their contents through the fusion pore.
Although biochemical studies revealed a set of proteins that are somehow
involved, the mechanisms of fusion are still obscure. Functional studies of
the fusion processes have revealed details of the dynamics of the fusion
events and the combination of functional and biochemical techniques will be
central to further elucidate the mechanism of exocytosis.
The highest resolution to investigate exocytotic fusion is obtained in
measurements of membrane capacitance using the patch clamp technique, since
exocytosis and endocytosis are associated with changes in plasma membrane
area leading to proportional changes of membrane capacitance. In addition,
release of oxidizable substances from single vesicles can be studied by
amperometry using a carbon fiber electrode. With these methods we can record
the opening of single fusion pores having molecular dimensions and the
dynamics of transmitter release during pore opening. We are developing and
improving these and other methods to resolve single exocytotic events.
The final aim of these experiments is the reduction of the exocytotic
systems such that eventually isolated granules will be fused with plasma
membrane patches and fusion among granules will be studied in vitro. The
molecular reconstitution of fusion between vesicles and plasma membrane
patches with properties similar to those of natural exocytotic events will
provide a clear picture of the molecular machinery analogous to the previous
demonstration that isolated protein complexes form functional ion channels
when reconstituted into lipid membranes.
Current Research Projects
- Characterization of Single Exocytotic Events in Cell: Attached Patches Using
Capacitance Measurements and Amperometry
- Reconstitution of Exocytosis in Excised Membrane Patches
- Direct Visualization of the Degranulation Process in White Blood Cells UsingModern Microscopic Imaging Methods
- Characterization of Docking and Fusion between Secretory Granules and
between Specific Components of the Exocytotic Machinery Using Optical
Tweezers