Exocytosis/endocytosis in Mast Cells

Exocytotic fusion occurs when a vesicle membrane fuses with the plasma membrane to form a fusion pore. Molecules (e.g. insulin, serotonin, proteases) stored in the vesicles are then “released” from the lumen of the vesicle into the extracellular mileau through this exocytotic fusion pore. Common key proteins found across the animal kingdom regulate fusion and release. These proteins are coined “the exocytotic machinery”. For example, they include proteins that target vesicles to the plasma membrane (e.g., muncs), bring the plasma and vesicle membranes into close apposition (SNARE proteins, VAMP, syntaxin and synaptobrevin), and bind calcium (e.g., synaptotagmin). Characterization of the exocytotic machinery is an active area of research. In mast cells this area of research is now being evaluated through isolation of the genes; identification of proteins; and subsequent production of mice with key proteins knocked-out. Secretory cells, including mast cells exhibit compound fusion; when the membranes of two vesicles fuse with each other, after which they either fuse with a third vesicle or fuse with the plasma membrane of the cell. The details of this process are not well understood.

Mast cells are secretory cells that participate in inflammation processes (e.g, asthma, antibody-induced rheumatoid arthritis) through exocytotic release of various pre-formed mediators (e.g., histamine, proteases, etc.) followed by the de novo synthesis of new products (e.g., platelet activating factor). The pre-formed mediators are stored within a proteoglycan gel contained within membrane bound granules. The discharge into the extracellular milleau involves both the release of soluble components and the “expansion” and “extrusion” of the gel from the vesicle. This process is coined “degranulation”. Of particular relevance is the storage and release of the active enzyme b -protease, because evidence suggests that this enzyme is responsible for much of the pathology associated with allergic and fibrotic diseases in humans. The proteases in humans and mice (e.g. mouse mast cell protease (mMCP)-4, mMCP-5 and carboxypeptidase A) are stored as active enzymes in mast cell granules in ionic complex with the heparin proteoglycan gel. Upon exocytosis the proteases are released with the gel stabilized by the ionic attachments with heparin. Understanding how and when the gel is extruded is relevant to disease. The mechanisms associated with exocytosis and endocytosis within these cells is less well understood (cf activation of the cells). Our goal is to describe and quantify mast cell ‘de-granulation’.

Experimental and theoretical evidence during transient or reversible fusion in mast cells suggest that there is a significant flux of membrane from the cell into the pore region. In our recent work, we estimate the tension of plasma membrane of a mast cell under passive conditions. We do this by monitoring the force acting on a membrane tether formed from plasma membrane of mast cell extracted from the peritoneum of mouse. From the measured force and radius of the tether we find that the tension is low our first estimates suggest it is about 3-6 m Nm -1.

Relevant Publications

Farrell, B. F. Qian, E. Merticoff, R. Adachi and B. Anvari. 2006 Membrane tethers formed from mast cells of the mouse with laser tweezers elucidate tension of plasma membrane”. Paper presented at Biophysical Society February 2006– Salt Lake City, Utah .

Farrell, B., and S.J. Cox. 2002. Estimating the time course of pore expansion during the spike phase of exocytotic release in mast cells of the beige mouse. Bulletin of Mathematical Biology 64: 979-1010.

Marszalek, P., B. Farrell, P. Verdugo and J.M. Fernandez. 1997. Kinetics of serotonin release from single secretory vesicles: (I) Amperometric detection of 5-hydroxytryptamine from electroporated granules. Biophysical Journal. 73:1160-1168.

Marszalek, P., B. Farrell, P. Verdugo and J.M. Fernandez. 1997. Kinetics of serotonin release from single secretory vesicles: (II) Ion exchange gel regulates the release of 5-hydroxytryptamine from electroporated granules. Biophysical Journal. 73:1169-1183.

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