EXOCYTOSIS OF SMALL AND LARGE VESICLES

Insulin exocytosis: A rise in blood glucose stimulates the secretion of insulin from b-cells, which increases glucose uptake and storage, and decreases glucose production.  The current view of glucose-stimulated insulin secretion is shown in Figure 1.  Increased glucose closes ATP-sensitive K⁺ (K_ATP) channels by increasing the intracellular ATP to ADP ratio.  Since these channels control the b-cell membrane potential, their closure depolarises the cell, thus opening voltage-dependent Ca²⁺ channels (VDCCs) which allow Ca²⁺ to flow into the cell.  The increased intracellular Ca²⁺ interacts with the machinery controlling exocytosis (SNARE proteins) to stimulate secretion of insulin containing large dense-core vecisles (LDCVs).  Repolarisation of the membrane, closure of VDCCs, and subsequent limitation of insulin secretion is mediated by the opening of voltage-dependent K⁺ (Kv) channels.  Recent studies show that both VDCCs¹ and Kv² channels bind the SNARE proteins in what is thought to be an important regulatory interaction.

Synaptic-like exocytosis: In addition to insulin, pancreatic b-cells secrete other molecules thought to be important regulators of islet function, including the inhibitory neurotransmitter g-aminobutyric acid (GABA) and ATP.  Of particular interest here, GABA is an important paracrine modulator of b-cells by inhibiting glucagon secretion³ and is secreted from b-cells through the exocytosis of small synaptic-like microvesicles (SLMVs)⁴.  In this way, pancreatic b-cells are similar to other neuro-secretory cells that also contain both SLMVs that contain small molecule neurotransmitters and LDCVs containing peptide hormones⁵.  SLMVs are similar to synaptic vesicles in that they contain small molecule transmitters and their contents appear clear in electron micrographs, however SLMVs are slightly larger (80-90 nm versus 40-50 nm diameter) and the mechanisms regulating their exocytosis are poorly understood.

Measuring exocytosis from single b-cells:  A number of methods have been established to measure exocytosis from single cells.  Standard whole-cell capacitance measurements have been used over the past 20 years to monitor the entire surface area of a cell, and have provided important information about exocytosis of LDCVs.  This method is performed when continuous electrical access is established between a patch-clamp pipette and the cell interior.  Entry of Ca²⁺ into the cell is either via the patch pipette (dialysis) or membrane Ca²⁺ channels (stimulated by depolarising the cell).  While this method provides a convenient measure of exocytosis, it suffers from the drawbacks of being unable to discern individual LDCVs or SLMVs, and by the possibility that endocytosis may reduce the observed effects.

Amperometric measurement of the release of oxidizable substrates has provided information about the exocytosis of single LDCVs, and the associated fusion pore that forms between vesicles and the cell membrane.  However, in b-cells it is generally necessary to pre-load the cells with serotonin which is more readily oxidized than insulin.  This method also does not provide information about the exocytosis of SLMVs or about endocytosis.

Fluorescent measures of exocytosis have also been developed, most notably systems in which fluorescent tagged proteins are over-expressed and monitored by total-internal reflection fluorescence (TIRF) or confocal microscopy.  This technique has allowed the visualization of single vesicle movements and, depending on the fluorescent markers used, vesicle acidification, fusion pore kinetics and ‘kiss-and-run’.  However this technique has been generally limited to LDCVs and requires the over-expression of proteins (often 2 or more different markers), which for studying primary tissues usually means constructing adenoviral transfection vectors.  As well, the expressed markers do not completely overlap with the vesicle population of interest (insulin vesicles for example).

While traditional membrane capacitance measurements monitor the increase in membrane surface area of the whole cell (in units of picofarad- pF), a modification of this technique will be used in the current proposal to measure the membrane capacitance of small membrane patches.⁶  This small membrane patch method can detect the fusion of vesicles as small as 60 aF (a vesicle diameter of approximately 45 nm), and can differentiate single LDCVs, SLMVs and endocytic vesicles.  Also, it is possible with this method to monitor the kinetics of fusion of a single vesicle and to calculate rates of exocytosis for the various groups of vesicles.  This technique can be used with metabolically intact cells, or with isolated membrane patches. 

General Aim: The general aim of this project is to examine mechanisms regulating exocytosis of single SLMVs and LDCVs in insulin secreting b-cells.  To accomplish this, cell-attached capacitance measurements will be performed on insulinoma cells and primary rodent b-cells.

Significance: Understanding pancreatic islet function under normal conditions is essential to the identification of defects that occur in type-2 diabetes and to developing effective treatments.  This study will provide important new information about the regulation of single vesicle exocytosis.  In particular, the cell-attached capacitance described here will allow the simultaneous study of, small synaptic-like microvesicles, large insulin containing vesicles and endocytosis for the first time.

 

      1.     D. Atlas, J.Neurochem. 77, 972-985 (2001).

      2.     P. E. MacDonald et al., Mol.Endocrinol. 16, 2452-2461 (2002).

      3.     A. Wendt et al., Diabetes In press, (2004).

      4.     M. Braun et al., J.Gen.Physiol In Press, (2004).

      5.     K. Langley and N. J. Grant, Neurochem.Int. 31, 739-757 (1997).

      6.     K. Lollike and M. Lindau, J.Immunol.Methods 232, 111-120 (1999).

 

 

 

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