Larbi, Karen Yvonne; (1997) Investigation of the machanism of exocytosis form the permeabilised guinea pig eosinophils. Doctoral thesis (Ph.D.), University College London (United Kingdom).

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Abstract

The regulatory pathways controlling exocytosis vary among cell types. The work described here concerns the regulation of exocytosis in eosinophils. In order to investigate the late steps regulating exocytosis, and to exclude complications arising from events mediated by cell surface receptors, it is helpful to work with permeabilised cells. The bacterial cytolysin, streptolysin-O generates large lesions in the plasma membranes of cells and so provides a means to access the interior. The cytosol can then be manipulated with precision allowing regulation of such diverse entities as divalent cations, nucleotides and even proteins which leak out, or which can be applied from the exterior, and their effect on exocytosis measured. When permeabilised with streptolysin-O, secretion of hexosaminidase from guinea pig eosinophils can be induced by Ca2+ and GTPγS both of which have been shown to be necessary. Extending this I have measured secretion of aryl sulphatase and peroxidase and in accompanying sub-cellular fractionations, I found that only the peroxidase is an exclusive component of the dense secretory (crystalloid) granules. If the application of the stimulus is delayed after permeabilisation, soluble proteins leak out and the secretory response runs down (over 30-50min). The rate at which the response runs down depends on the composition of the permeabilisation buffer. The inclusion of Ca2+ (10-5M), or soluble proteins derived from brain homogenates slows the rate of the rundown, and when both are applied together the run down is almost abolished. Using this technique as a basis of bio-assay, I have achieved a partial purification of two active factors from porcine brain homogenates. In addition, I have applied recombinant cytosolic proteins, in particular RhoGDI, to test their roles in the regulation of exocytosis.

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