Loss of cell surface microvilli on rat basophilic leukaemia cells precedes secretion and can be mimicked using the calmodulin antagonist trifluoperazine

Abstract.

Objective: We studied changes in cell surface morphology following treatment with secretagogue or trifluoperazine in a mast cell model.¶Materials and Methods: Rat basophilic leukaemia (RBL) cells were treated with antigen and or the calmodulin antagonist, 0–50 μM trifluoperazine (TFP). The release of a secretory granule enzyme, β-hexosaminidase, into the external medium was used as a measure of secretion. Quantitation of cell surface microvilli was determined by using a computer with input from a digitising tablet from scanning electron micrographs. Cytoskeletal proteins present in microvilli were analysed by confocal immunofluorescence.¶Results: When RBL cells are stimulated to secrete with an antigen, the cell surface is transformed from a microvillous morphology to a ruffled one. The cell surface rearrangement preceded β-hexosaminidase secretion: the majority of microvilli disappeared rapidly after stimulation (t_1/2 of 39 s) whereas secretion can only be measured after a lag of 47 s. The calmodulin antagonist, TFP did not inhibit antigen-induced secretion or loss of microvilli, however TFP alone caused a similar loss of microvilli but was unable to stimulate or potentiate secretion. The microvilli mostly disappeared within 30 s, and a half-maximal effect occurred at approximately 8 μM TFP. Using immunofluorescence, calmodulin was localized to punctate structures on the dorsal cell surface which presumably correspond to the microvilli, and which also stained for F-actin and myosin I.¶Conclusions: Loss of cell surface microvilli on RBL cells precedes secretion and could reflect a cytoskeletal rearrangement which facilitates fusion of secretory granules with the membrane. It can be mimicked using trifluoperazine and we suggest it may involve calmodulin-binding components of the microvillus cytoskeleton such as myosin I.