title: 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.
  
  
  METHODS
  Rat basophilic leukaemia (RBL) cells were treated with antigen and or the calmodulin antagonist, 0-50 microM trifluoperazine (TFP). The release of a secretory granule enzyme, beta-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 beta-hexosaminidase secretion: the majority of microvilli disappeared rapidly after stimulation (t1/2 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 microM 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.
authors.affiliation: Department of Anatomy and Cell Biology, Imperial College School of Medicine at St. Mary's, Imperial College of Science, Technology and Medicine, London, UK. a.edgar@ic.ac.uk
authors.forename: A J
authors.initials: AJ
authors.lastname: Edgar
authors.forename: G R
authors.initials: GR
authors.lastname: Davies
authors.forename: M A
authors.initials: MA
authors.lastname: Anwar
authors.forename: J P
authors.initials: JP
authors.lastname: Bennett
mesh_terms: D000818:Animals; D002147:Calmodulin / Q000037:antagonists & inhibitors*; D003594:Cytoplasmic Granules / Q000201:enzymology; D005455:Fluorescent Antibody Technique; D007700:Kinetics; D015471:Leukemia, Basophilic, Acute / Q000378:metabolism* / Q000473:pathology*; D008855:Microscopy, Electron, Scanning; D008871:Microvilli / Q000648:ultrastructure*; D051381:Rats; D014268:Trifluoperazine / Q000494:pharmacology*; D014407:Tumor Cells, Cultured; D001619:beta-N-Acetylhexosaminidases / Q000378:metabolism
chemical_list: D002147:Calmodulin; D014268:Trifluoperazine; D001619:beta-N-Acetylhexosaminidases