Kinetic measurements of fusion of phosphatidylserine-containing vesicles by electron microscopy and fluorometry

S. W. Hui*, S. Nir, T. P. Stewart, L. T. Boni, S. K. Huang

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

28 Scopus citations

Abstract

Large unilamellar vesicles (REV) containing phosphatidylserine and phosphatidylethanolamine at a ratio of 1:3 were induced to fuse by adding calcium (4 mM). The kinetics of fusion was monitored by fluorometry using terbium or dipicolinic acid-containing vesicles. The morphology and the states of vesicle aggregation and fusion were examined at approx. 2, 30, 60, 150 and 900 s after calcium addition, by rapid quenching and freeze-fracture electron microscopy. The size and the state of aggregation of vesicles are quantified from 4000 randomly selected vesicles. The aggregation and fusion kinetics as assayed by fluorescence volume mixing is very well simulated and predicted by the mass action model. The model essentially predicts the time course of the distribution of the aggregates and the increase in size of fused particles as measured by electron microscopy, although in some cases the predicted fusion rate exceeds that by morphometric measurement. No morphological features can be defined as fusion intermediates, although bead-like and rim-like materials may be attributed to the remnants of broken diaphragms between fusion partners.

Original languageEnglish
Pages (from-to)130-140
Number of pages11
JournalBiochimica et Biophysica Acta - Biomembranes
Volume941
Issue number2
DOIs
StatePublished - 22 Jun 1988

Keywords

  • Aggregation
  • Calcium ion
  • Fluorescence
  • Freeze-fracture
  • Fusion

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