Particle segregation in chromaffin granule membranes by forced physical contact.
Bovine chromaffin granules were exposed to different isotonic non-ionic and ionic solutions (sucrose; Ca2+- and Mg2+-free phosphate-buffered saline; Tris-HCl + NaCl; Ca2+- and Mg2+-free phosphate-buffered saline + sucrose; Tris-HCl + sucrose) at pH 7 and then frozen either in suspension or as firm pellets. Freezing was performed without prefixation or antifreeze treatments either by 'standard' techniques (approx. 1 mm3 suspended or pelleted material on gold specimen supports dipped into liquid Freon) or with increased cooling rates by spraying suspensions into liquid propane ('spray-freezing'). Regardless of the freezing method, membrane-intercalated particles were always randomly distributed when chromaffin granules were frozen in suspension. In contrast, forced physical contact between granules produced by centrifugation (12000 X g, 25 min) provoked dispersal of membrane-intercalated particles, but only in the presence of ions. Sucrose or EDTA in an ionic environment had no inhibitory effect. The following conclusions are derived: (1) Even below the reported phase transition region particle clustering is possible. (2) Chromaffin granule membranes are not liable to thermotropic segregation of membrane-intercalated particles. (3) Although the low freezing rates of 'standard' freezing techniques produce large-scale segregation artefacts (by which suspended chromaffin granules are pushed together within the segregated solute) this does not result in intramembraneous particle segregation. (4) Forced physical contact produces a Ca2+-independent particle segregation, but only when repulsive electrostatic forces of membrane components are partially screened in an ionic environment. (5) This does not invalidate results obtained by others, showing Ca2+-mediated chromaffin granules agglomeration and segregation of membrane-intercalated particles, but it might indicate the occurrence of another, not directly Ca2+-dependent particle segregation mechanism in a prefusional stage of close membrane-to-membrane contact during exocytosis.
D000818:Animals; D002118:Calcium; D002417:Cattle; D002498:Centrifugation; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002837:Chromaffin Granules / Q000502:physiology* / Q000648:ultrastructure; D002838:Chromaffin System / Q000502:physiology*; D005089:Exocytosis; D005614:Freeze Fracturing; D008560:Membrane Fluidity; D008722:Methods
D002118:Calcium