The extent to which a donor membrane will be retrieved, or if it is retrieved at all after it fuses with an acceptor membrane, is usually difficult to determine. We have studied the dynamics of membrane retrieval in the phagosome system of Paramecium multimicronucleatum using six monoclonal antibody markers. Our previous freeze-fracture and transmission electron microscopic studies have indicated that extensive changes take place in the membrane of the young phagosome as it progresses through its cycle. Using immunofluorescence and immunoelectron microscopy to determine the times of entry and exit of these individual antigens into the digestive vacuole system, we showed that two hydrophilic antigens, one located on the cytosolic and one on the lumenal side of the discoidal membrane (phagosome membrane precursor), were completely retrieved from the phagosome by tubulation within the first three minutes. At the same time that this membrane was retrieved, membrane from a second population of vesicles, the acidosomes, fused with the phagosome to produce the phagoacidosome. On the basis of immunogold localization on cryosections of a total of six antigens, the two specific for phagosome/discoidal vesicle membrane as well as four specific for the acidosome/phagoacidosome membrane, this replacement is total. We also showed that in the presence of the actin-active drug cytochalasin B, this replacement was essentially prevented. However, when vacuole acidification was neutralized by ammonium chloride, this replacement process continued unaffected after a lag. Consequently, acidification, per se, is not required to trigger the replacement of the phagosome membrane. We conclude, on the basis of these studies as well as our previous freeze-fracture studies that during phagoacidosome formation most of the acceptor membrane is retrieved and is replaced by the donor membrane. This shows that at least one cell type possesses the mechanisms needed to substantially replace the membrane of a phagosomal compartment when radical and rapid changes are needed to modulate the digestive and absorptive processes.

REFERENCES

Allen
R.D.
(
1974
).
Food vacuole membrane growth with microtubule-associated membrane transport in Paramecium.
J. Cell Biol
63
,
904
922
Allen
R.D.
,
Fok
A.K.
(
1980
).
Membrane recycling and endocytosis in Paramecium confirmed by horseradish peroxidase pulse-chase studies.
J. Cell Sci
45
,
131
145
Allen
R.D.
,
Fok
A.K.
(
1983
).
Phagosome fusion vesicles of Paramecium. I. Thin-section morphology. Eur.
J. Cell Biol
29
,
150
158
Allen
R.D.
,
Fok
A.K.
(
1983
).
Phagosome fusion vesicles of Paramecium. II. Freeze-fracture evidence for membrane replacement.
Eur. J. Cell Biol
29
,
159
165
Allen
R.D.
,
Fok
A.K.
(
1984
).
Stages of digestive vacuoles in Paramecium: membrane surface differences and location.
Eur. J. Cell Biol
35
,
149
155
Allen
R.D.
,
Ma
Le
,
Fok
A.K.
(
1993
).
Acidosomes: recipients of multiple sources of membrane and cargo during development and maturation.
J. Cell Sci
106
,
411
422
Bordier
C.
(
1981
).
Phase separation of integral membrane proteins in Triton X-114 solution.
J. Biol. Chem
256
,
1604
1607
Fok
A.K.
,
Allen
R.D.
(
1979
).
Axenic Paramecium caudatum. I. Mass culture and structure.
J. Protozool
26
,
463
470
Fok
A.K.
,
Ueno
M.S.
,
Allen
R.D.
(
1986
).
Differentiation of Paramecium phagosome membrane and stages using monoclonal antibodies.
Eur. J. Cell Biol
40
,
1
8
Hammarback
J.A.
,
Vallee
R.B.
(
1990
).
Antibody exchange immunochemistry.
J. Biol. Chem
265
,
12763
12766
Hausmann
K.
,
Allen
R.D.
(
1976
).
Membrane behavior of exocytic vesicles: II. Fate of the trichocyst membranes in Paramecium after induced trichocyst discharge.
J. Cell Biol
69
,
313
326
Ishida
M.
,
Aihara
M.S.
,
Allen
R.D.
,
Fok
A.K.
(
1993
).
Osmoregulation in Paramecium: the locus of fluid segregation in the contractile vacuole complex.
J. Cell Sci
106
,
693
702
Kearney
J.F.
,
Radbruch
A.
,
Liesegang
B.
,
Rajewsky
K.
(
1979
).
A new mouse myeloma cell line that has lost immunoglobulin expression but permits the construction of antibody-secreting hybrid cell lines.
J. Immunol
123
,
1548
1550
Kohler
G.
,
Milstein
C.
(
1975
).
Continuous cultures of fused cells secreting antibody of predefined specificity.
Nature
256
,
495
497
Laemmli
E.K.
(
1970
).
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature
227
,
680
685
Towbin
H.
,
Staehelin
T.
,
Gordon
J.
(
1979
).
Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications.
Proc. Nat. Acad. Sci. USA
76
,
4350
4354
von Gersdorff
H.
,
Matthews
G.
(
1994
).
Dynamics of synaptic vesicle fusion and membrane retrieval in synaptic terminals.
Nature
367
,
735
–.
Zimmermann
H.
,
Volknandt
W.
,
Henkel
A.
,
Bonzelius
F.
,
Janetzko
A.
,
Kanaseki
T.
(
1989
).
The synaptic vesicle membrane: Origin, axonal distribution, protein components, exocytosis and recycling.
Cell Biol. Int. Rep
13
,
993
1006
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