Allen R D, Bala N P, Ali R F, Nishida D M, Aihara M S, Ishida M, Fok A K
Pacific Biomedical Research Center, University of Hawaii, Honolulu 96822, USA.
J Cell Sci. 1995 Mar;108 ( Pt 3):1263-74. doi: 10.1242/jcs.108.3.1263.
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.
供体膜被回收的程度，或者它与受体膜融合后是否会被回收，通常很难确定。我们使用六种单克隆抗体标记物研究了多核草履虫吞噬体系统中膜回收的动力学。我们之前的冷冻蚀刻和透射电子显微镜研究表明，年轻吞噬体在其循环过程中，其膜会发生广泛变化。利用免疫荧光和免疫电子显微镜来确定这些单个抗原进入和离开消化液泡系统的时间，我们发现两种亲水性抗原，一种位于盘状膜（吞噬体膜前体）的胞质侧，另一种位于腔侧，在最初三分钟内通过微管形成从吞噬体中完全回收。在这种膜被回收的同时，来自第二群囊泡（酸性小体）的膜与吞噬体融合，形成吞噬酸性小体。基于对总共六种抗原的冷冻切片进行免疫金定位，两种针对吞噬体/盘状囊泡膜的特异性抗原以及四种针对酸性小体/吞噬酸性小体膜的特异性抗原，这种替换是完全的。我们还表明，在肌动蛋白活性药物细胞松弛素B存在的情况下，这种替换基本上被阻止。然而，当氯化铵中和液泡酸化时，这种替换过程在延迟后仍不受影响地继续进行。因此，酸化本身并非引发吞噬体膜替换所必需的。基于这些研究以及我们之前的冷冻蚀刻研究，我们得出结论，在吞噬酸性小体形成过程中，大部分受体膜被回收，并被供体膜所取代。这表明至少有一种细胞类型具备在需要剧烈且快速变化以调节消化和吸收过程时，大量替换吞噬体区室膜所需的机制。
