Réz G, Csák J, Fellinger E, László L, Kovács A L, Oliva O, Kovács J
Department of General Zoology, Eötvös University, Budapest, Hungary.
Eur J Cell Biol. 1996 Dec;71(4):341-50.
The time course of the vinblastine(-sulfate; 10 mg/kg body weight, single injection)-induced enlargement and subsequent regression of the autolysosomal compartment was studied by electron microscopic morphometrical and cell biochemical methods in order to gain information concerning some key problems of this major route of intralysosomal degradation of the cell's endogenous macromolecules and structures. Detailed analysis of the dynamics of the total autophagic vacuole (AV) compartment and its different subcompartments (early, advanced, late, and fused AVs), as well as of changes of rough-surfaced endoplasmic reticulum (RER) showed: 1. Pancreatic acinar cells react to vinblastine biphasically, i.e. two expansion phases of the AV compartment, the first in the 0 to 90 min and the second in the 2 to 8 h post-injectional periods, were detected. 2. Fusions of AVs are not inhibited by vinblastine, at least during the second expansion phase when cytoplasmic volume fraction (CVF) of fused AVs steadily increased until the 12th h. Fusion of early, advanced and late AVs or composition of fused complex vacuoles (AVc) are somehow regulated, as the proportion of the three AV stages from the CVF of AVc, was maintained constant throughout the second expansion phase. 3. Stimulation of autophagosome formation and resulting substrate overload seems to be the primary mode of action by which vinblastine causes the enormous expansion of the autolysosomal compartment. 4. Degranulation of the rough-surfaced endoplasmic reticulum (RER) membranes occurs in a biphasic fashion, similarly to the volume and surface changes of the AV compartment, thus supporting our previous hypothesis, that labilization or change of RER may have a role in the formation of autophagosomes. 5. Vinblastine-induced autophagocytosis is a selective process, as mitochondria, Golgi elements and zymogen granules are very much underrepresented, whereas RER is more than twice overrepresented in the volume of early AVs, when compared to their volume fraction in the whole cytoplasm. 6. Immunogold electron microscopy revealed the presence of ubiquitinylated proteins in advanced and late, but not in early AVs.
采用电子显微镜形态计量学和细胞生化方法，研究了长春碱（硫酸盐；10mg/kg体重，单次注射）诱导的自溶酶体区室增大及随后的消退过程，以获取有关细胞内源性大分子和结构的这种主要溶酶体内降解途径的一些关键问题的信息。对总自噬泡（AV）区室及其不同亚区室（早期、进展期、晚期和融合AV）的动态变化以及糙面内质网（RER）的变化进行详细分析，结果表明：1.胰腺腺泡细胞对长春碱的反应呈双相性，即检测到AV区室有两个扩张期，第一个在注射后0至90分钟，第二个在注射后2至8小时。2.长春碱不会抑制AV的融合，至少在第二个扩张期不会，此时融合AV的细胞质体积分数（CVF）持续增加直至12小时。早期、进展期和晚期AV的融合或融合复合泡（AVc）的组成受到某种调节，因为在第二个扩张期，AVc的CVF中这三个AV阶段的比例保持恒定。3.刺激自噬体形成并导致底物过载似乎是长春碱引起自溶酶体区室大量扩张的主要作用方式。4.糙面内质网（RER）膜的脱颗粒以双相方式发生，类似于AV区室的体积和表面积变化，从而支持了我们之前的假设，即RER的不稳定或变化可能在自噬体形成中起作用。5.长春碱诱导的自噬是一个选择性过程，因为线粒体、高尔基体元件和酶原颗粒的含量非常低，而与它们在整个细胞质中的体积分数相比，RER在早期AV的体积中含量超过两倍。6.免疫金电子显微镜显示，在进展期和晚期AV中存在泛素化蛋白，但在早期AV中不存在。
