Department of Cell Biology, University of Virginia, Charlottesville, Virginia 22908
Department of Cell Biology, University of Virginia, Charlottesville, Virginia 22908.
J Neurosci. 2022 Jun 1;42(22):4415-4434. doi: 10.1523/JNEUROSCI.2530-21.2022. Epub 2022 Apr 26.
In all cell types, endocytosed cargo is transported along a set of endosomal compartments, which are linked maturationally from early endosomes (EEs) via late endosomes (LEs) to lysosomes. Lysosomes are critical for degradation of proteins that enter through endocytic as well as autophagic pathways. Rab7 is the master regulator of early-to-late endosome maturation, motility, and fusion with lysosomes. We previously showed that most degradative lysosomes are localized in the soma and in the first 25 µm of the dendrite and that bulk degradation of dendritic membrane proteins occurs in/near the soma. Dendritic late endosomes therefore move retrogradely in a Rab7-dependent manner for fusion with somatic lysosomes. We now used cultured E18 rat hippocampal neurons of both sexes to determine which microtubule motor is responsible for degradative flux of late endosomes. Based on multiple approaches (inhibiting dynein/dynactin itself or inhibiting dynein recruitment to endosomes by expressing the C-terminus of the Rab7 effector, RILP), we now demonstrate that net retrograde flux of late endosomes in dendrites is supported by dynein. Inhibition of dynein also delays maturation of somatic endosomes, as evidenced by excessive accumulation of Rab7. In addition, degradation of dendritic cargos is inhibited. Our results also suggest that GDP-GTP cycling of Rab7 appears necessary not only for endosomal maturation but also for fusion with lysosomes subsequent to arrival in the soma. In conclusion, Rab7-dependent dynein/dynactin recruitment to dendritic endosomes plays multifaceted roles in dendritic endosome maturation as well as retrograde transport of late endosomes to sustain normal degradative flux. Lysosomes are critical for degradation of membrane and extracellular proteins that enter through endocytosis. Lysosomes are also the endpoint of autophagy and thus responsible for protein and organelle homeostasis. Endosomal-lysosomal dysfunction is linked to neurodegeneration and aging. We identify roles in dendrites for two proteins with links to human diseases, Rab7 and dynein. Our previous work identified a process that requires directional retrograde transport in dendrites, namely, efficient degradation of short-lived membrane proteins. Based on multiple approaches, we demonstrate that Rab7-dependent recruitment of dynein motors supports net retrograde transport to lysosomes and is needed for endosome maturation. Our data also suggest that GDP-GTP cycling of Rab7 is required for fusion with lysosomes and degradation, subsequent to arrival in the soma.
在所有细胞类型中，内吞的货物沿着一系列内体隔室运输，这些隔室从早期内体（EE）通过晚期内体（LE）成熟地连接到溶酶体。溶酶体对于通过内吞作用和自噬途径进入的蛋白质的降解至关重要。Rab7 是早期到晚期内体成熟、运动和与溶酶体融合的主调节因子。我们之前表明，大多数降解性溶酶体位于体部和树突的前 25 µm 处，并且树突膜蛋白的大量降解发生在体部附近/体内。因此，树突状晚期内体以 Rab7 依赖性方式逆行运动，与体部溶酶体融合。我们现在使用培养的 E18 大鼠海马神经元（雌雄两性）来确定哪种微管马达负责晚期内体的降解通量。基于多种方法（抑制动力蛋白/动力蛋白激活蛋白本身或通过表达 Rab7 效应物的 C 末端抑制动力蛋白向内体的募集），我们现在证明树突状晚期内体的净逆行通量由动力蛋白支持。动力蛋白的抑制也会延迟体部内体的成熟，这表现为 Rab7 的过度积累。此外，树突状货物的降解也受到抑制。我们的结果还表明，Rab7 的 GDP-GTP 循环不仅对于内体成熟，而且对于随后到达体部后的与溶酶体融合都是必要的。总之，Rab7 依赖性动力蛋白/动力蛋白激活蛋白向树突状内体的募集在树突状内体成熟以及晚期内体的逆行运输中发挥多方面的作用，以维持正常的降解通量。溶酶体对于通过内吞作用进入的膜和细胞外蛋白的降解至关重要。溶酶体也是自噬的终点，因此负责蛋白质和细胞器的动态平衡。内体-溶酶体功能障碍与神经退行性变和衰老有关。我们确定了与人类疾病相关的两种蛋白质（Rab7 和动力蛋白）在树突中的作用。我们之前的工作确定了一个需要在树突中进行定向逆行运输的过程，即短寿命膜蛋白的有效降解。基于多种方法，我们证明 Rab7 依赖性动力蛋白马达的募集支持向溶酶体的净逆行运输，并且是内体成熟所必需的。我们的数据还表明，Rab7 的 GDP-GTP 循环对于与溶酶体融合和随后在体部的降解是必需的。
