Studies of intraorganelle dynamics: the lysosome, the pre-lysosomal compartment, and the golgi apparatus

Abstract

The lysosome, a multi-copy organelle, was chosen as an example to study intraorganelle dynamics. Lysosomal contents and membrane proteins were shown to intermix rapidly in fused mammalian cells, with a t_½ of ~30 min. Lysosomal content intermixing, shown by a sensitive invertase-lysosome/[¹⁴C]-sucrose-lysosome pairing assay, was inhibited greatly by ATP inhibitors and partially by cytochalasin D. Lysosomal membrane protein intermixing was shown by the transfer of LAMP-2, a mouse specific lysosomal membrane antigen, from mouse lysosomes to hamster sucrosomes, sucrose-swollen lysosomes. Lysosomal membrane protein intermixing was also shown by the co-localization of LIMP I, a rat specific lysosomal membrane antigen, and LAMP-1, a mouse specific lysosomal membrane antigen. Co-localization was assessed by both double immunofluorescent staining and double immunogold labeling of thin cryosections. Both lysosomal content and membrane protein intermixing were inhibited by nocodazole, a microtubule disruptor. In fused cells, lysosomes remained small, punctate and scattered throughout the cytoplasm. In comparison to lysosomes, the prelysosomal compartment (PLC), a single copy organelle which is related to the lysosome, congregated together to form an extended PLC complex associated with clustered nuclei.

The intermixing of both resident and transient Golgi membrane proteins was studied in fused cells. Resident Golgi membrane protein intermixing was slow, with a t_½ of ~ 1.75 h; it was concomitant with the congregation of the Golgi units. In comparison, the transient Golgi membrane protein was transported much faster from Golgi units to the other Golgi units, with the t_½ ≤ 15 min. Transient Golgi membrane protein transport occurred between separate Golgi units. These results are consistent with two different pathways for resident and transient Golgi membrane protein transport: a slow, lateral diffusion along the Golgi connections transport pathway for resident Golgi membrane proteins; and a rapid, transient protein selective, vesicle-mediated transport pathway for transient Golgi membrane proteins.