Targeted delivery of lysosome-associated membrane proteins is important for lysosome stability and function. Here we identify a pathway for transport of lysosome-associated membrane proteins directly from the trans-Golgi network to late endosomes, which exists in parallel to mannose 6-phosphate receptor and clathrin-dependent transport of lysosomal enzymes to early endosomes. By immunoelectron microscopy we localized endogenous LAMP-1 and -2 as well as LAMP-1-mGFP to non-coated, biosynthetic carriers at the transGolgi network and near late endosomes. These LAMP carriers were negative for mannose 6-phosphate receptor, adaptor-protein complex-1, secretory albumin and endocytic markers, but contained the homotypic fusion and protein sorting complex component hVps41 and the soluble N-ethylmaleimide-sensitive factor attachment protein receptors protein VAMP7. Knockdown of hVps41 or VAMP7 resulted in the accumulation of lysosome-associated membrane protein carriers, whereas knockdown of hVps39 or hVps18 did not, indicating that the effect of hVps41 is independent of CORVET/HOPS. Mannose 6-phosphate receptor carriers remained unaffected upon hVps41 or VAMP7 knockdown, implicating that hVps41 and VAMP7 are specifically involved in the fusion of trans-Golgi network-derived lysosomeassociated membrane protein carriers with late endosomes.

The homotypic fusion and protein sorting (HOPS) complex is a multisubunit tethering complex that in yeast regulates membrane fusion events with the vacuole, the yeast lysosome. Mammalian homologs of all HOPS components have been found, but little is known about their function. Here, we studied the role of hVps41 and hVps39, two components of the putative human HOPS complex, in the endo-lysosomal pathway of human cells. By expressing hemagglutinin (HA)-tagged constructs, we show by immunoelectron microscopy (immunoEM) that both hVps41 and hVps39 associate with the limiting membrane of late endosomes as well as lysosomes. Small interference RNA (siRNA)-mediated knockdown of hVps41 or hVps39 resulted in an accumulation of late endosomes, a depletion in the number of lysosomes and a block in the degradation of endocytosed cargo. Lysosomal pH and cathepsin B activity remained unaltered in these conditions. By immunoEM we found that hVps41 or hVps39 knockdown impairs homotypic fusion between late endosomes as well as heterotypic fusion between late endosomes and lysosomes. Thus, our data show that both hVps41 and hVps39 are required for late endosomal-lysosomal fusion events and the delivery of endocytic cargo to lysosomes in human cells.

Live-cell correlative light-electron microscopy (live-cell-CLEM) integrates live movies with the corresponding electron microscopy (EM) image, but a major challenge is to relate the dynamic characteristics of single organelles to their 3-dimensional (3D) ultrastructure. Here, we introduce focused ion beam scanning electron microscopy (FIB-SEM) in a modular live-cell-CLEM pipeline for a single organelle CLEM. We transfected cells with lysosomal-associated membrane protein 1-green fluorescent protein (LAMP-1-GFP), analyzed the dynamics of individual GFP-positive spots, and correlated these to their corresponding fine-architecture and immediate cellular environment. By FIB-SEM we quantitatively assessed morphological characteristics, like number of intraluminal vesicles and contact sites with endoplasmic reticulum and mitochondria. Hence, we present a novel way to integrate multiple parameters of subcellular dynamics and architecture onto a single organelle, which is relevant to address biological questions related to membrane trafficking, organelle biogenesis and positioning. Furthermore, by using CLEM to select regions of interest, our method allows for targeted FIB-SEM, which significantly reduces time required for image acquisition and data processing.