Early onset effects of single substrate accumulation recapitulate major features of LSD in patient-derived lysosomes

Abstract

Summary Lysosome functions mainly rely on their ability to either degrade substrates or release them into the extracellular space. Lysosomal storage disorders (LSDs) are commonly characterized by a chronic lysosomal accumulation of different substrates, thereby causing lysosomal dysfunctions and secretion defects. However, the early effects of substrate accumulation on lysosomal homeostasis have not been analyzed so far. Here, we describe how the acute accumulation of a single substrate determines a rapid centripetal redistribution of the lysosomes, triggering their expansion and reducing their secretion, by limiting the motility of these organelles toward the plasma membrane. Moreover, we provide evidence that such defects could be explained by a trapping mechanism exerted by the extensive contacts between the enlarged lysosomes and the highly intertwined membrane structures of the endoplasmic reticulum which might represent a crucial biological cue ultimately leading to the clinically relevant secondary defects observed in the LSD experimental models and patients.

Lysosomal positioning diseases: beyond substrate storage

“…As such, the free diffusion of cargo molecules is restricted at stable MCSs, suggesting that this mechanism could play a role in cargo sorting. A recent work has shown that endolysosomes, whose size was expanded by the accumulation of intraluminal substrates, establish extended contact sites with the ER membrane through vesicle-associated membrane proteins VAP which strongly limit endolysosome motility and secretion [ 22 ], thereby confirming the need for transient interactions to drive endolysosomal motility, size control and function [ 87 ]. Accordingly, it has been recently demonstrated that also in neurons the ER establishes extended MCSs with enlarged and less motile lysosomes [ 36 ].…”

“…However, it has yet to be fully elucidated. Regardless, significant advances towards the understanding of lysosomal biology have provided novel and important insights into the type of cellular perturbations occurring in LSDs and their effects, thus paving the way for the development of new therapeutic approaches for the treatment of LSDs [21][22][23][24][25][26][27][28][29]. Currently, lysosomes are not merely considered terminal degradative organelles, but key metabolic hubs involved in nutrient sensing, secretion, gene regulation, plasma membrane (PM) repair, metal ion homeostasis, lipid transport and other fundamental cellular processes [30][31][32][33][34][35].…”

Protocol for labeling and fixation of intact lysosomes with esterified amino acid analogs to assess lysosomal expansion in living eukaryotic cells

“…However, the lysosomal compartment possesses a hydrolase that removes the methyl ester group causing the selective accumulation of the amino acid analog within the lysosomal lumen ( Reeves, 1979 ). As such, the aa-analog accumulation causes an osmotic effect, which ultimately triggers lysosome expansion via a membrane fusion-dependent process ( Desfougeres et al., 2016 ; Scerra et al., 2021 ). Consistent with this, we observed that after 2 h of amino acid analogs accumulation, lysosomes expand appearing like large and perfect spheres ( Figure 1 ).…”

“…We also present protocols to preserve lysosomal morphology, which yields a more accurate size measurement. For complete details on the use and execution of this protocol, please refer to Scerra et al. (2021) .…”

“…For complete details on the use and execution of this protocol, please refer to Scerra et al. (2021) .…”