Characterization of LAMP1-labeled nondegradative lysosomal and endocytic compartments in neurons

Abstract

Despite widespread distribution of LAMP1 and the heterogeneous nature of LAMP1-labeled compartments, LAMP1 is routinely used as a lysosomal marker, and LAMP1-positive organelles are often referred to as lysosomes. In this study, we use immunoelectron microscopy and confocal imaging to provide quantitative analysis of LAMP1 distribution in various autophagic and endolysosomal organelles in neurons. Our study demonstrates that a significant portion of LAMP1-labeled organelles do not contain detectable lysosomal hydrolases including cathepsins D and B and glucocerebrosidase. A bovine serum albumin-gold pulse-chase assay followed by ultrastructural analysis suggests a heterogeneity of degradative capacity in LAMP1-labeled endolysosomal organelles. Gradient fractionation displays differential distribution patterns of LAMP1/2 and cathepsins D/B in neurons. We further reveal that LAMP1 intensity in familial amyotrophic lateral sclerosis-linked motor neurons does not necessarily reflect lysosomal deficits in vivo. Our study suggests that labeling a set of lysosomal hydrolases combined with various endolysosomal markers would be more accurate than simply relying on LAMP1/2 staining to assess neuronal lysosome distribution, trafficking, and functionality under physiological and pathological conditions.

F‐actin patches associated with glutamatergic synapses control positioning of dendritic lysosomes

“…Run length and instant velocity were not affected (Fig M and N). Overall, LAMP1‐labeled vesicles were slightly more mobile than LysoTracker‐stained organelles, which probably has to do with the broader spectrum of endolysosomal compartments labeled by LAMP1 (Cheng et al , ). Altogether, these data indicate a contribution of the dendritic F‐actin cytoskeleton in directing trafficking of lysosomes.…”

“…In a recent study, Cheng and colleagues performed a rigorous characterization of LAMP1‐positive compartments in dendrites. They found that about half of the LAMP1‐labeled structures contained functional lysosomal proteases cathepsins B and D, markers for mature lysosomes (Cheng et al , ). It has also been shown that neuronal activity induces lysosomal exocytosis and release of cathepsins (Padamsey et al , ).…”

Mammalian Atg8 proteins regulate lysosome and autolysosome biogenesis through SNARE s

“…Since STX16 knockout resulted in reduced cellular content of LAMP1/2, which is in lysosomal and additional acidified endosomal compartments (Lippincott-Schwartz & Fambrough, 1987) (Cheng et al, 2018), we anticipated that the cells might have diminished content of acidified organelles. We used LysoTracker Red DND-99 (LTR) as a standard cell biological probe for acidification of intracellular compartments (Wubbolts et al, 1996).…”

F-actin patches associated with glutamatergic synapses control positioning of dendritic lysosomes

“…Similarly, we found that LAMP1-eGFP vesicles became more mobile following actin depolymerization ( Figure 5H-K) and the directional net flux was no longer biased towards anterograde trafficking ( Figure 5L). Overall, LAMP1-labelled vesicles were slightly more mobile than LysoTracker stained organelles, which probably has to do with the broader spectrum of endolysosomal compartments labeled by LAMP1 (Cheng et al, 2018). Altogether, these data suggest a contribution of the dendritic F-actin cytoskeleton in directing trafficking of lysosomes.…”

“…To address the question if dendritic actin patches influence organelle trafficking, we chose to look at lysosomes as a representative vesicular organelle, because they show very active, processive trafficking along dendrites, and recently more and more studies are emerging that highlight their significance in neuronal function (Cheng et al, 2018;Goo et al, 2017;Padamsey et al, 2017). First, we used 3-color STED to visualize the spatial relationship between F-actin, MTs and lysosomes, using antibodies against the lysosomal marker protein LAMP1.…”