Mechanisms of amine accumulation in, and egress from, lysosomes

Abstract

The human body is continuously exposed to small organic molecules containing one or more basic nitrogen atoms. Many of these are endogenous (i.e., neurotransmitters, polyamines and biogenic amines), while others are exogenously supplied in the form of drugs, foods and pollutants. It is well-known that many amines have a strong propensity to specifically and substantially accumulate in highly acidic intracellular compartments, such as lysosomes, through a mechanism referred to as ion trapping. It is also known that cells have acquired the unique ability to sense and respond to amine accumulation in lysosomes in an effort to prevent potential negative consequences associated with hyperaccumulation. We describe here methods that are used to evaluate the dynamics of amine accumulation in, and egress from, lysosomes. Moreover, we highlight specific proteins that are thought to play important roles in these pathways. A theoretical model describing lysosomal amine dynamics is described and shown to adequately fit experimental kinetic data. The implications of this research in understanding and treating disease are discussed.

Figure 1. pH partitioning mechanism of accumulation for weakly basic amine-containing compounds

There are three compartments separated by the presence of cellular membranes composed of a phospholipid bilayer for which the unionized weakly basic compound (B) is permeable and able to diffuse and therefore capable of attaining equivalent concentrations in the three compartments at steady state. By contrast, the ionized form of the compound (BH⁺) is unable to permeate through the lipid bilayer and becomes trapped within whichever compartment it resides. The relative concentration of B and BH⁺ in each compartment is dictated by the pH of the medium within that compartment as well as the pKa of the compound. The decrease in pH encountered within the lysosomal space compared with the cytosolic and extracellular spaces causes a shift in the ionic equilibrium favoring the membrane-impermeable ionized species, thus leading to an overall increase in compound accumulation within the lysosomal compartment.

Figure 2. Maintenance of the lysosomal pH gradient is required for accumulation of a weakly basic amine

Human fibroblasts were exposed to 100 nM of the weakly basic lysosomal probe LysoTracker™ Red with or without concomitant exposure to 100 nM of the vacuolar ATPase inhibitor concanamycin A. Inhibition of vacuolar ATPase is known to significantly elevate the intralysosomal pH and thus dissipate the pH gradient existing between the lysosomal and cytosolic lumens, thereby decreasing lysosomal accumulation of the probe.

Figure 3. Amine-induced vacuolization in human cells

Human fibroblasts treated with 70-μM neutral red or 100-μM chloroquine for 6 h show extensive vacuolization using light transmission microscopy or phase-contrast microscopy, respectively. Scale bars represent 10 μm.

Figure 4. Amine-induced vacuolization stems from late endosomal–lysosomal fusion

(A) Phase-contrast, (B) immunofluorescence and (C) merged images of normal fibroblasts after treatment with neutral red to induce vacuolization. Merged images confirm vacuoles consist of lysosomes (Lamp-1) and late endosomes (MPR), but not of Golgi (GM130) or early endosomes (EEA1). This is consistent with the hypothesis that amine-induced vacuoles represent enlarged hybrid organelles.

Figure 5. The size of hybrid organelles is decreased in cells with nonfunctional NPC1

Normal and NPC1^−/− cells were treated with 70-μM neutral red for 6 h. Phase-contrast images were acquired using confocal microscopy with an argon laser at 458 nm. Sizing was performed using ImageJ™ software. NPC1^−/− cells have impaired late endosome–lysosome fusion and, therefore, exhibit smaller hybrid organelles compared with normal fibroblasts.

Figure 6. Amines can influence lysosomal egress pathways

(A) Inhibitors of egress are, for the most part, amphiphilic molecules; possibly interacting with lysosomal membranes to inhibit retrograde trafficking. (B) Stimulators are capable of stimulating retrograde trafficking in a Niemann–Pick C1-dependent manner.

Figure 7. Hypothetical model depicting the NPC1-mediated trafficking of amines out of cells

Lysosomotropic amines enter into lysosomes via an ion-trapping mechanism. Certain amines can stimulate NPC1-mediated lysosome fusion with late endosomes to create expanded hybrid organelles. Hybrid organelles subsequently undergo fission, allowing them to condense their contents and reform dense lysosomal compartments. It is during this lysosome reformation that transport vesicles form containing lysosomal amines that are destined for release from the cell.

Figure 8. Kinetic model showing amine-trafficking pathways in cells

Rate constants are written so the first number in the parenthesis is the cargo destination compartment while the second number is the source compartment.

Figure 9. Radioactive dextran release is impaired in cells with nonfunctional NPC1

[³H]-dextran release was performed as described in ‘Experimental procedures’. Normal cells release approximately 80% of their dextran over 24 h, while cells with nonfunctional NPC1 release approximately 55%. The trend lines are the fits from SAAM modeling, the solid line represents normal dextran release, while the dotted line represents NPC1^−/− release.