Dissipation of lysosome pH impairs formation and collapses existing LPS-induced lysosome tubules in macrophages

Lysosomes are organelles responsible for degrading unwanted extracellular material that cells internalize, or intracellular material targeted for disposal. Typically, lysosomes are globular in morphology. However, in activated macrophages exposed to lipopolysaccharides, lysosomes undergo a dramatic reorganization to expand and become tubular in morphology. This tubulation phenomenon requires microtubules and dynein and kinesin motor activities regulated by lysosomal Rab7 and Arl8b GTPases. A key property of lysosomes is their acidic milieu established by the V-ATPase; the acidic gradient across the lysosomal membrane is ideal for hydrolytic activity, but this also powers other functions including membrane transport. Here, we sought to determine if V-ATPase and the acidic lysosomal pH were important for LPS-mediated lysosome tubulation in macrophages. We found that disruption of the V-ATPase prevented lysosome tubulation and collapsed preformed tubules. However, the V-ATPase also controls mTORC1 activity, which we previously showed promotes tubulation. In comparison, alkalization with NH4Cl did not interfere with mTORC1 activity, and yet, this also prevented lysosome tubulation. Moreover, clamping the pH to either acidic or alkaline values caused tubules to collapse, indicating that the gradient across the membrane is required for tubulation, rather than a specific pH value. Dissipation of lysosomal pH did not alter global microtubule organization, nor did it cause a collapse of lysosome movement, suggesting that motors remained associated with lysosomes. Interestingly, while LPS did not alter the global lysosomal pH nor the average pH of spherical and tubular lysosomes, we observed that growing tubules typically had a more acidic peripheral tip. We propose that localized gradients of acidic pH across the lysosomal membrane may directly or indirectly coordinate motor activity to enable tubulation.