Optimal lysosome function requires maintenance of an acidic pH maintained by proton pumps in combination with a counterion transporter such as the Cl À /H þ exchanger, CLCN7 (ClC-7), encoded by CLCN7. The role of ClC-7 in maintaining lysosomal pH has been controversial. In this paper, we performed clinical and genetic evaluations of two children of different ethnicities. Both children had delayed myelination and development, organomegaly, and hypopigmentation, but neither had osteopetrosis. Whole-exome and-genome sequencing revealed a de novo c.2144A>G variant in CLCN7 in both affected children. This p.Tyr715Cys variant, located in the C-terminal domain of ClC-7, resulted in increased outward currents when it was heterologously expressed in Xenopus oocytes. Fibroblasts from probands displayed a lysosomal pH approximately 0.2 units lower than that of control cells, and treatment with chloroquine normalized the pH. Primary fibroblasts from both probands also exhibited markedly enlarged intracellular vacuoles; this finding was recapitulated by the overexpression of human p.Tyr715Cys CLCN7 in control fibroblasts, reflecting the dominant, gain-of-function nature of the variant. A mouse harboring the knock-in Clcn7 variant exhibited hypopigmentation, hepatomegaly resulting from abnormal storage, and enlarged vacuoles in cultured fibroblasts. Our results show that p.Tyr715Cys is a gain-of-function CLCN7 variant associated with developmental delay, organomegaly, and hypopigmentation resulting from lysosomal hyperacidity, abnormal storage, and enlarged intracellular vacuoles. Our data supports the hypothesis that the ClC-7 antiporter plays a critical role in maintaining lysosomal pH.

The acidic luminal pH of lysosomes, maintained within a narrow range, is essential for proper degrative function of the organelle and is generated by the action of a V-type H+ ATPase, but other pathways for ion movement are required to dissipate the voltage generated by this process. ClC-7, a Cl-/H+ antiporter responsible for lysosomal Cl- permeability, is a candidate to contribute to the acidification process as part of this 'counterion pathway'. The signaling lipid PI(3,5)P2 modulates lysosomal dynamics, including by regulating lysosomal ion channels, raising the possibility that it could contribute to lysosomal pH regulation. Here we demonstrate that depleting PI(3,5)P2 by inhibiting the PIKfyve kinase causes lysosomal hyperacidification, primarily via an effect on ClC-7. We further show that PI(3,5)P2 directly inhibits ClC-7 transport and that this inhibition is eliminated in a disease-causing gain-of-function ClC-7 mutation. Together these observations suggest an intimate role for ClC-7 in lysosomal pH regulation.

The acidic luminal pH of lysosomes, maintained within a narrow range, is essential for proper degrative function of the organelle and is generated by the action of a V-type H+ ATPase, but other pathways for ion movement are required to dissipate the voltage generated by this process. ClC-7, a Cl-/H+ antiporter responsible for lysosomal Cl- permeability, is a candidate to contribute to the acidification process as part of this “counterion pathway”. The signaling lipid PI(3,5)P2 modulates lysosomal dynamics, including by regulating lysosomal ion channels, raising the possibility that it could contribute to lysosomal pH regulation. Here we demonstrate that depleting PI(3,5)P2 by inhibiting the PIKfyve kinase causes lysosomal hyperacidification, primarily via an effect on ClC-7. We further show that PI(3,5)P2 directly inhibits ClC-7 transport and that this inhibition is eliminated in a disease-causing gain-of-function ClC-7 mutation. Together these observations suggest an intimate role for ClC-7 in lysosomal pH regulation.

The lysosome is the terminal organelle in the endocytic pathway; its role is to degrade and recycle macromolecules. To function properly it must maintain an acidic pH. A V-type ATPase is necessary to drive protons into lysosomes, but the action of this pump generates a large voltage across the membrane, which limits further pumping. Fluxes of ions are therefore necessary to dissipate the electrochemical gradient, referred as the ''counterion pathway.'' It remains poorly understood which ions contribute to this pathway and facilitate net acidification of the lysosome. Our goal is to investigate the mechanism underlying the acidification of lysosomes and to evaluate the role of ClC-7, a lysosomal H þ /Cl-exchanger in the acidification process. We have probed the effects of external ion concentrations on acidification in isolated lysosomes using fluorescent ion-or voltage-sensing probes to measure internal pH or changes in membrane voltage.Using isolated lysosomes we demonstrate that the presence of external anions facilitates lysosomal acidification, suggesting a role in the counterion pathway. The anion specificity is similar to the anion selectivity observed for members of the ClC family (Cl->Br->I->NO3-). In contrast, the presence of cytoplasmic like concentrations of external K þ doesn't facilitate acidification; rather, at these levels (100mM), K þ ions induce alkalinization, which is enhanced in the presence of Valinomycin. Using the DisC3(5) membrane potential dye we monitored lysosomal membrane potential while varying external [K þ ]; we estimate the relative K þ permeability with and without Valinomycin. Finally, we used the null point titration approach to estimate the luminal concentration of ions, obtaining values in a range similar to those found using pH changes. This work helps constrain models of lysosomal acidification and suggests a limited role for K þ in the process.

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Diffusion of macromolecules and higher-order structures in the crowded interior of cells frequently shows an anomalous behavior with the mean-square displacement (MSD) increasing nonlinearly in time, MSDft a . Here we have probed to which extent also larger organelle structures show such an anomalous diffusion and how non-equilibrium contributions affect their diffusional motion [Phys. Rev. E 98, 012406 (2018), in press: Biophys. J. 115 (2018]. In particular, we have employed single-particle tracking to monitor the motion of tubular junctions in the endoplasmic reticulum (ER) network and of long-lived membrane domains on the ER, called ER exit sites (ERES). Our results show that both, ER junctions and ERES show a distinct anomalous diffusion with a significant anti-correlation of successive step increments that is reminiscent of fractional Brownian motion. Disrupting the microtubule cytoskeleton significantly altered the subdiffusive characteristics of both entities, highlighting that even anomalous diffusion is an actively driven process in living cells. While the diffusion behavior of ER junctions was seen to be directly dependent on the presence and activity of microtubules, ERES were only indirectly affected. ERES therefore can be seen as mobile membrane domains that perform a quasi-one-dimensional random walk on the shivering ER backbone. Within a few hours after infection of a cell by vesicular stomatitis virus (VSV), newly assembled VSV particles are released from the surface of the infected cell. In that time, the viral RNA and 5 viral proteins have travelled to the edge of the cell from the sites of synthesis near the nucleus, a radial distance of 5-10 mm. The modes of transport from the sites of synthesis to the edge of the cell are key questions. The movement of VSV ribonucleoprotein particles (nucleocapsids) in live A549 cells was recorded by fluorescence video microscopy at 100fps at 3 to 4h postinfection. Each nucleocapsid contains approximately 400 molecules of GFP-tagged P protein. Nucleocapsids are tracked to subpixel precision. About 60% of the tracks show spatially constrained, ATP-enhanced Brownian motion. These particles jiggle within an approximately circular area (''trap'') with 2s radii = 0.18 5 0.05 mm. Particles stay in one trap for 1-5 s, then move abruptly to an adjacent trap. Motion within a trap is not directional. 1-3% of tracks show short bouts (< 1s) of directed motion between traps. The velocity during directed motion is similar in magnitude to the frame-to-frame velocity in the traps. Bayesian analyses based on 2D location, particle velocity, and directional change are being used to identify the traps and the periods of directed motion. Simulations, biochemical perturbation (motor protein inhibitors) and colocalization of capsids with cytoskeletal fibers are being used to discern the physical basis for the observed modes of travel.