This project is using a combination of methods to analyze the ion transport properties of lysosomal membranes with particular focus on their role in generating the acid luminal pH of the organelle. Lysosomes are intracellular organelles that serve in most cells as digestive organelles although in some tissues they are used for other functions. Lysosomes utilize an ATP-driven proton pump to maintain an acidic luminal pH and facilitate their digestive function. Such a pump can only be effective if accompanied by additional ion transport to dissipate the transmembrane voltage built up by the ATPase, a counterion pathway. In the past, we used isolated lysosomes to identify and characterize a Chloride permeability in the lysosomal membrane which has the features required of such a counterion pathway and demonstrated that the chloride is transported by ClC-7, a Cl-/H+ antiporter specifically targeted to the lysosomal membrane. We have recently optimized the methods to use dual-wavelength ratiometric fluorophores linked to dextran to specifically target lysosomes. pH is measured by processing images of the cells taken at the two wavelengths. We have worked out methods to accurately calibrate the system to correlate image measurements with actual pH values. We have now analyzed the effects of ClC-7 gene knockdown on cells where siRNA controls do not affect lysosome pH and used those systems to demonstrate that ClC-7 knockdown compromises acidification in at least some cell types, we are currently completeing controls required to finalize this work. We have also nearly completed an analysis of the ion requirements of acidification in isolated lysosomes which will be and used that system to demonstrate that ClC-7 knockout severely impairs acidification in liver lysosomes isolated from knockout mice. We are also beginning to explore the influences of other metabolic processes on the pH of lysosomes, since lysosomes have recently been shown to serve as important monitoring stations for cellular metabolic stress. In addition to our work on lysosomes, we have begun to explore the acidification process in other organelles. We have completed work on analysis of chloride dependent acidification in clathrin coated vesicles in brain which has been accepted for publication this year. Finally, we have been collaborating with the Gahl lab in the Genome institute to analyze a mutant form of ClC-7 that they have found in a patient in the Undiagnosed Diseases Program who has a disease pattern completely unlike those found with other ClC-7 mutations. We have made substantial progress in understanding the effects of this mutation over the past year, with careful analysis of the effects of the mutation on ClC-7 function, the effects of these changes on pH in lysosomes, and possible approaches to treating this disease. We are currently preparing this work for publication. We demonstrated that the mutaion causes patient lysosomes to become hyperacidic, and that change in organellar pH leads to a host of disruptions of cellular metabolism. We also showed that we could correct the cellular defects by treating cells with agents known to alkalinize lysosomes. These approches suggest possibilities for therapy of the disease. We are currently preparing a manuscript for publication on this novel disorder.
| Marcoline, Frank V; Ishida, Yoichi; Mindell, Joseph A et al. (2016) A mathematical model of osteoclast acidification during bone resorption. Bone 93:167-180 |
| Mindell, Joseph A (2014) Cell biology. A SWELL channel indeed. Science 344:585-6 |
| Ishida, Yoichi; Nayak, Smita; Mindell, Joseph A et al. (2013) A model of lysosomal pH regulation. J Gen Physiol 141:705-20 |
| Mindell, Joseph A (2012) Lysosomal acidification mechanisms. Annu Rev Physiol 74:69-86 |
| Mindell, Joseph A (2008) The chloride channel's appendix. Nat Struct Mol Biol 15:781-3 |
