Date of Award
College of Natural Science and Mathematics, Biological Sciences
Scott A. Barbee
Michelle K. Knowles
Erich J. Kushner
Axonal transport, Lysosomes, Neurons, TRPML1, TRPML3, Zinc
Zinc (Zn2+) is crucial for proper cellular function, and as such it is important to measure and track Zn2+ dynamics in living cells. Fluorescent sensors have been used to estimate Zn2+ content of subcellular compartments, but little is known about endolysosomal Zn2+ homeostasis. Similarly, although numerous sensors have been reported, it is unclear whether and how Zn2+ can be released from intracellular compartments into the cytosol due to a lack of probes that can detect physiological dynamics of cytosolic Zn2+. My dissertation started with comparing and characterizing different Zn2+ sensors including the genetically encoded GZnP sensors developed in the Qin Lab, the commercially available small molecule sensor FluoZin-3, and a small molecule sensor from our collaborators. My results demonstrated that GZnP3 is able to detect cytosolic Zn2+ dynamics with sub-nanomolar sensitivity. Using small molecule sensors and GZnP3, we establish that TRPML1 and TRPML3 channels are permeable to physiological concentrations of Zn2+. Upon characterizing the location of these channels, we also provide the first direct evidence that TRPML channels can release Zn2+ from intracellular compartments (including endolysosomal vesicles) to the cytosol in primary hippocampal neurons. The TRPML-mediated Zn2+ signals are distinct from Ca2+ in that they are significantly higher in neurites as compared to the soma, sustain longer, and are cell type specific.
We then investigate the role of increased cytosolic Zn2+ in neurons. Accurate cargo delivery over long distances through axonal transport requires precise spatiotemporal regulation in neurons. Here we discover that lysosomal Zn2+ release through TRPML1 or Zn2+ influx via depolarization, can inhibit bidirectional axonal transport. Such inhibition is neither selective for cargo nor for cell type because elevated Zn2+ (IC50 ≈ 5 nM) reduces both lysosomal and mitochondrial motility in primary rat hippocampal neurons and HeLa cells. Zn2+ inhibits movement of peroxisomes artificially tethered to constitutively-active kinesin motors. In addition, Zn2+ binds to microtubules and inhibits both kinesin and dynein activity in vitro. Loss of TRPML1 function, which causes Mucolipidosis Type IV (MLIV) disease, impairs lysosomal Zn2+ release, disrupts Zn2+-mediated regulation of axonal transport, and increases overall mitochondrial motility. In addition, MLIV patient mutations in TRPML1 have decreased Zn2+ permeability, which parallels disease severity. Our results reveal that Zn2+ acts as a critical signal to locally pause axonal transport by directly blocking the progression of motor proteins on microtubules.
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Taylor Franklin Minckley
Received from ProQuest
Minckley, Taylor Franklin, "Organellar Zn2+ Homeostasis and the Role of TRPML Channels in Neuronal Lysosome Physiology and Axonal Transport" (2022). Electronic Theses and Dissertations. 2068.
Molecular biology, Neurosciences, Cellular biology