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.
","abstract_html":"<p>Zinc (Zn<sup>2+</sup>) is crucial for proper cellular function, and as such it is important to measure and track Zn<sup>2+</sup> dynamics in living cells. Fluorescent sensors have been used to estimate Zn<sup>2+</sup> 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 Zn<sup>2+</sup> can be released from intracellular compartments into the cytosol due to a lack of probes that can detect physiological dynamics of cytosolic Zn<sup>2+</sup>. My dissertation started with comparing and characterizing different Zn<sup>2+</sup> 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 Zn<sup>2+</sup> dynamics with sub-nanomolar sensitivity. Using small molecule sensors and GZnP3, we establish that TRPML1 and TRPML3 channels are permeable to physiological concentrations of Zn<sup>2+</sup>. Upon characterizing the location of these channels, we also provide the first direct evidence that TRPML channels can release Zn<sup>2+</sup> from intracellular compartments (including endolysosomal vesicles) to the cytosol in primary hippocampal neurons. The TRPML-mediated Zn<sup>2+</sup> signals are distinct from Ca<sup>2+</sup> in that they are significantly higher in neurites as compared to the soma, sustain longer, and are cell type specific.</p> <p>We then investigate the role of increased cytosolic Zn<sup>2+</sup> in neurons. Accurate cargo delivery over long distances through axonal transport requires precise spatiotemporal regulation in neurons. Here we discover that lysosomal Zn<sup>2+</sup> release through TRPML1 or Zn<sup>2+</sup> influx via depolarization, can inhibit bidirectional axonal transport. Such inhibition is neither selective for cargo nor for cell type because elevated Zn<sup>2+</sup> (IC50 ≈ 5 nM) reduces both lysosomal and mitochondrial motility in primary rat hippocampal neurons and HeLa cells. Zn<sup>2+</sup> inhibits movement of peroxisomes artificially tethered to constitutively-active kinesin motors. In addition, Zn<sup>2+</sup> 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 Zn<sup>2+</sup> release, disrupts Zn<sup>2+</sup>-mediated regulation of axonal transport, and increases overall mitochondrial motility. In addition, MLIV patient mutations in TRPML1 have decreased Zn<sup>2+</sup> permeability, which parallels disease severity. Our results reveal that Zn<sup>2+</sup> acts as a critical signal to locally pause axonal transport by directly blocking the progression of motor proteins on microtubules.</p>","abstract_has_math":false,"creators":["Minckley, Taylor Franklin"],"institution":null,"degree_name":"Ph.D.","degree_level":"Dissertation","degree_discipline":null,"degree_department":null,"school":null,"contributors":["Yan Qin","Scott A. Barbee","Michelle K. Knowles","Erich J. Kushner"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2022,"date_issued":"2022-01-01T08:00:00Z","date_published":"2022-01-01T08:00:00Z","updated_at":"2026-07-24T02:02:30Z","subjects":["Axonal transport","Lysosomes","Neurons","TRPML1","TRPML3","Zinc","Biochemistry, Biophysics, and Structural Biology","Cell and Developmental Biology","Cell Biology","Life Sciences","Molecular Biology","Neurology"],"languages":["en"],"rights":["Copyright is held by the author. User is responsible for all copyright compliance.
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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.
"]},{"key":"dc:format","label":"Dc Format","values":["application/pdf"]},{"key":"dc:title","label":"Title","values":["Organellar Zn2+ Homeostasis and the Role of TRPML Channels in Neuronal Lysosome Physiology and Axonal Transport"]}]}],"canonical_facts":{"dc:contributor":["Yan Qin","Scott A. Barbee","Michelle K. Knowles","Erich J. Kushner"],"dc:creator":["Minckley, Taylor Franklin"],"dc:date.available":["2023-07-21T07:00:00Z"],"dc:description.abstract":["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.
"],"dc:format":["application/pdf"],"dc:identifier":["https://digitalcommons.du.edu/etd/2068"],"dc:language":["en"],"dc:rights":["Copyright is held by the author. User is responsible for all copyright compliance.
"],"dc:subject":["Axonal transport","Lysosomes","Neurons","TRPML1","TRPML3","Zinc","Biochemistry, Biophysics, and Structural Biology","Cell and Developmental Biology","Cell Biology","Life Sciences","Molecular Biology","Neurology"],"dc:title":["Organellar Zn2+ Homeostasis and the Role of TRPML Channels in Neuronal Lysosome Physiology and Axonal Transport"],"thesis:degree_level":["Dissertation"],"thesis:degree_name":["Ph.D."]},"updated_at":"2026-07-24T02:02:30Z"}