Organellar Zn2+ Homeostasis and the Role of TRPML Channels in Neuronal Lysosome Physiology and Axonal Transport

<p>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.</p> <p>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.</p>

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Year dc:date.available

2022

Author dc:creator

• Minckley, Taylor Franklin

Contributors dc:contributor

• Yan Qin
• Scott A. Barbee
• Michelle K. Knowles
• Erich J. Kushner

• Axonal transport
• Lysosomes
• Neurons
• TRPML1
• TRPML3
• Zinc
• Biochemistry, Biophysics, and Structural Biology
• Cell and Developmental Biology
• Cell Biology
• Life Sciences
• Molecular Biology
• Neurology

Statement dc:rights

• <p>Copyright is held by the author. User is responsible for all copyright compliance.</p>

Language dc:language

en

Repository record dc:identifier

https://digitalcommons.du.edu/etd/2068

OAI identifier oai:identifier

oai:digitalcommons.du.edu:etd-3059