Date of Award

2022

Document Type

Dissertation

Degree Name

Ph.D.

Organizational Unit

College of Natural Science and Mathematics, Biological Sciences

First Advisor

Yan Qin

Second Advisor

Scott A. Barbee

Third Advisor

Michelle K. Knowles

Fourth Advisor

Erich J. Kushner

Keywords

Axonal transport, Lysosomes, Neurons, TRPML1, TRPML3, Zinc

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.

Publication Statement

Copyright is held by the author. User is responsible for all copyright compliance.

Rights Holder

Taylor Franklin Minckley

Provenance

Received from ProQuest

File Format

application/pdf

Language

en

File Size

220 pgs

Discipline

Molecular biology, Neurosciences, Cellular biology

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