Super-resolution imaging reveals mechanisms of glutamate transporter localization near neuron-astrocyte contacts
| dc.contributor.author | Leek, Ashley N., author | |
| dc.contributor.author | Tamkun, Michael M., advisor | |
| dc.contributor.author | Hentges, Shane T., committee member | |
| dc.contributor.author | Tjalkens, Ronald B., committee member | |
| dc.contributor.author | Tsunoda, Susan, committee member | |
| dc.date.accessioned | 2021-09-06T10:26:01Z | |
| dc.date.available | 2022-09-03T10:26:01Z | |
| dc.date.issued | 2021 | |
| dc.description.abstract | Astrocytes contact neurons at several locations, including somatic clusters of Kv2.1 potassium channels and synapses across the brain. A primary function of astrocytes at these locations is to limit the action of extracellular glutamate. Astrocytic glutamate transporters, such as Glt1, ensure the fidelity of glutamic neurotransmission by spatially and temporally limiting glutamate signals. Additionally, they act to limit glutamate induced hyperexcitability by preventing the spread of glutamate to extrasynaptic receptors. The role of Glt1 in limiting neuronal hyperactivity relies heavily on the localization and diffusion of the transporter in the membrane, however, little is known about the mechanisms governing these properties. The work presented in this dissertation examines the mechanisms of Glt1 localization near Kv2.1-mediated neuron-astrocyte contact sites. To that end, in Chapter 2, we used super-resolution imaging to analyze the localization of two splice forms of Glt1, Glt1a and Glt1b. In cultures of primary astrocytes, we find that Glt1a, but not Glt1b, is specifically localized over cortical actin filaments. We go on to discover that this localization is dependent on the Glt1a C-terminus, where Glt1a and Glt1b differ, as exogenous expression of the Glt1a C-terminus was able to prevent localization of Glt1a to cortical actin filaments. In the somatosensory cortex, astrocyte Glt1 forms net-like structures around neuronal Kv2.1 clusters, however the cause of this Glt1 localization pattern is unknown. In Chapter 3, using super-resolution imaging of mixed cultures of astrocytes and neurons, we replicate findings of astrocyte Glt1 in a net-like localization around neuronal Kv2.1 clusters. We discover that both astrocyte actin and ER were excluded from the region across from neuronal Kv2.1 clusters. The actin-Glt1a relationship discussed in Chapter 2 is likely responsible for the net-like appearance of Glt1, as astrocytic Glt1 and actin colocalize in nets around Kv2.1 clusters at points of neuron-astrocyte contact. Neuronal control over the astrocyte cytoskeleton appears central to this Glt1a localization, although the mechanism of this control is still unknown. Together, these data describe a novel interaction between the Glt1a C-terminus and cortical actin filaments, which localizes Glt1 near neuronal structures involved in detecting ischemic insult. Although the mechanism of neuronal control over the astrocyte cytoskeleton remains a mystery, presumably cell-cell contact has a major influence. Contacts between neurons and astrocytes at Kv2.1 clusters could be mediated by the Kv2.1 β-subunit, AMIGO, which acts a cell adhesion molecule. Only one member of the AMIGO family of proteins is known to be an auxiliary β-subunit for Kv2 channels and to modulate Kv2.1 electrical activity. However, the AMIGO family has two additional members of ∼50% similarity that have not yet been characterized as Kv2 β-subunits. In Chapter 4, we show that the surface trafficking and localization of all three AMIGOs are controlled by their interaction with both Kv2.1 and Kv2.2 channels. Additionally, assembly of each AMIGO with either Kv2 alters important electrophysiological properties of these channels. The coregulatory effects of Kv2s and AMIGOs likely fine-tune both electrical and cell adhesion properties of the neurons in which they are expressed. Altogether, the work presented in this dissertation further defines the composition of Kv2.1-induced neuron-astrocyte contact sites, representing the first significant addition to this field in more than a decade. | |
| dc.format.medium | born digital | |
| dc.format.medium | doctoral dissertations | |
| dc.identifier | Leek_colostate_0053A_16661.pdf | |
| dc.identifier.uri | https://hdl.handle.net/10217/233802 | |
| dc.language | English | |
| dc.language.iso | eng | |
| dc.publisher | Colorado State University. Libraries | |
| dc.relation.ispartof | 2020- | |
| dc.rights | Copyright and other restrictions may apply. User is responsible for compliance with all applicable laws. For information about copyright law, please see https://libguides.colostate.edu/copyright. | |
| dc.subject | astrocytes | |
| dc.subject | Kv2.1 | |
| dc.subject | synapses | |
| dc.subject | cell-cell contact | |
| dc.subject | AMIGO | |
| dc.subject | neurons | |
| dc.title | Super-resolution imaging reveals mechanisms of glutamate transporter localization near neuron-astrocyte contacts | |
| dc.type | Text | |
| dcterms.embargo.expires | 2022-09-03 | |
| dcterms.embargo.terms | 2022-09-03 | |
| dcterms.rights.dpla | This Item is protected by copyright and/or related rights (https://rightsstatements.org/vocab/InC/1.0/). You are free to use this Item in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you need to obtain permission from the rights-holder(s). | |
| thesis.degree.discipline | Biomedical Sciences | |
| thesis.degree.grantor | Colorado State University | |
| thesis.degree.level | Doctoral | |
| thesis.degree.name | Doctor of Philosophy (Ph.D.) |
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