Visualizing synaptic orientation using super-resolution localiazation microscopy

AMPA receptors (AMPARs) mediate synaptic transmission. An open question in the field is what percentage of AMPARs are primed for release of synaptic vesicles and lie within the synaptic cleft? To answer this question, AMPAR, and markers for both presynaptic and postsynaptic markers need to be visualized at high resolution. This poses technical challenges as it requires transfection of neighboring neurons. Here, we explore using the synaptic adhesion molecule Neuroligin 1 to serve as an indicator for synaptic orientation of the presynapse to the postsynapse. The vector from the postsynaptic marker Homer1 to Neuroligin1 may be sufficient to determine the vector from pre to postsynapse. This scheme allows for transfection of only one neuron rather than the pair that make up the synapse. We utilized both new and old super resolution techniques to label synaptic markers and present preliminary data towards getting the synaptic orientation in a living cell using Neuroligin1. The introduction and methods are presented in chapters 1 and 2. Chapter 3 demonstrates why PAINT using peptides rather than DNA oligos proves to be more promising. It also reveals how a single spectral channel can be shared and used to image multiple proteins. We present technical challenges on imaging only the surface rather than the total Neurolgin1 pool. In Chapters 4 and 5, we explore two side projects undertaken during the course of my thesis. Chapter 4 is a single-particle trafficking (SPT) study done on AMPAR receptors. AMPARs indirectly bind to PSD-95. PSD-95 has many binding partners, one of which is Pin1, an isomerase protein found to interact with phosphorylated PSD-95. We explored AMPAR mobility under the overexpression and knockdown of Pin. Using SPT on AMPARs, we found Pin1 overexpression to decrease the number, but not the strength of functional synapses. Chapter 5 delves into the construction of a custom-built super-resolution microscope and software. We showcase various applications of the microscope’s flexibility in both instrumentation (when applied to IR quantum dots) and software (when applied to adaptive optics).

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Physics

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2022

Author dc:creator

• Nall, Duncan

Contributors dc:contributor

• Selvin, Paul R
• Kim, Sangjin
• Saif, Tahir A
• Chemla, Yann R

• Ampar
• Storm
• Palm
• Super Resolution
• Neuroligin
• Pin1
• Quantum Dots
• Paint
• Spines

Statement dc:rights

• Copyright 2022 Duncan Nall

Language dc:language

en, eng

Handle dc:identifier

https://hdl.handle.net/2142/117661

OAI record oai:identifier

https://www.ideals.illinois.edu/oai-pmh?verb=GetRecord&metadataPrefix=etdms&identifier=oai%3Awww.ideals.illinois.edu%3A2142%2F117661