Advancements in Nanobody Applications: From Live Synaptic Imaging to Multiplexed Fluorescence Microscopy and Nanoscale Topography Mapping

by Nikolaos Mougios
Doctoral thesis
Date of Examination:2024-05-13
Date of issue:2024-12-09
Advisor:Dr. Luis Felipe Opazo Dávila
Referee:Prof. Dr. Stefan Jakobs
Referee:Prof. Dr. Jörg Enderlein
Persistent Address: http://dx.doi.org/10.53846/goediss-10917

 

 

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Abstract

English

Fluorescence microscopy facilitates biological research by enabling straightforward visualization of fluorescently labeled proteins in live and fixed samples. Despite significant progress in microscopy techniques, including those enabling molecular-level resolution, challenges persist in the availability of small, minimally invasive affinity probes, such as nanobodies, and in the simultaneous detection of multiple targets. These challenges are particularly significant when studying small and complex subcellular compartments such as neuronal synapses. Therefore, new small molecular tools and labeling strategies compatible with modern fluorescence microscopy are essential to reveal target proteins. To address these limitations, in this work, I initially produced and characterized a novel nanobody against synaptotagmin 1 (NbSyt1) offering precise imaging of synapses without perturbing synaptic function. NbSyt1 also functions as an intrabody in living neurons, exhibiting minimal invasiveness and enabling the generation of protein-based fluorescent reporters for super- resolution imaging. Thereafter, to enable the detection of multiple targets within single samples, I developed NanoPlex, a versatile method for multiplexing based on repetitive imaging cycles facilitated by engineered nanobodies that bind selectively to primary antibodies (secondary nanobodies) and carry adaptable cleavable fluorescent groups. Finally, I utilized some of these technologies to investigate neuronal physiology and study the dynamics of trafficking proteins within living postsynaptic compartments, as well as to resolve the effect of liquid-liquid phase separation (LLPS) on the neuronal localization of several proteins in individual neurons.
Keywords: Single-domain antibodies; Cleavable probes; Multiplexing; FRAP; Super-resolution microscopy; Neuronal imaging; Neuroscience
 
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