Development of Nanobodies to Image Synaptic Proteins in Super-Resolution Microscopy
von Manuel Maidorn
Datum der mündl. Prüfung:2017-11-15
Erschienen:2018-10-17
Betreuer:Dr. Luis Felipe Opazo Dávila
Gutachter:Prof. Dr. Silvio O. Rizzoli
Gutachter:Prof. Dr. Peter Rehling
Gutachter:Prof. Dr. Mikael Simons
Dateien
Name:PhD-Thesis_MMaidorn_publish_final.pdf
Size:6.85Mb
Format:PDF
Zusammenfassung
Englisch
In the recent decades, super resolution microscopy substantially contributed to the investigation of subcellular structures. A prominent example is the molecular organization of the synapse which is known to be vital for neuronal signal transmission. Synapses contain a plethora of different proteins which interact in fine-tuned mechanisms on molecular level to provide synaptic function. The observation of those proteins at nanoscale resolution thus provides important information on synaptic mechanisms. To identify different synaptic proteins they need to be marked with a label, which can be used for detection in microscopy. Commonly, antibodies are used in immunofluorescence microscopy to specifically detect target antigens. However in recent studies, antibodies have been shown to exhibit several drawbacks in super-resolution imaging, primarily imposed by their comparable large size and divalent nature. In contrast, small probes such as nanobodies gain increasing interest in the field of molecular imaging as they bypass several drawbacks of antibodies. Nanobodies are single-domain binders derived from an antibody subtype devoid of the light chain. They show high thermodynamic stability and can be produced in scalable amounts using bacterial expression systems. In this project, I selected and characterized two novel nanobodies binding with high affinity and specificity two neuronal SNARE proteins, SNAP 25 and syntaxin 1A. The nanobodies were subsequently used in immunofluorescence microscopy to investigate the organization of these proteins in super resolution. I found that the obtained fluorescence image using nanobodies significantly differs from the stainings observed if primary and secondary antibodies are used. Furthermore, the obtained nanobodies show several advantages over conventional antibodies including increased tissue penetration, detection of more epitopes and the absence of artificial clustering effects. Taking together the findings, I conclude that nanobodies are versatile tools for super-resolution microscopy to study small but complex biological structures such as synapses. The small size and biochemical properties of nanobodies direct the fluorophore in direct proximity to the antigen. This eventually also increases the attainable resolution and thus to the amount of detail observed in microscopy.
Keywords: nanobody; alpaca; microscopy; phage display; sdAb; STED; super-resolution; VHH; SNAP25; syntaxin1A