Antibody technology identifies distinct mechanisms controlling the lengths of filopodia-like structures

Filopodia are finger-like cytoplasmic protrusions that contain filamentous actin and extend beyond the leading edge of migrating cells. Filopodia are thought to facilitate both sensing of signaling molecules and the movement of cells towards them. Profuse filopodia formation is seen in metastatic cancer cells and in diseases causing intellectual disability. Conversely, in the central nervous system, growth cone filopodial architecture fails to reform after injury, limiting healing. Despite the major impact that filopodia formation has on embryonic development and pathology, our understanding of the molecular mechanisms underlying filopodia formation remain incomplete and controversial. Using a collection of antibodies isolated by phage display antibody phenotypic screening on an *in vitro* system of filopodia-like structures (FLS) I aimed to discover new molecular mechanisms underlying filopodia formation. To identify the antigens of the phage display antibodies I combined Western blotting, immunoprecipitation and immunostaining approaches with different proteomic techniques and transient expression. The antibody panel was strikingly split into two groups: (1) Antibodies that lengthen FLS and do not recognise specific proteins in their eluates or (2) Antibodies that shorten FLS and show specific candidate proteins. Immunoprecipitation revealed a strong association with actin itself either directly or indirectly for the long phenotype subset of the antibodies. I used multiple mass spectrometry proteomic strategies including quantitative (tandem mass tag liquid chromatography mass spectrometry (TMT LC-MS/MS)) and qualitative (LC-MS/MS) for both immunoprecipitated proteins and excised bands. I developed immunostaining protocols and determined the localisation of the target antigens by immunostaining in FLS as well as in Xl177, U20S, HEK 293 cell lines, retinal ganglion cell growth cones and mouse intestinal organoids. To verify the candidate proteins, I combined Western blotting against purified proteins, sedimentation assays with purified actin, immunostaining and transient expression of proteins of interest, confirming the identity of two target antigens. I also sought to develop an *in vitro* system for filopodia triggered by SARS-CoV2 nucleocapsid protein, with limited success, but promising results for further study. This work shows that novel actin regulatory mechanisms can be identified based on antibody reactivity in the FLS system and gives new candidate molecules and mechanisms underlying filopodia formation and other actin assemblies during development and in different pathologies.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2022

Author dc:creator

• Ioannou, Pantelis Savvas

Advisors dc:contributor.advisor

• Gallop, Jennifer
• Lilley, Kathryn

• Actin
• Actin cytoskeleton
• Antibody characterisation
• AstraZeneca
• Filopodia

Licence

• https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/cd4f87d4-674f-43d7-a6f9-7507a61f3374/download
• https://www.rioxx.net/licenses/all-rights-reserved/

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.100090

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/354192