Abstract
Filopodia are finger-like actin-rich protrusions that extend from the cell surface and are important for cell-cell communication and pathogen internalization. The small size and transient nature of filopodia combined with shared usage of actin regulators within cells confounds attempts to identify filopodial proteins. Here, we used phage display phenotypic screening to isolate antibodies that alter the actin morphology of filopodia-like structures (FLS) in vitro. We found that all of the antibodies that cause shorter FLS interact with SNX9, an actin regulator that binds phosphoinositides during endocytosis and at invadopodia. In cells, we discover SNX9 at specialized filopodia in Xenopus development and that SNX9 is an endogenous component of filopodia that are hijacked by Chlamydia entry. We show the use of antibody technology to identify proteins used in filopodia-like structures, and a role for SNX9 in filopodia.
Highlights
Assemblies of actin filaments (F-actin) are major dynamic superstructures required for cell motility, intercell communication, and force generation
By selecting the antibodies that generated shorter filopodia-like structures (FLS), we identified the antigen recognized by three independent antibodies as sorting nexin 9 (SNX9) and subsequently demonstrated its role by immunodepletion and rescue experiments
Antibody-mediated modifications of FLS by phage display phenotypic screening To isolate antibodies targeting novel components of FLS, a phage display library was incubated with a cocktail of purified actin regulatory proteins that are known to localize to FLS to adsorb and deselect phage against known FLS components
Summary
Assemblies of actin filaments (F-actin) are major dynamic superstructures required for cell motility, intercell communication, and force generation.
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