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A direct role for SNX9 in the biogenesis of filopodia.

The Journal of cell biology (2020-04-25)
Iris K Jarsch, Jonathan R Gadsby, Annalisa Nuccitelli, Julia Mason, Hanae Shimo, Ludovic Pilloux, Bishara Marzook, Claire M Mulvey, Ulrich Dobramysl, Charles R Bradshaw, Kathryn S Lilley, Richard D Hayward, Tristan J Vaughan, Claire L Dobson, Jennifer L Gallop
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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
DL-Cysteine, technical grade
Sigma-Aldrich
Maleimide, 99%
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Dulbecco′s Modified Eagle′s Medium/Nutrient Mixture F-12 Ham, With 15 mM HEPES and sodium bicarbonate, without L-glutamine, liquid, sterile-filtered, suitable for cell culture
Avanti
18:1 PI(3,4)P2, 1,2-dioleoyl-sn-glycero-3-phospho-(1′-myo-inositol-3′,4′-bisphosphate) (ammonium salt), powder
Sigma-Aldrich
Wortmannin, from Penicillium funiculosum, ≥98% (HPLC and TLC)
Sigma-Aldrich
Protease Inhibitor Cocktail, for use with mammalian cell and tissue extracts, DMSO solution