We quantified FLS parameters using our image analysis pipeline FLSAce and used the resulting median number, lengths and areas both with and without additional actin as the input for the t-distributed stochastic neighbor embedding (tSNE) algorithm to visualize similarities between the actions of scFvs ( Fig. Remarkably, a range of altered FLS phenotypes was evident upon coincubation of different scFvs ( Fig. We performed the screen with and without additional unlabeled actin as lengthening phenotypes could be constrained by limitations in the actin, whereas shortening phenotypes could be suppressed by greater actin concentrations. We purified 22 scFvs, which were individually preincubated with the assay mix and resulting FLS visualized by spinning disk confocal microscopy. A single phage that bound to the cocktail of known proteins was also selected (scFv8). Phage ELISA screening of each selection output was performed against their respective condition and the bilayer alone, and a panel of highly specific, strongly binding clones was selected from each condition ( Fig. We excluded phages that bound under all three conditions as they were more likely to bind residual proteins from the extracts. Phages selected from each condition were eluted, cloned, and sequenced. We performed screens against mature fully formed FLS, mature FLS subjected to an additional washing step to relax the bundled FLS architecture, and at an early time point where FLS were fixed rapidly to ensure accessibility to the earliest arriving proteins. A further deselection step was performed against the supported lipid bilayer and experimental setup, followed by positive selection of phage on FLS ( Fig. 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 (actin, TOCA-1, Ena, VASP, N-WASP, fascin, and the Arp2/3 complex Lee et al., 2010).
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