Background

Cells develop ruffles or erect veils of membrane on their surfaces during migration and in responses to growth factors or other mediators. As innate immune cells, macrophages ruffle both constitutively and upon stimulation by growth factors, pathogens or other activating stimuli. Ruffles can take on different forms. Dorsal ruffles on the upper surface of adherent cells are typically large erect. F-actin-rich veils which can be circular (hence the alternative name ‘circular dorsal ruffles’) usually rise up transiently and then collapse back onto the surface. Ruffles often form macropinosomes as described in Condon et al., 2018. Macropinosomes internalise the ruffle membrane, simultaneously ingesting fluid and soluble or small particulate cargo (Kerr and Teasdale, 2009; Commisso et al., 2013). Dorsal ruffles or circular dorsal ruffles have been described in depth in macrophages, epithelial cells and fibroblasts where they have gained recognition as sites for immune cell activation (Luo et al., 2014; Wall et al., 2017), growth factor signaling (Buccione et al., 2004) and activated receptor clustering, and internalisation that play pivotal roles in cancer cell signaling (Buccione et al., 2004; Orth and McNiven, 2006). Dorsal ruffles are also induced by a number of pathogens, including Salmonella entericia serovar typhimurium at sites of pathogen entry (Ly and Casanova, 2007).

The large, circular dorsal ruffles that have been observed on different cell types are dependent on the transient production of F-actin, and inhibitors of F-actin polymerisation such as cytochalasin D abolish their formation (Hedberg et al., 1993; Buccione et al., 2004). As dorsal ruffles are highly enriched in F-actin and contribute a much greater area than other F-actin rich projections, such as filopodia, on the dorsal surface, fluorescent markers of F-actin are relatively enriched in dorsal ruffles. Many studies utilize the presence of dorsal ruffles as a qualitative read-out of cellular responses (Gurung et al., 2003; Gandhi et al., 2004; Singla et al., 2019). The ability to quantify ruffling or F-actin cell surface protrusions, would provide useful analysis for a wide range of biological research ranging from stimulation assays to high-throughput screening. Ruffling has been assessed in images by taking whole-cell, whole field of view F-actin measurements (Schlam et al., 2016) or by performing manual selection and user-interactive regions of interest (Venter et al., 2015).

Here, we have developed a robust and automated assay for dorsal ruffle quantification that requires no user input for cell selection or segmentation of both basal and dorsal regions, defined by the mid-point of the nucleus. Measurements for ruffles are also made on Sum-slices images, meaning depth information is taken into account (i.e., taller ruffles contribute to higher intensity counts). This assay enables batch processing of multiple images to analyse large cell numbers and provide tabulated results for easy statistical analysis using third party software. This robust, automated assay is well suited for screening cell populations to detect changes in dorsal ruffling induced by extracellular stimuli such as pathogens, growth factors, hormones or cytokines and/or large scale screening of pharmacological inhibitors, natural products, or nutritional conditions.