JCB_201705113 3405..3422

Cells form an extensive network of actin-rich protrusions to move through tissues, including veil-like lamellipodia and finger-like filopodia (Mattila and Lappalainen, 2008; Bisi et al., 2013; Krause and Gautreau, 2014; Jacquemet et al., 2015). Filopodia play key roles throughout embryonic development, including in the developing nervous system. Filopodia mediate the sensing of attractant and repellent guidance cues by growth cones (Zheng et al., 1996; McConnell et al., 2016) and the guidance of axons through the developing nervous system (Bentley and Toroian-Raymond, 1986; O’Connor et al., 1990; Chien et al., 1993). Once neurites arrive at their destination, filopodia are involved in the formation of branches (Dwivedy et al., 2007) and synaptic connections (Ziv and Smith, 1996; Lohmann and Bonhoeffer, 2008). Despite the overwhelming importance of filopodia, many questions about their biology remain unanswered. First, the molecular mechanism of their formation is not clear. Multiple models have been proposed and experimentally supported, including (1) elongation of converging filaments within an underlying branched network of lamellipodial actin, fundamentally dependent on the Arp2/3 complex (Svitkina et al., 2003); (2) de novo nucleation of filopodial filaments at their tip by proteins also capable of promoting actin polymerization, such as formins (Faix and Rottner, 2006); and (3) initial clustering of membrane-binding proteins that subsequently recruit other factors required for filopodium formation (Lee et al., 2010; Saarikangas et al., 2015). However, the molecular identities of protein assemblies driving filopodium formation according to these models are not clear-cut. ENA/VASP proteins localize to filopodia tips upon their protrusion and are implicated in regulating the number and length of filopodia in a variety of cell types, through diverse mechanisms that structurally fit the “convergent elongation” and “tip nucleation” models (Lanier et al., 1999; Svitkina et al., 2003; Barzik et al., 2014). In retinal ganglion cells (RGCs), which we use here, ENA/VASP proteins are important for growth and stabilization of filopodia and terminal arborization of the axon (Dwivedy et al., 2007). Moreover, it also appears that cells use multiple filopodial actin filament elongating proteins. Studies in Drosophila melanogaster and fibroblasts suggest that filopodia driven by ENA/ VASP proteins or by formins (Dia/mDia2) have distinct properties (Barzik et al., 2014; Bilancia et al., 2014; Nowotarski et al., 2014). To better understand which proteins actively contribute to filopodia growth in various contexts, and to differentiate between models of filopodia formation, it would be critical to quantify the amount of proteins of interest within filopodia concurrently with their dynamic behavior. This approach has been very fruitful in understanding the protrusion of lamellipodia and membrane blebs (Charras et al., 2006; Machacek et al., 2009; Barry et al., 2015; Lee et al., 2015; Martin et al., 2016). A comprehensive image analysis pipeline is essential for these goals. Recently, computational tools have been developed for automated segmentation and analysis of filopodia. These include MAT LAB applications FiloDetect, which measures the Filopodia have important sensory and mechanical roles in motile cells. The recruitment of actin regulators, such as ENA/ VASP proteins, to sites of protrusion underlies diverse molecular mechanisms of filopodia formation and extension. We developed Filopodyan (filopodia dynamics analysis) in Fiji and R to measure fluorescence in filopodia and at their tips and bases concurrently with their morphological and dynamic properties. Filopodyan supports high-throughput phenotype characterization as well as detailed interactive editing of filopodia reconstructions through an intuitive graphical user interface. Our highly customizable pipeline is widely applicable, capable of detecting filopodia in four different cell types in vitro and in vivo. We use Filopodyan to quantify the recruitment of ENA and VASP preceding filopodia formation in neuronal growth cones, and uncover a molecular heterogeneity whereby different filopodia display markedly different responses to changes in the accumulation of ENA and VASP fluorescence in their tips over time. Filopodyan: An open-source pipeline for the analysis of filopodia