A new tool for the quantitative analysis of dendritic filopodial motility

Abstract

Dendritic filopodia are tiny and highly motile protrusions formed along the dendrites of neurons. During the search for future presynaptic partners, their shape and size change dynamically, with a direct impact on the formation, stabilization and maintenance of synaptic connections both in vivo and in vitro. In order to reveal molecular players regulating synapse formation, quantitative analysis of dendritic filopodia motility is needed. Defining the length or the tips of these protrusions manually, however, is time consuming, limiting the extent of studies as well as their statistical power. Additionally, area detection based on defining a single intensity threshold can lead to significant errors throughout the image series, as these small structures often have low contrast in fluorescent images. To overcome these problems, the open access Dendritic Filopodia Motility Analyzer, a semi-automated ImageJ/Fiji plugin was created. Our method calculates the displacement of the centre of mass (CoM) within a selected region based on the weighted intensity values of structure forming pixels, selected by upper and lower intensity thresholds. Using synthetic and real biological samples, we prove that the displacement of the weighted CoM reliably characterizes the motility of dendritic protrusions. Additionally, guidelines to define optimal parameters of live cell recordings from dendritic protrusions are provided. V C 2014 International Society for Advancement of Cytometry Key terms neuron; dendritic filopodia; motility analysis; time-lapse MOST excitatory synapses in the mammalian brain terminate on dendritic spines.During neuronal development, transient actin-rich protrusions named dendritic filopodia appear first along the dendrites. Filopodia are highly motile structures, which actively search for future presynaptic contact sites. In the absence of a suitable axonal contact, filopodia retract. On the other hand, initial contact triggers the formation of synaptic machinery and the appearance of dendritic spines (1). Filopodia can change their shape by a nodding motion when the angle between the neck and the head or dendritic shaft is changing. In addition, their length or the width of their head can also alter in time (2-4). Developmental regulation of filopodial motility is required for synapse formation: initial movements and elongations are needed to get into contact with the future presynaptic partner, while motility is attenuated for the successful maintenance of the synaptic connections (5).In contrast to filopodia, dendritic spines have established synaptic connections, possessing postsynaptic components within the head region of dendritic spines. Stubby spines are shorter, while thin and mushroom spines are usually longer than 1 mm with a well-extended head region in case of mushroom spines. The size and shape of established dendritic spines can also alter during development, in correlation with the stability and the strength of the synapse (1).To characterize dendritic filopodia and spine motility, som...

Automated analysis of filopodial length and spatially resolved protein concentration via adaptive shape tracking

“…Unlike previously published work, which is capable of analyzing either growth parameters such as elongation speed and filopodial shape (Styner et al , 2003; Costantino et al , 2008; Fanti et al , 2011; Nilufar et al , 2013; Hendricusdottir and Bergmann, 2014; Tsygankov et al , 2014; Tarnok et al , 2015) or relative spatiotemporal protein concentration (Machacek et al , 2009; Barry et al , 2015), this approach allows us to track filopodial dynamics and combine these shape measurements with spatiotemporal information on relative protein concentration along filopodia. However, although the experiments provide evidence for the versatility and robustness of the software, we stress that the image analysis is only as good as the quality of the images.…”

“…, 2011; Nilufar et al , 2013; Hendricusdottir and Bergmann, 2014; Tsygankov et al , 2014; Tarnok et al , 2015). Similarly, image analysis tools are available to measure spatiotemporal protein concentration in cellular structures such as the leading edge (Machacek et al , 2009; Barry et al , 2015).…”

Filopodyan: An open-source pipeline for the analysis of filopodia

“…The ImageJ plugin ADAPT identifies filopodia, among its suite of other functions, for automated quantification of cell migration and the dynamic behavior of lamellipodia and blebs in relation to their fluorescence intensities ( Barry et al, 2015 ). Software has also been specifically designed for the automated detection of filopodia in dendrites, with a focus on their longitudinal and lateral movement ( Hendricusdottir and Bergmann, 2014 ; Tárnok et al, 2015 ). Saha et al (2016) pioneered concurrent analysis of fluorescence and filopodium behavior, developing MATLAB software for semiautomated analysis of filopodia dynamics and fluorescence.…”

Ras and Rab interactor 1 controls neuronal plasticity by coordinating dendritic filopodial motility and AMPA receptor turnover

“…The morphology of motile filopodia and that of the more stable, thin spines with already established synaptic connections are quite similar and can be distinguished only by the presence of postsynaptic machinery within the protrusion head in fixed cultures. However, live-cell imaging can provide direct observations on protrusion motility (Tárnok et al ., 2015). Therefore we transfected Rin1 − / − neurons with fluorescently tagged RIN1 constructs and analyzed the protrusion motility on secondary dendritic branches on DIV 12–13, within 24 h of posttransfection time (Supplemental Movies S1 and S2).…”