Abstract
Cellular protrusions are highly dynamic structures involved in fundamental processes, including cell migration and invasion. For a cell to migrate, its leading edge must form protrusions, and then adhere or retract. The spatial and temporal coordination of protrusions and retraction is yet to be fully understood. The study of protrusion dynamics mainly relies on live-microscopy often coupled to fluorescent labeling. Here we report the use of an alternative, label-free, quantitative and rapid assay to analyze protrusion dynamics in a cell population based on the real-time recording of cell activity by means of electronic sensors. Cells are seeded on a plate covered with electrodes and their shape changes map into measured impedance variations. Upon growth factor stimulation the impedance increases due to protrusive activity and decreases following retraction. Compared to microscopy-based methods, impedance measurements are suitable to high-throughput studies on different cell lines, growth factors and chemical compounds. We present data indicating that this assay lends itself to dissect the biochemical signaling pathways controlling adhesive protrusions. Indeed, we show that the protrusion phase is sustained by actin polymerization, directly driven by growth factor stimulation. Contraction instead mainly relies on myosin action, pointing at a pivotal role of myosin in lamellipodia retraction.
Highlights
A challenging feature of studying protrusion dynamics is the ability to provide quantitative as well as time-resolved data
Impedance reading variations can be quantitatively mapped into cell protrusion dynamics
We investigated protrusive activity in MCF10A cells by imaging cells stably transduced with LifeAct-GFP by total internal reflection fluorescence (TIRF) microscopy
Summary
A challenging feature of studying protrusion dynamics is the ability to provide quantitative as well as time-resolved data. Atomic force microscopy has been used to measure lamellipodia dynamics and thickness in adenocarcinoma cells or in migrating keratocytes[18,19] These approaches are powerful as they all allow single cell or even subcellular resolution, and represent the method of choice to study protrusion dynamics. Such methods present a few drawbacks: i) they often require complex image and/or mathematical processing to obtain quantitative results, ii) they are hardly suitable for high throughput studies such as biochemical functional or drug screening and iii) are subject to cell to cell variability. We employ the impedance reading (IR) technique to quantitatively measure protrusion dynamics and validate the results by direct comparison with quantitative data of cell surface variation, obtained through image analysis of live TIRF microscopy. By direct comparison of microscopy data with IR data we can dissect the different parts of the response into molecularly distinct events mediated by actin polymerization and myosin contraction that can be inhibited separately by specific drugs
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