The time-lapse video shows live-cell imaging of an actin-GFP expressing mouse fibroblast responding to PDGF with circular dorsal ruffles. Circular dorsal ruffles are dynamic, F-actin-enriched plasma membrane structures that transiently occur on the dorsal surface of fibroblasts in response to mitogenic stimuli such as platelet-derived growth factor (PDGF). These dynamic structures form within minutes of stimulation and generally disappear within 30 minutes. The proposed functions of these dorsal ruffles include the internalization of growth factor receptors, macropinocytosis/fluid phase uptake, and cytoskeletal rearrangements required for cell movement. Note that ruffles expand into circles that eventually coalesce, in this unusual case to generate the image of a heart.
Methods: Mouse fibroblasts transiently expressing actin-GFP (Clontech, Foster City, CA) were plated on 35mm glass-bottom culture dishes (MatTek, Ashland, MA) and starved overnight in serum-free DMEM medium (Invitrogen, Carlsbad, CA). Cells were washed three times with phosphate buffered saline (PBS) and maintained in imaging buffer (136mM NaCl, 2.5mM KCl, 2mM CaCl2, 1.3mM MgCl, 10mM HEPES) at 37C. Time-lapse fluorescence images were acquired with a Leica AM6000 inverted microscope equipped with a 40X oil immersion objective (HCX PL HP APO 40X/ 1.25-0.75, Leica, Germany), and a 12-bit charge-coupled device (CCD) camera (CoolSnap HQ). Image capture and data acquisition were performed using Metamorph software (V7.0r1, Molecular Devices, Downingtown, PA). Images were acquired in binning 2x2 modes at 8s internals. Image sequences were processed with ImageJ software
Acknowledgements: Celldance 2008, Honorable Mention Video: A 'Lovely' Example of Growth Factor-Induced Membrane Ruffles Hongying (Hoy) Shen (Yale University) Thank Dr. Pietro De Camilli for mentoring, Shawn Ferguson for generating cell lines and providing actin-GFP constructs, Min Wu for assisting with image acquisition and software usage. Pietro De Camilli (Yale university, New Haven, CT), Shawn Ferguson (Yale University, New Haven, CT), Min Wu (Yale University, New Haven, CT)