ArhGAP44 is recruited to collapsing artificial lamellipodia in neurons. ( A ) Rapamycin-induced dimerization assay. Neurons were quadruple-transfected with a membrane-anchored FRB, a fluorescently labeled FKBP that was associated with the Rac-GEF TIAM, a cytosolic reference, and the fluorescently labelled N-BAR domain of ArhGAP44. Addition of rapamycin triggered dimerization of FRB and FKBP, which led to a rapid translocation of TIAM to the plasma membrane. Enrichment of TIAM at the plasma membrane augmented local Rac activity and actin dynamics. ( B ) Neurons quadruple-transfected with the constructs before and after addition of rapamycin. Note the formation of ectopic actin-rich structures. ( C ) Time lapse images show enrichment of the N-BAR domain of ArhGAP44 at retracting actin-rich structures. Cells were quadruple-transfected with Lyn-FRB, CFP-FKBP-TIAM, YFP-ArhGAP44(N-BAR), and the empty mCherry plasmid as a cytosolic reference. Note the enrichment of the N-BAR domain of ArhGAP44 at the collapsing lamellipodia. ( D ) Kymograph of ArhGAP44(N-BAR) (top panel) and the cytosolic reference mCherry (middle panel) show relative enrichment of the N-BAR domain of ArhGAP44 at a retracting actin-rich structures (bottom panel). ( B ), 10 µm; ( C ), 2 µm. DOI: http://dx.doi.org/10.7554/eLife.03116.039