Live-cell imaging and FRET analysis

Stable macrophage cell line RAW/LR5 used previously in ref. ²⁷ with inducible expression of Cdc42 or Rac1 biosensors were used for live cell imaging. Repression of biosensor expression was maintained by culturing cells in RAW medium supplemented with 2 µg/ml doxycycline (MP Biomedicals). Induction of biosensor expression was performed as previously described in). Briefly, 48 hours prior to imaging, cells were washed with PBS to remove doxycycline medium then trypsinized and replated at low density. For optimal induction of biosensor expression, cells were cultured in RAW medium supplemented with 10% tetracycline-free serum. After 24 hours, cells were briefly trypsinized and replated at low density for another 24hrs. Then cells were lifted again and plated in 35 mm MatTek dish at a concentration of 2 × 10⁵ overnight. The MatTek dish was then mounted on a heated stage maintained at 37 °C during imaging. To image TNTs, imaging was performed in BWD supplemented with 5% FBS to achieve optimal signal-to-noise ratio. Following temperature stabilization, images were acquired 10 s intervals for 10 min at 60X magnification. Both FRET and mCer1 emission channels were acquired simultaneously at each frame using two side-mounted cameras in order to eliminate motion artifacts as described in ref. ⁶², followed by an acquisition of the differential interference contrast (DIC) channel at the third camera mounted on the bottom port of the microscope. mVen emission was captured to confirm biosensor expression at first frame only to minimize photobleaching during imaging session. Detailed descriptions of live-cell imaging, microscope settings and image processing are provided in refs ⁶² and ⁶³. FRET/donor ratiometric analysis at every frame was performed using Metamorph and MatLab software. GTPase activity was determined in an area representing the base, within or the tip of each TNT structure, then compared to area within the cell body. Frequency of GTPase activity was then determined over ≥10 frames.