Septin2 is recruited into nascent podosomes following assembly of the precursor complex. (A) Src-mediated actin rearrangement in the presence and absence of Septin2. HPAECs were treated with siRNA for Septin2 (SEPT2) or control siRNA (NT) for 72 h. Cells were transduced with adenoviral constructs for expression of RapR-Src and FRB. Cells were fixed and stained for F-actin following 0.5 h Src activation. Epifluorescence images were analyzed using an ImageJ “invadopodia colocalization” macro to count F-actin foci. Data were collected from five independent experiments (n = 5), with each dot representing one field of view. Error bars represent SE. Statistical significance was determined using a two-sample t test. (B–E) HPAECs coexpressing RapR-Src with Tks5-GFP and fTractin-iRFP670 (F-actin marker; B) or Tks5-GFP and Septin2-tagRFP (D) were imaged live following Src activation (using total internal reflection fluorescence microscopy; scale bar = 8 µm). Representative time-lapse images (C and E) show the order of arrival of Septin2 and F-actin relative to the known podosomal precursor Tks5 (Tks5 arrival time = 0 s). Arrival of the protein was detected using Invadopodia Tracker V3 plugin for ImageJ software. Time of arrival is marked by appearance of the red circle. Arrowheads indicate podosome foci. (F) Arrival times of F-actin and Septin2 with respect to Tks5. Data show arrival times of each protein (n = 8 for F-actin and n = 6 for Septin2) with respect to Tks5 ± SE, with each dot representing an individual focus. Statistical significance was determined using a Student’s t test for pairwise comparison of F-actin with Septin2.