Localization and dynamics of ARF1 puncta in TGFβ1-stimulated THP1 cells. (A) TIRF image of the ventral surface of cell with podosomes labeled by mCherry-vinculin (left) and ARF1 puncta labeled by GFP-ARF1 (middle). Merged image (right) shows nonrandom distribution of ARF1 puncta with a tendency to colocalize to podosome periphery (see white arrowheads). Bar, 5 µm. Boxed area (2.5 × 2.5 µm²) contains a podosome whose colocalization dynamics with ARF1 puncta is presented in B. (B, left) Kymograph representing fluorescent intensities in a line scan through the podosome boxed in A. Although mCherry-vinculin is stably labeled in the podosome ring (top), GFP-ARF1 was transiently concentrated at one side of the ring (middle, merged image at bottom). See also Video 1. (B, right) The time course of fluorescence intensity of GFP-ARF1 at the podosome ring. (C) Each dot corresponds to a single cell and represents percentage of podosome rings (labeled by vinculin) contacted by either ARF1-containing puncta or Rab6-containing vesicles within 5 min of image acquisition. Mean ± SD is indicated. (D) Frequency distribution of the durations of podosome contacts (in seconds) with ARF1-containing puncta (35 podosomes from 10 cells were filmed as shown in kymograph B). (E and F) GFP-ARF1 puncta moving along microtubules. (E) Left, microtubule labeling with 125-kD microtubule-associated protein, ensconsin (mCherry-ensconsin); middle, GFP-ARF1 puncta in the same cell; right, merged image. The dynamics of microtubules and ARF1 puncta in the boxed area (8 × 7.5 µm²) of C is shown in F. Bars, 5 µm. Movement of puncta along the microtubule is indicated by the arrowhead. See also Video 2.