Prolonged cilia contacts form in cultured cells. (A) MDCK cells were cultured in a matrix to form a three-dimensional cyst, then fixed and stained. The arrow indicates an area of overlapping contact (acetylated tubulin - green; Hoechst - blue; actin - red). (B, C) MDCK cells were grown on transwell filters, fixed, stained, and imaged using a confocal microscope. These maximum intensity projection images show overlapping (B) and point (C) contacts between cilia (acetylated tubulin - green; Hoechst - blue; ZO1 - red). (D) Filter grown MDCK cells were stained and imaged on a confocal microscope (Hoechst - blue, anti-detyrosinated tubulin - green, and anti-gamma tubulin - red). The image presented is a maximum intensity projection (the contrast in the red channel was changed to make the centrosomes more visible). (E, F) Filter grown MDCK cells stably expressing tubulin-GFP were imaged by confocal microscopy and used to generate projection images. Cilia from nearby cells appear to be coming together. (G) We monitored cells during contact formation and measured how long cilia remained together to determine whether the contacts are transient chance encounters or more persistent inter-cellular interactions. Each point on the graph represents a contact event. The shortest contact duration observed was just over 2 h. (H) To test whether primary cilia can move we collected rapid z stacks of primary cilia from TubGFP expressing cells at 4.5 s intervals using a spinning disk confocal microscope. We generated a time series of the maximum intensity projections of the z stacks and then subtracted the preceding frame from each image; thus, the shadow indicates the position of the cilium at the previous time point. Yellow arrowheads in the first panel indicate the base of each cilium. The end panel is a projection of the position of the cilia at every time point for 11.9 min (scale bar is 10 μm in B, C, and D; 5 μm in G).