BBS4 is moved by IFT. (A) BBS4-GFP undergoes IFT. (a) Cell attached to the glass surface by its flagella (arrowheads). (b) TIRFM image of the two flagella of a BBS4-GFP1 cell. (c) Kymogram revealing the anterograde and retrograde movement of BBS4-GFP in the flagella. (B) Western blot of flagella isolated from wild-type control (g1), bbs4-1, IFT20-mCherry, and strain MxG1.3 expressing BBS4-GFP and IFT20-mCherry. The blots were probed as indicated; the positions of the immunoreactive proteins and standard proteins and their molecular masses in kilodaltons are marked. (C) The velocity of fluorescent protein–tagged IFT20 and BBS4 in flagella, as determined by TIRFM. In the BBS4-GFP/IFT20-mCherry strain, only particles with both tags were scored. n, number of particles analyzed. (D) Frequency of BBS4-GFP and fluorescent protein–tagged IFT20, as determined by TIRFM. n, number of flagella analyzed. (C and D) SDs are indicated. (E) Single frame from simultaneous recording of BBS4-GFP and IFT20-mCherry. Flagella are marked by arrowheads, and the cell body is marked by an arrow. (F) Kymograms showing the movement of BBS4-GFP (a) and IFT20-mCherry (b) in one flagellum. The image in c is a merger of a and b. Arrowheads and arrows indicate anterograde and retrograde cotransport of both proteins, respectively. Several tracks representing IFT particles are devoid of BBS4-GFP. Note the fission of a BBS4-GFP signal (arrowhead 1), a part of which is later picked up by a different IFT particle (arrowhead 2). (G) A BBS4-GFP particle falls off (arrowheads) a moving IFT20-mCherry particle (arrows). See Video 7. Bar, 10 µm.