Figure 1.

The intraflagellar transport (IFT) machinery of gliding Chlamydomonas can be observed and manipulated in total internal reflection microscopy (TIRFM)

(A) ① Anterograde trains, driven by the heterotrimeric motor kinesin-II, move towards the ciliary tip. ② At the ciliary tip, kinesin-II dissociates from the train, and the trains convert into retrograde form by an unknown mechanism. ③ Reassembled retrograde trains move away from the tip towards the cell body by the dynein-1b motor specific for IFT.

(B) A sharp but soft wedge is made by drop casting silicone onto a supporting capillary, followed by trimming off excess after curing.

(C) Illustration of mechanical blockage by lowering a silicone wedge onto the cilium of a Chlamydomonas reinhardtii cell by micro-manipulator on a TIRF microscope.

(D) Individual frames from a TIRFM movie show how IFT is locally and reversibly blocked inside a cilium by the force applied using a silicone wedge (dashed line, non-blocking in blue, blocking in red). See also Video S1.