Fig. 2.

Cin8 accumulates at and tracks along the minus ends of dynamic MTs. (A) Schematic presentation of a MT dynamics assay. Yellow, GMPCPP-stabilized seed adsorbed onto the surface via an anti-biotin antibody; red, dynamic MTs; green, Cin8. ‘B’, biotin. (B–F) Representative kymographs of Cin8 movements on dynamic (dyn) MTs. In the merged images: red, dynamic MTs; yellow, GMPCPP-stabilized seeds; white, Cin8. The plus and minus ends of the MTs are indicated at the bottom, based on the dynamics characteristics at the two ends. Scale bars: 10 µm (horizontal); 100 s (vertical). (B,C) Long-term and (D) short-term imaging. Vertical red arrows point to Cin8 attachment and tracking along the minus-end of dynamic MTs. MT 1 in Movie 2 is shown in B, left panels. (C) Example of lack of Cin8 attachment to the MT minus end. Yellow arrows indicate the tracking of Cin8 along MT plus ends. (E) Examples of bi-directional and plus-end-directed movements of Cin8 clusters on dynamic MTs and GMPCPP-stabilized seeds. White asterisks indicate slow bi-directional and plus-end-directed bright Cin8 particles. Green asterisks indicate Cin8 cluster-splitting events. The splitting of the Cin8 cluster shown in the upper-left panel is shown in Movie 4. (F) Two sporadic events of Cin8 tracking on a depolymerizing plus end. Minus ends of the MTs were not observed in these examples. The plus end was assigned based on the dynamics of this end. Note: Cin8 tracking on growing and shrinking MT plus ends was extremely rare and was only observed on one occasion (Fig. 2B, right MT).