Fig. 5. Motion of EEs in the kin3 deletion strain RWS13. (A–C) Co-expression of a GFP–Tub1 fusion protein and Yup1–GFP in a Δkin3 strain allowed us to analyze the role of MTs in the remaining motility of EEs. Brightly stained EE clusters move along MTs (A; arrows mark the MT). In the absence of Kin3, they were located at the ends of MTs. In all cases (n = 31), MTs rapidly shortened towards these cluster (B; end of MT marked by asterisk), suggesting that EEs are located at the minus-ends (labeled ‘+’ and ‘–’). Consistent with this, MT elongation was found to be directed away from the BSDs (C; ends of MTs marked by asterisks, orientation indicated by ‘+’ and ‘–’). Time between frames is 1.4 s. Bar: 2 µm. (D) Endosome organization in the temperature-sensitive dynein–Δkin3 double mutant (RWS14). After 2 h at 33°C, endosome clusters (arrowheads) are smaller or even absent from the cell (D1; right cell). Note that cells have a separation defect due to the deletion of kin3. In these structures, almost no EE motility was found (D2, D3; time in seconds is given in the bottom right corner). Bars: 3 µm. (E) Expression of a dominant-negative kin3 mutant allele that is described to result in a rigid binding of the motor to the MT leads to ‘pearl-string’-like arrangement of EEs (arrows) and abolished almost all motion (compare E1 and E2). Bar: 2 µm; time in seconds is given in the bottom right corner. Movies are available as Supplementary data.