Kinesin-3 numbers drop before anterograde to retrograde turning of EEs. (A) Numbers of Hok1-GFP and kinesin-3–GFP in signals moving bidirectionally. Bars are means ± SE of two experiments; sample size is indicated. No significant difference is found, P = 0.391. Numbers were estimated using an internal calibration standard (Schuster et al., 2011c) and assume proteins are dimers (Xu et al., 2008; Hammond et al., 2009). (B) Numbers of kinesin-3–GFP in anterograde and retrograde signals. Bars are means ± SE of two experiments; sample size is indicated. No significant difference is found, P = 0.811. Numbers were estimated using an internal calibration standard (Schuster et al., 2011c) and assume proteins are dimers (Hammond et al., 2009). (C) Pausing time of EEs and dynein before retrograde motility. Bars are means ± SE of two experiments; sample size is indicated. (D) Kymographs showing the binding of GFP3-dynein (green) to a mCherry-Rab5a–labeled EE (red). An anterograde (antero)-moving organelle pauses (pause) before it binds dynein and turns to retrograde motility (retro). (E) False-colored kymographs showing Hok1-GFP and kinesin-3-GFP during anterograde to retrograde turning. Kinesin-3 signals drop just before the organelle pauses (arrowheads). Recovery of the fluorescent signal begins during pause (yellow arrowhead). Note signal variations caused by changes in focal plane and that stationary signals are brighter as they are more focused. (F) Mean intensities of Hok1-GFP and kinesin-3-GFP during anterograde to retrograde turning of EEs. Hok1 numbers remain stable, whereas kinesin-3 signals drop before the pausing phase. Data points are means ± SE from a representative experiment, and sample sizes are indicated. Significant difference is indicated: *, P < 0.05; ***, P < 0.0001. Images in D and E were adjusted in brightness, contrast, and γ settings. Horizontal bars are in micrometers, and vertical bars are in seconds.
