Fig. 1: 3D tracking of MKLP1 kinesin-6 movement around the lattice surface of freely suspended microtubules.
From: CYK4 relaxes the bias in the off-axis motion by MKLP1 kinesin-6
a A schematic of the domain structures of the dimeric N-terminal piece of CYK4 (C2, 1–120 a.a. of C. elegans CYK-4), dimeric MKLP1 kinesin-6 (M2, ZEN-4 (1–555 a.a.)-SNAP tag-His tag), monomeric MKLP1 (M1, ZEN-4 (1–439 a.a.)-SNAP tag-His tag), and M2C2 (the complex of M2 and C2). CC: coiled coil. b SDS-PAGE analysis of the purified M2, M2C2 and M1. Coomassie blue-stained SDS-polyacrylamide gel (12%). Lane 1: molecular size markers (in kDa), Lanes 2–4: purified M2C2, M2, and M1. The arrows indicate the positions of the individual proteins. c A schematic of the experimental setup. Glass was patterned into 2 µm-high, 10 µm-wide parallel walls with a 10 µm gap. Microtubules were suspended between them using streptavidin-biotin interaction. This allowed beads coated with multiple kinesin molecules to move all around the microtubule surface. d A schematic drawing of a 3D prismatic optical tracking (termed tPOT) microscope. The z position of kinesin-coated bead motion as well as the x–y position are obtained from a pair of images split by the prism. The temperature of the sample is maintained at 23 °C by the combined temperature management unit. e Pairs of split images by the tPOT microscope. The top panel shows a pair of split images of an Alexa488-biotin-labeled microtubule (marked by blue rectangles). The other panels show the time series of paired images of an M2-coated bead moving along the suspended microtubule (pairs of solid and open red arrowheads). (Scale bar, 1.5 µm; 5 s interval between images).
