Dataset FigS3AB_MTcompact
Project
191-Shima-KIF5Cmove
Title
KIF5C running on various states of microtubules
Description
Kinesin-1, the founding member of the kinesin superfamily of proteins, is known to use only a subset of microtubules for transport in living cells. This biased use of microtubules is proposed as the guidance cue for polarized transport in neurons, but the underlying mechanisms are still poorly understood. Here, we report that kinesin-1 binding changes the microtubule lattice and promotes further kinesin-1 binding. This high-affinity state requires the binding of kinesin-1 in the nucleotide- free state. Microtubules return to the initial low-affinity state by washing out the binding kinesin-1 or by the binding of non-hydrolyzable ATP analogue AMPPNP to kinesin-1. X-ray fiber diffraction, fluorescence speckle microscopy, and second- harmonic generation microscopy, as well as cryo-EM, collectively demonstrated that the binding of nucleotide-free kinesin-1 to GDP microtubules changes the conformation of the GDP microtubule to a conformation resembling the GTP microtubule.
Funding
This work was supported by the Ministry of Education, Cul- ture, Sports, Science and Technology through a Grant-in-Aid for Specially Promoted Research (grant 23000013 to N. Hi- rokawa) and Grant-in-Aid for Scientific Research (KAKENHI grant 16H06372 to N. Hirokawa; grants 16H05119, 15H01334, 26115721, 26650069, and 25293046 to Y. Okada; grants 15H01656 and 17H05897 to H. Shigematsu; and grant 15K08168 to R. Nitta), the Uehara Memorial Foundation (Y. Okada), the Takeda Science Foundation (Y. Okada and R. Nitta), the Mochida Memorial Foun- dation for Medical and Pharmaceutical Research (R. Nitta), the RIKEN Special Postdoctoral Researchers Program (T. Shima), the All RIKEN Research Project on Single Cell (Y. Okada), and the RIKEN Pioneering Project on Dynamic Structural Biology (Y. Okada, H. Shigematsu, and M. Shirouzu).
Title
Time-series images of GDP-MT and KIF5C before and after washout of KIF5C
Description
Time-series images of GDP-MT (GDP-microtubules) and monomeric KIF5C (K351) before and after washout of KIF5C, which were obtained by fluorescent speckle microscopy.
Description
Immobilized microtubule seeds, which were labeled with tetramethylrhodamine, on a glass, and KIF5C were labeled with DY-647
Gene ontology: Biological processes
Gene ontology: Cellular components
Gene ontology: Molecular functions
Dimensions
512x512x1x2x71
X scales
60 nanometer/pixel
Y scales
60 nanometer/pixel
T scales
2 seconds per time interval,
5 seconds per time interval
Detection methods
electron multiplying CCD (EMCCD)
Visualization methods
Tetramethylrhodamine (probe for microtubules) and DY-647 (probe for KIF5C)
Illumination methods
electron multiplying CCD (EMCCD)
Contrast enhancing methods
optical method
Resolution enhancing methods
Sample preparation methods
Body
Olympus-Evident IX83
Light source
Coherent, Obis LX 532 (for microtubule) and Cooherent, CUBE 640 (for KIF5C)
Detector
iXon3 EM CCD camera
Objective
UP- lanSApo, NA 1.40, oil; Olympus