Dataset Fig4EG_MTelongation_1
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 x-ray fiber diffraction measuring axial pitch of GDP-MT before and after addition of K11C or AMPPMP
Description
Time-series images of x-ray fiber diffraction measuring axial pitch of GDP-MT (GDP-microtubules) before and after addition of K11C or AMPPMP. K11C is a mutant KIF5C, the L11-alpha4 junction was replaced with the corresponding sequence of KIF1A. A synchrotron radiation x-ray beam at BL45XU beamline of SPring-8 (Japan Synchrotron Radiation Research Institute) irradiated the oriented microtubules. K11C was added after time00001, and AMPPMP was applied after time00011.
Description
Polymerized tubulin in vitro.
Extrinsic variables
K11C, 1 mM AMPPNP
Gene ontology: Biological processes
Gene ontology: Cellular components
Gene ontology: Molecular functions
Dimensions
1475x1679x1x1x26
X scales
0.017 nanometer^{-1}/pixel
Y scales
0.017 nanometer^{-1}/pixel
T scales
15 seconds per time interval
Contrast enhancing methods
Resolution enhancing methods
Sample preparation methods
Light source
A synchrotron radiation x-ray beam at BL45XU beamline of SPring-8
Detector
Pilatus 300K-W detector system