Neck linker docking is critical for Kinesin-1 force generation in cells but at a cost to motor speed and processivity
- University of Michigan, United States
- Vanderbilt University, United States
- University of California, San Diego, United States
- Vanderbilt University School of Medicine, United States
- University of Michigan Medical School, United States
Figures
Figure 1 with 2 supplements
MD simulations identify key interactions between the kinesin-1 NL and motor domain.
(A) Surface representation of the kinesin-1 (RnKIF5C) motor domain in the nucleotide-free (apo) state (top, PDB 4LNU) or ATP-bound, post-power stroke state (bottom, PDB 4HNA). The neck linker (NL, …
Figure 1—figure supplement 1
CS and NL interactions in the no nucleotide (apo) and ATP-bound, post-power stroke states of the kineisn-1 motor domain bound to tubulin.
(A, B) Nucleotide-free (apo) state. (A) Cartoon representation of the kinesin-1 motor domain (PDB 4LNU) in the nucleotide-free (apo) state. Secondary structure elements: coverstrand (CS, purple), α1 …
Figure 1—figure supplement 2
Sequence alignment of the motor domain reveals subtle sequence changes that may alter CNB formation and NL docking across the kinesin superfamily.
Sequence alignment of the motor domain from kinesin-1,–2, −3,–4, −5,–6 families across species (Dm, Drosophila melanogaster; Ce, Caenorhabditis elegans, Xl, Xenopus laevis; Rn, Rattus norvegicus; Hs,…
Figure 2 with 1 supplement
MD simulations predict that CNB+Latch mutations alter CNB formation and NL docking.
(A–F) The kinesin-1 motor domain in the ATP-bound, post-power stroke state is shown as a cartoon representation (PDB 4HNA). Secondary structure elements are colored: coverstrand (CS, purple), α1 …
Figure 2—figure supplement 1
MD simulations predict that mutations of the N-Latch alter CNB formation and NL docking.
(A,B) Differences in residue-residue distances between (A) WT and CNB+Latch mutant motors or (B) WT and Latch mutant motors based on MD simulations of tubulin-bound motors in the ATP-bound state. …
Figure 3
CNB and N-Latch formation are critical for force generation by single kinesin-1 motors.
(A) Schematic of single-molecule optical trap assay. Cell lysates containing FLAG-tagged KIF5C(1-560) motors were incubated with beads functionalized with anti-FLAG antibodies and subjected to …
Figure 4 with 1 supplement
CNB and Latch mutants display enhanced motility properties under single-molecule, unloaded conditions.
(A) Motility properties of WT or mutant motors tagged with three tandem monomeric Citrines (3xmCit) at their C-termini were analyzed in standard single-molecule motility assays using TIRF …
Figure 4—figure supplement 1
CNB+Latch mutants exhibit fast reattachment events during processive runs.
(A) Motility properties of kinesin-1 KIF5C(1-560) CNB+Latch mutant or the highly processive kinesin-3 motor KIF1A(1-393)-LZ. The motors were tagged with either a HaloTag and labeled with an JF649 …
Figure 5 with 1 supplement
CNB+Latch mutations enhance microtubule binding and catalytic site closure.
(A) Ribbon representation of the kinesin-1 motor domain in the ATP-bound, post-power stroke state (PDB 4HNA). Secondary structure elements critical for nucleotide binding and hydrolysis are colored …
Figure 5—figure supplement 1
Interactions between nucleotide coordinating elements (P Loop, Switch 1, Switch 2, and α0) in WT, CNB+Latch, and Latch mutant motors.
Differences in residue-residue distances based on MD simulations between (A–C) the nucleotide-free (apo) and ATP-bound states of WT kinesin-1 associated with tubulin, (D–F) WT and CNB+Latch mutants …
Figure 6 with 2 supplements
CNB and Latch mutations do not affect transport of peroxisomes (low-load cargo) by teams of kinesin-1 motors in cells.
(A) Schematic of the inducible motor recruitment assay. A kinesin motor tagged with monomeric NeonGreen (mNG) and an FRB domain (KIF5C-mNG-FRB) is coexpressed with a cargo targeting sequence (CTS) …
Figure 6—figure supplement 1
Peroxisome dispersion (low-load cargo) by teams of WT or CNB and/or NL docking mutant motors.
(A–D) Representative images of peroxisome dispersion before (-Rap) and after (+Rap) motor recruitment to the peroxisome surface. Blue lines indicate the nucleus and periphery of each cell. Blue …
Figure 6—figure supplement 2
Analysis of cargo dispersion in cells.
(A) Qualitative analysis of cargo dispersion. Cargo localization was scored as: black: clustered (cargo is tightly clustered near the nucleus of the cell); dark gray: partially dispersed (cargo is …
Figure 7 with 3 supplements
CNB and NL docking mutations impair transport of Golgi elements (high-load cargo) by teams of kinesin-1 motors in cells.
(A) Schematic of inducible Golgi dispersion assay. A variety of mechanisms, including the action of cytoplasmic dynein motors (black), maintain the Golgi in a compact cluster near the nucleus. Thus, …
Figure 7—figure supplement 1
Validation of peroxisome and Golgi as low- and high-load cargoes, respectively.
(A,D) Representative images of (A) peroxisome dispersion or (D) Golgi dispersion before (-Rap) and after (+Rap) recruitment of teams of KIF18A motors. (A) COS7 cells were cotransfected with plasmids …
Figure 7—figure supplement 2
Golgi dispersion (high-load cargo) by teams of WT or NL docking mutant motors.
(A–D) Representative images of Golgi dispersion before (-Rap) and after (+Rap) recruitment of teams of motors to the Golgi surface. COS7 cells were cotransfected with plasmids encoding for the …
Figure 7—figure supplement 3
Kinesin-1 CNB and/or Latch mutants can drive transport of reduced-load Golgi elements.
(A) Schematic of Golgi dispersion assay with reduced-load. A truncated dynein intermediate chain (IC2), which acts as a dominant negative (DN) for dynein function, was expressed to interfere with …
Videos
Video 1
NL docking in WT kinesin-1.
Representative simulation of WT kinesin-1 KIF5C motor domain in the ATP-bound, post-power stroke state. The N-terminal half of the NL (β9) and the N-latch residue N334 (shown as a sphere) are docked …
Video 2
NL undocking in the Latch mutant.
Representative simulation of the Latch mutant motor domain in the ATP-bound state. The C-terminal half of the NL (N-latch residue N334 and β10) undock followed by complete undocking of the NL. …
Video 3
NL undocking and disruption of CNB in CNB+Latch mutant.
Representative simulation of CNB+Latch mutant motor domain in the ATP-bound state. The C-terminal half of the NL (N-latch residue N334 and β10) undocks from the core motor domain followed by …
Tables
Key resources table
| Reagent type | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line | COS-7 (Cercopithecus aethiops) male | AATC | Cat. #: CRL-1651, RRID: CVCL_0224 | |
| Transfected construct | KIF5C(1-560) -mNG-FRB | (Schimert et al., 2019) PMID: 30850543 | ||
| Transfected construct | PEX3-mRFP-FKBP | (Kapitein et al., 2010) PMID: 20923648 | ||
| Transfected construct | GMAP-mRFP-FKBP | (Engelke et al., 2016) PMID: 27045608 | ||
| Transfected construct | dyneinIC2-N237 | (King et al., 2003) PMID: 14565986 | ||
| Transfected construct | KIF18A(1-452) | (Weaver et al., 2011) PMID: 21885282 | ||
| Antibody | cis-Golgi marker giantin | BioLegend | Cat. #: 924302 RRID: AB_2565451 | IF(1:1200) |
| Antibody | β-tubulin | Developmental Studies Hybridoma Bank | Cat. #: E7, RRID: AB_528499 | IF(1:2000) |
| Antibody | goat anti-rabbit Alexa680 | Jackson Immuno Research Labs | Cat. #: 111-625-144, RRID: AB_2338085 | IF(1:500) |
| Antibody | goat anti-mouse Alexa350 | Molecular Probes | Cat. #: A-11045, RRID: AB_142754 | IF(1:500) |
| Drug | rapamycin | Calbiochem, Millipore Sigam | Cat. #: 553210 | |
| Software | GraphPad Prism | GraphPad Software Inc | RRID: SCR_002798 | Version 7.0 c |
| Software | MATLAB, code used for single molecule analysis | MathWorks | RRID: SCR_001622 | R2016b, PMID: 25365993 |
| Software | Origin 2017 | OriginLab | RRID: SCR_014212 | |
| Software | RStudio, code used for quantitative dispersion analysis | Rstudio | RRID: SCR_000432 | Version 3.4.1, manuscript in preparation |
| Software | ImageJ | NIH | RRID: SCR_003070 | |
| Software | PyMOL | PyMOL Molecular Graphics System, Schrödinger | RRID: SCR_000304 | Version 2.2.0 |
| Software | MODELLER | PMID: 8254673 | RRID: SCR_0083595 | Version 9.18 |
| Software | AMBER 14 Package | PMID: 16200636 | RRID: SCR_014230 |
Additional files
-
Supplementary file 1
List of residue-residue distances for WT KIF5C in the apo versus ATP-bound states.
Differences in residue-residue distances based on MD simulations of tubulin-bound motors in the ATP-bound, post power stroke state.
- https://doi.org/10.7554/eLife.44146.022
-
Supplementary file 2
List of residue-residue distances for WT versus Latch mutant motors in the tubulin- and ATP-bound states.
Differences in residue-residue distances are based on MD simulations of tubulin-bound motors in the ATP-bound, post power stroke state.
- https://doi.org/10.7554/eLife.44146.023
-
Supplementary file 3
List of residue-residue distances for WT versus CNB+Latch mutant motors in the tubulin- and ATP-bound states.
Differences in residue-residue distances are based on MD simulations of tubulin-bound motors in the ATP-bound, post power stroke state.
- https://doi.org/10.7554/eLife.44146.024
-
Supplementary file 4
List of structures used for PCA analysis.
- https://doi.org/10.7554/eLife.44146.025
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Transparent reporting form
- https://doi.org/10.7554/eLife.44146.026
