Single molecule FRET observation of kinesin-1's head-tail interaction on microtubule

Abstract

Kinesin-1 (conventional kinesin) is a molecular motor that transports various cargo such as endoplasmic reticulum and mitochondria in cells. Its two head domains walk along microtubule by hydrolyzing ATP, while the tail domains at the end of the long stalk bind to the cargo. When a kinesin is not carrying cargo, its motility and ATPase activity is inhibited by direct interactions between the tail and head. However, the mechanism of this tail regulation is not well understood. Here, we apply single molecule fluorescence resonance energy transfer (smFRET) to observe this interaction in stalk-truncated kinesin. We found that kinesin with two tails forms a folding conformation and dissociates from microtubules, whereas kinesin with one tail remains bound to the micro-tubule and is immobile even in the presence of ATP. We further investigated the head-tail interaction as well as head-head coordination on the microtubule at various nucleotide conditions. From these results, we propose a two-step inhibition model for kinesin motility.

Keywords: autoinhibition; cargo transport; molecular motor; smFRET; tail-regulation.

Figure 1

Kinesin constructs and their ATPase activity. (A) A schematic drawing of the full-length kinesin heavy chain dimer. The three coiled coil regions are represented by the oscillating lines. Stars denote the position of the introduced cysteine residues used for dye labeling. Dashed lines indicate the truncated regions. (B) Schematic representation of the stalk-truncated kinesin constructs. Full-length kinesin consists of a motor head, neck-linker (NL), three coiled-coil regions (CC1–CC3), two hinges (H1, H2) and tail. Stalk-truncated kinesin constructs are variably truncated at the end of the stalk region. GFP was fused to the C-terminus of the tail construct (904–963), as too was a His₆-tag (or Strep-tag). Mutations are omitted in the schematics. (C) MT-stimulated ATPase activity of dimer constructs bound or unbound to cargo. Maximum ATPase (k_cat) was determined from Michaelis-Menten fits to the MT-stimulated ATPase activities versus MT concentrations at 1 mM ATP, 25°C. Solid bar represents k_cat without cargo, shaded bar represents k_cat with cargo (kinesin:cargo = 1:6). Percentages indicate the inhibition ratio (k_cat without cargo)/(k_cat with cargo). (D) MT-stimulated ATPase activity of monomer constructs (without cargo). Error bars represent±SE.

Figure 2

Kinesin motility. (A) Schematic representation of the experimental setup for single-molecule imaging of kinesin motility (not to scale). PLL-g-PEG (black wavy lines), κ-casein (black spheres), Pluronic F-127 (blue-short wavy lines) and Tween 20 (blue spheres) were used to prevent nonspecific adsorption of the tail to the glass surface. An axoneme is fixed nonspecifically to the PLL-g-PEG coated glass surface. (B) Typical kymograph for the tail-less construct K490CLM. (C) Typical kymographs for each tail-conjugated construct without (left) and with (right) cargo (kinesin:cargo = 1:6). Movements were observed at 1 mM ATP, 22°C. Scale bar, 4 μm (vertical), 10 s (horizontal). (D) Proportion of the motility modes for each construct (n = 178–459). See text for definitions of modes. (E) Typical kymograph for tail-GFP. Scale bar, 4 μm (vertical), 5 s (horizontal).

Figure 3

SmFRET observations of the head-tail interaction in two-tail kinesin. (A) Scheme of the experimental geometry for smFRET observations of 2T-K490CLM with the 215–964 sensor (not to scale). His₆-tagged kinesin is fixed onto the glass surface via a penta-His anti-body (Y shape) immobilized with biotin-Neutravidin linkages. (B) Histograms of smFRET efficiencies from each frame of the images. (C) 2T-K490CLM smFRET efficiencies at 200 mM KCl and (D) at 800 mM KCl. Values in parentheses are the number of analyzed molecules.

Figure 4

SmFRET observations of the head-head configuration and head-tail interaction in one-tail kinesin. (A) Histograms of smFRET efficiencies (from each frame) for the 215–43 sensor of 1T-K490CLM, (B) 215–964 sensor of 1T-K490CLM and (C) 43–964 sensor of 1T- K490CLM at 50 U/ml apyrase (top), 75 nM ADP (middle) and 1 mM ATP (bottom). Values in parentheses are the number of analyzed molecules.

Figure 5

A two-step model for the tail regulation of kinesin. (A) Without cargo, kinesin heads cannot bind to MT due to interactions within their respective tails. (B) When a cargo binds to the tail, the interaction is relieved and kinesin binds to and walks along the micro-tubule. (C) When the cargo detaches from the tail, kinesin can still walk along MT. (D) Just after this detachment, one tail binds to the detached head in (C), which prevents further ATP hydrolysis and motility. The other tail then binds to the other head causing kinesin to detach from MT, returning to state (A).