Figure 6.
In vitro analysis of KHC motility. (A) Diagram of in vitro motility experiment. mNG::KHC (red) and Halo::PLP584-1811 (green) were transfected into S2 cells. Cleared lysate was then flowed onto MTs (blue) for TIRF analysis. (B) Co-migration of mNG-KHC (green) with Halo-PLP584-1811 (red) on HiLyte-647 MTs (blue). (C) Velocities of KHC motors and PLP584-1811 cargos on MTs. Kinesin mean velocity = 202 (± 113, SD) nm/s (n = 1,051). PLP584-1811 mean velocity = 177 (± 115, SD) nm/s (n = 395). Mann-Whitney test P value = <0.0001, ****. (D) Characteristic run lengths of KHC motors and PLP584-1811 cargo. PLP584-1811 run length = 3.1 μm, (n = 1,051). KHC run length = 3.7 μm (n = 395). Mann-Whitney test P value = 0.08, ns. (E) Quantification of the landing rates of 25 nM KHC + LC with PLP584-1811 at indicated concentrations in comparison with that of 25 nM KHCΔh2 + LC. Error bars represent the SD of landing rates determined from three or four independent movies. For each condition, several thousand landing events were quantified on a total MT length of several millimeters. Unpaired, two tailed t test P value = <0.001 (**), KHCΔh2 + LC vs. KHC + LC + PLP (at varying concentrations). (F) Kymographs showing landing and movement of KHC on MTs in the absence and presence of PLP and compared with the activated KHCΔh2. (G) Steady-state ATPase activities of different Kinesin-1 constructs in the presence or absence of 2 μM PLP584-1811 as a function of MT concentration. Data were presented as mean ± SD and fitted with Michaelis-Menten kinetics. N = 3 independent titrations. (H) Steady-state analysis of Kinesin-1 to PLP binding affinity using biolayer interferometry (BLI). KHC, KHC + LC, or KHCΔh2 + LC were loaded onto the biosensors and exposed to WT PLP584-1811 or PLP584-1811PD mutant at the indicated concentrations. BLI signals at equilibrium were plotted against PLP concentrations, and dissociation constant (Kd) were determined by fitting the data with nonlinear regression single-site binding: WT PLP584-1811 vs. KHC, 4.4 μM; vs. KHC + LC, 1.7 μM; vs. KHCΔh2 + LC, 65 nM; PLPPD vs. KHCΔh2 + LC, 281 nM. (I) Kymograph analysis showing events of PLP584-1811 comigrating with KHCΔh2 + LC. (J) Diagram highlighting how the interaction between the motor domain and cargo binding tail could inhibit PLP interaction.

In vitro analysis of KHC motility. (A) Diagram of in vitro motility experiment. mNG::KHC (red) and Halo::PLP584-1811 (green) were transfected into S2 cells. Cleared lysate was then flowed onto MTs (blue) for TIRF analysis. (B) Co-migration of mNG-KHC (green) with Halo-PLP584-1811 (red) on HiLyte-647 MTs (blue). (C) Velocities of KHC motors and PLP584-1811 cargos on MTs. Kinesin mean velocity = 202 (± 113, SD) nm/s (n = 1,051). PLP584-1811 mean velocity = 177 (± 115, SD) nm/s (n = 395). Mann-Whitney test P value = <0.0001, ****. (D) Characteristic run lengths of KHC motors and PLP584-1811 cargo. PLP584-1811 run length = 3.1 μm, (n = 1,051). KHC run length = 3.7 μm (n = 395). Mann-Whitney test P value = 0.08, ns. (E) Quantification of the landing rates of 25 nM KHC + LC with PLP584-1811 at indicated concentrations in comparison with that of 25 nM KHCΔh2 + LC. Error bars represent the SD of landing rates determined from three or four independent movies. For each condition, several thousand landing events were quantified on a total MT length of several millimeters. Unpaired, two tailed t test P value = <0.001 (**), KHCΔh2 + LC vs. KHC + LC + PLP (at varying concentrations). (F) Kymographs showing landing and movement of KHC on MTs in the absence and presence of PLP and compared with the activated KHCΔh2. (G) Steady-state ATPase activities of different Kinesin-1 constructs in the presence or absence of 2 μM PLP584-1811 as a function of MT concentration. Data were presented as mean ± SD and fitted with Michaelis-Menten kinetics. N = 3 independent titrations. (H) Steady-state analysis of Kinesin-1 to PLP binding affinity using biolayer interferometry (BLI). KHC, KHC + LC, or KHCΔh2 + LC were loaded onto the biosensors and exposed to WT PLP584-1811 or PLP584-1811PD mutant at the indicated concentrations. BLI signals at equilibrium were plotted against PLP concentrations, and dissociation constant (Kd) were determined by fitting the data with nonlinear regression single-site binding: WT PLP584-1811 vs. KHC, 4.4 μM; vs. KHC + LC, 1.7 μM; vs. KHCΔh2 + LC, 65 nM; PLPPD vs. KHCΔh2 + LC, 281 nM. (I) Kymograph analysis showing events of PLP584-1811 comigrating with KHCΔh2 + LC. (J) Diagram highlighting how the interaction between the motor domain and cargo binding tail could inhibit PLP interaction.

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