Figure 4.
Figure 4. Complexes of two kinesin-1 motors show minimal cooperative behavior. (A) Schematic of the experimental setup. COS7 cells were transfected with plasmids for self-assembly of kin1-mNeGr on the scaffold by the split GFP linker (Transfection 1) or for expression of kin1-2×mCh (Transfection 2). The cell lysates were mixed and the kin1-2×mCh component was recruited to the kin1-splitGFP-scaffold via the split EF Hand linker. (B–D) Motors in lysates were imaged by single molecule microscopy at saturating ATP (2 mM). (B) Representative kymographs. In the absence of scaffold, only individual red or green events are observed. In the presence of scaffold, the colocalizing red and green events (yellow) represent assembled two-motor complexes. Time is on the x axis (bar, 1 s) and distance is on the y axis (bar, 1 µm). The “No scaffold” data are shown again in Fig. S4 A. (C and D) The run lengths (C) and velocities (D) were determined for single kin1-mNeGr motors in the absence of scaffold (green bars, n = 622 events) and for two-color two-motor events in the presence of scaffold (yellow bars, n = 318 events). The population data from two independent experiments were plotted in histograms. The inset in the run length graph shows the same data fit to a CDF. (E) Probability densities for the experimentally obtained velocities of one motor (kin1-mNeGr, green dotted line, n = 622 events) and two-motor (kin1-mNeGr + kin1-mCh, yellow dotted line, n = 1,454) events. Solid lines show fits to theoretically derived distribution functions for two-motor motility events driven by only one motor (green line), both motors (blue line), or a mixed state where either one or two motors can contribute (yellow line). (F–H) Motors in lysates were imaged by single molecule microscopy at limiting ATP (20 µM). (F) Representative kymographs. Time is on the x axis (bar, 1 min) and distance is on the y axis (bar, 5 µm). (G and H) The run lengths (G) and velocities (H) were determined for single kin1-mNeGr motors in the absence of scaffold (green bars, n = 840 events) and for two-color two-motor events in the presence of scaffold (yellow bars, n = 116 events). The population data from two independent experiments were plotted in histograms. The inset in the run length graph shows the same data fit to a CDF.

Complexes of two kinesin-1 motors show minimal cooperative behavior. (A) Schematic of the experimental setup. COS7 cells were transfected with plasmids for self-assembly of kin1-mNeGr on the scaffold by the split GFP linker (Transfection 1) or for expression of kin1-2×mCh (Transfection 2). The cell lysates were mixed and the kin1-2×mCh component was recruited to the kin1-splitGFP-scaffold via the split EF Hand linker. (B–D) Motors in lysates were imaged by single molecule microscopy at saturating ATP (2 mM). (B) Representative kymographs. In the absence of scaffold, only individual red or green events are observed. In the presence of scaffold, the colocalizing red and green events (yellow) represent assembled two-motor complexes. Time is on the x axis (bar, 1 s) and distance is on the y axis (bar, 1 µm). The “No scaffold” data are shown again in Fig. S4 A. (C and D) The run lengths (C) and velocities (D) were determined for single kin1-mNeGr motors in the absence of scaffold (green bars, n = 622 events) and for two-color two-motor events in the presence of scaffold (yellow bars, n = 318 events). The population data from two independent experiments were plotted in histograms. The inset in the run length graph shows the same data fit to a CDF. (E) Probability densities for the experimentally obtained velocities of one motor (kin1-mNeGr, green dotted line, n = 622 events) and two-motor (kin1-mNeGr + kin1-mCh, yellow dotted line, n = 1,454) events. Solid lines show fits to theoretically derived distribution functions for two-motor motility events driven by only one motor (green line), both motors (blue line), or a mixed state where either one or two motors can contribute (yellow line). (F–H) Motors in lysates were imaged by single molecule microscopy at limiting ATP (20 µM). (F) Representative kymographs. Time is on the x axis (bar, 1 min) and distance is on the y axis (bar, 5 µm). (G and H) The run lengths (G) and velocities (H) were determined for single kin1-mNeGr motors in the absence of scaffold (green bars, n = 840 events) and for two-color two-motor events in the presence of scaffold (yellow bars, n = 116 events). The population data from two independent experiments were plotted in histograms. The inset in the run length graph shows the same data fit to a CDF.

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