DOI: 10.5061/dryad.44j0zpcvz
title: Enhanced processivity and collective force production of kinesin-1 at low radial forces

description: Kinesin-1 is a robust motor that carries intracellular cargos towards the
 plus ends of microtubules. However, optical trapping studies reported that
 kinesin-1 is a slippery motor that quickly detaches from the microtubule,
 and multiple kinesins are incapable of teaming up to generate large
 collective forces. This may be due to the vertical (z) forces that the
 motor experiences in a single bead trapping assay, accelerating the
 detachment of the motor from a microtubule. Here, we substantially lowered
 the z-force by using a long DNA handle between the motor and the trapped
 bead and characterized the motility and force generation of single and
 multiple kinesin-1s. Contrary to previous views, we show that kinesin-1 is
 a robust motor that resists microtubule detachment before it reaches high
 hindering forces, but it quickly detaches under assisting forces even at
 low z-forces. We also demonstrate highly efficient collective force
 generation by multiple kinesin-1 motors. These results provide an
 explanation for how multiple kinesins team up to perform cellular
 functions that require higher forces than a single motor can bear.
description: # Enhanced processivity and collective force production of kinesin-1 at
 low radial forces Dataset DOI:
 [10.5061/dryad.44j0zpcvz](https://doi.org/10.5061/dryad.44j0zpcvz) ##
 Description of the data and file structure **The data falls into 3 broad
 categories:** Optical Trapping TIRF Microscopy Refeyn 
 
 **Refeyn:** The raw Refeyn data was taken on a TwoMP mass photometer, and
 the calibrated files has been exported in the .csv format. **TIRF:** The
 TIRF microscopy data covers three experiments. In the first, oligo-labeled
 FL KIF5B is mixed with a complementary fluorescent DNA oligo and allowed
 to walk on microtubules decorated with MAP7. The 488 channel is KIF5B-GFP,
 and the other channel is the Cy3 oligo. This data was used exclusively for
 illustrative purposes and was not analyzed. In the second, FL KIF5B is
 attached to streptavidin quantum dots via a DNA handle and allowed to walk
 on axonemes decorated with MAP7. This assay was used as a control for the
 DNA handle attachment system and to measure the velocity and run length of
 KIF5B on axonemes. Lastly, k560-GFP was allowed to walk on axonemes in the
 absence of MAP7. These movies were used only to measure the velocity and
 run length of k560 on axonemes. **Optical Trapping:** Two types of optical
 trapping experiments were performed. In one type, motors were allowed to
 walk against a fixed trap. These experiments were performed for k560
 attached directly to a bead, k560 attached to a bead via a DNA tether, FL
 KIF5B attached to a bead via a DNA tether, and chassis experiments where
 two or three copies of k560 were attached to a DNA chassis which was then
 either directly attached to the bead or via a DNA tether. This data is
 presented as .csv files where one column each corresponds to time, force
 parallel to the axoneme, trap position perpendicular to the axoneme, force
 perpendicular to the axoneme, and trap position parallel to the axoneme.
 In the other type, the trap position was updated to provide a fixed load.
 These experiments were performed for k560 attached directly to beads and
 FL KIF5B attached via a DNA tether. This data is presented as .csv files,
 where one column each corresponds to time, displacement along the axoneme,
 trap position perpendicular to the axoneme (always zero), displacement
 perpendicular to the axoneme, and trap position along the axoneme. ###
 Files and variables #### File: Raw_Data_for_Z-Force_Project.zip
 **Description:** Raw data for entire publication 
 
 **Fixed Trap:** All files are called 'TraceX.csv' and contain
 time in seconds (first column), force along the axis of the axoneme in pN
 (column 2), trap position along the axis perpendicular to the axoneme in
 nm (column 3), force along the axis perpendicular to the axoneme in pN
 (column 4), and trap position along the axis parallel to the axoneme in nm
 (column 5). Since this is a fixed trap assay, the values in columns 3 and
 5 are always zero. \--&gt;Chassis \--&gt;--&gt;High Z-Force 2 Binding
 Sites 3 Binding Sites \--&gt;--&gt;Low Z-Force 1 Binding Site 2 Binding
 Sites 3 Binding Sites \--&gt;Single Motor \--&gt;--&gt;High Z-Force
 \--&gt;--&gt;Low Z-Force **Force Feedback:** All files are called
 'TraceX.csv' and contain time in seconds (first column),
 displacement along the axis of the axoneme in nm (column 2), trap position
 along the axis perpendicular to the axoneme in nm (column 3), displacement
 along the axis perpendicular to the axoneme in nm(column 4), and trap
 position along the axis parallel to the axoneme in nm (column 5). Since
 force feedback is only performed on the axis of the axoneme, the values in
 column 3 are always zero. \--&gt;High Z-Force +0.5 pN +2 pN +3 pN +4 pN -1
 pN -2 pN -4 pN -6 pN -10 pN \--&gt;Low Z-Force +0.5 pN +2 pN +3 pN +4 pN
 -1 pN -2 pN -4 pN -6 pN -10 pN **Refeyn:** All files are .csv format. The
 first column is the frame. Note that most frames have more than one
 particle. Columns 2 and 3 are the x- and y- coordinates of a particle,
 respectively, sub-pixel localized from a Gaussian fit of the interference
 pattern. Column 4 is the change in light intensity at the particle
 (unitless ratio). The fifth column is the fitted molecular weight in kD.
 The sixth column is the fit error in kD. The seventh column is error
 related to a differential blurring filter, which was not used. As such,
 the seventh column is uniformly nan. The eighth column is nearest neighbor
 distance in pixels. The ninth column is a Boolean for whether or not the
 particle is chosen for analysis, which always has a value of 1.
 \--&gt;High Z - 2 Binding Sites \--&gt;High Z - 3 Binding Sites
 \--&gt;Kinesin Alone \--&gt;Low Z - 2 Binding Sites \--&gt;Low Z - 3
 Binding Sites **TIRF:** All files are in .tif format. \--&gt;FL KIF5B
 \--&gt;--&gt;Handle Control w QDots on Axonemes (pixel size 43 nm)
 \--&gt;--&gt;Oligo Label Control w Complementary Oligo on MTs (pixel size
 160 nm) \--&gt;K560 \--&gt;--&gt;Motility on Axonemes (pixel size 160 nm)
 ## Code/software ImageJ is commonly used to analyze .tif files. Users may
 find custom MATLAB software helpful for analyzing force feedback
 measurements
 ([https://github.com/Yildiz-Lab/ForceFeedbackAutoAnalyzer](https://github.com/Yildiz-Lab/ForceFeedbackAutoAnalyzer)). ## Access information Other publicly accessible locations of the data: * 
 
 Data was derived from the following sources: *