Dynactin p150 promotes processive motility of DDB complexes by minimizing diffusional behavior of dynein Public

ABSTRACT: Cytoplasmic dynein is activated by forming a complex with dynactin and the adaptor protein BicD2. We used interferometric scattering (iSCAT) microscopy to track dynein–dynactin–BicD2 (DDB) complexes in vitro and developed a regression-based algorithm to classify switching between processive, diffusive, and stuck motility states. We find that DDB spends 65% of its time undergoing processive stepping, 4% undergoing 1D diffusion, and the remaining time transiently stuck to the microtubule. Although the p150 subunit was previously shown to enable dynactin diffusion along microtubules, blocking p150 enhanced the proportion of time DDB diffused and reduced the time DDB processively walked. Thus, DDB diffusive behavior most likely results from dynein switching into an inactive (diffusive) state, rather than p150 tethering the complex to the microtubule. DDB–kinesin-1 complexes, formed using a DNA adapter, moved slowly and persistently, and blocking p150 led to a 70 nm/s plus-end shift in the average velocity of the complexes, in quantitative agreement with the shift of isolated DDB into the diffusive state. The data suggest a DDB activation model in which dynactin p150 enhances dynein processivity not solely by acting as a diffusive tether that maintains microtubule association, but rather by acting as an allosteric activator that promotes a conformation of dynein optimal for processive stepping. In bidirectional cargo transport driven by the opposing activities of kinesin and dynein–dynactin–DDB, the dynactin p150 subunit promotes retrograde transport and could serve as a target for regulators of transport.

README

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General Information


Title:

Dynactin p150 promotes processive motility of DDB complexes by minimizing diffusional behavior of dynein

Author Information:

  • Principal Investigator: William O. Hancock

Date of data collection/creation:

2018-2019

Geographic location of data collection:

Hallowell building
Penn State University

Sharing/Access Information


License & restrictions on data reuse:

Creative Commons Attribution 4.0 International (CC BY 4.0)

Recommended citation for the data:

Dynactin p150 promotes processive motility of DDB complexes by minimizing diffusional behavior of dynein. Q Feng, AM Gicking, WO Hancock. Molecular Biology of the Cell 31 (8), 782-792
https://www.molbiolcell.org/doi/full/10.1091/mbc.E19-09-0495

Data & File Overview


Study design:
The research is designed to study the motility states of DDB (Dynein-Dynactin-BicD2) and the role of p150 (part of dynactin) in directing the motility states of DDB.
We used the antibody to block the function of p150 as the experimental group while the control group is without the treatment of antibody.

Parameters:
1. Percent of DDB in a processive, diffusive and stuck states
2. Switching rates between processive, diffusive and stuck states.

Measurements:
1. Microscope imaging and 2-D gaussian fitting to get the time series of position of the DDB molecule
2. Regression of time VS. position to get velocity (as slopes), standard deviation of the residuals and standard deviation of positions

Analysis:
1. Motility states of control and experimental groups are defined as follows:
using three cutoffs: Vcut, SDposcut, and SDrescut
Processive state (P): V > Vcut and SDres < SDrescut
Stuck state (S): V < Vcut and SDpos < SDpos
cut
Diffusive state (D) SD > SDposcut or SDres > SDrescut
2. Calculate the percent of time DDB is in a processive, diffusive and stuck states and calculate the switching rates between the three states.

Manual of the Matlab code:
1. Function of the code: The code is to detect the motility states for DDB molecules
2. Order of the code: The code should be used following the sequence from 1 to 3.
3. Input of the code: In code1, a mat file is required as the input. This mat file is usually generated by Fiesta; No more inputs are required other than the Fiesta file.
4. The output of the code:
Code 1 generates plots for every molecule. The plots showed:
Top left: traces with missing position filled by extrapolation
Top right: decomposition of traces into longitudinal and perpendicular to the microtubules (tracks)
Bottom left: velocity, velocity SD, SD residuals and SD position
Bottom right: kernel density of residuals
Code 2 generates plots for every molecule with motility states detected
Code 3 generates plots for every molecule with motility states detected while removes the segments that are shorter than the length of the window size.
Conclude all traces, code 3 returns the mean duration of processive, diffusive and stuck segment; and returns the number of switches detected.

Description of the data files:
Apolock: This is the data for the apolock experiment related to figure 3 and figure S1 C.
On the left is the DDB experiment and on the right is the Abp150 treatment group.
controlAntibody: This is the data to confirm the specificity of p150 antibody. We used a non-specific antibody to do the landing experiment compared with the landing results without antibody. This data is for figure S1.
Landing: This is the data for figure 2 and figure S1 B.
Dcompare: is for figure S8.
DDB_32: This is the mat file from Fiesta. It includes 32 traces for DDB motility. It is related to figure 5 and figure 6.
p150_32: This is the mat file from Fiesta. It includes 32 traces for DDB motility with the treatment of p150 antibody. It is related to figure 5 and figure 6.

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