Direct imaging of intraflagellar-transport turnarounds reveals that motors detach, diffuse, and reattach to opposite-direction trains

Intraflagellar transport (IFT), a bidirectional intracellular transport mechanism in cilia, relies on the cooperation of kinesin-2 and IFT-dynein motors. In Caenorhabditis elegans chemosensory cilia, motors undergo rapid turnarounds to effectively work together in driving IFT. Here, we push the envelope of fluorescence imaging to obtain insight into the underlying mechanism of motor turnarounds. We developed an alternating dual-color imaging system that allows simultaneous single-molecule imaging of kinesin-II turnarounds and ensemble imaging of IFT trains. This approach allowed direct visualization of motor detachment and reattachment during turnarounds and accordingly demonstrated that the turnarounds are actually single-motor switching between opposite-direction IFT trains rather than the behaviors of motors moving independently of IFT trains. We further improved the time resolution of single-motor imaging up to 30 ms to zoom into motor turnarounds, revealing diffusion during motor turnarounds, which unveils the mechanism of motor switching trains: detach–diffuse–attach. The subsequent single-molecule analysis of turnarounds unveiled location-dependent diffusion coefficients and diffusion times for both kinesin-2 and IFT-dynein motors. From correlating the diffusion times with IFT train frequencies, we estimated that kinesins tend to attach to the next train passing in the opposite direction. IFT-dynein, however, diffuses longer and lets one or two trains pass before attaching. This might be a direct consequence of the lower diffusion coefficient of the larger IFT-dynein. Our results provide important insights into how motors can cooperate to drive intracellular transport.

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Single-Molecule Analysis of Intraflagellar Transport in Live Chlamydomonas Cells

Biophysical Journal ◽

10.1016/j.bpj.2010.12.269 ◽

2011 ◽

Vol 100 (3) ◽

pp. 11a ◽

Cited By ~ 1

Author(s):

Sheng-Min Shih ◽

Fatih Kocabas ◽

Ahmet Yildiz

Keyword(s):

Single Molecule ◽

Intraflagellar Transport ◽

Single Molecule Analysis

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Roles of motor on-rate and cargo mobility in intracellular transport

10.1101/2020.07.13.201434 ◽

2020 ◽

Cited By ~ 1

Author(s):

Matthew J. Bovyn ◽

Babu J.N. Reddy ◽

Steven P. Gross ◽

Jun F. Allard

Keyword(s):

Modeling And Simulation ◽

Single Molecule ◽

Molecular Motors ◽

Intracellular Transport ◽

Microtubule Associated Proteins ◽

Single Motor ◽

Two Factors ◽

Associated Proteins ◽

Binding Time ◽

The Difference

AbstractMolecular motors like kinesin are critical for cellular organization and biological function including in neurons. There is detailed understanding of how they move and how factors such as applied force and the presence of microtubule-associated proteins can alter this single-motor travel. In order to walk, the cargo-motor complex must first attach to a microtubule. This attachment process is less studied. Here, we use a combination of single-molecule bead experiments, modeling, and simulation to examine how cargos with kinesin-1 bind to microtubules. In experiment, we find that increasing cargo size and environment viscosity both signficantly slow cargo binding time. We use modeling and simulation to examine how the single motor on rate translates to the on rate of the cargo. Combining experiment and modeling allows us to estimate the single motor on rate as 100 s−1. This is a much higher value than previous estimates. We attribute the difference between our measurements and previous estimates to two factors: first, we are directly measuring initial motor attachment (as opposed to re-binding of a second motor) and second, the theoretical framework allows us to account for missed events (i.e. binding events not detected by the experiments due to their short duration). This indicates that the mobility of the cargo itself, determined by its size and interaction with the cytoplasmic environment, play a previously underestimated role in determining intracellular transport kinetics.

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