Rotational dynamics of cargos at pauses during axonal transport

Abstract

Direct visualization of axonal transport in live neurons is essential for our understanding of the neuronal functions and the working mechanisms of microtubule-based motor proteins. Here we use the high-speed single particle orientation and rotational tracking technique to directly visualize the rotational dynamics of cargos in both active directional transport and pausing stages of axonal transport, with a temporal resolution of 2 ms. Both long and short pauses are imaged, and the correlations between the pause duration, the rotational behaviour of the cargo at the pause, and the moving direction after the pause are established. Furthermore, the rotational dynamics leading to switching tracks are visualized in detail. These first-time observations of cargo's rotational dynamics provide new insights on how kinesin and dynein motors take the cargo through the alternating stages of active directional transport and pause.

Correlative live-cell and superresolution microscopy reveals cargo transport dynamics at microtubule intersections

“…Lysosome pauses and slowly passes through the first intersection (frames 1-4) and pauses for an extended period at the second intersection (frames 4-6). pausing, which might correspond to the rearrangement of motors on the microtubule surface (22). Future studies that combine high-precision 3D tracking with superresolution microscopy can potentially reveal whether motors rearrange on the microtubule surface at intersections as opposed to other possibilities (cargo squeezing, microtubule deformation, etc.…”

The development of optical microscopy techniques for the advancement of single-particle studies

“…43 Since then TIRS microscopy has been used to measure colloidal and hydronamic forces, [44][45][46] and eventually became used to monitor the cellular uptake of sub-micron non-fluorescent particles. 47 Recently TIRS microscopy has been applied to single metallic nanoparticle orientation determination.…”

“…The intensity ratio between the bright and dark part of the particle have a periodic cos 4 and sin 4 relationship, respectively that can be used to monitor rotational dynamics on a cell membrane, 45 and investigate the rotational motions of cargo during intracellular transport. 46 While the DIC configuration previously described is an ideal microscope to study systems on transparent substrates, most nanoparticles and nanostructures created through lithographic techniques are made on reflective surfaces such as silicon wafers. In 1979 Lessor et.…”

Multishell Au/Ag/SiO2 Nanorods with Tunable Optical Properties as Single Particle Orientation and Rotational Tracking Probes

“…S ingle particle tracking (SPT) is a powerful tool to study the dynamics of cellular and molecular processes, such as membrane dynamics, 1,2 viral infection, 3 and intracellular transport. 4,5 A large collection of imaging probes, including fluorescent molecules, 6,7 quantum dots, 8,9 and plasmonic nanoparticles, 5,10−16 have been visualized with the aid of various optical microscopy techniques. While it has become a routine practice in SPT experiments to identify the trajectory of an imaging probe, it is more difficult to resolve the dipole orientation of the probe in real time, which may be essential in understanding the underlying biological functions.…”

“…The relative bright and dark intensity measurement errors were increased from ∼1% at 50 ms to ∼5% at 0.5 ms for both nanorods (Table S1 in the Supporting Information). Because of the LSPR enhancement due to the silver coating, the core− shell hybrid nanorods were detectable at a temporal resolution as fast as 0.5 ms, which is approximately an order of magnitude faster than the previously reported fastest temporal resolution of 2−5 ms. 5,19 A 360°rotation study of the Au/Ag/SiO 2 −NRs was carried out by rotating the sample stage with 10°increments to position the nanorods in different orientations while exciting at the longitudinal LSPR wavelength of 563 nm. The orientation angle φ was defined as the angle between the long axis of the nanorod and the "dark" polarization direction of the DIC microscope ( Figure S5A in the Supporting Information).…”