Nano-Particles Carried by Multiple Dynein Motors Self-Regulate Their Number of Actively Participating Motors

Abstract

Intra-cellular active transport by native cargos is ubiquitous. We investigate the motion of spherical nano-particles (NPs) grafted with flexible polymers that end with a nuclear localization signal peptide. This peptide allows the recruitment of several mammalian dynein motors from cytoplasmic extracts. To determine how motor–motor interactions influenced motility on the single microtubule level, we conducted bead-motility assays incorporating surface adsorbed microtubules and combined them with model simulations that were based on the properties of a single dynein. The experimental and simulation results revealed long time trajectories: when the number of NP-ligated motors Nm increased, run-times and run-lengths were enhanced and mean velocities were somewhat decreased. Moreover, the dependence of the velocity on run-time followed a universal curve, regardless of the system composition. Model simulations also demonstrated left- and right-handed helical motion and revealed self-regulation of the number of microtubule-bound, actively transporting dynein motors. This number was stochastic along trajectories and was distributed mainly between one, two, and three motors, regardless of Nm. We propose that this self-regulation allows our synthetic NPs to achieve persistent motion that is associated with major helicity. Such a helical motion might affect obstacle bypassing, which can influence active transport efficiency when facing the crowded environment of the cell.

Smart-design of universally decorated nano-particles for drug delivery applications driven by active transport

“…A single component is added at each step (Figure 1A). 66 The NP core consists of a carboxyl polystyrene (PS-COOH) microsphere of defined diameter. In this work we use both 40 and 200 nm diameter microspheres.…”

“…Having confirmed the proper integration of the various added components, we now turn to quantify the amount of recruited dynein motors per NP. Recruitment of the mammalian dynein motor proteins machinery, which is mediated by NLS, 66 is achieved by incubating the PEG-NLS-coated NPs in Hela cytoplasmic cells extract (CE). 74,75 The amount of recruited dynein motors, which we expect to depend on the number of bound NLSs, and thus on [NLS], is quantified using WB assays.…”

“…Since many PEG-NLSs cover the NP surface, even if just a fraction of them recruit dynein, the intracellular run-length and run-time for the NP motion over an MT are expected to be greatly improved, as we have showen recently. 66 Therefore, if the NP core will be made from a bio-compatible drug carrier material (instead of the polystyrene used here), it is high-likely that the drug delivery to the nucleus will be amplified.…”

“…66 Green symbols and connecting line: model simulations results for the mean longitudinal velocity against the number of NP-bound motors taken from Ref. 66 tached by Trypsin-EDTA solution, washed with Phosphate Buffered Saline (PBS) (10 mM Phosphate buffer, 2.7 mM KCl, 137 mM NaCl, pH = 7.4), and pelleted for 5 min at RT and 500 g. The cells were then incubated on ice with a lysis buffer (12 mM Pipes, 2 mM MgCl 2 , 1 mM EGTA, 0.4 % Triton X-100, 10 % protease inhibitor cocktail, pH = 6.8) for 15 min. Finally, the lysates were cleared by centrifugation at 2,700 g and then at 100,000 g, Nuclear Localisation Signal (NLS) peptide.…”

“…As we have shown previously, our designed universally decorated nano-particles, with multiple binding sites for dynein motors, can move actively towards the MT-minus end for distances that are much longer than the single dynein run-length, thereby enhancing the NP transport towards the nucleus. 66…”