Tubulins show susceptibility to magnetic nanoparticle interaction.

Abstract

Low piconewton forces can induce axonal specification and outgrowth in neurons. However, the secondary mechanism underlying force-mediated axon specification is poorly understood. Within the axon shaft, microtubules, composed of α and β tubulin dimers, are stabilized by the protein Tau. These microtubules polymerize toward the axonal tip, where actin provides a tensile force against the membrane to maintain or extend the structure of the tip. We hypothesize that exogenous forces manipulate one or more of these structural proteins, which could induce axonal lineage. Our previous work has shown that hTau40, the brain's most ubiquitous isoform of Tau, can be directly dragged by internalized magnetic nanoparticles (MNPs) within primary neurons. Here, we sought to establish whether this direct relationship between the nanoparticles and Tau applied to other axonal structural proteins. We transduced (BacMam) dissociated primary cortical neurons (rat, E18) at the day in vitro two (DIV 2) with one of three vectors containing a gene for a fluorescently labeled structural protein: α-tubulin-mCherry, β-tubulin-GFP, or β-actin-GFP. At DIV 4, the neurons were incubated for 24 h with starch-NH2-coated, far-red fluorescent magnetic nanoparticles (d: 100 nm) at 10 µg/mL. Cells were then washed and imaged using a Leica DMi8 inverted fluorescent microscope to determine whether there was high colocalization, indicating direct interaction, between the MNPs and the fluorescent proteins. Compared to the high-intensity protein spots, α-tubulin-mCherry showed the greatest percentage of correlated MNP clusters (45.7 ± 6.8 %, n = 11 cells), followed by β-tubulin-GFP, with 36.9 ± 11.7 % (n = 6) of high protein spots correlated to MNP clusters. Only 8.27 ± 5.32% (n = 7) of β-actin-GFP spots correlating to MNP clusters. These preliminary data suggest tubulins may be more susceptible to direct nanomagnetic force manipulation (mean ± SEM).