Associate Professor Montana State University Bozeman, Montana, United States
Introduction:: Numerous studies have shown that applying a force on a neuron can produce and direct the growth of an axon, which is the neurite responsible for transmitting messages to subsequent cells [1-5]. This finding has been replicated by applying tension on the exterior of cells [1,2] by pulling with microneedles, as well as applying pressure from the interior [4,5] using magnetic nanoparticles (MNPs) pulled by magnetic fields (nanomagnetic forces, NMF). The ability to engineer axons is particularly interesting to biomedical engineers who want to arrange neuronal circuits to replace neural tissues, returning cognition or motor function lost to disease and injury. However, the underlying mechanism by which an axon is generated by these forces, rather than a dendrite, is currently unknown [6].
This study sought to examine how NMF might influence the dynamics of particular structural proteins within the axon. The main cytoskeletal component, microtubules (MT), comprise α- and β-tubulin subunits and are oriented with their growing end and β-tubulin subunits toward the axon tip [7]. Tau is an axon-specific MT-associated protein that helps maintain structure and intracellular transport along MTs [8]. Finally, actin is a protein that exerts a force on the tip of neurites, preserving the shape of the tip and aiding in growth [9]. We hypothesized that NMFs might be manipulating the transport dynamics of one or more of these proteins, which are central to the specification and elongation of the axon, thus enabling the engineered growth of that neurite.
Materials and Methods:: To examine the magneto-mechanical manipulation of structural protein dynamics within neurons, fluorescently tagged proteins were imaged in primary neurons at high magnification in the absence or presence of cell-internal NMF.
Embryonic day 18 (E18) cortical neurons were dissociated from whole brains (Transnetyx), according to [4]. Cells were seeded on 35-mm PLL-treated glass-bottom Petri dishes (day in vitro (DIV) = 0). On DIV 2, each plate was transduced with one of the following BacMam probes (Montana Molecular): hTau40-tdTomato (2.5E3 VG/cell), hα-tubulin-mCherry (2.6E4 VG/cell), hβ-tubulin-GFP (8.8E3 VG/cell), or hβ-actin-GFP (1.8E4 VG/cell) for 24 hours. Cell media was then exchanged every two days. Cells were incubated with 10 μg/mL starch-NH2-coated fluorescent MNPs (MicroMod) for 12 hours on DIV 8. On DIV 9, cells were imaged using a Leica DMi8 fluorescent microscope at 1000x total magnification at a 0.5 fps frame rate for one minute. Cells were imaged either in the absence or presence of a spatially variable static magnetic field using the ForceApplicator platform (NanoMagnetic Solutions). GFP proteins were imaged using a GFP filter (Exposure = 500 ms), mCherry (100 ms) and tdTomato (500 ms) with TXR filter, and far-red MNPs (300 ms) with Y5 filter.
The movement of proteins was tracked using FIJI ImageJ Manual Tracker software. The magnitude and direction of forces were calculated by first tracking 8.3 μm magnetic microparticles in the exact field location where cells had been imaged. This magnitude was then scaled to the nanoparticle force using the formula in [4].
Results, Conclusions, and Discussions:: To examine how nanomagnetic forces may be impacting the dynamics of each of these proteins, at least 30 protein puncta were tracked, moving in either the anterograde (away from the soma) or retrograde (toward the soma) direction in each cell (n ≥ 60 puncta/cell). We then calculated the mean velocity of protein puncta moving in each direction within each cell. We established a ratio between each cell's mean anterograde velocity and the mean retrograde velocity of the tracked proteins (A/R); if A/R > 1, dynamics were biased in the anterograde direction, whereas A/R < 1 indicated that dynamics were limited in the retrograde movement.
Our findings indicate that hTau40 and β-tubulin dynamics appear particularly susceptible to internally applied mechanical forces above a threshold of 1.5 pN (Figure 1). Beyond this threshold, the dynamics of these proteins shifted increasingly in the anterograde direction as the magnitude of force increased. β-tubulin showed a particularly linear trend of increasingly anterograde-directed dynamics, where hTau40 exposed to > 1.5 pN generally showed more anterograde-biased dynamics than proteins exposed to forces below that threshold. No proteins showed distinct trends below this threshold.
Acknowledgements (Optional): : This work is supported by the National Institute of Aging at NIH (A.K., Grant# 1R21AG071691 ). AK and ML declare inventorship on a provisional US patent application No. 63/446,770 (Prov #2) and co-foundership of NanoMagnetic Solutions, Inc. No financial support was received from NanoMagnetic Solutions, Inc. for this research study.
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