Promoting filopodial elongation in neurons by membrane-bound magnetic nanoparticles

doi:10.1016/j.nano.2014.11.011

Nanomedicine: Nanotechnology, Biology and Medicine

Volume 11, Issue 3, April 2015, Pages 559-567

https://doi.org/10.1016/j.nano.2014.11.011 Get rights and content

Abstract

Filopodia are 5-10 μm long processes that elongate by actin polymerization, and promote axon growth and guidance by exerting mechanical tension and by molecular signaling. Although axons elongate in response to mechanical tension, the structural and functional effects of tension specifically applied to growth cone filopodia are unknown. Here we developed a strategy to apply tension specifically to retinal ganglion cell (RGC) growth cone filopodia through surface-functionalized, membrane-targeted superparamagnetic iron oxide nanoparticles (SPIONs). When magnetic fields were applied to surface-bound SPIONs, RGC filopodia elongated directionally, contained polymerized actin filaments, and generated retrograde forces, behaving as bona fide filopodia. Data presented here support the premise that mechanical tension induces filopodia growth but counter the hypothesis that filopodial tension directly promotes growth cone advance. Future applications of these approaches may be used to induce sustained forces on multiple filopodia or other subcellular microstructures to study axon growth or cell migration.

From the Clinical Editor

Mechanical tension to the tip of filopodia is known to promote axonal growth. In this article, the authors used superparamagnetic iron oxide nanoparticles (SPIONs) targeted specifically to membrane molecules, then applied external magnetic field to elicit filopodial elongation, which provided a tool to study the role of mechanical forces in filopodia dynamics and function.

Graphical Abstract

What is the role of mechanical tension in promoting filopodia elongation and axon growth? Here we induce filopodia elongation using superparamagnetic iron oxide nanoparticles (SPIONs) functionalized with molecules that bind neuronal membranes and direct SPIONs to the neuronal growth cone. In response to applied magnetic fields, SPIONs exerted mechanical tension at the filopodia tip and induced filopodia elongation. This technique permitted us to answer questions about whether filopodia tension and elongation are sufficient to induce axon growth.

Section snippets

SPION functionalization and characterization

Coupling of 40 nm SPIONs was performed by Ocean Nanotech. Briefly, 0.2 mg of anti-Thy1 antibody (Millipore, Billerica, MA), or Cholera toxin b (Sigma-Aldrich, St Louis, MO) was coupled to 1 mg of 40 nm carboxyl SPIONs (Ocean NanoTech, Springdale, AR) using the carbodiimide method. The SPION coupling was verified by agarose electrophoresis by the manufacturer. SPION hydrodynamic size and homogeneity after functionalization were measured using a Light Scattering Device (Wyatt Technology Corporation,

Results

For this study, we used two types of SPIONs with iron core diameters of either 10 or 40 nm. Due to the greater iron content, the larger SPIONs generated forces about 10-fold greater at 10-100 μm distances from our electromagnet tip (Figure 1, A). To target SPIONs to the RGC surface, we took advantage of GM1-ganglioside³⁵ and Thy-1³⁶ expression on RGC membranes by functionalizing SPIONs with moieties that bind these molecules specifically. Cholera Toxin B and anti-Thy1 antibody were chemically

Discussion

In this study, we have used SPIONs and magnetic fields to demonstrate that mechanical tension applied at the tip of neuronal growth cone filopodia is able to induce filopodia elongation, supporting the idea that an axial force against the tip of the filopodia is sufficient to effectively induce filopodia growth, consistent with the Brownian ratchet model.¹⁸ In our experiments, the application of magnetic fields to SPIONs located at the RGC growth cone membrane produced accumulation of SPIONs at

Acknowledgment

We thank Amber Hackett for helpful comments on the manuscript and Margaret Bates for technical assistance with electron microscopy.

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: Financial support: Department of Defense USAMRAA (W81XWH-12-1-0254, JLG), NEI (EY017971, JLG and P30s EY022589 and EY014801), National Institutes of Health NRSA T32NS007044 (MBS), the James and Esther King Foundation (Technology Transfer Feasibility Grant #2KF03), as well as an unrestricted grant from Research to Prevent Blindness, Inc. These funding sources did not play a role in designing and conducting the research, interpreting the data or writing the manuscript.

: Competing financial interests: The authors declare no competing financial interests.

View full text

Original ArticlePromoting filopodial elongation in neurons by membrane-bound magnetic nanoparticles

Abstract

From the Clinical Editor

Graphical Abstract

Section snippets

SPION functionalization and characterization

Results

Discussion

Acknowledgment

Neuron

Brain Res

Neuron

Brain Res Bull

Neuron

Exp Neurol

Int Rev Cell Mol Biol

Biophys J

Prog Neurobiol

Dev Biol

Biophys J

Neuron

Neurochem Int

J Neurosci Methods

Biophys J

Amacrine-signaled loss of intrinsic axon growth ability by retinal ganglion cells

Science

In-vivo single neuron axotomy triggers axon regeneration to restore synaptic density in specific cortical circuits

Nat Commun

Lesioned corticospinal tract axons regenerate in myelin-free rat spinal cord

Proc Natl Acad Sci U S A

Oncomodulin is a macrophage-derived signal for axon regeneration in retinal ganglion cells

Nat Neurosci

Neuroprotective and axon growth-promoting effects following inflammatory stimulation on mature retinal ganglion cells in mice depend on ciliary neurotrophic factor and leukemia inhibitory factor

J Neurosci

Effects of ocular injury and administration of brain-derived neurotrophic factor on survival and regrowth of axotomized retinal ganglion cells

Proc Natl Acad Sci U S A

Nano neuro knitting: peptide nanofiber scaffold for brain repair and axon regeneration with functional return of vision

Proc Natl Acad Sci U S A

Self-assembling nanofibers inhibit glial scar formation and promote axon elongation after spinal cord injury

J Neurosci

Promoting axon regeneration in the adult CNS by modulation of the PTEN/mTOR pathway

Science

Sustained axon regeneration induced by co-deletion of PTEN and SOCS3

Nature

Original Article
Promoting filopodial elongation in neurons by membrane-bound magnetic nanoparticles