Devrim Kilinc

Low Piconewton Towing of CNS Axons against Diffusing and Surface-Bound Repellents Requires the Inhibition of Motor Protein-Associated Pathways

Growth cones, dynamic structures at axon tips, integrate chemical and physical stimuli and translate them into coordinated axon behaviour, e.g., elongation or turning. External force application to growth cones directs and enhances axon elongation in vitro; however, direct mechanical stimulation is rarely combined with chemotactic stimulation. We describe a microfluidic device that exposes isolated cortical axons to gradients of diffusing and substrate-bound molecules, and permits the simultaneous application of piconewton (pN) forces to multiple individual growth cones via magnetic tweezers. Axons treated with Y-27632, a RhoA kinase inhibitor, were successfully towed against Semaphorin 3A gradients, which repel untreated axons, with less than 12 pN acting on a small number of neural cell adhesion molecules. Treatment with Y-27632 or monastrol, a kinesin-5 inhibitor, promoted axon towing on substrates coated with chondroitin sulfate proteoglycans, potent axon repellents. Thus, modulating key molecular pathways that regulate contractile stress generation in axons counteracts the effects of repellent molecules and promotes tension-induced growth. The demonstration of parallel towing of axons towards inhibitory environments with minute forces suggests that mechanochemical stimulation may be a promising therapeutic approach for the repair of the damaged central nervous system, where regenerating axons face repellent factors over-expressed in the glial scar.

Bio-Nano-Magnetic Materials for Localized Mechanochemical Stimulation of Cell Growth and Death

Magnetic nanoparticles are promising new tools for therapeutic applications, such as magnetic nanoparticle hyperthermia therapy and targeted drug delivery. Recent in vitro studies have demonstrated that a force application with magnetic tweezers can also affect cell fate, suggesting a therapeutic potential for magnetically modulated mechanical stimulation. The magnetic properties of nanoparticles that induce physical responses and the subtle responses that result from mechanically induced membrane damage and/or intracellular signaling are evaluated. Magnetic particles with various physical, geometric, and magnetic properties and specific functionalization can now be used to apply mechanical force to specific regions of cells, which permit the modulation of cellular behavior through the use of spatially and time controlled magnetic fields. On one hand, mechanochemical stimulation has been used to direct the outgrowth on neuronal growth cones, indicating a therapeutic potential for neural repair. On the other hand, it has been used to kill cancer cells that preferentially express specific receptors. Advances made in the synthesis and characterization of magnetic nanomaterials and a better understanding of cellular mechanotransduction mechanisms may support the translation of mechanochemical stimulation into the clinic as an emerging therapeutic approach.

Magnetic Tweezers-Based Force Clamp Reveals Mechanically Distinct apCAM Domain Interactions

Cell adhesion molecules of the immunoglobulin superfamily (IgCAMs) play a crucial role in cell-cell interactions during nervous system development and function. The Aplysia CAM (apCAM), an invertebrate IgCAM, shares structural and functional similarities with vertebrate NCAM and therefore has been considered as the Aplysia homolog of NCAM. Despite these similarities, the binding properties of apCAM have not been investigated thus far. Using magnetic tweezers, we applied physiologically relevant, constant forces to apCAM-coated magnetic particles interacting with apCAM-coated model surfaces and characterized the kinetics of bond rupture. The average bond lifetime decreased with increasing external force, as predicted by theoretical considerations. Mathematical simulations suggest that the apCAM homophilic interaction is mediated by two distinct bonds, one involving all five immunoglobulin (Ig)-like domains in an antiparallel alignment and the other involving only two Ig domains. In summary, this study provides biophysical evidence that apCAM undergoes homophilic interactions, and that magnetic tweezers-based, force-clamp measurements provide a rapid and reliable method for characterizing relatively weak CAM interactions.

Microtechnologies for studying the role of mechanics in axon growth and guidance.

The guidance of axons to their proper targets is not only a crucial event in neurodevelopment, but also a potential therapeutic target for neural repair. Axon guidance is mediated by various chemo- and haptotactic cues, as well as the mechanical interactions between the cytoskeleton and the extracellular matrix (ECM). Axonal growth cones, dynamic ends of growing axons, convert external stimuli to biochemical signals, which, in turn, are translated into behavior, e.g., turning or retraction, via cytoskeleton–matrix linkages. Despite the inherent mechanical nature of the problem, the role of mechanics in axon guidance is poorly understood. Recent years has witnessed the application of a range of microtechnologies in neurobiology, from microfluidic circuits to single molecule force spectroscopy. In this mini-review, we describe microtechnologies geared towards dissecting the mechanical aspects of axon guidance, divided into three categories: controlling the growth cone microenvironment, stimulating growth cones with externally applied forces, and measuring forces exerted by the growth cones. A particular emphasis is given to those studies that combine multiple techniques, as dictated by the complexity of the problem.

Neuron Subpopulations with Different Elongation Rates and DCC Dynamics Exhibit Distinct Responses to Isolated Netrin-1 Treatment

Correct wiring of the nervous system requires guidance cues, diffusible or substrate-bound proteins that steer elongating axons to their target tissues. Netrin-1, the best characterized member of the Netrins family of guidance molecules, is known to induce axon turning and modulate axon elongation rate; however, the factors regulating the axonal response to Netrin-1 are not fully understood. Using microfluidics, we treated fluidically isolated axons of mouse primary cortical neurons with Netrin-1 and characterized axon elongation rates, as well as the membrane localization of deleted in colorectal cancer (DCC), a well-established receptor of Netrin-1. The capacity to stimulate and observe a large number of individual axons allowed us to conduct distribution analyses, through which we identified two distinct neuron subpopulations based on different elongation behavior and different DCC membrane dynamics. Netrin-1 reduced the elongation rates in both subpopulations, where the effect was more pronounced in the slow growing subpopulation. Both the source of Ca(2+) influx and the basal cytosolic Ca(2+) levels regulated the effect of Netrin-1, for example, Ca(2+) efflux from the endoplasmic reticulum due to the activation of Ryanodine channels blocked Netrin-1-induced axon slowdown. Netrin-1 treatment resulted in a rapid membrane insertion of DCC, followed by a gradual internalization. DCC membrane dynamics were different in the central regions of the growth cones compared to filopodia and axon shafts, highlighting the temporal and spatial heterogeneity in the signaling events downstream of Netrin-1. Cumulatively, these results demonstrate the power of microfluidic compartmentalization and distribution analysis in describing the complex axonal Netrin-1 response.

Neuronal Cell Bodies Remotely Regulate Axonal Growth Response to Localized Netrin-1 Treatment via Second Messenger and DCC Dynamics

Netrin-1 modulates axonal growth direction and speed. Its best characterized receptor, Deleted in Colorectal Cancer (DCC), is localized to growth cones, but also observed in the cell bodies. We hypothesized that cell bodies sense Netrin-1 and contribute to axon growth rate modulation, mediated by the second messenger system. We cultured mouse cortical neurons in microfluidic devices to isolate distal axon and cell body microenvironments. Compared to isolated axonal treatment, global Netrin-1 treatment decreased the axon elongation rate and affected the dynamics of total and membranous DCC, calcium, and cyclic nucleotides. Signals induced by locally applied Netrin-1 propagated in both anterograde and retrograde directions, demonstrated by the long-range increase in DCC and by the increased frequency of calcium transients in cell bodies, evoked by axonal Netrin-1. Blocking the calcium efflux from endoplasmic reticulum suppressed the membranous DCC response. Our findings support the notion that neurons sense Netrin-1 along their entire lengths in making axonal growth decisions.