A diffusion-based neurite length-sensing mechanism involved in neuronal symmetry breaking

Shootin1, one of the earliest markers of neuronal symmetry breaking, accumulates in the neurite tips of polarizing neurons in a neurite length-dependent manner. Thus, neurons sense their neurites' length and translate this spatial information into a molecular signal, shootin1 concentration. Quantitative live cell imaging of shootin1 dynamics combined with mathematical modeling analyses reveals that its anterograde transport and retrograde diffusion in neurite shafts account for the neurite length-dependent accumulation of shootin1. The neurite length-dependent shootin1 accumulation and shootin1-induced neurite outgrowth constitute a positive feedback loop that amplifies stochastic shootin1 signals in neurite tips. Quantitative mathematical modeling shows that the above positive feedback loop, together with shootin1 upregulation, constitutes a core mechanism for neuronal symmetry breaking.
Cell morphology and size must be properly controlled to ensure cellular function. Although there has been significant progress in understanding the molecular signals that change cell morphology, the manner in which cells monitor their size and length to regulate their morphology is poorly understood. Cultured hippocampal neurons polarize by forming a single long axon and multiple short dendrites (Craig and Banker, 1994; Arimura and Kaibuchi, 2007), and symmetry breaking is the initial step of this process. This symmetry-breaking step reproduces even when the neuronal axon is transected; the longest neurite usually grows rapidly to become an axon after transection, regardless of whether it is the axonal stump or another neurite (Goslin and Banker, 1989). Elongation of an immature neurite by mechanical tension also leads to its axonal specification (Lamoureux et al, 2002). These results suggest that cultured hippocampal neurons can sense neurite length, identify the longest one, and induce its subsequent axonogenesis for symmetry breaking. However, little is known about the mechanism for this process. Shootin1 is one of the earliest markers of neuronal symmetry breaking (Toriyama et al, 2006). During the symmetry-breaking step, it undergoes a stochastic accumulation in neurite tips, and eventually accumulates predominantly in a single neurite that subsequently grows to become an axon. In this study, we demonstrated that shootin1 accumulates in neurite tips in a neurite length-dependent manner, regardless of whether it is the axonal stump or another neurite (Figure 3A, C–F). Thus, morphological information (neurite length) is translated into a molecular signal (shootin1 concentration in neurite tips). We previously reported that shootin1 is transported from the cell body to neurite tips as discrete boluses and diffuses back to the cell body (Toriyama et al, 2006). The boluses containing variable amounts of shootin1 traveled repeatedly but irregularly along neurites, and their arrival caused large stochastic fluctuations in shootin1 concentration in the neurite tips. To understand the mechanism of length-dependent shootin1 accumulation, we performed quantitative live cell imaging of the anterograde transport and retrograde diffusion of shootin1 and fitted the obtained data into mathematical models of the anterograde transport and retrograde diffusion. The parameters of these two models were derived entirely from quantitative experimental data, without any adjustment. Shootin1 concentration at neurite tips, calculated by integrating the two models, was neurite length dependent (Figure 3B) and showed good agreement with the experimental data (Figure 3A). These results suggest that the neurite length-dependent accumulation of shootin1 is quantitatively explained by its anterograde transport and retrograde diffusion. This length-dependent shootin1 accumulation constitutes a positive feedback interaction with the previously reported shootin1-induced neurite outgrowth (Shimada et al, 2008). To analyze the functional role of this feedback loop, we quantified shootin1 upregulation (Toriyama et al, 2006) and shootin1-induced neurite outgrowth, and integrated them, together with the above model of length-dependent shootin1 accumulation, into a model neuron (Figure 7A). Furthermore, the parameters of the model components were chosen to give the best fit to the quantitative experimental data without any adjustment. Integrating the three components into a model neuron resulted in spontaneous symmetry breaking (Figure 7B and C). Furthermore, there are a total of 15 agreements between the model predictions and the experimental data, including the neurite length-dependent axon specification and regeneration (Goslin and Banker, 1989; Lamoureux et al, 2002). These data suggest that the three components in our model—namely, diffusion-based neurite length sensing system, shootin1-induced neurite outgrowth and shootin1 upregulation—are sufficient to induce neuronal symmetry breaking. Bolus-like transport of shootin1 caused large stochastic fluctuations in shootin1 concentration in neurite tips. Interestingly, the generation of continuous shootin1 transport in our model neuron impaired the symmetry-breaking process (Figure 7D). This is consistent with theoretical models in which feedback amplification of fluctuations in signaling can give rise to robust patterns (Turing, 1952; Meinhardt and Gierer, 2000; Kondo, 2002), and underscores the importance of the stochastic fluctuating signals in spontaneous neuronal symmetry breaking. The combination of quantitative experimentation and mathematical modeling is regarded as a powerful strategy for attaining a profound understanding of biological systems (Hodgkin and Huxley, 1952b; Lewis, 2008; Ferrell, 2009). By focusing on a simple system involving one of the earliest markers of neuronal symmetry breaking, shootin1, we were able to evaluate here the core components of neuronal symmetry breaking on the basis of quantitative experimental data. The present model may thus provide a core mechanism of neuronal symmetry breaking, to which other possible mechanisms can be added to increase the model's complexity in future studies.
Although there has been significant progress in understanding the molecular signals that change cell morphology, mechanisms that cells use to monitor their size and length to regulate their morphology remain elusive. Previous studies suggest that polarizing cultured hippocampal neurons can sense neurite length, identify the longest neurite, and induce its subsequent outgrowth for axonogenesis. We observed that shootin1, a key regulator of axon outgrowth and neuronal polarization, accumulates in neurite tips in a neurite length-dependent manner; here, the property of cell length is translated into shootin1 signals. Quantitative live cell imaging combined with modeling analyses revealed that intraneuritic anterograde transport and retrograde diffusion of shootin1 account for its neurite length-dependent accumulation. Our quantitative model further explains that the length-dependent shootin1 accumulation, together with shootin1-dependent neurite outgrowth, constitutes a positive feedback loop that amplifies stochastic fluctuations of shootin1 signals, thereby generating an asymmetric signal for axon specification and neuronal symmetry breaking.