Motor neuron boutons remodel through membrane blebbing
Posted on: 22 June 2021
Preprint posted on 7 March 2021
Article now published in Nature Communications at http://dx.doi.org/10.1038/s41467-023-38421-9
Muscle contraction promotes membrane blebbing during high potassium stimulation in Drosophila motor neurons.
Selected by Mónica Quiñones-FríasCategories: cell biology, neuroscience
Background:
Neurons regulate the activity of other excitable cells by forming stereotypical connections called synapses. Neuronal communication at synapses occurs at axonal swellings called boutons, which are enriched with proteins that regulate synaptic vesicle fusion and trigger neurotransmitter release. However, developing neurons lack boutons, and their formation is triggered when the neuron finds its synaptic partner. In order to guide the neuron towards its target, developing neurons contain specialized structures called growth cones at their axon tips that act as a “GPS”. At growth cones, actin-rich structures called filopodia probe the local environment of the cell to guide it.
Interestingly, mature synapses still possess the ability to remove or add boutons in a phenomenon known as synaptic structural plasticity. In this preprint, Fernandes et al., studied bouton formation in Drosophila neuromuscular junctions, because these neurons display robust structural plasticity following strong stimulation (Ataman et al., 2008; Yoshihara et al., 2005). Previous work found that various signaling pathways regulate the structural plasticity observed in Drosophila motor neurons. However, the physical mechanism that drives the formation of new boutons was unknown. One hypothesis was that new boutons could arise from filipodialike structures (as observed in developing neurons), but these were not commonly observed at nerve terminals, suggesting that new boutons likely formed through an alternate mechanism. Here, Fernandes et al. discovered that Drosophila motor neurons form new boutons through membrane blebbing, a mechanism only previously observed in migrating cells.
Summary:
Figure 1. Time-lapse images of a new bouton forming during high potassium stimulation at the Drosophila neuromuscular junction. (Modified from Figure 1 in Fernandes et al., 2021)
Fernandes et al., 2021 discovered that Drosophila motor neurons form new boutons through membrane blebbing following strong stimulation. In order to investigate this, they used live imaging during nerve stimulation to visualize the formation of new boutons. These experiments showed that new boutons formed within a few minutes of stimulation without signs of filopodia-like structures. However, during their live imaging experiments they observed that new boutons lacked F-actin. This result suggests new boutons are membrane blebs, as previously observed in other cell types.
Another key observation was that the live distribution of F-actin was restricted to the base of new boutons. However, F-actin was later recruited to boutons once they reached full size. This led them to further explore the role of actin in this process. Various drug treatments to disrupt actin polymerization were used to test the role of actin dynamics in bleb formation. Inhibition of actin polymerization led to the formation of larger boutons upon stimulation. In contrast, stabilizing actin polymerization led to the formation of very small boutons that clustered together upon stimulation. Interestingly, neither manipulation affected the total number of boutons formed. Taken together, these results suggest that actin polymerization regulates bleb size and has no role in bouton formation.
Next, they probed the role of Myosin II in bleb formation at the Drosophila neuromuscular junction. Previous studies have shown that blebs can be triggered in conditions of high cortical tension regulated by Myosin II. First, they studied the distribution of Myosin II during stimulation and discovered that boutons formed faster with low levels of Myosin II. This suggests that Myosin II regulates the dynamics of bouton formation but not bouton growth. To test this, they performed a neuron specific knockdown of Myosin II. Low levels of Myosin II led to a significant increase in the number of boutons formed after stimulation. These results suggest that cortical tension generated by Myosin II blocks membrane bending upon muscle contraction.
They next explored if muscle contraction is the main trigger of membrane blebbing at the Drosophila neuromuscular junction. To directly test if forces generated by muscle contraction regulate membrane blebbing, they inhibited it by blocking glutamate receptors or stretching the larval body wall. The formation of new boutons was blocked with both protocols in wild-type and Myosin II knockdowns. These experiments suggest that muscle contraction plays a crucial role in the formation of new boutons upon stimulation.
Taken together, this study discovered that new boutons are formed through membrane blebbing at the Drosophila neuromuscular junction and that the size of these blebs is regulated by actin polymerization. The formation of blebs is triggered by mechanical forces generated from muscle contraction and this is counteracted by cortical tension generated from presynaptic Myosin II. This study sheds light on how new boutons may be formed at mature neuromuscular junctions.
Questions to authors:
- What role does exo/endocytosis play in the formation of new boutons? How would sites of active endocytosis or active zones affect membrane bleb formation?
- Do you think there are mechanosensors that could regulate the acto-myosin cytoskeleton at nerve terminals?
- Previous work has shown that a variety of anterograde and retrograde signaling pathways (Wingless, BMP, and Syt4) are crucial for activity-dependent structural plasticity. Muscle contraction is not altered in many of these mutants, but they all fail to form new boutons. How do you think what you have discovered is regulated by anterograde and retrograde signaling at this synapse?
Bibliography:
Ataman B, Ashley J, Gorczyca M, Ramachandran P, Fouquet W, Sigrist SJ, Budnik V. 2008. Rapid Activity-Dependent Modifications in Synaptic Structure and Function Require Bidirectional Wnt Signaling. Neuron 57:705–718. doi:10.1016/j.neuron.2008.01.026
Yoshihara M, Adolfsen B, Galle KT, Littleton JT. 2005. Retrograde signaling by Syt 4 induces presynaptic release and synapse-specific growth. Science 310:858–863. doi:10.1126/science.1117541
doi: https://doi.org/10.1242/prelights.29719
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