Motor neuron boutons remodel through membrane blebbing

Andreia R. Fernandes, César S. Mendes, Edgar R. Gomes, Rita O. Teodoro

Preprint posted on 7 March 2021 https://www.biorxiv.org/content/10.1101/2021.03.07.434250v2?ct=

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-Frías

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

Posted on: 22 June 2021

doi: https://doi.org/10.1242/prelights.29719

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Author's response

Andreia Fernandes and Rita Teodoro shared

It was previously suggested in Piccioli and Littleton (2014) that local endosomal dynamics occur during activity-dependent bouton formation. They observed syntaxin-13, a t-SNARE protein that decorates endocytic compartments and presynaptic membrane, puncta near synaptic growth sites. Additionally, when we expressed CD4-Tom in neurons (using nSybGal4 driver) to label the membrane we could detect on many occasions a bright membrane punctum at the base of newly formed boutons. Our data suggests that local intracellular vesicle dynamics may be associated with bouton growth. Interestingly, Vasin et. (2019) showed that activity mainly promotes formation of boutons filled with synaptic vesicles (SVs) and these boutons are capable of exo/endocytosis (>30% of new boutons demonstrated prominent FM1-43 loading, which was comparable to that in mature boutons, and all the boutons loaded with FM1-43 destained by ~80% during subsequent stimulation with no dye added). Blebs are membrane protrusions that are produced in conditions of high cortical contractility, high confinement, and low adhesion. Since we show that boutons form by blebbing, it is plausible that active sites of exo/endocytosis may contribute to local membrane addition, which would support membrane expansion. For instance, one can envisage a scenario where fusion of SVs or other vesicles, either at active zones, or peri-synaptic sites can accommodate growth. Additionally, it is also possible that cell adhesion proteins need to be recycled at the bouton formation sites, allowing downregulation of adhesion at these sites, therefore favoring blebbing.
Blebs nucleate at sites where there is weakening of membrane-actin cortex interactions, and, if there is enough cortical tension to generate intracellular pressure in the cytoplasm, expand as round membrane protrusions driven by fluid flow. Our hypothesis is that muscle contraction has a mechanical role during activity-dependent bouton formation at the NMJ by providing confinement to motor neurons (and therefore increasing neuronal cortical tension) during elevated activity. Additionally, muscle contraction can also act as mechanical cue that (together with chemical cues) helps to determine/specify where new boutons will be formed. This would be possible by coupling actin dynamics to muscle contraction. In this scenario we can envisage a mechanosensor that could regulate the acto-myosin cytoskeleton at nerve terminals. For instance, by allowing membrane detachment from the cortex in regions of high muscle force.
We show that muscle contraction is required for activity induced bouton formation. It is well established that activity-dependent structural plasticity requires several signaling pathways (cAMP/PKA, Wingless, BMP, Syt4). The fact that muscle contraction is not altered in many of these mutants, but they all fail to form new boutons suggests that muscle contraction per si is not enough to induce bouton formation. Accordingly, in our movies we sometimes observe muscle contractions without formation of new boutons. Moreover, it indicates that, in addition to mechanical cues, a chemical signal is required to prime plasticity at the NMJ. Thereby, one hypothesis is that in response to elevated activity, and in response to a still unclear signal to initiate bouton growth, neurons add new boutons by membrane blebbing using a mechanism where muscle contraction provides an external source of force being required to increase neuronal confinement, and consequently cortical tension, which powers bouton outgrowth at primed regions. This hypothesis is in agreement with blebs being favored in compressive 3-D environments. However, if the signal/s required to prime plasticity at Drosophila NMJ is neuron derived, muscle derived, or from both cells is yet to be determined. As has been proposed for blebbing cells in Asante-Asamani et. (2020), it is possible that an external signal, for instance a muscle derived factor released in regions of higher activity, can polarize the recruitment of presynaptic molecules required to/or that facilitate bouton formation by blebbing in regions of high membrane tension.

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Motor neuron boutons remodel through membrane blebbing

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