Written by - Wang Yalong Responsible Editor - Wang Sizhen Editor - Yang Binwei

The continuous neural activity of neurons requires the participation of a large number of synaptic vesicles, and the cell body cannot replenish the consumed vesicles in time, so a variety of vesicle circulation methods are formed within the synapses to ensure the rapid recovery of the membrane and protein components of the synaptic vesicles for reuse[1].


Previous studies have shown that the clathrin mediated endocytosis (CME) is the primary way to recover intrasynaptic vesicle components[2], but in recent years the reported ultrafast endocytosis has attracted increasing interest [3,4].


Launch protein 1 (Dynamin 1, Dyn1) is an important protein involved in multiple endocytosis pathways, exerting GTPase activity during the separation of vesicles from the presynaptic membrane (fission
) [5].

However, Dyn1 often takes longer to recruit from the cytoplasm to the cell membrane to the cell membrane (10-30 s)[6], while ultrafast endocytosis can be done in 50-300 ms at physiological temperature [3,4].


Therefore, this difference in time scale has aroused the interest
of scientists.

Recently, Professor Shigeki Watanabe's research group from the Center for Cellular Biology of Hopkins University and the research group of Professor Christian Rosenmund of Scharetty Medical University in Germany jointly published a research paper entitled "Dynamin is primed at endocytic sites for ultrafast endocytosis" at Neuron.
It was revealed that the dyn1 protein within the synapse can aggregate in advance at the endocytosis site to form a Dyn1 enrichment region, thereby quickly starting the ultrafast endocytosis mode and making it complete
within 100 ms.

They reported that Dyn1A is the primary Dyn1 subtype that plays a role in ultrafast endocytosis, forming a biomolecular condensate on cell membranes by interacting with another cell-eating protein, Syndapin 1
.

By blocking their interactions through point mutations, this molecular dense no longer forms and the ultrafast endocytosis slows down by about 100 times
.

Thus, the Syndapin 1 protein, as a adapter protein, enriches and anchors Dyn1A to the cell membrane at the cell membrane, skipping the usual rate-limiting step of protein recruitment, thereby speeding up the rate of endocytosis
within the synapse.

Dyn1's involvement in ultrafast endocytosis has been reported in previous studies [4].

Figure 1.
The Dyn1A subtype mediates rapid endocytosis
.

(Source: Imoto Y et al.

Now that it is microstructureally clear that Dyn1A is involved in the rapid endocytosis, how is Dyn1A distributed within synapses? The authors overexpressed Dyn1A/B-GFP in primary cultured hippocampal neurons, using Synaptobrevin 2 (Syb2) as a presynaptic marker, and then observed under confocal microscopy that more Dyn1A signals were distributed presynapses (51.
1% vs 22.
4%), while Dyn1B was more diffuse throughout the axons (Figure 2A-C).


And by locating the peak distributions of Dyn1A and Syb2 signals, it was found that the Dyn1A signals were mainly clustered near the active region (<100 nm) (Figure 2E-F).


The Dyn1A/B-GFP knock-in method using CRISPR/Cas9 can eliminate the non-specificity caused by overexpression, and ultra-high-resolution STED microscopy has obtained similar results (Figure 2G-L
).

Not only that, by using a weak detergent Digitonin, increasing the permeability of the cell membrane so that small molecules in the cytoplasm can spread out of the cell and retain the organelle components in the cell, and it was found that although the Dyn1A signal intensity was reduced by about 40%, the signal spot (puncta) was still distributed near Syb2, and the tdTomato signal expressed in the cytoplasm was quickly washed out (Figure 2M-O).
Indicates that Dyn1A signaling is attached to the
cell membrane.

Similar results were observed
in living neurons.

The synthesis showed that the Dyn1A protein prematurely aggregated in the endocytotic region
on the cell membrane.

Figure 2.
The Dyn1A subtype is enriched in presynaptic structures
.

(Source: Imoto Y et al.

Next, the authors used live-cell imaging techniques and FRAP techniques to examine the behavior of overexpressed Dyn1A-GFP neuron synapse Dyn1A puncta signals
.

The results showed that the Dyn1A fluorescence signal quenched on the axon could recover quickly.
The recovery of the fluorescence signal was significantly slower when the Dyn1A signal of the entire synapse was quenched (Figure 3A-D), indicating that the synaptic Dyn1A puncta had a potential droplet state and suggesting that Dyn1A may be able to form a molecular condensates
.

Similar results were observed
in COS-7 viable cells.

In order to further explore whether Dyn1A's activity has phase separation characteristics, the authors used a variety of fatty alcohols to interrupt weak hydrophobic binding and found that the Dyn1A signal was diffused along the axons within 30-60 s, while the Dyn1A signal in the control group could remain stable for more than 60 s (Figure 3 E&F), indicating that the Dyn1A signal did have phase
。
At the same time, follow-up experiments further showed that Dyn1A puncta is droplet-like, and a small part of the Dyn1A molecule may bind
to proteins or membranes within puncta.

Figure 3.
Dyn1A is distributed presynaptically with phase-separated features
.

(Source: Imoto Y et al.
, Neuron, 2022)
Multiple endocytotic proteins, such as Dyn1, are involved in the modification state
of cytosis dependent on phosphorylation and dephosphoric acid.

GSK3b and CDK5 can be phosphorylated at the S774 and S778 sites of the proline-rich motif module (proline-rich motif) of Dyn1, respectively.
Calcineurin can be dephosphorylated at the S774/S778 site of Dyn1, increasing the interaction between Dyn1 and another endocytotic protein, Syndapin 1 [7].


To demonstrate whether the phosphorylation or dephosphorylation state of Dyn1 is involved in phase isolation regulation of Dyn1A, the authors constructed two mutant types of Dyn1A: Dyn1A S774/778D, which is persistently phosphorylated, and Dyn1A S774/778A
, which cannot be phosphorylated (i.
e.
, maintained dephosphorylation).

Observe the distribution
of mutant Dyn1A in neurons by expressing it in synapses.

It was found that the total fluorescence level and distribution of the dephosphorylated type Dyn1A S774/778A were consistent with WT neurons, while the signal of the phosphorylated type Dyn1A S774/778D was more diffuse and widely distributed on axons, and the fluorescence intensity of the signal puncta was also significantly reduced (Figure 4).


Shows that the enrichment of Dyn1A before synapses forms a signal puncta that relies on the dephosphorylation
of Dyn1A.
Figure 4.
Dephosphorylation of the dyn1A proline enrichment module is necessary for phase separation
.

(Credit: Imoto Y et al.
, Neuron, 2022)
So, how does Dyn1A aggregate next to the active region on the cell membrane? Syndapin 1 is an important interacting protein of Dyn1A that binds to Dyn1A's proline enrichment module through the SH3 domain, while Syndapin 1 contains a BAR domain capable of binding to cell membranes [8].


To explore whether Syndapin 1 is also involved in presynaptic enrichment of Dyn1A puncta, the authors first performed Syndapin 1 knockdown
in neurons.

In the Syndapin 1 knockdown neurons, not only did the Dyn1A signal begin to spread along the axons, but the remaining Dyn1A puncta signal strength within the synapses also weakened
significantly.

Not only that, but under transmission electron microscopy, the endocytosis of ferritin by synapses is significantly slowed down, and the formation of vesicles from LEVs is also blocked
.

Reexpressing wild-type Syndapin 1 was able to salvage these phenotypes, while Syndapin 1 ID/ME, which could not bind to the cell membrane, and Syndapin 1 P434L, which could not bind to Dyn1A, could not salvage these phenotypes, indicating that Syndapin 1 is likely to act as an important core protein, enriching Dyn1A to the site next to the active region before endocytosis, greatly accelerating the process of endocytosis
。 Figure 5.
Syndapin 1 is necessary for ultrafast endocytosis
.

The left side of the EM is the unstimulated control group, the middle is after 1 s, and the right side is after
10 s.

(Source: Imoto Y et al.
, Neuron, 2022)
Figure 6.
Illustrating the difference in time scale between CME and ultrafast endocytosis
.

(Source: Imoto Y et al.
, Neuron, 2022)
Conclusion and discussion, inspiration and prospects

Combining the results of the above series of experiments, the authors propose a reasonable hypothesis to explain the difference in time scales between different types of endocytotic pathways (Figure 6
).

In the meshin-mediated endocytosis pathway (CME), the initiator protein (Dynamin) and other endocytotic proteins need to be recruited from the cytoplasm, and this process is often relatively slow, taking more than ten seconds or even tens of seconds; But for ultrafast endocytosis, the Dyn1A protein is prematurely enriched before synapses to form the Dyn1A protein puncta, the maintenance of which relies on the interaction of Dyn1A's proline enrichment module with Syndapin 1's SH3 module, and anchoring Dyn1A puncta on
the cell membrane next to the active region.

A series of mutations or drug treatments interrupting their interactions blocks this enrichment state, allowing proteins to be re-recruited from the cytoplasm, ultimately slowing down the rate of endocytosis
.

Through a series of work, this paper explains the working mechanism of ultrafast endocytosis from multiple angles: the Dyn1A protein aggregates in advance at the endocytosis site to form a Dyn1A enrichment region, which greatly accelerates the ultrafast endocytosis (Figure 6).


The proposed theory provides an important clue
to the interpretation of the time-scale differences between different vesicle recovery methods in the vesicle cycle.

However, previous literature has reported that ultrafast endocytosis can only be performed at physiological temperature, and CME is also considered to be mainly performed
at room temperature.

Therefore, the regulation of cytosis pathways such as ultrafast endocytosis at different temperatures is also a rather interesting topic, and may become the next hot topic
of argument.
Original link: https://doi.
org/10.
1016/j.
neuron.
2022.
06.
010
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[2] Trailer | Conference on Neuromodulation and Brain-Computer Interface (October 13-14, Beijing Time)

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References (swipe up and down to read)

[1] Chanaday N L, Cousin M A, Milosevic I, et al.
The Synaptic Vesicle Cycle Revisited: New Insights into the Modes and Mechanisms[J].
J Neurosci, 2019, 39(42): 8209-8216.

[2] Granseth B, Odermatt B, Royle S J, et al.
Clathrin-mediated endocytosis is the dominant mechanism of vesicle retrieval at hippocampal synapses[J].
Neuron, 2006, 51(6): 773-86.

[3] Watanabe S, Liu Q, Davis M W, et al.
Ultrafast endocytosis at Caenorhabditis elegans neuromuscular junctions[J].
Elife, 2013, 2: e00723.

[4] Watanabe S, Rost B R, Camacho-Perez M, et al.
Ultrafast endocytosis at mouse hippocampal synapses[J].
Nature, 2013, 504(7479): 242-247.

[5] Praefcke G J, McMahon H T.
The dynamin superfamily: universal membrane tubulation and fission molecules? [J].
Nat Rev Mol Cell Biol, 2004, 5(2): 133-47.

[6] Taylor M J, Perrais D, Merrifield C J.
A high precision survey of the molecular dynamics of mammalian clathrin-mediated endocytosis[J].
PLoS Biol, 2011, 9(3): e1000604.

[7] Anggono V, Smillie K J, Graham M E, et al.
Syndapin I is the phosphorylation-regulated dynamin I partner in synaptic vesicle endocytosis[J].
Nat Neurosci, 2006, 9(6): 752-60.

[8] Dharmalingam E, Haeckel A, Pinyol R, et al.
F-BAR proteins of the syndapin family shape the plasma membrane and are crucial for neuromorphogenesis[J].
J Neurosci, 2009, 29(42): 13315-27.

End of article