For all its mysteries, the brain can sometimes be described through simple metaphors. For example, communication between neurons works in many ways like a busy shipping depot: components come in, others go out, and everything needs to be standardized to prevent total chaos.
In addition, cargos need to be shipped. Chemical messengers between neurons aren’t just floating around in interstitial space. Instead, they are packaged in membraned vesicles that transport them from one neuron to another.
“Synaptic vesicles are the fundamental units of neuronal communication, packaging neurotransmitters for release,” explains Dr. Jihong Bai. The goal of his lab in the Basic Sciences Division is to understand how neurons communicate.
Vesicles play a key role in this; disruptions to the way vesicles are formed or how frequently they traverse between cells can profoundly disrupt essential life processes from basic movement to learning and memory.
One striking feature of synaptic vesicles is their remarkably uniform size. While the contents of each package vary, vesicles are consistently 30-50 nm in diameter. This is not only a rule for one organism but is constant across animal species from mice to flies to worms.
Vesicles are formed through endocytosis which recycles proteins, amino acids, and membranes. Proteins involved in this process include endocytic adaptor AP180, which facilitates efficient synaptic transmission. Mutations or loss of AP180 in mice and flies dramatically impact vesicle size and have deleterious impacts on synaptic signaling. Furthermore, mutation of the C. elegans ortholog unc-11 results in similar vesicle defects and dramatically reduced worm movement.
However, the mechanism by which AP180 performs this crucial vesicle quality control is not clear. In their recent study in PLOS Biology, the Bai Lab set out to answer this question.
One way to understand the function of any protein is by breaking it into its component parts. For AP180 or its orthologs, there are two main domains: an N-terminal structured domain (called ANTH for AP180 N-terminal Homology domain) and an intrinsically disordered C-terminal Assembly Domain (AD).
The Bai Lab began their study by deleting unc-11 completely in worms. Total knockout of unc-11 resulted in larger and less consistently sized vesicles and reduced worm locomotion, as expected.
This ability of the worms to move around is a readout, or a downstream impact of how efficiently the synapses can signal. To measure the fidelity of synaptic signaling, you need to perform electrophysiology to measure neuronal currents after vesicle fusion. The Bai team is an expert in this technique, and they were able to precisely measure the both the frequency of vesicle fusion as well as the amplitude of the signal stimulated by a fusion event.
The authors then re-introduced unc-11 back into the worms in three different forms: full-length, a truncated version lacking the assembly domain (∆AD), or a version containing only AD. The AD-only construct did not improve the knockout phenotype at all. The control—the full-length unc-11 construct—was able to restore both frequency and amplitude back to wild-type levels, and restored worm movement as well.
Interestingly, the ∆AD construct significantly increased worm movement. It also elevated both frequency and amplitude of post-fusion signaling beyond wild-type levels.
However, the one thing the ∆AD construct did not restore was uniform vesicle size; vesicles in these worms remained as large and irregularly sized as in the unc-11 mutant animals. This suggests that the assembly domain is needed to form properly sized packages.
Importantly, neither the deletion of unc-11 or the restoration of UNC-11 full-length or ∆AD impacts vesicle number. However, it seems as though the size impacts how often vesicles fuse with target neurons and the amplitude of the signal the target neuron sends as a result.
“Our work shows that their size is not merely structural but directly influences how often they release, revealing a mechanism by which vesicle morphology shapes the fidelity of presynaptic signaling,” explains Dr. Bai.
Because the AD domain is intrinsically disordered, there is no structure available to help the authors understand how it controls for size. However, through clever genetic work, the authors were able to untangle this mystery.
In one experiment, they spliced the AD domain of other species into the unc-11 locus. The AP180 AD of both mouse and Drosophila completely restored normal vesicle size. This indicates that the AD domain has a conserved function to ensure consistent vesicle size.
In another experiment, they looked at other endocytic proteins with similar domain organization as AP180. A few of these proteins had large, C-terminal domains that the authors hypothesized may interact with AP180 or other important factors to help shape vesicle size. They tested this by fusing the C-terminal domains of the other endocytic proteins to the C-terminus of UNC-11, and they found that this fusion also rescues vesicle size.
Finally, they were able to home in on a mechanism. Because many other endocytic proteins – including the ones in the paragraph above – interact with actin, they hypothesized that the AD domain interacts directly with actin to shape vesicle size. They showed through genetic and biochemical studies that UNC-11 interacts with actin and that restoring its actin-binding ability alone can allow properly sized vesicles to form.
“These findings elucidate a molecular mechanism by which UNC-11 links the actin cytoskeleton to endocytic membranes through complementary functions of its AD and ANTH domains,” the authors write.
“Going forward, we want to understand how neurons use curvature-sensing mechanisms to fine-tune synaptic transmission with high precision,” says Dr. Bai. “We are also interested in how actin dynamics are regulated at presynaptic terminals and how these processes, together with other protein machinery, help maintain the fidelity of neurotransmission.”
Fred Hutch/University of Washington/Seattle Children’s Cancer Consortium Member Jihong Bai contributed to this research.
The spotlighted research was funded by the National Institute of General Medical Sciences and the National Cancer Institute.
Wang Y, Wu L, Zhang L, Dong Y, Pant A, Liu Y, Bai J. Endocytic protein AP180 assembly domain regulates synaptic vesicle size and release in Caenorhabditis elegans. 2026. PLoS Biol. doi: 10.1371/journal.pbio.3003643
Hannah Lewis is a postdoctoral research fellow with Jim Boonyaratanakornkit’s group in the Vaccine and Infectious Disease Division (VIDD). She is developing screens to find rare B cells that produce protective antibodies against human herpesviruses. She obtained her PhD in molecular and cellular biology from the University of Washington.
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